Volume / tomas 27(2) - Sodininkystė ir daržininkystė - Sodininkystės ...

Lietuvos SODININKYSTĖs ir daržininkystės instituto ir
lietuvos žemės ūkio universiteto mokslo darbai
Scientific works of the Lithuanian institute of
horticulture AND lITHUANIAN UNIVERSITY OF AGRICULTURE
SODININKYSTĖ ir daržininkystė
27(2)
Eina nuo 1983 m.
Published since 1983
Babtai 2008

UDK 634/635 (06)
Redaktorių kolegija
Editorial Board
Doc. dr. Česlovas BOBINAS – pirmininkas (LSDI, biomedicinos mokslai, agronomija),
prof. habil. dr. Pavelas DUCHOVSKIS (LSDI, biomedicinos mokslai, agronomija),
dr. Edite KAUFMANE (Latvija, Dobelės sodo augalų selekcijos stotis, biomedicinos mokslai, biologija),
dr. Aleksandras KMITAS (LŽŪU, biomedicinos mokslai, agronomija),
dr. Laimutis RAUDONIS (LSDI, biomedicinos mokslai, agronomija),
prof. habil. dr. Vidmantas STANYS (LSDI, biomedicinos mokslai, agronomija),
prof. habil. dr. Andrzej SADOWSKI (Varрuvos ŽŪA, biomedicinos mokslai, agronomija),
dr. Audrius SASNAUSKAS (LSDI, biomedicinos mokslai, agronomija),
prof. habil. dr. Algirdas SLIESARAVIČIUS (LŽŪU, biomedicinos mokslai, agronomija).
Redakcinė mokslinė taryba
Editorial Scientific Council
Doc. dr. Česlovas BOBINAS – pirmininkas (Lietuva),
prof. habil. dr. Pavelas DUCHOVSKIS (Lietuva), dr. Kalju KASK (Estija),
dr. Edite KAUFMANE (Latvija), prof. habil. dr. Zdzisław KAWECKI (Lenkija),
prof. habil. dr. Albinas LUGAUSKAS (Lietuva), habil. dr. Maria LEJA (Lenkija),
prof. habil. dr. Lech MICHALCZUK (Lenkija), prof. habil. dr. Andrzej SADOWSKI (Lenkija),
dr. Audrius SASNAUSKAS (Lietuva), prof. dr. Ala SILAJEVA (Ukraina),
prof. habil. dr. Algirdas SLIESARAVIČIUS (Lietuva),
prof. habil. dr. Vidmantas STANYS (Lietuva), prof. dr. Viktor TRAJKOVSKI (Švedija).
Redakcijos adresas:
Lietuvos Sodininkystės ir daržininkystės institutas
LT-54333 Babtai, Kauno r.
Tel. (8 37) 55 52 10
Faksas (8 37) 55 51 76
El. paрtas institutas@lsdi.lt
Pilnas tekstas http://vddb.library.lt/obj/LT-eLABa-0001:J.02~1983~ISSN_0236-4212
Address of the Editorial Office:
Lithuanian Institute of Horticulture
LT-54333 Babtai, Kaunas district, Lithuania
Phone: +370 37 55 52 10
Telefax: +370 37 55 51 76
E-mail institutas@lsdi.lt
Full text http://vddb.library.lt/obj/LT-eLABa-0001:J.02~1983~ISSN_0236-4212
Leidinio adresas internete www.lsdi.lt
Leidinys cituojamas CAB Internacional ir VINITI duomenų bazėse
© Lietuvos Sodininkystės ir daržininkystės institutas, 2008
© Lietuvos žemės ūkio universitetas, 2008

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
The vicennial of scientific searches in the field of plant
physiology
Pavelas Duchovskis
Lithuanian Institute of Horticulture, Kauno 30, 54333 Babtai, Kaunas distr.,
Lithuania, e-mail: p.duchovskis@lsdi.lt
The Laboratory of Plant Physiology at LIH was established on 16 th of April, 1988. From
the beginning the striving was to create the modern scientific base and an actual topic for
the researches. Today there is established the phytotron complex with 10 climate chambers,
experimental greenhouses and vegetative field. The laboratory is provided with automatic
equipment for analysis of plant physiological processes, there are high-performance liquid and
gas chromatography systems and other analytical equipment.
The following main scientific directions were formed in laboratory: plant morphogenesis,
flowering initiation, ecophysiology, photophysiology and plant productivity physiology.
The main subjects of Plant Physiology laboratory are designed for the development of plant
flowering initiation theory and for investigations of plant morphogenesis. The model of flowering
initiation in wintering and biennial plants was developed in the laboratory (P. Duchovskis). The
mechanisms of photo and thermo induction, evocation and flower initiation and differentiation
are investigated. The role of phytohormones, carbohydrates and other metabolites is analysed in
these processes (P. Duchovskis, G. Samuolienė, A. Urbonavičiūtė, G. Šabajevienė, A. Kurilčik).
The possibility to understand and manipulate the processes of plant growth and development
permits to intensify the technologies in horticulture.
The photophysiological investigations are carried out on purpose to design the new
generation semiconductor lamps for plant irradiation in greenhouses, phytotron and for in vitro
cultivation systems (P. Duchovskis, A. Urbonavičiūtė, G. Samuolienė, A. Kurilčik, A. Brazaitytė,
R. Ulinskaitė). The parameters of such lamps can be very easily changed or programmed, thus
allows to control plant photosynthetic, photomorphogenetic, phototropic processes and to affect
the trend of plant metabolic processes. These works are carried out together with physics of
Vilnius University (prof. A. Žukauskas). The significant results presumed to attract the private
capital for the patent of these data and for the expanding of new plant irradiation technology
business. The offset company UAB HORTILED was established.
Very important direction of our investigation is the influence of unfavorable natural and
anthropogenic factors on plant physiological and biochemical indexes. Also it is important
to determine the tolerance rate and adaptivity for various stresses and concurrent capacity in
conditions of volatile climate and environmental pollution (A. Brazaitytė, J. Sakalauskaitė,
S. Sakalauskienė, G. Samuolienė, A. Urbonavičiūtė, G. Šabajevienė, P. Duchovskis). These are
the complex works, which are carried out with other institutions of agricultural and biological
profile. The final goal of these investigations is to formulate the recommendations for the
stability increasing in agriculture and forestry, rural and forest ecosystems.
The physiological investigations of field vegetables and other crop and garden plants are
expanded in our laboratory (A. Brazaitytė, J. B. Šikšnianienė, G. Šabajevienė, P. Duchovskis). It
allows to notice on time the variation of photosynthetic parameters and to control these processes
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y technological means on purpose to improve plant productivity and quality of production.
It was published over 500 scientific publications, whereof 250 in international, Lithuanian
and foreign publications. There was defended 1 degree of habilitated doctor and 4 doctoral
degrees.
Currently there are working tree research workers, two technicians and six PhD students
are studying in the laboratory of plant physiology.
Key words: flowering initiation, photophysiology, phytomonitoring, stress physiology.
Introduction. The methods of plant physiology play an important role solving
theoretical problems and these of the applied agriculture. The knowledge of the
physiological regularities of plants allows solving the questions of plant production
quality. The main problems, which were solved in recent decades by the physiologists of
LIH increasing the efficiency of agriculture were as follows: plant morphogenesis and
flowering initiation (Duchovskis, 1995, 1996, 1998 a , 2000, 2004, 2006; Duchovskienė,
1996, 2000; McCall, Brazaitytė 1997; Bandaravičienė et al., 1999; Duchovskienė
et al., 1999, 2000, 2004; Duchovskis et al., 2000 a , 2000 b , 2003 b , 2004; Šikšnianienė
et al., 2000,; Šikšnianienė, 2002, 2005, 2006; Samuolienė et al., 2005 a , 2005 b , 2006 a ,
2006 b , 2006 c , 2007, 2008; Samuolienė, 2007), the physiology of plant productivity
(Brazaitytė, 1996, 1998, 1999, 2000; Čelkienė, 1998, 1991; Brazaityje, Jankauskiene,
2000; Tarvydienė et al., 2004 a , 2004 b ; Baranauskis et al., 2005; Šabajevienė et al.,
2005, 2006 b , 2007; Šikšnianienė et al., 2006, 2007; Tranavičienė et al., 2007), and plant
production quality (Karitonas et al., 1996, Rubinskienė et al., 2006), optimization of
photophysiological processes (Brazaitytė et al., 1994, 2004, 2006; Jankauskienė et al.,
2001, 2002; Bliznikas et al., 2004; Samuoliene et al., 2004, 2006 d ; Tamulaitis et al.,
2004 a , 2004 b , 2005; Ulinskaitė et al., 2004; Grishenkova et al., 2006; Urbonavičiūtė
et al., 2007 a , 2007 b ; Kurilčik et al., 2007, 2008), resistance to ecological stress and
adaptivity (Duchovskis, 1998 b ; Gelvonauskis et al., 1999, 2000; Duchovskis et al.,
2001, 2003 a , 2005, 2006, 2007; Brazaitytė et al., 2001, 2005, 2007; Bashmakov et al.,
2005, 2007; Raklevičienė et al., 2005; Juknys et al., 2006; Sakalauskaitė et al., 2006 a ,
2006 b ; Urbonavičiūtė et al., 2006 a , 2006 b ; Baranauskis et al., 2006; Ramaškevičienė
et al., 2006; Šabajevienė et al., 2006 a ; Lukatkin et al., 2007; Juozaitytė et al., 2007;
Sakalauskienė et al., 2007, 2008), the increase of seed growing efficiency and seed
quality (Stašelis et al., 1996, 1999; Orentienė et al., 1998, 2000; Duchovskis et al.,
2001; Šikšnianienė et al., 2006).
Productivity of plants and the desirable quality indices realize themselves in
ontogenesis. Therefore, it is very important to learn to control plant physiological
parameters in the different stages of growth and development. The theory of plant
flowering initiation created in Plant Physiology Laboratory of LIH formed the basis
for the control of plants morphogenesis by technological means in the desirable
direction (Duchovskis, 1995, 1996, 2000, 2004; Duchovskienė, 2000; Šikšnianienė,
2002; Samuolienė, 2007).
Very important investigations are carried out in order to optimize photosynthetic
indices of field and vegetable crops and orchards. They allow establishing the
possibilities to increase plant productivity by technological means and to improve
production quality (Brazaitytė, 2000; Tarvydienė et al., 2004 a , 2004 b ; Šabajevienė
et al., 2005, 2006 b , 2007; Tranavičienė et al., 2007; Šikšnianienė, 2006, 2007).
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When the energetic expenditures increase, the problem of the improvement of
greenhouse plant growing technologies becomes more and more actual. Scientists of
laboratory participates in the national project of high technology program PHYTOLED.
The lamps made on the basis of the semiconductor illuminators (LED) present unique
possibility to coordinate the optimal light spectrum, flux, photoperiod and frequency
of impulses for the effective control of photosynthesis, morphogenesis and quality
parameters for every plant variety (Samuolienė et al., 2004, 2006 d ; Tamulaitis et al.,
2004 a , 2004 b , 2005, Urbonavičiūtė et al., 2007 a , 2007 b ; Kurilčik et al., 2007, 2008).
The new technical possibilities in the field of plant irradiation gave a big impulse to
expand the photophysiological investigations of the greenhouse plants.
During the recent decades the problems of the influence of climate and
anthropogenic environmental factors on plants become still deep relevant. They are
connected with the disintegration of ozone layer of stratosphere, increase of the flux
of ultraviolet rays, the rise of temperature. Ecophysiological works are conducted at
the laboratory from 2000 (Duchovskis et al., 2003). It is intend that these results will
influence the development of the conception of the ecological agriculture and will form
the basis for the creation of the strategy of plant selection in the future.
Lately the significant progress in biology and technique allowed the creation
of automatic plant investigation systems, which help physiologists to realize the
conception of “conversation with the plant”. This field in plant physiology is called
phytomonitoring (Rrazaitytė, 1996, 1998, 1999, 2000; Brazaitytė, Duchovskis,
1999; Brazaitytė, Jankauskienė, 2000; Duchowski, Brazaitytė, 2001). Methodology
and equipment of phytomonitoring allow the conducting of investigations in vivo,
without plant injures, in dynamics, at the same time observing several necessary
parameters of plant and its environment.
The aim of the investigation was to present the trends of physiological scientific
works, carried out at LIH and connected with the increase of the efficiency of
horticulture and the improvement of production quality.
Object, methods and conditions. Ontogenetic aspect of investigations is
characteristic to all works of laboratory. Method of vegetation experiments (Żurbicki,
1974), morphophysiological method (Куперман, 1977), methodology and methods
of phytomonitoring (Ton, 1996; Ильницкий et al., 1997), methods of liquid
chromatography (Fernandez-Orozco et al., 2003; Wang et al., 2003) are applied
in the laboratory on a large scale; spectrophotometric methods for various indices
(Гавриленко, Жыгалова, 2003; Ragane et al., 2006; Janghel et al., 2007;) and the
other substances were investigated.
There was established the phytotrone complex with 10 climate chambers,
experimental greenhouses, vegetative and ex situ fields at the laboratory. Besides the
equipment characteristic to the Plant Physiology Laboratory, the investigations were
conducted with the unique automatic plant investigation systems “Ekoplant-11” and
“LPS-3”. There are two systems of chromatography of liquids with fluorescence,
diode array and refractive index detectors and gas chromatographer with MS at the
laboratory.
M o r p h o g e n e s i s a n d fl o w e r i n g i n i t i a t i o n o f p l a n t s. The
control of plant morphogenesis is important for the technological aspects. Moreover,
the acceleration of the change of plant generations in phytotrons, greenhouses, climate
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chambers, the other installations of the artificial climate allows accelerating significantly
the selection process. The investigations of plant ontogenesis are intensively carried
out in Plant Physiology Laboratory of LIH. The new plant flowering initiation theory
was created (Duchovskis, 1995, 1996, 2000, 2004; Duchovskienė, 2000; Šikšnianienė,
2002; Samuolienė, 2007). According to this theory, the stages of flowering initiation
are as follows:
Flowering induction → evocation → flower initiation → gamete initiation →
destruction of flowering stimulus
On the basis of this theory the new conception about the character of two periods
– flowering induction and evocation of wintering plants was created. According to this
conception, the first flowering induction period under natural conditions is stipulated by
photoperiod (photoinduction period) and realizes itself in leaves through phytochrome
system. The metabolites of this period are transported to the apical meristems and as a
result of their activity the expression of organogenesis genes of inflorescence axis occurs
(the first evocation stage). The first evocation period ends at the complete development
of inflorescence axis (III rd organogenesis stage according to F. Kuperman (1977)).
The second flowering induction period is conditioned by low positive temperatures
(thermoinduction period) and occurs in the growth apex. The stimulus of the second
flowering induction stage de-blocks the genes of the second evocation period. The
morphological expression of evocation period is the development of the elements of
inflorescence axis (IV th organogenesis stage). Dependently on the type and degree of
development of the wintering plant, these processes may occur in autumn, winter, but
most often – in spring. The first and the second periods of flowering induction may
occur together (in the II nd organogenesis stage) or in consecutive order (in the II nd
and III rd organogenesis stages), or thermoinduction period may pass even earlier than
photoinduction period, but the periods of evocation pass only in consecutive order
(Duchovskis, 2000).
Lately a lot of attention is paid to the role of pigments photosynthetic system,
system of phytohormones, sugars and other metabolites, also on the light spectrum and
flux in the different periods of flowering initiation (Samuolienė et al., 2005 a,b , 2006 a-d ,
2007, 2008; Samuolienė, 2007).
These works are good theoretical basis to control the plant growth and development
and to create the methods of the accelerated growing of plant generations under the
conditions of the artificial climate.
Plant productivity and quality realizes itself in ontogenesis. Plant requirements
to the environmental factors and the elements of nutrition differ in ontogenesis.
Investigations showed that it is possible to optimize the growth factors in the different
periods of development by technological means (Brazaitytė, 1998; Tarvydienė et al.,
2004 a,b ; Žebrauskienė et al., 2001).
P h o t o p h y s i o l o g i c a l i n v e s t i g a t i o n s. The photophysiological
investigations are carried out on purpose to design the new generation semiconductor
lamps for plant irradiation in greenhouses, phytotrone and for in vitro cultivation
systems (Bliznikas et al., 2004; Brazaitytė et al., 2004, 2006; Samuoliene et al., 2004,
2006 d ; Tamulaitis et al., 2004 a , 2004 b , 2005; Ulinskaitė et al., 2004; Grishenkova et al.,
2006; Urbonavičiūtė et al., 2007 a , 2007 b ; Kurilčik et al., 2007, 2008). The parameters
of such lamps can be very easily changed or programmed, thus allows to control plant
6

photosynthetic, photomorphogenetic, phototropic processes and to affect the trend of
plant metabolic processes. These works are carried out together with physics of Vilnius
University (prof. A. Žukauskas). The significant results presumed to attract the private
capital for the patent of these data and for the expanding of new plant irradiation
technology business. The offset company UAB HORTILED was established.
Physiology of plant productivity. In recent years a lot of attention is paid to the
investigation of photosynthetic parameters of field crops (Brazaitytė, 1998; Tarvydienė
et al., 2004 a,b ; Šikšnianienė et al., 2006, 2007; Tranavičienė et al., 2007) and orchards
(Šabajevienė et al., 2005, 2006 b , 2007). These investigations allow to notice the factors
during vegetation timely, which reduce the productivity and quality and to optimize
them by technological means.
Methodology of phytomonitoring allows effectively observe plant physiological
parameters during vegetation and control them by the technological means. This
presents the opportunity operatively to correct the technologies of growing and to
obtain more and qualitative production. This methodology of investigation especially
justified itself in the closed systems of growing, for example, in greenhouses, where
there is an opportunity to control more parameters of growing. Especially good results
were obtained with tomatoes and cucumbers in greenhouses (Brazaitytė, 1996, 1998,
1999, 2000; Brazaitytė at al., 1999; Uselis et al., 2007).
Ecophysiological investigations. The laboratory participates in the ecophysiological
projects of the national program of priority trends. The aim of these investigations
was to establish plant reaction on the stress of ozone, CO 2
, temperature, heavy metals,
acidity of substratum, UV-B and their complex influence, to reveal the possibilities of
plant adaptation and competition in the background of the increased environmental
stress and to prevision plant growing strategy for the nearest decades.
Many stress factors arouse homeostasis mechanisms and plants become more
resistant to the additional stress. Nevertheless, in different cases, the stress influence
on the environmental factors sums up (Duchovskis et al., 2003 a ; Brazaitytė et al., 2005,
2006, 2007; Baranauskis et al., 2006; Sakalauskaitė, 2006 a , 2006 b ; Sakalauskienė et al.,
2007, 2008). Plant sensitivity to the different stress factors isn’t the same (Bashmakov
et al., 2005; Brazaitytė et al., 2006; Juknys et al., 2006). Weeds most often are more
resistant to the environmental pollution, what helps them to compete for nutrients and
light (Kavaliauskaitė et al., 2005). We hope that the results of these investigations will
allow modelling the strategy of agriculture plant selection for the nearest decades.
The applied physiological investigations may effectively contribute to the plant
productivity enlargement and quality improvement.
In laboratory there was published over 500 scientific publications, whereof 250
in international, Lithuanian and foreign journals. There was defended 1 degree of
habilitated doctor and 4 doctoral degrees.
Currently there are working tree research workers, two technicians and six PhD
students are studying in the laboratory.
Gauta 2008 04 02
Parengta spausdinti 2008 04 24
7

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31. Duchovskis P., Bobinas Č., Orentienė V. 2001. Valgomųjų morkų (Daucus sativus
Röhl.) sėklų augimas ir vystymasis ontogenezėje. Sodininkystė ir daržininkystė,
20(2): 48–53.
32. Duchovskis P., Juknys R., Brazaitytė A. & Žukauskaitė I. 2003 a . Plant Response
to Integrated Impact of Natural and Anthropogenic Stress Factors. Russian Journal
of Plant Physiology, 50(2): 147–154.
33. Duchovskis P., Kviklys D., Kawecki Z., Petronis P., Kviklienė N. 2000 a . Impact
of rootstock and irrigation on apple bud differentiation and flowering initiation.
Sodininkystė ir daržininkystė, 19(3)–1: 352–358.
34. Duchovskis P., Stanys V., Aliukevičiūtė R. 2001. Naminės slyvos (Prunus
domestica L.) ir kaukazinės slyvos (Prunus cerasifera Ehrh.) atsparumas šalčiams.
Sodininkystė ir daržininkystė, 20(2): 3–17.
35. Duchovskis P., Stanys V., Sasnauskas A., Bobinas Č. 2007. Cold Resistance of
Prunus domestica L. and Prunus cerasifera Ehrh. in Lithuania. Acta Horticulturae,
734: 299–303.
36. Duchovskis P., Šikšnianienė J., Bobinas Č., Viškelis P. 2000 b . Dynamics
of peroxidase and orto-diphenoloxidase isoensymes and other organic
substances in the evocation stage of white cabbage. Sodininkystė ir daržininkystė,
19(3)–1: 309–317.
37. Duchovskis P., Šikšnianienė J. B., Duchovskienė L., Kviklys D., Sakalauskaitė J.,
Deltuvas R., Kupčinskienė E. 2005. Pramonės taršos poveikis obelų fotosintetiniams
pigmentams ir atsparumui ligoms bei kenkėjams. Sodininkystė ir daržininkystė,
24 (4): 65–71.
38. Duchovskis P., Žebrauskienė A., Šikšnianienė J. B., Viškelis P., Samuolienė G.,
Bobinas Č., Kmitienė L., Kmitas A.. 2004. Evocation of edible onions.
Sodininkystė ir daržininkystė, 23 (2): 125–136.
39. Duchovskis P., Žukauskas N., Šikšnianienė J. B., Samuolienė G. 2003 b . Valgomųjų
morkų (Daucus sativus Röhl.) juvenalinio periodo, žydėjimo indukcijos ir
evokacijos procesų ypatumai. Sodininkystė ir daržininkystė, 22 (1): 86–93.
40. Duchowski P., Brazaitytė A. 2001. Tomato Photosynthesis Monitoring
in Investigations on Tolerance to Low Temperatures. Acta Horticulturae,
562: 335–339.
41. Fernandez-Orozco R., Zielinski H., Piskula M. K. 2003. Contribution of lowmolecular-weight
antioxidants to the antioxidant capacity of raw and processed
lentil seeds. Nahrung/Food, 47(5): 291–299.
42. Gelvonauskis B., Duchovskis P., Bandaravičienė G. 1999. Estimation of apple
seedlings winterhardiness and its inheritance. Biologija, 3: 36–38.
43. Gelvonauskis B., Duchovskis P., Bandaravičienė G. 2000. Investigation of
winter hardiness and cold hardiness in apple progenies. Acta Horticulturae,
538: 277–280.
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44. Grishenkova N. N., Lukatkin A. S., Stanys V. A., Duchovskis P. V. 2006. Light
condition effects on chilling adaptation of maize seedlings. Russian Agricultural
Sciences, 5: 12–14.
45. Janghel E. K., Gupta V. K., Rai M. K., Rai J. K. 2007. Micro determination of
ascorbic acid using methyl viologen. Talanta, 72: 1 013–1 016.
46. Jankauskienė J., Brazaitytė A. 2002. Įvairios spinduliuotės lempų įtaka agurkų
daigų augimui bei produktyvumui. Sodininkystė ir daržininkystė, 21(1): 63–71.
47. Jankauskienė J., Brazaitytė A., Kazėnas V. 2001. Įvairios spinduliuotės šviesos
lempų įtaka pomidorų daigams. Sodininkystė ir daržininkystė, 20(4)-1: 25–34.
48. Juknys R., Duchovskis P., Sliesaravičius A., Šlepetys J., Martinavičienė J.,
Brazaitytė A., Juozaitytė R., Lazauskas S., Dėdelienė K., Sakalauskaitė J.,
Romaneckienė R., Kadžiulienė Ž., Januškaitiennė I. 2006. Anglies dioksido ir
temperatūros diferencijuotas bei kompleksinis poveikis žemės ūkio augalams.
Sodininkystė ir daržininkystė, 25(2): 3–13.
49. Juozaitytė R., Ramaškevičienė A., Sliesaravičius A., Brazaitytė A., Duchovskis P.,
Burbulis N. 2007. Growth and physiological features of pea (Pisum sativum L.)
of different morphotypes under ozone exposure. Biologija, 53(3): 71–74.
50. Karitonas R., Viškelis P., Brazaitytė A., Žebrauskienė A. 1996. Nitratų, vitamino C
ir pigmentų tyrimai įvairaus produktyvumo (Brassica oleracea var. italika) veislių
ir hibridų brokoliuose. Teorinės ir praktinės problemos šiuolaikinėje kultūrinių
augalų fiziologijoje (mokslinių straipsnių rinkinys). Babtai: 153–157.
51. Kavaliauskaitė D., Duchovskis P., Bobinas Č. 2005. Competitive influence of white
goose-foots (Chenopodium album L.) on biological productivity and chemical
composition on red beets (Beta vulgaris L. var. conditiva Alef.). Sodininkystė ir
daržininkystė, 24(3): 127–137.
52. Kurilčik A., Miklušytė-Čanova R., Dapkūnienė S., Žilinskaitė S., Kurilčik G.,
Tamulaitis G., Duchovskis P., Žukauskas A. 2008. In vitro culture of
Chrysanthemum plantlets using light-emitting diodes. Central European Journal
of Biology, 3(2): 161–167.
53. Kurilčik A., Miklušytė-Čanova R., Žilinskaitė S., Dapkūnienė S., Duchovskis P.,
Kurilčik G., Tamulaitis G., Žukauskas A. 2007. In vitro cultivation of grape culture
under solid-state lighting. Sodininkystė ir daržininkystė, 26(3): 235–245.
54. Lukatkin A. S., Gracheva N. V., Grishenkova N. N., Duchovskis P. V.,
Brazaitytė A. A. 2007. Cytokinin-like growth regulators mitigate toxic action of
zinc and nickel ions on maize seedlings. Russian Journal of Plant Physiology,
54(3): 381–387.
55. McCall D., Brazaitytė A. 1997. Salinity effects on seedling growth and floral
initiation in tomato. Acta Agriculturae Scandinavica, 47: 248–252.
56. Orentienė V., Duchovskis P., Bobinas Č. 2000. Morphophysiological investigation
of cabbage seed ripening. Sodininkystė ir daržininkystė, 19(3)–1: 212–216.
57. Orentienė V., Duchovskis P., Zalatorius V., Bobinas Č., Stašelis A. 1998. Impact
of mechanic, physical, and chemical factors on germination power of carrot seeds.
Sodininkystė ir daržininkystė, 17(2): 104–110.
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58. Ragaee S., El-Sayed M., Abdel-Aal, Maher Noaman. 2006. Antioxidant activity
and nutrient composition of selected cereals for food use. Food Chemistry,
95: 32–38.
59. Raklevičienė D., Duchovskis P., Švegždienė D., Rančelienė V., Brazaitytė A. 2005.
Response of cress (Lepidium sativum L.) seedlings to impact of ultraviolet-B
and to elevated ozone levels under controlled environmental conditions. Acta
Physiologiae Plantarum, 27(4) (supplement): 83–84.
60. Ramaškevičienė A., Burbulis N., Duchovskis P., Sliesaravičius A., Pilipavičius V.,
Kuprienė R., Blinstrubienė A., Urbonavičiūtė A., Sakalauskaitė J., Baranauskis K.
2006. Impact of substrate acidity and heavy metals (Cu, Cd) on pea plants growth
and pollen germination. Ekologija, 2: 8–14.
61. Rubinskienė M., Viškelis P., Jasutienė I., Duchovskis P., Bobinas Č. 2006. Changes
in biologically active constituents during ripening in black currants. Journal of
Fruit and Ornamental Plant Research, 14 (Suppl. 2): 237–246.
62. Sakalauskaitė J., Stanienė G., Stanys V., Duchovskis P., Samuolienė G.,
Baranauskis K., Urbonavičiūtė A., Revin V., Lukatkin A. 2006 b . Cadmium
Resistance of Apple Rootstocks M.9 and B.396 In vitro. Sodininkystė ir
daržininkystė, 25(3): 273–282.
63. Sakalauskaitė J., Kviklys D., Lanauskas J., Duchovskis P. 2006 b . Biomass
Production, Dry Weight Partitioning and Leaf Area of Apple Rootstocks under
Drought Stress. Sodininkystė ir daržininkystė, 25(3): 283–291.
64. Sakalauskienė S., Šabajevienė G., Duchovskis P., Lazauskas S.,
Urbonavičiūtė A., Brazaitytė A., Samuolienė G., Ulinskaitė R., Sakalauskaitė J.,
Povilaitis V. 2007. Skirtingo drėgmės ir temperatūros režimo kompleksinis
poveikis žemės ūkio augalų augimo rodikliams. Sodininkystė ir daržininkystė,
26(4): 317–325.
65. Sakalauskienė S., Šabajevienė G., Lazauskas S., Brazaitytė A., Samuolienė G.,
Urbonavičiūtė A., Sakalauskaitė J., Ulinskaitė R., Duchovskis P. 2008. Skirtingo
drėgmės ir temperatūros režimo kompleksinis poveikis ridikėlių fotosintetiniams
rodikliams III–IV organogenezės etapuose. Sodininkystė ir daržininkystė,
27(1): 97–104.
66. Samuolienė G. 2007. The Physiological and Biochemical Aspects of Flowering
Initiation in Edible Carrot (Daucus sativa (Hoffm.) Röhl.). Summary of Doctoral
Dissertation. Babtai, 26 p.
67. Samuolienė G., Duchovskis P. 2006 a . Angliavandenių dinamika ir vaidmuo
paprastojo kmyno žiedų iniciacijos ir diferenciacijos metu. Sodininkystė ir
daržininkystė, 25(1): 199–206.
68. Samuolienė G., Duchovskis P. 2006 b . Fitohormonų dinamika ir vaidmuo
po paprastojo kmyno žydėjimo indukcijos. Sodininkystė ir daržininkystė,
25(4): 278–285.
69. Samuolienė G., Duchovskis P. 2006 c . Variation of ABA and carotenoids during
flowering initiation in carrots. Acta Biologica Szegediensis, 50(3–4): 121–125.
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70. Samuolienė G., Duchovskis P., Šikšnianienė J., Brazaitytė A., Ulinskaitė R.,
Tamulaitis G., Bliznikas Z., Kurilčik G., Žukauskas A. 2006 d . Flowering
initiation in carrots by tailoring the illumination spectrum. Acta Horticulturae,
711: 279–284.
71. Samuolienė G., Duchovskis P., Ulinskaitė R. 2005 a . Dynamics and role of
carbohydrates in carrot during different flowering initiation stages. Sodininkystė
ir daržininkystė, 24(3): 147–154.
72. Samuolienė G., Duchovskis P., Urbonavičiūtė A. 2005 b . Phytohormones dynamics
during flowering initiation in carrots. Acta Biologica Szegediensis, 49(3–4): 33–
37.
73. Samuolienė G., Šabajevienė G., Ulinskaitė R., Duchovskis P. 2008. Fotosintezės
rodiklių kitimas paprastojo kmyno žydėjimo iniciacijos metu. Sodininkystė ir
daržininkystė, 27(1): 131–138.
74. Samuolienė G., Šabajevienė G., Urbonavičiūtė A., Duchovskis P. 2007. Carrot
flowering initiation: light effect, photosynthetic pigments, carbohydrates. Acta
Biologica Szegediensis: 51(1): 39–42.
75. Samuolienė G., Šikšnianienė J. B., Duchovskis P., Brazaitytė A., Ulinskaitė R.,
Baranauskis K. 2004. Šviesos kokybės įtaka valgomųjų morkų fiziologiniams
procesams evokacijos bei žiedų iniciacijos tarpsniais. Sodininkystė ir daržininkystė,
23(1): 78–87.
76. Stašelis A., Duchovskis P., Brazaitytė A. 1999. Elektromagnetinių laukų poveikis
daržovių sėklų daigumui bei daigų morfogenezei. Inžinerija, 4(1): 77–85.
77. Stašelis A., Duchovskis P., Optažas R., Meškelis L. 1996. Elektromagnetinių
laukų įtaka pomidorų ir agurkų sėklų daigumui bei daigų morfogenezei. Žemės
ūkio inžinerija, 28(2): 121–130.
78. Šabajevienė G., Kviklys D., Samuolienė G., Sasnauskas A., Duchovskis P.
2007. Seasonal patterns of carbohydrates in apple tree cv. ‘Auksis’ on different
rootstocks. Sodininkystė ir daržininkystė, 26(3): 159–165.
79. Šabajevienė G., Sakalauskaitė J., Šlapakauskas V., Uselis N., Duchovskis P. 2006 a .
Chlorophyll Fluorescence Characteristics of Cultivar ‘Auksis’ on Rootstocks P 22
and P 60 in High Density Orchards of Different Construction. Sodininkystė ir
daržininkystė, 25(3): 364–370.
80. Šabajevienė G., Uselis N., Duchovskis P. 2005. Auksio obelų fotosintezės pigmentų
tyrimai įvairių konstrukcijų intensyviuose soduose. Sodininkystė ir daržininkystė,
24(4): 57–64.
81. Šabajevienė G., Uselis N., Duchovskis P. 2006 b . ‘Auksis’ veislės obelų su P 22
poskiepiu fotosintetinės pigmentų sistemos formavimasis įvairių konstrukcijų
soduose. Sodininkystė ir daržininkystė, 25(1): 23–28.
82. Šikšnianienė J. B. 2002. Eucoreosma sekcijos serbentų peroksidazės ir
polifenoloksidazės tyrimai skirtinguose ontogenezės tarpsniuose. Daktaro
disertacijos santrauka, Babtai, 24 p.
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83. Šikšnianienė J. B. 2005. Raudonųjų burokėlių (Beta vulgaris L. var.
Conditiva Alef.) peroksidazės ir polifenoloksidazės izofermentų dinamika
žydėjimo indukcijos ir evokacijos tarpsniais. Sodininkystė ir daržininkystė,
24(4): 112–119.
84. Šikšnianienė J. B., Bundinienė O., Brazaitytė A., Duchovskis P. 2007. Trąšų su
nitrifikacijos inhibitoriumi poveikis raudonųjų burokėlių fotosintezės rodikliams.
Sodininkystė ir daržininkystė, 26(2): 52–59.
85. Šikšnianienė J. B., Duchovskis P., Bundinienė O., Brazaitytė A. 2006. Skirtingų
azoto trąšų formų ir ceolito įtaka burokėlių pasėlio fotosintezės rodikliams.
Sodininkystė ir daržininkystė, 25(1): 116–122.
86. Šikšnianienė J., Duchovskis P., Stanys V., Šikšnianas T. 2000. Peroxidase and ortodiphenoloxidase
isoenzymic spectra of Eucoreosma section currant species and
black currant cultivars in the stages of flower and gamete initiation. Sodininkystė
ir daržininkystė, 19(3)–1: 168–179.
87. Šikšnianienė J. B., Duchovskis P., Stašelis A. 2006. Elektromagnetinių laukų
poveikis baltosios balandos augimui bei sėklų daigumui. Sodininkystė ir
daržininkystė, 25(1): 192–198.
88. Šikšnianienė J. B., Duchovskis P., Viškelis P., Petronienė D. 2006. Raudonųjų
burokėlių Ilgiai angliavandenių ir azoto medžiagų dinamika žydėjimo indukcijos
ir evokacijos tarpsniais. Sodininkystė ir daržininkystė, 25(1): 100–109.
89. Tamulaitis G., Duchovskis P., Bliznikas Z., Breivė K., Ulinskaitė R., Brazaitytė A.,
Novičkovas A., Žukauskas A. 2004 a . High-power solid-state lighting facility
for greenhouse vegetable cultivation. Institute of Physics Conference Series,
182: 317–318.
90. Tamulaitis G., Duchovskis P., Bliznikas Z., Breivė K., Ulinskaitė R., Brazaitytė A.,
Novickovas A., Žukauskas A., Shur M. S. 2004 b . High-power LEDs for plant
cultivation. Proc. SPIE, 5 530: 165–173.
91. Tamulaitis G., Duchovskis P., Bliznikas Z., Breivė K., Ulinskaitė R.,
Brazaitytė A., Novičkovas A., Žukauskas A. 2005. High-power light-emitting
diode based facility for plant cultivation. Journal of Physics D: Applied Physics,
38(17): 3 182–3 187.
92. Tarvydienė A., Duchovskis P., Šiuliauskas A. 2004 a . Raudonųjų burokėlių
fotosintetinių rodiklių formavimosi dinamika skirtingai tręštame pasėlyje.
Sodininkystė ir daržininkystė, 23(3): 76–88.
93. Tarvydienė A., Duchovskis P., Šiuliauskas A. 2004 b . Skirtingų raudonųjų burokėlių
(Beta vulgaris L. var. conditiva) morfotipų fotosintetinių rodiklių formavimosi
dinamika įvairaus tankumo pasėlyje. Vagos, 62(15): 44–52.
94. Ton Y. 1996. Basics of phytomonitoring. Phytomonitoring, 1: 3–5.
95. Tranavičienė T., Šikšnianienė J. B., Urbonavičiūtė A., Vagusevičienė I.,
Samuolienė G., Duchovskis P., Sliesaravičius A. 2006. Effects of Nitrogen
Fertilizers on Wheat Photosynthetic pigment and carbohydrate contents. Biologija,
53(4): 80–84.
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96. Ulinskaitė R., Duchovskis P., Brazaitytė A., Jankauskienė J., Viškelis P.,
Šikšnianienė J. B., Samuolienė G., Šabajevienė G., Bliznikas Z., Breivė K.,
Novičkovas A., Tamulaitis G., Žukauskas A. 2004. Influence of illumination
spectrum on growth and quality of onion leaves. Sodininkystė ir daržininkystė,
23(3): 44–53.
97. Urbonavičiūtė A., Pinho P., Samuolienė G., Duchovskis P., Vitta P., Stonkus A.,
Tamulaitis G., Žukauskas A., Halonen L. 2007 a . Effect of short-wavelength
light on lettuce growth and nutritional quality. Sodininkystė ir daržininkystė,
26(1): 157–165.
98. Urbonavičiūtė A., Pinho P., Samuolienė G., Duchovskis P., Vitta P., Stonkus A.,
Tamulaitis G., Žukauskas A., Halonen L. 2007 b . Influence of bicomponent
complementary illumination on development of radish. Sodininkystė ir
daržininkystė, 26(4): 309–316.
99. Urbonavičiūtė A., Samuolienė G., Sakalauskaitė J., Duchovskis P., Brazaitytė A.,
Šikšnianienė J. B., Ulinskaitė R., Šabajevienė G., Baranauskis K. 2006 a . The
Effect of Elevated CO 2
Concentrations on Leaf Carbohydrate, Chlorophyll
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15(6): 921–925.
100. Urbonavičiūtė A., Ulinskaitė R., Samuolienė G., Sakalauskaitė J., Duchovskis P.,
Brazaitytė A., Šikšnianienė J. B., Šabajevienė G., Baranauskis K. 2006 b . The
response of radish phytohormone system to ozone stress. Sodininkystė ir
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101. Uselis N., Duchovskis P., Viškelis P., Brazaitytė A., Švagždys S., Petronis P.
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ir daržininkystė, 26(4): 37–47.
102. WangY., Mopper S., Hasenste I. K. H. 2003. Effects of salinity on endogenous
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342.
103. Žebrauskienė A., Duchovskis P., Bobinas Č. 2001. Ropinių svogūnų (Allium
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15

SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Dvidešimt metų mokslinių ieškojimų augalų fiziologijos srityje
P. Duchovskis
Augalų fiziologijos laboratorija LSDI įkurta 1988 m. balandžio 16 dienа. Nuo pat pradžių
buvo siekiama sukurti šiuolaikinę mokslinę bazę bei aktualią tyrimų tematiką. Šiuo metu prie
laboratorijos įkurtas fitotroninis kompleksas su 10 klimato kamerų, eksperimentiniais šiltnamiais,
vegetacine aikštele. Laboratorija aprūpinta automatinėmis augalų fiziologinių procesų tyrimo,
skysčių ir dujų chromatografijos sistemomis, kita analitine įranga.
Laboratorijoje susiformavo tokios svarbiausios mokslinio darbo kryptys: augalų
morfogenezė, žydėjimo iniciacija, ekofiziologija, fotofiziologija, augalų produktyvumo
fiziologija. Svarbiausios laboratorijos temos skirtos augalų žydėjimo iniciacijos teorijos bei
morfogenezės tyrimams plėtoti. Laboratorijoje sukurtas žiemojančių, dvimečių ir daugiamečių
augalų žydėjimo iniciacijos modelis (P. Duchovskis). Tiriami foto- ir termoindukcijos,
evokacijos, žiedų iniciacijos ir diferenciacijos mechanizmai, fitohormonų, angliavandenių ir
kitų medžiagų vaidmuo šiuose procesuose (P. Duchovskis, G. Samuolienė, A. Urbonavičiūtė,
G. Šabajevienė, A. Kurilčik). Galimybė pažinti ir valdyti augalų augimo ir vystymosi procesus
sudaro puikias prielaidas intensyvinti sodininkystės ir daržininkystės technologijas.
Siekiant sukurti naujos kartos puslaidininkes lempas augalams švitinti šiltnamiuose,
in vitro kultivavimo sistemose bei fitotronuose vykdomi fotofiziologiniai tyrimai (P. Duchovskis,
A. Urbonavičiūtė, G. Samuolienė, A. Kurilčik, A. Brazaitytė, R. Ulinskaitė). Tokių lempų
šviesos parametrai lengvai keičiami bei programuojami, o tai leidžia valdyti augalų fotosintezės,
fotomorfogenezės, fototropinius procesus bei veikti augalų metabolinių procesų kryptingumą.
Šie darbai vykdomi su Vilniaus universiteto fizikais (prof. A. Žukauskas). Pasiekti reikšmingi
darbo rezultatai leido pritraukti privatų kapitalą šiems rezultatams patentuoti bei naujam augalų
švitinimo technologijos verslui plėtoti. Įkurta dukterinė įmonė UAB HORTILED.
Svarbi tyrimo kryptis – nepalankių gamtinių ir antropogeninių veiksnių poveikis augalų
fiziologiniams ir biocheminiams rodikliams, jų tolerancijos ribos ir adaptyvumas įvairiems
stresams bei konkurencinis pajėgumas besikeičiančio klimato ir aplinkos taršos sąlygomis
(A. Brazaitytė, J. Sakalauskaitė, S. Sakalauskienė, G. Samuolienė, A. Urbonavičiūtė,
G. Šabajevienė, P. Duchovskis). Tai kompleksiniai darbai, kurie vykdomi su kitomis žemės
ūkio ir biologinio profilio institucijomis ir kurių galutinis tikslas – parengti rekomendacijas
žemės ir miškų ūkiui agro- ir miškų ekosistemų tvarumui didinti.
Laboratorijoje plėtojami lauko daržovių ir kitų augalų pasėlio bei sodų fiziologijos tyrimai
(A. Brazaitytė, J. B. Šikšnianienė, G. Šabajevienė, P. Duchovskis). Jie leidžia laiku pastebėti
pasėlio fotosintetinių rodiklių kitimа ir veikti šiuos procesus technologinėmis priemonėmis
siekiant didesnio augalų produktyvumo ir geresnės produkcijos kokybės.
Per 20 darbo metų laboratorijoje paskelbta daugiau kaip 500 mokslinių publikacijų, iš jų
250 tarptautiniuose, Lietuvos ir užsienio recenzuojamuose leidiniuose. Apgintas 1 habilituoto
daktaro ir 4 daktaro disertacijos moksliniam laipsniui įgyti.
Šiuo metu laboratorijoje dirba 3 mokslo darbuotojai, 2 laborantai bei studijuoja 6
doktorantai.
Reikšminiai žodžiai: augalų streso fiziologija, fitomonitoringas, fotofiziologija, žydėjimo
iniciacija.
16

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Flowering initiation in carrot and caraway
Giedrė Samuolienė 1, 2 , Akvilė Urbonavičiūtė 1, 2 ,
Gintarė Šabajevienė 1 , Pavelas Duchovskis 1, 2
1
Lithuanian Institute of Horticulture, Kauno 30, 54333 Babtai, Kaunas distr.,
Lithuania, e-mail: g.samuoliene@lsdi.lt
2
Lthuanian university of agriculture, LT-53361, Akademija, Kaunas distr.,
Lithuania
The aim of this paper was to analyze the influence of external factors such as short and
long day, vernalization and temperature on the generative development rate in biannual plants
and on sucrose and gibberelic acid metabolism in apical meristems during flowering initiation.
The researches were carried out in 2004–2007 in phytotron complex of the Lithuanian Institute
of Horticulture according to vegetative assay methodology under controlled conditions. Edible
carrot (Daucus sativus (Hoffm.) Röhl.) var. ‘Garduolės’ and co mmon caraway (Carum carvi L.)
var. ‘Gintaras’ with 9 leaves in rosette were kept in a phytotron chambers with different
photo and thermo periods for 120 days: 0 h – 4 °C; 8 h – 4 °C; 16 h – 4 °C; 8 h – 21/17 °C;
16 h – 21/17 °C. Different developmental rates and ways in two disputed Apiaceae species
were observed in subject to environmental factors. Thus the peculiar sucrose supply in shoot
apex and differences in GA 3
concentration during evocation under particular environmental
conditions influenced the formation rate of inflorescence stem in carrot and caraway. We deduced
that vernalization makes stronger positive effect on carrot flowering initiation, whereas high
temperature blocks the formation of generative organs. The flowering initiation in carrots is
more dependent on temperature than on photoperiod regimes during different ontogenesis stages.
Long day and vernalization determines almost full flowering, high temperatures independently
from photoperiod results in partial flowering and short day and vernalization is the limiting
factor of caraway flowering.
Key words: caraway, carrot, gibberellic acid, photoperiod, sucrose, temperature,
vernalization.
Introduction. The Apiaceae are mostly temperate herbs almost always with
umbellate inflorescences comprising about 300 genera and 3,000 species (Pimenov
and Leonov, 1993). Daucus carota subsp. sativus (Hoffm.) Arcang., the co mmon
cultivated carrot, is by far its most economically important member (Downie et al.,
2000). Other familiar vegetables, flavorings or garnishes include angelica, caraway,
celery, dill, parsley and etc.
The timing of the transition from vegetative growth to flowering is of paramount
importance in agriculture, horticulture, and plant breeding because flowering is the first
step of sexual reproduction (Bernier et al., 1993). Factors, which may induce flowering
in biennial plants, are complex ones. Differences in the vegetation length and induction
requirement have their genetical and physiological background (Németh, 1998). In
17

several species, cold effect is the major factor stimulating flower initiation. Its value
and length are of basic importance, however, they are satisfactorily cleared up only
for a few species (Németh, 1998). The required vernalization length is in connection
with the development and size of the plants, more developed ones demanding a shorter
period. Also other factors are involved, in several cases the interaction of temperature
and photoperiods are proved (Ramin and Atherton, 1994). Beside temperature,
illumination length may also play a basic role in flower initiation (Rünger, 1977).
Those inductive factors stay in tight correlation with each other (Booij and Meurs,
1994). Illumination may act on flowering through its length during the day (the ratio of
light and dark is important), its length during the plant life and sometimes its intensity
(Németh, 1998). The different flowering-promoting factors are perceived by different
parts of the plant. Temperature is perceived by all plant parts, vernalization mainly
by the shoot apex, and photoperiod – by matured leaves. Therefore, this implies that
these parts interact and that the fate of the apical meristem – remaining vegetative
or becoming reproductive – is controlled by an array of long-distance signals from
the entire plant (Bernier et al., 1993). The transport of sucrose from leaves to apical
meristem (Bernier et al., 1993) and increase in gibberellins concentration is observed
during flowering induction (Blázquez et al., 1998). Multiple lines of evidence indicate
that many of plant developmental and physiological processes are regulated in response
to other signaling molecules, such as sucrose or gibberellins (Gibson, 2004). Therefore,
the theory was raised that flowering initiation acts as multicomponent and multistep
mechanism and without other endogenous and egzogenous factors, depends on solid
action of phytohormones and sugars (Bernier, 1988).
The aim of this paper was to analyze the influence of external factors such as
short and long day, vernalization and temperature on the generative development rate
in biannual plants and on sucrose and gibberelic acid metabolism in apical meristems
during flowering initiation.
Object, methods and conditions. The researches were carried out during
2004–2007 in phytotron complex of the Lithuanian Institute of Horticulture according
to vegetative assay methodology (Zubricki, 1974). Edible carrot (Daucus sativus
(Hoffm.) Röhl.) var. ‘Garduolės’ and co mmon caraway (Carum carvi L.) var.
‘Gintaras’ were initially grown in vegetative tumbler, 54 × 34 × 15 cm in size, placed
in a greenhouse until particular developmental level needed for special experiment
(16-hour photoperiod and 21/16 °C day/night temperature) was reached. Peat (pH ≈ 6)
was used as a substrate.
Carrots and caraway with 9 leaves in rosette were kept in a phytotron
chambers with different photo and thermo periods for 120 days: 0 h – 4 °C;
8 h – 4 °C; 16 h – 4 °C; 8 h – 21/17 °C; 16 h – 21/17 °C. After the exposure,
evocation, flower initiation and differentiation processes were investigated under
illumination with the photoperiod of 16-hour and 21/16 ± 2 °C day/night temperatures.
Determined parameters: organogenesis stage (Куперман, 1982); flowering initiation
stage (Duchovskis, 2000). Analyses of gibberellic acid (GA 3
) were performed using
a Shimadzu HPLC model 10A chromatographer equipped with DAD detector
(SPD-M 10A VP), the detection wavelength – 254 nm. Separations were performed
on an Inertsil ODS-2 column (150 × 4.6 mm 2 ). Mobile phase: of 45 % methanol
18

containing 1 % acetic acid. Analyses of sucrose were performed on the same Shimadzu
HPLC model 10A equipped with refractive index detector (RID 10A). Separations
were performed on an Adsorbosil NH 2
-column (150 mm × 4.6 mm). Mobile phase:
75 % acetonitrile. Limits of detection: for GA 3
0.87 µg ml -1 , for sucrose 0.05 µg ml -1 .
Statistical analysis was performed using Excel (version 7.0). The data presented in
figures are given as the mean ± standard error.
Results. As it is shown in Table, the best developmental rate was observed
in carrots. In opposite to high temperature, low positive temperature caused faster
development rate independently from duration of photoperiod. Under dark conditions
carrots and caraway didn’t develop and rooted away. Vernalization and long day (in
opposite to short day) influenced the most intensive formation of generative organs in
caraway. Meanwhile under high (21/17 °C) temperature the duration of photoperiod
didn’t cause any restriction of caraway flowering rate (table).
Table. The intensity level of different flowering initiation stages in common
caraway and edible carrot
Lentelė. Valgomosios morkos ir paprastojo kmyno skirtingų žydėjimo iniciacijos tarpsnių
intensyvumo lygis
Under treatment with high (21/17 °C) temperature, independent from
photoperiod, there was no sucrose detected after evocation in carrot apical meristems.
Still the amount of sucrose increased during flower initiation especially under
treatment with long day (16 h) (Fig. 1). While in caraway the concentration of
sucrose was higher under long day (16 h) than under short day (8 h), treatment was
independent from temperature regime during evocation stage II (Fig. 1). During
flower initiation it decreased under treatment with high temperature. During flower
initiation and differentiation under treatment with long day (16 h) and low (4 °C)
temperature there was no sucrose detected in caraway apical meristems. Besides, no
19

sucrose (8 h – 4 °C; 8 h – 4 °C), or very low concentrations (8 h – 21/17 °C; 16 h –
21/17 °C) were detected in caraway during flower differentiation (Fig. 1). Also the
decrease in sucrose amount was observed in carrot apical meristems during this period
(Fig. 1).
Fig 1. The amount of sucrose in apical meristems of edible carrot and co mmon
caraway during different periods of flowering initiation
1 pav. Sacharozės kiekis valgomosios morkos ir paprastojo kmyno apikalinėse meristemose
skirtingais žydėjimo iniciacijos tarpsniais
Higher concentrations of GA 3
were accumulated in caraway than in carrot apical
meristems. Nevertheless, analysing data in carrot apical meristems (see Fig. 2 A) it
was noticed that short day (8 h) and vernalization promoted higher accumulation of
gibberellic acid. The increase in gibberellic acid concentration was observed during
all flowering initiation stages under these conditions (8 h – 4°C). The highest GA 3
amount (19.14 µg g -1 ) was detected during second evocation stage under long day
(16 h) and vernalization treatment. During flowering initiation the amount of GA 3
was lower than the limit of detection under all conditions except short day and low
temperature treatment where it increased during flower differentiation (Fig. 2 A). The
same downtrend in GA 3
concentration was observed in caraway (see Fig. 2 B) during
flowering initiation; it also dramatically increased during flowering differentiation.
Short day and low temperature as well as long day and high temperature (in opposite
of 16 h – 4°C and 8 h – 21/17°C) caused low GA 3
accumulation in caraway apical
meristems during second evocation and flower initiation periods. The increase in
GA 3
concentrations was observed during flower differentiation under all conditions
(Fig. 2 B).
20

Fig. 2. The amount of gibberellic acid (GA 3
) in apical meristems of edible carrot
(A) and co mmon caraway (B) during different periods of flowering initiation
2 pav. Giberelo rūgšties (GA 3
) kiekis valgomosios morkos (A) ir paprastojo kmyno (B)
apikalinėse meristemose skirtingais žydėjimo iniciacijos tarpsniais
Discussion. During juvenile period plants are insensitive to any flowering inductive
factor and aren’t able to form reproductive organs. The minimal developmental level to
accept photo and thermo inductive factors for flowering induction differs (Duchovskis
et al., 2003). The formation of inflorescence axis (5 formed leaves in rosette for carrots)
means that photo induction ended and after that the processes of second evocation
stage began (Duchovskis, 2000). The thermo induction conditioned the formation of
inflorescence axis elements (IV th organogenesis stage) when carrots had 8–9 leaves
21

in rosette (Duchovskis et al., 2003). Therefore, modulating the flowering initiation
processes, carrots were placed into inductive regime with 9 leaves in rosette when
plants were able to accept both stimulus of photo and thermo induction. However,
in opposite to high temperature, low positive temperature caused faster development
rate independently from duration of photoperiod (Table). As for caraway, it seems that
juvenile period is longer than in carrots. According to Rьnger (1977), for the majority
of the most important vegetables of the Apiacea family temperature between 5–10 °C
proved to be the most effective for flowering, however both lower (5 °C) and higher
(15 °C) temperatures might have an inductional effect.
E. Németh (1998) noticed that caraway optimal induction regime might lie between
5 °C and 8 °C, which is effective when lasting more than two weeks. Both a shorter
period as well as high temperatures results in partial flowering. In case of caraway,
scientific data are very few. Putievsky (1983) examined the effect of day length and
temperatures on the flowering of three Apiacea species: caraway, dill and coriander.
The tree spices exhibited different reactions to the treatments. Caraway developed
flowers under all experimental circumstances (18/12 °C or 24/12 °C day and night
temperatures, with 10 h or 16 h photoperiods). Pursuant to other authors, a longer
vegetative growth at lower (4 °C) temperature and short day (8 h) occurred, whereas
earlier flowering was preceded by long day (16 h) and low temperature. The duration of
photoperiod didn’t affect flowering rate under treatment with high temperature (Table).
It might mean either that caraway does not need any short day induction for flower
initiation at all, or that any photoperiodic response is effective only with interaction
of low temperatures (Németh, 1998).
In agreement with other authors (Borisjuk et al., 2002), the highest sucrose
concentrations were determined in cells which can actively divide straight before
VI th organogenesis stage, when the formation of inflorescence axis elements begins
(Fig. 1). Carrots with 9 leaves in rosette can accept the both stimulus of photo and
thermo induction. According to our data (Fig. 1), during second evocation stage, high
temperature disturbs sugar metabolism in carrot but not in caraway apical meristems.
Such sugar metabolism and transport to apical meristems, influenced by photo and
thermo periods, could determine the differences in plant development processes (Table).
A lot of scientists investigated the sucrose distribution in apex and in other plant tissues
(Chailackhyan, 1936; Bodson, 1997; Bodson, Outlaw, 1985; Lejeune et al., 1993; King,
Ben-Tal, 2001). It is presumed that the supply of sucrose to apical meristemic tissues
is important for flower induction. Although it is not the specific flowering induction
stimulus, it is independent from the response to the photoperiod duration.
Analysing the distribution of gibberellic acid in carrot apex and the flowering
effects it was noticed that short day (8 h) and vernalization conditioned constant
increase in GA 3
amount during all flowering initiation stages (see Fig. 2 A). The
same trend was observed in sucrose accumulation in carrot apical meristems (Fig. 1).
Such increase in sucrose and gibberellic acid concentrations shows the co mmon
action of metabolic processes, which induce flower formation. Furthermore, the
GA regulated increased sucrose transport to apex could be not short-term (King,
Ben-Tal, 2001). Under these conditions (8 h – 4 °C) the fastest carrot development
rate was observed (table). Regrettably, such correlation between photo and thermo
22

stimulus and sucrose and GA 3
accumulation wasn’t observed in caraway apical
meristems (Fig. 1; Fig. 2 B). An increase in GA 3
concentration under special inductive
conditions was observed in both carrot (8 h – 4 °C, 16 h – 4 °C) and caraway
(16 h – 4 °C, 8 h – 21/17 °C) apical meristems before flower initiation and it may be
connected with developmental rate of these plants (Fig. 2, Table). Eriksson with colleges
(Eriksson et al., 2006) in experiments with Arabidopsis show that during growth in
short days shoot apical levels of active gibberelins and sucrose increase dramatically
before floral initiation occurs and that the expression patterns of the genes involved
in GA metabolism suggests that this increase in GAs possibly originates from sources
outside the shoot apex. Reeves and Coupland (2001) also maintained that GAs play a
central role in the control of flower initiation under short days, a role that is much less
important under long days, in which the flowering is delayed. As it was mentioned,
different developmental rates and ways in two disputed Apiaceae species were observed
subject to environmental factors, thus determined peculiar accumulation of sucrose
and GA 3
in apical meristems during flowering initiation.
Conclusions. 1. Vernalization makes stronger positive effect on carrot flowering
initiation, whereas high temperature blocks the formation of generative organs. The
flowering initiation in carrots is more dependent on temperature than on photoperiod
regimes during different ontogenesis stages.
2. Long day and vernalization determines almost full flowering, high temperatures
independently from photoperiod results in partial flowering and short day and
vernalization is the limiting factor of caraway flowering.
3. The sucrose supply in shoot apex and differences in GA 3
concentration during
evocation under particular environmental conditions influenced the formation rate of
inflorescence stem in carrot and caraway.
References
Gauta 2008 04 11
Parengta spausdinti 2008 04 17
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signals that induce flowering. The Plant Cell, 5: 1 147–1 155.
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7. Borisjuk L., Walenta S., Rolletschek H., Muller-Klieser W., Wobus U., Weber H.
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12. Eriksson S., Böhlenius H., Moritz T., Nilsson O. 2006. GA 4
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in the regulation of LEAFY transcription and Arabidopsis floral initiation. The
Plant Cell, 18: 2 172–2 181.
13. Gibson S. I. 2004. Sugar and phytohormone response pathways: navigating a
signalling network. Journal of Experimental Botany, 55: 253–264.
14. King R. W., Ben-Tal Y. 2001. A florigenic effect of sucrose in Fuchsia hybrida
is blocked by gibberellin-induced assimilate competition. Plant Physiology,
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24

SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Žydėjimo iniciacija morkose ir kmynuose
G. Samuolienė, A. Urbonavičiūtė, G. Šabajevienė, P. Duchovskis
Santrauka
Šio darbo tikslas – ištirti išorinių faktorių, kaip trumpos ir ilgos dienos, vernalizacijos
ir temperatūros įtaką dvimečių augalų generatyviniam išsivystymo tempui bei sacharozės ir
giberelo rūgšties metabolizmui apikalinėse meristemose žydėjimo iniciacijos metu. Tyrimai
atlikti 2004–2007 metais Lietuvos sodininkystės ir daržininkystės instituto fitotroniniame
komplekse pagal vegetacinių bandymų metodikа. Suformavę 9 lapus skrotelėje dvimečiai
augalai, valgomosios morkos (Daucus sativus (Hoffm.) Röhl.) veislė ‘Garduolės’ ir paprastojo
kmyno (Carum carvi L.) veislė ‘Gintaras’, 120 parų buvo veikiami skirtingais foto ir termo
periodais: 0 val. – 4 °C, 8 val. – 4 °C, 16 val. – 21/17 °C, 8 val. – 4 °C, 16 val. – 21/17 °C. Dviejose
aptatose Apiaceae šeimos rūšyse stebėti skirtingi išsivystymo tempai ir keliai priklausomai
nuo aplinkos sąlygų, o tai apsprendė savitą aprūpinimą sacharoze ir GA 3
kaupimаsi apkalinėse
meristemose žydėjimo iniciacijos metu bei įtakojo žiedynstiebio formavimąsi morkose ir
kmynuose. Mes nustatėme, kad vernalizacija turi stipresnį teigiamą efektą morkų žydėjimo
iniciacijai, o aukšta temperatūra stabdo generatyvinių organų formavimąsi. Žydėjimo iniciacija
morkose labiau priklauso nuo temperatūros nei nuo fotoperiodo sąlygų skirtingais ontogenezės
tarpsniais. Ilga diena ir vernalizacija kmynuose lėmė beveik pilnа žydėjimа, aukšta temperatūra,
nepriklausomai nuo fotoperiodo apsprendė dalinį žydėjimą, o trumpa diena ir vernalizacija yra
limituojantis kmynų žydėjimą veiksnys.
Reikšminiai žodžiai: giberelo rūgštis, fotoperiodas, kmynas, morka, sacharozė,
temperatūra, vernalizacija.
25

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
H + -ATPase functional activity in plant cell plasma
membrane
Jūratė Darginavičienė, Sigita Jurkonienė, Nijolė Bareikienė,
Vaidevutis Šveikauskas
Institute of Botany, Žaliųjų Ežerų 49, LT-08406 Vilnius, Lithuania
E-mail: jurate.darginaviciene@botanika.lt
The aim of the present work was to characterize ATPase activity of wheat coleoptiles cell
plasmalemma and check up its coupling with ATP-dependent H + transport under the influence
of indole-3-acetic acid and environmental stresses (salt and cold).
ATPase activity was controlled in plasmalemma isolated from four-day-old spring wheat
(Triticum aestivum L. ‘Nandu’) coleoptiles by the method of differential ultracentrifugation and
purification on sucrose density gradient. Plasmalemma marker enzyme K + Mg 2+ -ATPase activity
and its suppression by sodium orthovanadate, diethylstilbestrol, dicyclohexylcarbodiimide and
inhibitors of possibly contaminating ATPases – olygomicin and nitrate, also proton pumping
activities lead to the conclusion that isolated plasmalemma fraction contains Mg 2+ -dependent,
K + activated vanadate-sensitive H + -ATPase (EC 3.6.1.35).
Artificially created transmembrane electrochemical potential on plasmalemma vesicles
activated indole-3-acetic acid influence on plasmalemma ATP-dependent H + transport and
response reactions in nuclei. Cold and salt stresses induced changes in coupling of these
processes. The data lead to the supposition concerning plasmalemma H + -ATPase participation
in stresses signals transduction and cell response processes.
Key words: cold and salt stresses, H + -ATPase, IAA, plasmalemma.
Introduction. Plant plasma membrane (plasmalemma) proton pump (H + -ATPase)
is a single polypeptide with molecular mass of about 100 kDa (Arango et at., 2003).
It plays a central role in transport processes across the plasmalemma and controls
essential functions of plant organism such as nutrient uptake, intracellular pH
regulation, cell elongation and leaf movements (Osses, Godoy, 2006). Regulation of
the activity of H + -ATPase has been proposed to mediate broad range of physiological
responses related with growth and development of plants. According to “acid growth”
hypothesis indole-3-acetic acid (IAA) enhances H + pumping which lowers cell wall
pH, activates pH-sensitive enzymes and proteins within the wall, and initiates all-wall
loosening and extension growth. These processes, induced by IAA or fusicoccin, can
be blocked instantly and specifically by the removal of K + ions or the addition of K +
channel blockers. Vice versa, H + pumping and growth are immediately “switched on”
by the addition of K + ions (Hager, 2003). Such data lead to the supposition that these
processes could take part in the final realization phases of IAA-dependent plant cell
growth by elongation.
Besides the above-mentioned facts, plasmalemma H + -ATPase can participate
27

in early cellular signalling events, as it is an integral plasma membrane protein and
for this reason can be significant for perception and transduction of hormonal and
environmental signals to cell nuclei.
Broad spectrum of functional activities of H + -ATPases in plant cell is associated
with large number of enzyme activity regulating factors. Basic function of the enzyme,
that of coupling ATP hydrolysis and H + -pumping need for fine complex of regulation
(Arango et at., 2003) and could be changed as response to different endogen and
environmental factors. Among them – phytohormones and changes aroused by signals
of environmental stress. The maximal electrochemical potential gradient for H + (∆µH + )
that can be formed by the H + -ATPase is a function of free energy of hydrolysis of
ATP and stoichiometry of H + transported per ATP hydrolyzed (Bennet, Spanswick,
1984).
Investigations of functioning of isolated from membrane H + -ATPases are possible
only after enzyme reactivation and on the models of artificial membranes. More
natural and convenient is the model systems created on isolated plasma membrane
fragments – sealed vesicles, where ATPases are in their naturally surrounding membrane
components. The last model was used in our work.
The aim of the present work was to characterize ATPase activity of wheat
coleoptiles cell plasmalemma and check up its coupling with ATP-dependent H +
transport under the influence of indole-3-acetic acid and environmental stresses (salt
and cold). ATPase activity was controlled in plasmalemma of spring wheat coleoptiles
cells intensively growing by elongation – classical test object in phytohormone IAA
investigations.
Object, methods and conditions. The tested object of the work – ethyolated
decapitated 4-day spring wheat (Triticum aestivum L. cv. ‘Nandu’) coleoptiles. The
fraction enriched by sealed plasmalemma vesicles was obtained by the method of
differential centrifugation modified for wheat coleoptiles cells. For the identification
of H + -ATPase in plasmalemma membrane fraction inhibitors of different origin
ATPases and their phosphorylated intermediates were used: diethylstilbestrol (50 µM),
dicyclohexylcarbodiimide (50 µM), sodium orthovanadate (50 µM), oligomycin
(5 mg ml -1 ). H + -ATPase activity in plasmalemma samples was assessed according to
accumulation of inorganic phosphate (Pi) (Maksimov et at., 2002). For the evaluation of
orientation of plasmalemma vesicles, the total ATPase activity in isolated plasmalemma
vesicles was obtained by exposing them to alamethycine (50 µg·ml -1 ). Plasmalemma
vesicle permeability to monovalent cations was determined using potential-sensitive
positively charged dye dis-C 3
-(5) (λ excit.
= 570 nm; λ fluor.
= 670 nm; 3.3 × 10 -7 M).
Fluorescence was measured by spectrofluorimeter. Sodium diffusion potential was
induced by a specific ionophore valinomycine (8.3 nM) and this served as a starting
point for electrochemical potential (∆µH + ) calculations.
The physiological activity of IAA-protein complexes formed in the plasmalemma
(pH 7.2) was evaluated from RNA-polymerase II (RNP-II) activity in a system of nuclei
isolated from wheat coleoptiles cells (Кулаева et al., 1979). Triphosphates CTP, GTP,
UTP and 8- 14 C-ATP (ammonium salt, 2.11 GBq mmole -1 , Hartmann Analytic) at final
concentration of 0.1 mM were added to the system. The label content was determined
with the aid of an LS 1801 scintillation counter (Beckman, USA).
28

The figures represent mean arithmetical values of 3–5 tests (with no less than 2–3
replications each) and their standard deviations.
Results. 1. A c t i v i t y o f w h e a t c o l e o p t i l e s c e l l p l a s m a l e m m a
H + -ATPase. Membrane fraction isolated from wheat coleoptiles cells located in sucrose
interphase with d 1.11–1.15 g cm -3 contained K + -activated Mg 2+ - dependent ATPase.
As it is shown in Fig. 1 this ATPase is characterized by exact optimum in acid part of
pH 5.5 and high substrate specificity to ATP.
Fig.1. Optimal pH for K + Mg 2+ ATPase activity of wheat coleoptiles cell
plasmalemma vesicles enriched fraction
1 pav. Kviečių koleoptilių ląstelių plazmolema praturtintos frakcijos ląstelių
K + Mg 2+ ATPazės aktyvumo optimalus pH
K m
for ATP was 0.3 ± 0.02 mM, for ADP – 0.9 ± 0.07 mM. The range of substrates
exhibiting the lowering dephosphorylating activity was the following: ATP ≥ ADP =
paranytrophenylphosphate ≥ AMP > glycerophosphate.
K + Mg 2+ -ATPase activity comprised in wheat coleoptile cell plasmalemma fraction
was inhibited by sodium orthovanadate, diethylstilbestrol and dicyclohexilcarbodiimide
up to 50–73 % (Fig. 2). Nitrate slightly inhibited this ATPase (up to 15 %), olygomicin
even at pH 8.0 did not influence it.
These data show that the plasmalemma fractions are slightly contaminated with
tonoplast vesicles but without mitochondria contaminations. The investigated K + Mg 2+ -
ATPase is vanadate sensitive and has phosphorylated intermediates. On the model
of sealed vesicles isolated from wheat coleoptiles cell plasmalemma we were able
to show the transport function of K + Mg 2+ -ATPase and check up the electrochemical
potential created due to proton extrusion from the cell imitating processes coupled
with K + transport to the cell. These data will be showed below. The treatment with
alamethycine revealed that the isolated plasmalemma fraction consists from 50 % right
side and 50 % inverted vesicles. All above mentioned characteristics of ATPase in plants
glycophytes, and wheat among them, are attributed to proton pumping plasmalemma
H + -ATPase using the energy of ATP hydrolysis (Максимов, 1989).
29

Fig 2. Inhibitor analysis of plasmalemma K + Mg 2+ -ATPase activity.
1 – Control, 2 – diethylstilbestrol (50 µM),
3 – dicyclohexylcarbodiimide (50 µM), 4 – Na 3
VO 4
(50 µM),
5 – NO 3į
(50 mM), 6 – oligomycin (5 mg ml -1 , pH 8.0).
1–5 – pH of incubation medium 5.5
2 pav. Plazmolemos K + Mg 2+ -ATPazės inhibitorinė analizė. 1 – Kontrolė,
2 – dietilstilbestrolis (50 µM), 3 – dicikloheksilkarbodiimidas (50 µM),
4 – Na 3
VO 4
(50 µM), 5 – NO 3į
(50 mM), 6 – oligomicinas (5 mg ml -1 , pH 8,0).
1–5 – Inkubacinės terpės pH 5,5
2. I A A a n d H + -ATPase functional activity. IAA treatment in vivo activated
wheat coleoptiles cell growth by elongation. IAA used in vitro slightly raised
transmembrane ion transport in plasmalemma vesicles. The latter processes were
expressed in more considerable extent (up to 22 %) when treatment with IAA underwent
in experimental conditions favouring the creation of IAA-protein plasmalemma
complexes (Fig. 3). IAA-dependent processes undergoing in the cell nuclei do not take
part in the models with isolated plasmalemma vesicles, which were used for studies of
electrochemical cations gradient formation. Consequently here we have only effects of
IAA on plasmalemma level without any IAA-dependent protein synthesis activation.
Then the question arises whether IAA created ion transport changes on plasmalemma
level participate in nuclei IAA-dependent responses. The plasmalemma H + -ATPase
excrudes H + from cell to generate a proton motive force with membrane potential of
-120 to -160 mV (negative inside) and pH gradient of 1.5 to 2 units (acid ouside) (Sze
et at., 1999). We found the abilities to create artificially transmembrane potential on the
plasmalemma vesicles: by the method elaborated by Maksimov (1989) plasmalemma
vesicles loaded by K + ions were transferred into a Na + -medium and K + permeability
on membrane induced by valinomycine.
30

Fig. 3. Influence of IAA (5 · 10 - 8 M) on the active proton transport through
membrane of plasmalemma vesicles according to fluorescence of dis – C 3
– (5).
1 – Control, 2 – IAA, 5 · 10 -8 M,
3 – IAA, 5 · 10 - 8 M + the protein fraction of plasmalemma
(~ 20 kDa). 100 % – K + – diffusion potential.
3 pav. IAR (5 · 10 - 8 M) poveikis aktyviam protonų transportui per plazmolemą
pagal dis – C 3
– (5) fluorescenciją. 1 – Kontrolė, 2 – IAR, 5 · 10 - 8 M,
3 – IAR, 5 · 10 - 8 M + plazmolemos baltymų frakcija (~ 20 kDa).
100 % – K + – difuzinis potencialas.
The transmembrane potential calculated by titration according to the Nernst
equation in such conditions reaches about 100 mV (Maksimov, 1989). The generated
K + -diffusion potential was checked by potential-sensitive dye dis-C 3
-(5). IAA-dependent
changes in the nuclei were studied according to the changes of RNR-polymerase II
activity in the system of isolated nuclei. The obtained data have shown that the addition
to the system of the plasmalemma fraction treated with IAR to the enzyme activity
provoking medium arouse more pronounced activity of RNA-polymerase II under
conditions of artificially created electrochemical gradient (Fig. 4). These data allow
the suggestion that electrochemical gradient on membrane which naturally creates at
a great deal by functioning of H + -pump could influence processes of IAA perception
and realization.
31

Fig. 4. Activity of RNA-polymerase II in the system of isolated nuclei with addition
of IAA (5 · 10 -6 M) treated plasmalemma vesicles (1 and 2).
1 – Electrochemical potential not created on plasmalemma vesicles,
2 – Under conditions of created electrochemical gradient
4 pav. RNR-polimerazės II aktyvumas izoliuotų branduolių RNR sistezės sistemoje,
pridėjus IAR (5 · 10 -6 M) paveiktų plazmolemos vezikulių (1 ir 2).
1 – be elektrocheminio potencialo plazmolemos vezikulėse,
2 – sukurto elektrocheminio potencialo sąlygomis
3. H y d r o l y t i c a n d t r a n s p o r t f u n c t i o n s o f p l a s m a l e m m a
H + -ATPase under conditions of environmental stresses. Changes in plasmalemma
H + -ATPase dephosphorylating activity and H + transport are main markers of functional
state of this membrane. Could the environmental stresses, for example, cold and salt
stresses, arise the changes in plant cell plasmalemma related with the maintaining of
plant cell stability directed to its survival under unfavourable conditions For answering
this question we restricted this very broad field of investigations by determination of
H + -ATPase functioning properties under two fixed conditions – one hour long treatment
by -8 °C or 250 mM NaCl.
The changes in plasmalemma H + -ATPase activity under the above-mentioned
environmental stresses are shown in Fig. 5. Sub lethal treatment in vivo by cold
(-8 °C) exhibited only tendency for altering of dephosphorylating activity in wheat
coleoptiles cell plasmalemma, but short treatment of coleoptiles by 250 mM NaCl
lowered it up to 17 %. At the same time we were not able to elucidate pronounced
changes in plasmalemma H + transport processes. ∆µH + of plasmalemma in cold treated
coleoptiles was slightly lowered, but more interesting situation was exhibited in salt
treated variants – there was no lowering of ∆µH + in spite of the fact that ATPase
dephosphorylating activity, as it was shown in Fig. 5, was inhibited up to 17 %
(Fig. 6).
32

Fig. 5. Activity of wheat coleoptile cell plasmalemma H + -ATPase.
1 – control, 2 – -8 °C, 1 h, 3 –250 mM NaCl, 1 h. All treatments in vivo.
5 pav. Kviečių koleoptilių ląstelių plazmolemos H + -ATPazės aktyvumas.
1 – kontrolė, 2 – -8 °C, 1 val., 3 – 250 mM NaCl, 1 val. Visi poveikiai in vivo.
Fig. 6. ATP-dependent electrochemical potential ∆µH + in wheat coleoptiles
plasmalemma vesicles. 1 – control, 2 – -8 °C, 1 h,
3 – 250 mM NaCl, 1 h. All treatments in vivo.
6 pav. Nuo ATP priklausomas elektrocheminis potencialas
∆µH + kviečių koleoptilių plazmolemos vezikulėse. 1 – kontrolė,
2 – -8°C, 1 val., 3 – 250 mM NaCl, 1 val. Visi poveikiai in vivo.
So, the above data show that plasmalemma properties, which are directly related
with coleoptiles cell homeostasis, even at sub lethal stress conditions could be
maintained at the optimal level for cell living processes.
Discussion. The studies described above were planned in order to show how
wheat coleoptile cell plasmalemma H + -ATPase activity is exhibited in two main
fields of possible participation in the processes cell growth and responses to sublethal
stresses.
33

Highly purified plasmalemma fraction was used in the studies. According to
inhibitory analysis of K + -activated Mg 2+ -dependent ATPase activity, slight contamination
with tonoplast vesicles (slight inhibition by nitrate) and no contamination by ATPases
from mitochondria (no inhibition by oligomycine) were determined. Activity, pH
optimum, specificity to ATPase as substrate, functioning peculiarities, sensitivity to
orthovanadate and dicyclohexilcarbodiimide lead us to the conclusion that we have
elucidated the plasmalemma H + -ATPase – H + pump, which according to literature data
is vanadate sensitive K + – activated Mg 2+ – dependent ATPase (EC 3.6.1.35) located in
plant plasmalemma (Osses, Godoy, 2006).
One of the main functions of plasmalemma H + -ATPase is H + extrusion from cell
cytosol to cell wall area and loosening of cell wall microfibriles leading to cell growth
by elongation (Hager, 2003). These processes can be controlled by IAA and fusicoccine
and possibly reveal the last phases of hormone controlled growth realization. More
complicated are the situations with early events of growth and development controlling
processes, which could be influenced by changing plasmalemma H + -ATPase activity. It
is well known that in plasmalemma of functioning cells 100–150 mV transmembrane
potential is generated and maintained. Many if not all processes taking place in
plasmalemma are dependent on this transmembrane potential. The last is maintained by
the work of H + pump, functioning of ion canals (for example – K + and Ca 2+ canals), cell
growth-regulating processes. Plasmalemma H + -ATPase activity is closely related with
seasonal course of plant growth: plasmalemma H + -ATPase activity in Festuca pratensis
Huds showed specific fluctuation when ATPase activity peaks were determined early in
spring – just before renewal of growth, and the lowest ATPase activity was in root at the
time approaching to flowering and in shoot basal nodes during the transition to growth
cessation (Darginavičienė et at., 2007). There are literature data about plasmalemmal
regulator protein 14-3-3 interaction with H + -ATPase, which leads to strong stimulation
of H + -ATPase activity. The processes are interrelated with phosphorylation events in
plasmalemma (Garufi et at., 2007) during primary signal transduction steps.
Plant H + -ATPase can also be regulated independently of 14-3-3 proteins. An
auxin-binding protein isolated from rice was found to stimulate H + -ATPase activity
directly, and auxin binding increased the affinity of the auxin-binding protein for H + -
ATPase (Arango et at., 2003). There is no definite opinion about direct influence of
IAA on H + -ATPase activity. According to Erdel (1979) very small IAA concentrations
(10 -10 –10 -8 M) induced ATPase activity of microsomal fraction. In the work with wheat
it was suggested that plasmalemma H + -ATPases is a component of IAA signalling
cascade that may direct pattern formation in embrios (Rober-Kleber et at., 2003).
Our data show that in wheat coleoptiles (growing by elongation) transmembrane ATP
dependent potential is able to activate IAA response reactions in nuclei. These data
are also in agreement with possible role of plasmalemma H + -ATPase functioning in
the first phases of IAA signal perception and transduction processes.
Changes in plasmalemma H + -ATPase activity was observed as response to cold
stress. So when cucumber under exposure at 8 °C temperature for 1 day reduced
plasma membrane H + -ATPase activity from 30 to 16 µmol P i
mg -1 protein 1 h -1 (Lee
et at., 2004). The mentioned changes could be attributed to indirect enzyme activity
changes to temperature lowering, but there are suppositions that cold-induced reactive
34

oxygen species may activate a mitogen activated protein-kinase cascade that regulates
tolerance to freezing and other abiotic stresses.
Cucumber seedlings treated by 200 mM NaCl one day showed increased activity
of plasmalemma and tonoplast H + -ATPases which correlated with altered H + transport.
The alignment between H + -ATPase functional processes – enzyme hydrolytic activity
and ATP-dependent H + transport – could undergo changes (Bennet, Spanswick, 1984).
Such changes could be observed under the influence of different stress conditions.
Moreover our investigations with cold tolerant and non-tolerant Festuca pratensis
Huds genetic lines revealed that cold tolerance is characteristic for Festuca plant in the
cell plasmalemma –membranes of cold tolerant lines are able to maintain H + transport
level ensuring its homeostasis in spite of cold stress dependent changes in ATPase
dephosphorylating activity (Darginavičienė et at., 2007). Partial confirmation achieved
in present work is that spring wheat coleoptiles, which usually can be influenced by
spring cold stresses, are able to stabilize H + transport levels.
Conclusions. 1. Plasmalemma fraction isolated from spring wheat coleoptiles
contains Mg 2+ -dependent, K + -activated vanadate sensitive H + - ATPase – proton
pump.
2. Artificially created transmembrane electrochemical potential activated an IAA
influenced ATPase dependent H + transport in plasmalemma and response reactions in
nuclei.
3. The supposition is made that cell plasmalemma of more tolerant for
environmental stresses plants is able to change the coupling between ATPase hydrolytic
and H + transport activities and so maintain the H + transport levels ensuring the cell
homeostasis.
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Šveikauskas V., Bareikienė N. 2007. Changes in plasmalemma K + Mg 2+ -ATPase
dephosphorylating activity and H + transport in relation to seasonal growth and
freezing tolerance of Festuca pratensis Huds. Journal of Plant Physiology, doi:
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4. Erdel L., Toth I., Zsoldos F. 1979. Hormonal regulation of Ca 2+ stimulated K +
influx and Ca 2+ , K-ATPase in rice roots: in vivo and in vitro effects of auxins and
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5. Garufi A., Visconti S., Camoni L., Adduci P. 2007. Poyamines as physiological
regulators of 14-3-3 interaction with plant plasma membrane H + -ATPase. Plant
Cell Physiology, 48(3): 434–440.
6. Hager A. 2003. Role of the plasma membrane H + -ATPase in auxin-induced
elongation growth: historical and new aspects. Journal of Plant Research,
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7. Lee S. H., Singh A. P., Chung G. C., Ahn S. J., Noh E. K., Steudle E. 2004.
Exposure of roots of cucumber (Cucumis sativus) to low temperature severely
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8. Maksimov G., Šveikauskas V., Darginavičienė J., Jurkonienė S., Banienė J.,
Šiemaitė J. 2002. The usage of plasmalemmal vesicles inverted by Brij treatment
for studying processes, which occur on cytosolic membrane surface. Russian
Journal of Plant Physiology, 49(6): 761–765.
9. Osses L. R., Godoy C. A. 2006. Characterizing plasma membrane H + -ATPase in
two varieties of coffee leaf (Coffea arabica L.) and its interaction with an elicitor
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Physiology and Biochemistry, 44: 226–235.
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Spech V., Neuhaus G., Fischer-Iglesias Ch. 2003. Plasma membrane H-ATPase is
involved in auxin-mediated cell elongation during wheat embryo development.
Plant Physiology, 131: 1302–1312.
11. Sze H., Li H., Palmgren M. G. 1999. Energization of plant cell membranes by H + -
pumping ATPases: regulation and biosynthesis. The Plant Cell, 11: 677–689.
12. Кулаева О., Селиванкина С. Ю., Романко Е. Г., Николаева М. К.,
Ничипорович А. А.1979. Активация РНК-полимеразы изолированных ядер
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и роль цитокининов в его регуляции у растений: диссертация доктора
биологических наук. Москва.
Acknowledgements. The work was partly supported by Lithuanian State Science
and Studies Foundation. The help of assistant Liudmila Chramova is gratefully
acknowledged.
36

SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Funkcinis augalų ląstelių plazmolemos H + -ATPazės aktyvumas
J. Darginavičienė, S. Jurkonienė, N. Bareikienė, V. Švetkauskas
Santrauka
Pateikiamo darbo tikslas – charakterizuoti ATPazės hidrolitinį aktyvumа vasarinių kviečių
koleoptilių plazmolemoje ir jo santykį su nuo ATP-priklausomu H + transportu indolil-3-acto
rūgšties ir išorinių stresų (druskų ir šalčio) poveikyje.
Darbui naudotos keturių parų etioliuotos kviečių (Triticum aestivum L., ‘Nandu’)
koleoptilės, iš kurių ląstelių diferencinio ultracentrifugavimo būdu buvo Išskiriama plazmolema
ir išvaloma sacharozės tankio gradiente. Plazmolemos markerinio fermento K + Mg 2+ -ATPazės
aktyvumas ir natrio ortovanadato, dietilstilbestrolio, dicikloheksilkarbodiimido bei galimų
užterрiančių ATPazių inhibitorių (oligomicino ir nitrato) poveikis, o be to, protonų perneрimo
per membranа ypatybės leido padaryti išvadа, kad išskirtoji plazmolemos frakcija talpina nuo
Mg 2+ -priklausomą, K + -aktyvuojamą vanadatui jautrią H + -ATPazę (EC 3.6.1.35).
Dirbtinai sukurtas plazmolemos vezikulėse elektrocheminis potencialas sustiprino indolil-
3-acto rūgšties poveikį plazmolemos ATP-priklausomam H + transportui ir atsako reakcijoms
branduolyje. Šalčio ir druskų stresai sukėlė pakitimus santykyje tarp ATPazės hidrolitinio
aktyvumo ir ATP-priklausomo H + transporto ir parodė, kad augalų ląstelių plazmolemos H + -
ATPazė gali dalyvauti stresinių signalų transdukcijos ir atsako į juos procesuose.
Reikšminiai žodžiai: šalčio ir druskų stresai, H + -ATPazė, IAR, plazmolema.
37

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Effect of the photoperiod duration on the growth of
Chrysanthemum plantlets in vitro
Anželika Kurilčik 1, 2 , Stasė Dapkūnienė 2 , Genadij Kurilčik 3 ,
Silva Žilinskaitė 2 , Artūras Žukauskas 3, 4 , Pavelas Duchovskis 1, 4
1
Lithuanian Institute of Horticulture, Kauno 30, 54333 Babtai, Kaunas distr.,
Lithuania, e-mail: a.kurilcik@lsdi.lt
2
Botanical Garden of Vilnius University, Kairėnų 43, LT-10239, Vilnius, Lithuania
3
Institute of Materials Science and Applied Research, Vilnius University,
Saulėtekio 9-III, LT-10222 Vilnius, Lithuania
4
UAB HORTILED, Breslaujos 3, LT-44403 Kaunas, Lithuania
We report on the influence of the photoperiod duration on chrysanthemum growth
that was studied using light-emitting diode (LED)-based illuminator. After transplantation,
culture of chrysanthemum (Chrysanthemum morifolium Ramat. ‘Ellen’) was grown
in vitro in Murashige & Skoog modified nutrient medium in a phytotron for 42 days at
26/22 °C day/night temperature. Five groups of plants were simultaneously grown under
independently set different photoperiod regimes: 8 h, 12 h, 16 h, 20 h and 24 h, respectively.
All treatments were illuminated using an illumination system consisting of four groups
of LEDs emitting in the blue (450 nm), red (640 and 660 nm), and far-red (735 nm)
spectral regions. The intensity ratio of the light components was fixed at 14 % for the
450 nm, 36 % for 640 nm, 36 % for 660 nm, and 14 % for 735 nm components, respectively.
The total photon flux density (PFD) in all treatments was maintained at the same level
(56 ± 5 µmol m 2 s 1 ). Morphological and biometric parameters and concentration of
photosynthetic pigments in the plantlets were measured after the experiment. With an increase
of photoperiod duration from 8 h to 24 h, the dry and fresh weight (DW and FW, respectively)
as well the number of leaves and DW to FW ratio continually increases. The highest values of
the length of shoots and roots, and number of roots were observed in plantlets grown at 16 h
photoperiod. Meanwhile, differences in concentration of photosynthesis pigments were not
significant.
Key words: Chrysanthemum morifolium, in vitro plant cultivation, light-emitting diodes,
photoperiod.
Introduction. Photoperiod, light intensity, and light quality influence plant growth
and development from seed germination to flowering. Photoperiod has an effect on
the development of some plant species, and no effect on growth of other plants. The
influence of the photoperiod duration on flowering and rooting of ornamental plants
in vivo received a lot of attention (Runkle and Heins, 2006; Cameron et al., 2005).
However, few results were published on the growth under in vitro conditions. In
particular, the influence of 8 h and 16 h photoperiod on the microtuberization and
growth of potato plantlets in vitro (Seabrook et al., 1993; Kozai et al., 1995) as well
39

as on subsequent yield of greenhouse-grown potato tubers (Seabrook et al., 1995) was
described. Some research on the influence of photoperiod on in vitro growth and floral
initiation of Nicotiana tabacum and chicory (Altamura et al., 1991; Demeulemeester
et al., 1995), on stem elongation and growth of Mentha rotundifolia (Jeong et al.,
1996), and on the bulb formation of garlic and Hyacinthoides paivae (Takagi and Qu,
1995; Iglesias et al., 1999) was also conducted. In the majority of papers, only two
photoperiod regimes in vitro were checked: short day (SD) at 8 h and long day (LD) at
16 h, respectively. A few works are devoted to the investigation of other photoperiod
regimes (Lu et al., 2004; Vaz et al., 2004).
From an application standpoint, plant morphogenesis can be influenced by an
appropriate choice of lighting, which may affect photoreceptors of the plants. The
common sources of light currently used for in vitro plant cultivation are fluorescent
lamps. However, they have no possibility to vary illumination parameters (spectrum
and time characteristics). LED-based illuminators provide an alternative to fluorescent
lamps, as a light source with a tailored spectrum, which can meet specific needs of
plants (Bula et al., 1991; Brown et al., 1995; Žukauskas et al., 2002; Bliznikas et al.,
2004; Tamulaitis et al., 2005). Investigation of the effect of illumination intensity and
spectrum on plant growth in vitro has been carried out by applying LED illumination
to a few species of plants (Tanaka et al., 1998; Lian et al., 2002; Nhut et al., 2003; Jao
et al., 2005; Heo et al., 2006).
The Chrysanthemum is the second economically most important floricultural
(cut-flower) crop following the Rose (Teixeira da Silva, 2004). Micropropagation of
chrysanthemum shoots grown using LEDs were reported. Kim et al. (2004) showed that
shoot growth, stem and internode elongation, the net photosynthetic rate, and stomatal
characteristics of chrysanthemum plantlets are affected by light quality. Shimizu and Ma
(2006) showed that blue light from LEDs inhibits stem elongation of chrysanthemum
in vivo. However, the effect of photoperiod on growth and morphogenesis of in vitro
cultured chrysanthemum explants under LEDs has not been carried out so far.
The present study was aimed at the analysis of the growth of chrysanthemum
plantlets that were cultured in vitro under illumination at photoperiods of 8 h : 16 h,
12 h : 12 h, 16 h : 8 h, 20 h : 4 h, and 24 h light : 0 h darkness, respectively. An LEDbased
illumination system containing four groups of LEDs emitting in blue, red and
far-red regions was used.
Object, methods and conditions. P l a n t m a t e r i a l s a n d c u l t u r e
c o n d i t i o n. Chrysanthemum plantlets (Chrysanthemum morifolium Ramat. ‘Ellen’)
were grown in vitro in Murashige and Skoog (1962) modified nutrient medium (MS +
IAA 0.2 mg/l + BAP 0.05 mg/l, Ѕ NH 4
NO 3
, Ѕ KNO 3
, without vitamins, mio-inositol and
glycine) at 26/22 °C (day/night) temperatures maintained within 1 °C. Five milliliters
of medium were dispensed in 16 × 150 mm tubes covered with PVC caps with air
exchange. The pH of the medium was adjusted to 5.8 before autoclaving at 121 °C for
20 min. One explant per tube was planted and 36 tubes per treatment were prepared.
Light treatments. The cultures of in vitro plantlets were illuminated using red
(at the wavelenghts of 660 nm and 640 nm), blue (450 nm), and far-red (735 nm)
40

LEDs powered by a self-designed driver. The total photon flux density (PFD) in all
treatments was maintained at the same level (56 ± 5 µmol m -2 s -1 ). The intensity ratio
of the light components was fixed at 14 % for the 450 nm, 36 % for 640 nm, 36 %
for 660 nm, and 14 % for 735 nm components, respectively. Selection of the range of
the PFDs used in the experiments was based on our previous studies (Kurilčik et al.,
2008). The photoperiod duration in different treatments was maintained at 8 h, 12 h,
16 h, 20 h, and 24 h, respectively
D a t a c o l l e c t i o n a n d s t a t i s t i c a l a n a l y s i s. The fresh and dry
weight (FW and DW, respectively), stem and root length, number of leaves and roots,
and amount of photosynthetic pigments of the chrysanthemum plantlets were studied
after 42 days of cultivation. 35–36 replicates were used for the biometrical analysis.
Among those, 18 replicates were randomly selected for the dry weight measurement.
To determine the dry weight, the plantlets were oven-dried at 105 °C until a constant
mass was reached. The other 17–18 replicates were used for the measurement of the
photosynthetic pigment concentrations. After extraction with 100 % acetone according
to the Wettstein method (Гавриленко, Жыгалова, 2003), the total chlorophyll a and b
and carotenoid content in leaf tissues per one gram of green foliage mass was analysed
by a double-array spectrophotometer (model Genesys 6, Thermospectronic, USA).
Organogenesis stages of chrysanthemum plantlets were also determined (Куперман,
1982). After 42 days, the regenerantes were at the II nd organogenesis stage. All the data
were evaluated for significance by the analysis of variance (ANOVA).
Results. The biometric parameters of the chrysanthemum plantlets grown in vitro
under different photoperiod regimes are shown in Fig. The estimated parameters exhibit
the dependence on the photoperiod duration that was varied from 8 h to 24 h per day.
The length of the shoots shows a tendency to increase with increasing photoperiod from
8 h to 16 h (Fig. a). The further increase of the photoperiod duration to 24 h showed a
tendency for a decrease of the length of shoots. The length and number of roots followed
the same trend (Fig. a–b). Meanwhile, the leaf number, fresh and dry weight, and
DW/FW ratio continually increased with the increase of the photoperiod from 8 h to
24 h (Fig. b–d). At round-the-clock irradiation, the regenerants of chrysanthemum had
by two leaves more, than those grown at 8 h photoperiod. They also have accumulated
up to one and a half times more fresh weight and twice as more dry weight. Meanwhile,
the variation of the amount of photosynthetic pigments in treatments with different
photoperiods was not significant (Fig. e).
41

Fig. Parameters of chrysanthemum regenerants grown under different photoperiod
regimes: shoot and roots length (a), number of leaves and roots (b), fresh and dry
weight (c), DW/FW ratio (d),
contents of photosynthetic pigments in leaves (e)
Pav. Chrizantemų regenerantų parametrai skirtingo fotoperiodo sąlygomis:
stiebo ir šaknų ilgis (a), lapų ir šaknų skaičius (b), žalioji ir sausoji masė (c),
sausosios ir žaliosios masių santykis (d), fotosintezės pigmentų kiekis lapuose (e)
Discussion. This experiment was aimed at the determination of the optimal
photoperiod for the growth and development of the chrysanthemum plantlets in vitro
under LEDs. Our research has shown that a change of the photoperiod influences the
estimated parameters in different ways. For the plantlets height and for the development
of roots, the optimum photoperiod of 16 h was established (Fig. a–b). Note that Kozai
et al. (1995) also showed suppressed root growth of potato plantlets under conditions
of 8 h photoperiod in comparison to 16 h photoperiod; however, other photoperiod
42

treatments have not been investigated by these authors.
In our study, the development of leaves and the accumulation of fresh and dry
weight were faster at 24 h photoperiod (Fig. b–c). This is in line with the observations
of Adams and Langton (2005). These authors revealed that long-day (LD = 16 h)
treatments usually promote an increase in dry weight of various plants that otherwise
grow in short days (SD = 8 h).
In this study the largest DW/FW ratio was also observed at 24 h photoperiod
(Fig. d). Meanwhile, Kozai et al. (1995) showed that 16 h photoperiod in comparison
to 8 h photoperiod led to an increase in the fresh and dry weights of potato plantlets
but maintained similar percentage of dry matter (DW/FW ratio). We suppose that in
our study the dry matter content depends on the duration of dark period. A decrease
of the dark period resulted in an increase of the DW to FW ratio.
In addition, our study shows that the concentration of photosynthetic pigments
per 1 g of leaves FW does not depend on photoperiod duration (Fig. e). Meanwhile,
Adams and Langton (2005) showed that chlorophyll amount per unit leaf area is
occasionally increased by LD (16 h) treatment, which may increase photosynthesis
and constitute a second mechanism that increases dry weight. Lu et al. (2004) also
recorded significantly negative correlations between chlorophyll content in leaves of
potato plantlets and darkness length.
The stage of the development of chrysanthemum plantlets after the experiment
was similar for various photoperiods (data not shown). After 42 days, the regenerants
were at the II nd organogenesis stage (Куперман, 1982) and no distinctions have
been established for different photoperiods. Our results are in line with the review
of Carvalho and Heuvelink (2001), where data on the influence of various factors
on flower formation were summarized. According to these data, at the temperature
of 27 °C that was maintained in our study, the development of flowers lasts about
120 days. Therefore, at the 42 nd day, no distinctions between the treatments could be
expected. Meanwhile the same review infers that at 18 °C, the development of flowers
lasts about 60 days. Therefore we assume that in our study the morphogenesis of
flowers was hindered by a higher temperature and was less sensitive to the duration
of photoperiod. An additional study including variation of temperature is necessary
for a deeper understanding of this phenomenon.
Conclusions. Taking into account all differences observed while changing the
photoperiod duration, the optimal photoperiod for growth of shoots and roots of
chrysanthemum plantlets in vitro was estimated to be 16 h. However, morphogenesis of
new leaves and accumulation of DW and FW was most prominent at 24 h photoperiod.
This data may be helpful for improving of the efficiency of micropropagation of
chrysanthemum.
Acknowledgment. The authors would like to acknowledge the support from the
Lithuanian Science and Studies Foundation.
Gauta 2008 04 05
Parengta spausdinti 2008 05 05
43

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45

28. Vaz A. P. A, Figueiredo-Ribeiro R. C. L. and Kerbauy G. B. 2004. Photoperiod
and temperature effects on in vitro growth and flowering of P. pusilla, an epiphytic
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Москва, Академия. 256.
31. Куперман Ф. М., Ржанова Е. И., Мурашев В. В., Львова И. Н., Седова Е. А.,
Ахундова В. А., Щербина И. П. 1982. Биология развития культурных
растений. Москва, Высшая школа. 343.
SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Fotoperiodo trukmės poveikis chrizantemų eksplantų augimui
in vitro
A. Kurilčik, S. Dapkūnienė, G. Kurilčik, S. Žilinskaitė, A. Žukauskas,
P. Duchovskis
Santrauka
Aptariamas fotoperiodo trukmės poveikis chrizantemų eksplantų augimui ir vystymuisi.
Tyrimo objektas – chrizantemų veislė ‘Ellen’ (Chrysanthemum morifolium Ramat. ‘Ellen’)
auginama in vitro 16 × 150 mm mėgintuvėliuose su 5 ml maitinamosios terpės. Augalai 42 paras
buvo auginami kietakūnio apšvietimo šviestuvuose. Kiekviename iš jų atskirai programiškai
valdomi 450, 640, 660 ir 735 nm spektrinių komponenčių fotonų srautai, ir tomis pačiomis
sąlygomis auginami 36 augalai. Bendras fotonų srauto tankis palaikomas 56 ± 5 µmol m 2 s 1
ribose, temperatūra 26 °C/22 °C. Dienos/nakties fotoperiodo trukmė penkiuose apšvietimo
deriniuose atitinkamai buvo 8/16 val., 12/12 val., 16/8 val., 20/4 val. ir 24/0 val. Eksperimento
pabaigoje buvo vertinami kiekvieno augalo augimo parametrai bei nustatomas fotosintezės
pigmentų kiekis.
Nustatyta, kad ilgėjant fotoperiodui nuo 8 val. iki 24 val. per parа, chrizantemų eksplantų
sausoji ir žalioji masės, o taip pat vidutinis lapų kiekis bei sausos ir žalios masių santykis
nuosekliai didėja. Stiebų ir šaknų ilgis bei šaknų skaičius buvo didžiausi 16/8 val. fotoperiodo
sąlygomis. Fotoperiodo trukmės pokyčiai neturėjo reikšmingos įtakos fotosintezės pigmentų
kiekiui lapuose.
Reikšminiai žodžiai: augalų kultivavimas in vitro, Chrysanthemum morifolium,
fotoperiodas, šviestukai.
46

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Light and gravity-related tropistic responses of garden
cress leaves
Regina Losinska, Danguolė Raklevičienė, Danguolė Švegždienė
Institute of Botany, Laboratory of Plant Physiology, Sector of Gravitational
Physiology, Žaliųjų Ežerų 49, LT-80406 Vilnius, Lithuania
E-mail: regina.losinska@botanika.lt
The growth and spatial orientation of garden cress (Lepidium sativum L.) leaves in
response to spectral components of light in weightlessness simulated by slow (3-rpm)
horizontal clino-rotation were investigated. Unidirectional monochromatic blue (450 nm,
4.5 µmol·m -2 s -1 ) and red (660 nm, 8.5 µmol·m -2 s -1 ) illumination was applied separately or
simultaneously (9.5 µmol·m -2 s -1 ). Seedlings were cultivated for five days on the clinostat and
in a vertical stationary position (control, 1 g), either under 8 h/d and 24 h/d lighting or in the
dark. Length of leaves and bending of hypocotyls, leafstalks and laminas caused by altered
gravity and light were evaluated and compared with control samples at 1 g or in the dark. No
gravity-related alterations have been determined in the elongation of leafstalks and laminas in
the dark-growing leaves. Blue light in 8 h/d did not affect significantly the growth of lamina;
however, it promoted the elongation of leafstalks by 65–70 % in both gravity conditions. In
simulated weightlessness, the bending of hypocotyls from the upward growth direction was
enhanced. This curvature has been corrected considerably by used lighting. The data shows
that under continuous illumination gravitropism can be masked by phototropism. Red and blue
light applied separately did not significantly affect the angles between the two primary leaves.
However, their complex action promoted the opening of leaves more considerably. Lightrelated
positioning of leafstalks and laminas was determined when the applied light restored
the disorientation evoked by elimination of unidirectional gravitropic stimulus. The obtained
results suggest the interaction between phototropism and gravitropism in leaves.
Key words: clinostat, curvature, garden cress, gravity, gravitropism, leaf, light,
phototropism.
Introduction. Plants control leaf orientation in response to many environmental
stimuli to optimize their photosynthetic potentiality. Light and gravity are the most
critical factors for maintaining optimal positioning of leaves for maximal capturing of
light energy. However, current knowledge of the events related to mutual interaction
of light and gravity signals in the orientation control of plant organs (Kiss et al., 2003;
Lariquet, Frankhauser, 2004; Raklevičienė et al., 2005, 2007) and especially of leaves
(Inoue et al., 2008; Mano et al., 2006; Stutte et al., 2005) is sparse. In natural gravity
(1 g), phototropic response of every organ involves bending towards or away from the
light gradient with consequent deviation from the gravity vector. As a result, the actual
degree of growth movement depends on the phototropic response and a counteracting
gravitropic response. In real weightlessness (microgravity in space) or simulated by a
47

horizontal clinostat, where unidirectional action of gravity is eliminated, this process
is disturbed without making obvious alterations in plant development and growth
(Merkys, Laurinavičius, 1990; Soga et al., 2002; Stutte et al., 2005, 2006). Sufficient
lighting could help restore optimal spatial orientation of developing plant organs in
that environment. In addition to being the energy source for photosynthesis, light
spectral composition regulates many phototropic and photomorphogenic aspects of
plant development and morphology (Liscum, 2002; Nemhauser, Chory, 2002; Sullivan,
Deng, 2003). Previous studies showed that blue and red spectral components could
be effective for respective orientation of plant axial organs in altered gravity (Correll
et al., 2003; Kiss et al., 1997; Lariquet, Frankhauser, 2004; Raklevičienė et al., 2007;
Vitha et al., 2000); however, their impact on supporting optimal growth of leaves still
remains unclear.
Therefore, the aim of the present research was to determine and compare the effect
of 450 nm blue (B) and 660 nm red (R) light, and their simultaneous action (R + B)
on the growth and orientation of leafstalks and laminas in weightlessness simulated
by 3-rpm horizontal clinostat and at 1 g.
Object, methods and conditions. The object of the study was the seedlings of
garden cress (Lepidium sativum L.) growing in a vertical stationary position (control,
1 g) and on a horizontal clinostat rotating at 3 rpm for elimination of unidirectional
gravity action. They were cultivated for 5 days on solid MS medium with Ѕ salts
(Murashige, Skoog, 1962) and 0.2 % (w/v) gelrite (Sigma) under continuous or 8 h/d
illumination as well as without light. Light emitting diodes (LEDs) were used for
lighting from above with monochromatic blue (B, peak wavelength 450 nm, photon flux
density 4.5 µmol m -2 s -1 ), red (R, 660 nm, 8.5 µmol m -2 s -1 ) light or their combination
(R + B, 9.5 µmol m -2 s -1 ). The fluence rate of light was measured by a quantum sensor
(PM-20, Sonopan, Poland). Ambient temperature was 23 ° ± 1 °C. At the end of each
experiment, the over ground parts of seedlings were photographed with the PENTAX
*ist D digital camera, the leaves were scanned. The digital images were analyzed using
SigmaScan Pro 5 (Jandel Scientific Software) and Motic Images Plus 2.0 ML on the
PC platform. The influence of illumination on the length of leafstalks and laminas,
their spatial positioning as well as on flattening of leaves was studied. Measurements
of seedlings grown on the clinostat under light were compared with these of control
ones. Comparison has been made also between the measurements of light and dark
grown seedlings. Statistical analysis was performed using Excel (version 7.0). The data
presented in figures are given as the mean ± standard error (SE). Statistical significance
was set at p Ј 0.05.
Results. Illumination with B or R light from above and their combined action with
B + R under natural and altered gravity conditions revealed differences in leafstalk
and leaf lamina growth and tropistic responses. In the dark, parameters of leafstalks
and laminas did not differ significantly (Fig. 1a).
48

Fig. 1. Length of leafstalks and lamina of garden cress seedlings grown under
8 h/d (a) and continuous (b) illumination by monochromatic blue (B),
red (R) light or their combination (B + R) on horizontal clinostat
(HC) and control 1 g conditions
1 pav. Lapkočių ir lapalakščių ilgiai sėjamosios pipirnės daigų, augusių šviečiant
8 val./per parа (a) ir pastoviai (b) mėlyna (M), raudona (R) šviesa
ar kartu (M + R) natūralios gravitacijos (1 g) ir pakeisto svarumo sąlygomis
Illumination with 8 h/d B light promoted considerably the leaf growth. In
comparison with the dark-grown samples, the leafstalks and laminas were longer on the
clinostat and at 1 g approximately by 30 % and 20 %, respectively (Fig. 1 a). Continuous
B lighting enhanced the elongation of leafstalks and laminas more significantly than
at 8 h/d photoperiod (Fig. 1 b). However, the difference between the average length of
1-g and clino-rotated leaves was statistically insignificant under both, continuous and
8 h/d B-light conditions. Next set of experiments revealed the positive R-light-effect
on elongation of 1-g and clino-rotated leaves. R light, independently of illumination
duration, promoted the elongation of leafstalks by 55–60 % in both gravity conditions.
Nevertheless, the clino-rotated laminas in R light were much shorter in comparison
with the control ones. R + B illumination enhanced the length of both leafstalks and
laminas of 1-g leaves under 8 h/d and continuous illumination approximately by 35 %
and by 55 %, respectively. Data on parameters of leaves clino-rotated under 8 and
24 h/d illumination are contradictory.
Tropistic responses of leaves were tested in respect to hypocotyl position (Fig. 2).
The mode of the measurement of the bending of hypocotyl upper part, i. e. the hook,
is shown in Fig. 2 a.
In the dark, significant difference between the curvature of clino-rotated and 1-g
hypocotyls was determined (Fig. 2 b). In 1 g the hypocotyls were orientated vertically,
while they were more hooked after clino-rotation. This difference decreased in B light,
especially under continuous illumination. Orientation of 1 g hypocotyls in R light was
the same as in the dark and independent on the duration of lighting. Clino-rotation
under 8 h/d R light increased the curvature of hypocotyls from the originally position;
however, under continuous irradiation, the hypocotyls have been straightened. Lightrelated
bending of hypocotyls was the same in R + B and R light as on the clinostat
as well as at 1 g, too.
49

Fig. 2. Measurement of curvature of hypocotyls (a) and the data on their bending
(b) in the dark (D / T), under different lighting and gravity conditions
2 pav. Hipokotilio nuokrypio matavimo schema (a) ir duomenys apie hipokotilių nuokrypių
kampus (b) tamsoje (D / T), skirtingomis apšviestumo ir gravitacijos sąlygomis
Gravitropic responses of 1-g leafstalks appeared in plants grown in the dark
(Fig. 3). Fig. 3a illustrates the mode of the performed measurement of the leafstalk
curvature (angle X).
Fig. 3. Measurement of curvature (angle X) of the first leafstalks
(a) and data on their bending
(b) under different lighting and gravity conditions
3 pav. Pirmojo lapo lapkočių nuokrypio (kampas X) matavimas
(a) ir duomenys apie jų nuokrypį
(b) skirtingomis apšviestumo ir gravitacijos sąlygomis
In the dark, difference between the curvature of 1-g and clino-rotated leafstalks
amounted to approximately 10° (Fig. 3 b). Under B light, the leafstalks grown in both
gravity conditions oriented towards light source. Therefore, a more vertical orientation
50

has been achieved by the first leafstalk. Illumination with R light, independently on the
alterations of gravity, also promoted a vertical positioning of leafstalks, i. e. orientation
towards the red LED from above. However, the curvature angle of leafstalks decreased
more significantly under 8 h/d than in continuous lighting. Combination R + B lights
effectively directed the growth of leafstalks irrespective of gravity action.
Increment of the angle between two leaves is particularly important in seedling
development. Measuring scheme of the angle between leafstalks and obtained data
are presented in Fig. 4 a and b, respectively.
Fig. 4. Measurement of the angle between two leafstalks (a) and data on this angle
(b) under different lighting and gravity conditions
4 pav. Lapkočių prasivėrimo kampo matavimas (a) ir duomenys apie lapkočių prasivėrimа
(b) skirtingomis apšviestumo ir gravitacijos sąlygomis
Opening of leafstalks in 1-g and clino-rotated leaves was not responsive to applied
B light, because it was the same as in the dark. However, R light and its combined action
with B light slightly increased the angles between the leafstalks on the clinostat in 8 h/d
photoperiod. This effect was more pronounced under continuous illumination.
In the dark, apices of hypocotyls with leaves are interconnected and show natural
gravity-related bending in the initial stage of seedling development. The study of
light-related movement of leaf lamina in 1-g and clino-rotated leaves was based on
the measurement of X angle performed according to the illustration in Fig. 5 a. After
5-day cultivation in the dark, laminas of 1-g leaves were curved more significantly in
respect to the direction of gravity action than these of clino-rotated ones (Fig. 5 b).
51

Fig. 5. Measurement of the curvature angle of the first leaf laminas
(a) and data on this angle
(b) under different lighting and gravity conditions
5 pav. Pirmojo lapo lapalakščio nuokrypio matavimas
(a) ir nuokrypio kampai
(b) skirtingomis apšviestumo ir gravitacijos sąlygomis
Usually, the laminas are positioned horizontally or move towards light after the
opening of leafstalks. According to our data, in B light they moved toward the blue LED
and obtained the orientation between horizontal and vertical positioning. Interestingly,
R or R + B light in 8 h/d oriented the laminas close to the vertical position. Curvature
of 1-g and clino-rotated laminas was quite similar under separate R or B lighting with
the exception of that of leaves in R + B in 8 h/d photoperiod of light.
Discussion. In previous studies, interactions between light-based and gravityrelated
responses in roots (Corell et al., 2003; Boccalandro et. al., 2008) and stem-like
portions of plants (Fukaki, Tasaka, 1999; Corell, Kiss, 2002; Lariquet, Fankhauser,
2005) have been shown. In higher plants, stems and roots show negative and positive
gravitropism, respectively. However, current knowledge of gravi-response of leaves
is insufficient.
Data presented in this paper show the correlation between elongation, positioning
and movement of leaves of garden cress in the dark or in the both blue and red light
when gravity-induced tropistic response of seedlings is inhibited by clino-rotation.
In the dark, gravity alteration suppressed slightly, i. e. statistically insignificantly,
the elongation of leafstalks and promoted the elongation of laminas (Fig. 1a). Blue
light applied continuously enhanced the length of both leaf parts more significantly
than that applied in 8 h/d illumination. The positive effect of red light or its combined
action with blue light on leaf growth exceeded blue-light-induced response. It is shown
blue and red light invokes extracellular acidification and expansion of epidermis cells
in young pea leaf laminas (Staal et al., 1994; Elzega et. al., 2000), which is directly
connected with elongation.
Seedlings or young plants control leaf position in response to environmental
stimuli, such as light or gravity, to optimize their photosynthetic performance and to
undergo adaptive changes in growth. The current model proposes that gravity perception
52

the hypocotyl is initiated by sedimentation of amyloplasts in endodermis cells of the
apical zone (Kiss et al., 1997) and of the basal part of leafstalk (Mano et al., 2006).
Positioning of rosette leaves correlates primarily with hypocotyl orientation. We have
also shown that gravity alteration by clino-rotation in the dark caused the change in
the positioning of hypocotyl apex. The increase of the curvature of 1-g hypocotyls
was determined in garden cress seedlings germinating under photoperiodical or
uninterrupted illumination with unilateral blue light. Blue-light-induced phototropism
can be dependent on the promotion of growth and inhibition to develop a differential
growth gradient (Whipo, Hangarter, 2003). In our experiments, hypocotyls did not
respond phototropically to red light at 1 g; however, they curved toward red LEDs
under continuous illumination on the clinostat. Thus, only continuous red lighting
eliminated the effect of simulated weightlessness on the curvature of hypocotyls
(Fig. 2). This supports the finding that weak phototropic response can be evident in
plants with reduced gravitropism (Corell, Kiss, 2002).
On the basis of the analysis of leaves positioning in blue and red light and the
obtained results we can assert that phototropic responses of leafstalks are evident under
both applied gravity conditions. Simultaneous action of blue and red light oriented the
leafstalks vertically toward the light from above (Fig. 3). Leaf flattening is important
for optimizing the efficiency of light perception and depends on the angle between
leafstalks and position of laminas in the rosette of young leaves. In the dark, the angle
between leafstalks was not affected by performed clino-rotation (Fig. 4.), but clinorotation
promoted the flattening of lamina (Fig. 5). Simultaneous action of blue and red
light enhanced the opening of leafstalks in the both gravitational conditions. Orientation
of laminas was strongly governed by the action direction of light. According to the
data obtained on rosette leaves of Arabidopsis thaliana (L.) Heynh., the direction
of gravity under continuous white light did not affected their position (Mano et al.,
2006). In contrast, when the plants were shifted to darkness, the leaves moved upward,
suggesting negative gravitropism. Besides, there was determined sedimentation of
amyloplasts in leafstalks, important in gravity perception.
Finally, these results indicate the interaction between light-related control of leaf
positioning and gravity-induced responses in leaves. Further experiments will attempt
to separate and reveal the light- and gravity-related responses for optimization of leaf
flattening and growth.
Conclusions. 1. Gravity-related alterations in the elongation of leafstalks and
laminas in the dark-grown leaves were not determined. The applied lighting stimulated
the growth of leaves. Uninterrupted, i.e. continuous, illumination with red and blue
light promoted the elongation of leafstalks more significantly than in 8 h/d photoperiod
at 1 g and in altered gravity.
2. In simulated weightlessness, the bending of hypocotyls from the upward
growth direction was enhanced. This curvature has been corrected considerably by
used lighting. The data show that under continuous illumination gravitropism could
be masked by predominant phototropism.
3. Light-related orientation of leafstalks and laminas was demonstrated when
the applied light restored their disorientation what was evoked by elimination of a
unidirectional gravitropic stimulus.
53

Acknowledgements. This work was supported by the Lithuanian State Science
and Studies Foundation.
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Gauta 2008
Parengta spausdinti 2008 04 15
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2. Corell M. J., Kiss J. Z., 2002. Interactions between gravitropism and phototropism
in plants. Journal of Plant Growth Regulation, 21: 89–101.
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2003. Phytochromes play a role in phototropism and gravitropism in Arabidopsis
roots. Advances in Space Research, 31(10): 2 203–2 210.
4. Elzega J. T. M., Staal M., Prins H. B. A. 2000. Modulation by phytochrome of
the blue light-induced extracellular acidification by leaf epidermal cells of pea
(Pisum sativum L.): a kinetic analysis. The Plant Journal, 22(5): 377–389.
5. Fukaki H., Tasaka M. 1999. Gravity perception and gravitropic response of
inflorescence stems in Arabidopsis thaliana. Advances in Space Research,
24: 763–770.
6. Inoue S., Kinoshita T., Takemiya A., Doi M., Shimazaki K. 2008. Leaf positioning
of Arabidopsis in response to blue light. Molecular Plant, 1(1): 15–26.
7. Kiss J. Z., Guisinger M. M., Miller A. J., Stackhouse K. S. 1997. Reduced
gravitropism in hypocotyls of starch-deficient mutants of Arabidopsis. Plant and
Cell Physiology, 97: 237–244.
8. Kiss J. Z., Mullen J. L., Correll M. J., Hangarter R. P. 2003. Phytochromes A and
B mediate red-light-induced positive phototropism in roots. Plant Physiology,
131: 1 411–1 417.
9. Lariquet P., Fankhauser C. 2004. Hypocotyl growth orientation in blue light
is determined by phytochrome A inhibition of gravitropism and phototropin
promotion of phototropism. The Plant Journal, 40: 826–834.
10. Lariquet P., Fankhauser C., 2005. The effect of light and gravity on hypocotyl
growth orientation. In: Wada M. Shimazaki K., Iino M. (eds.), Light sensing in
plants. Springer-Verlag Tokyo Berlin Heidelberg New York, 277–284.
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SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Tropinės sėjamosios pipirinės lapų reakcijos į šviesą ir gravitaciją
R. Losinska, D. Raklevičienė, D. Švegždienė
Santrauka
Tirtos sėjamosios pipirnės (Lepidium sativum L.) lapų augimo ir erdvinės orientacijos
reakcijos į šviesos spektrines komponentes nesvarumo, imituoto lėtu (3 aps./min.) horizontaliu
klinostatu, sąlygomis. Vienakryptė monochromatinė mėlyna (450 nm, 4,5 µmol m -2 s -1 ), raudona
(660 nm, 8,5 µmol m -2 s -1 ) šviesa arba abi kartu (9,5 µmol m -2 s -1 ) buvo panaudotos apšvietimui.
Daigai augo penkias paras klinostate ir vertikaliai stacionarioje padėtyje (kontrolė, 1 g) 8 ir
24 val./per parą šviesos arba tamsos sąlygomis. Buvo įvertinti lapų ilgiai ir hipokotilių, lapkočių
55

ei lapalakščių linkiai, kuriuos indukavo pakeisto svarumo ir apšvietimo sąlygos, bei palyginti
su kontroliniais pavyzdžiais, augusiais 1 g ir tamsos sąlygomis. Klinostatavimas tamsoje nekeitė
lapkočių ir lapalakščių tįstamojo augimo. Mėlyna šviesa (8 val./per parą) spartino lapkočių tįsimą
65–70 % kaip klinostate, taip ir 1 g sąlygomis. Pastovaus apšvietimo poveikis buvo stipresnis,
negu šviečiant 8 val./per parą. Klinostatu imituotas nesvarumas sustiprino hipokotilių nuokrypį.
Šviesa reguliavo hipokotilių augimo kryptį klinostate. Gauti rezultatai rodo, kad pastovaus
apšvietimo sąlygomis fototropizmas gali dominuoti ir maskuoti gravitropizmą. Raudonos
arba mėlynos šviesos poveikis pirmųjų lapų prasiskleidimui nebuvo esminis, tačiau suminis jų
apšvietimas ženkliai paskatino šį procesа. Nustatytas šviesos fototropinis poveikis lapkočiams ir
lapalakščiams, nes šviesa atstatė dėl vienakrypčio gravitacinio stimulo nebuvimo sutrikusia lapų
orientacijа. Gauti rezultatai rodo sаveikas tarp lapo fototropinių ir gravitropinių reakcijų.
Reikšminiai žodžiai: fototropizmas, gravitacija, gravitropizmas, klinostatas, lapas, linkis,
sėjamoji pipirnė, šviesa.
56

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Effect of 2-chlorethylphosphonic acid application on the
growth and development of Actinidia kolomikta
Laima Česonienė
Kaunas Botanical Garden of Vytautas Magnus University, Ž. E. Žilibero 6,
LT-46324 Kaunas, Lithuania, e-mail: l.cesoniene@bs.vdu.lt
The effect of foliar applied 2-chlorethylphosphonic acid was studied in male clone
M3 and female cultivar ‘Landė’ of Actinidia kolomikta. This plant regulator shortened the
vegetation period for 9–15 days compared to the control. Plants of A. kolomikta treated with
the 2-chlorethylphosphonic acid revealed strong morphological changes. The length of shoots
treated with 0.2 % solution was significantly shorter at the end of vegetation period. The
application of 2-chlorethylphosphonic acid significantly reduced the number of flowers and
berries per metre length of branch. This growth regulator influenced changes in the parameters
of berries and their mass also.
Key words: clone, cultivar, fruiting shoot, growth regulator, vegetation period.
Introduction. The genus Actinidia Lindl. is comprised of perennial climbing
species, which vegetative shoots grow very rapidly and achieve from 2 to 3 m length
per year. These shoots produce large amounts of hormones and absorb considerable
quantity of nutrients (Inglese, Gulo, 1992; Salinger, Kenny, 1995). Hereby, these
physiological changes disimprove growth and development of fruit (Manson, Snelgar,
1995; Snowball, 1995).
Plant growth regulators are frequently used with a purpose to maintain a balance
between growth and productivity of horticultural plants. The growth regulator
2-chlorethylphosphonic acid induces anatomical and biochemical changes of cambium
and enhances stability of bark. Cell division becomes more intensive in the radial
direction, accordingly the shoots growth slows down (Pakasorn et al., 1995).
As several authors (Henzell et al., 1986; Manson, Snelgar, 1995; Salinger, Kenny,
1995) reported, an alternative approach to mechanically remove summer growth would
be the application of plant growth regulators. Evaluation of different horticultural plants,
such as high bush blueberry, peach, and apricot, demonstrated that different number and
time of plant regulators treatment could increase winter hardiness and change duration
of flowering and fruit ripening (Davies, 1992, Walton et al., 2000; Williamson, Moust,
1996). This quality of plant regulators is very relevant by cultivation of some Actinidia
species, which flowering coincides with a period of late spring frosts.
The aim of this study was to investigate the feasibility to regulate growth
and productivity characteristics of Actinidia kolomikta (Maxim.) Maxim. using
2-chlorethylphosphonic acid.
57

Object, methods and conditions. Two-year-old male clone M3 and female
cultivar ‘Landė’ were planted at 40–50 cm space within three replications by 20 plants.
The plants were sprayed with 2-chlorethylphosphonic acid solution of 0.2 and 0.1 %
concentration. The control plants were sprayed with water. Suitable conditions for
applying growth regulator occurred in the last decade of May, when the temperature
ranged to 24.1 °C. The first full-developed leaves were formed on the shoots, whereas
the fixed length of one-year-old shoots reached 16.3 ± 1.4 cm.
The phenological phases were observed using the methods of assessing seasonal
development of plants being introduced (Коропачинский, 1982; Лапин, 1982). The
collection of A. kolomikta was observed and assessed twice a week on the same days
of the week. The following main phases of seasonal development were recorded: the
beginning of shoot growth, the beginning of budding, the beginning of flowering, the
end of flowering, the beginning of ripening, the end of ripening, the end of growth of
mixed shoots, and the end of vegetation. Length of one-year-old shoots was measured
after 14 and 28 days as well as at the end of vegetation period. Number of fruiting
shoots, flowers, and berries per metre length of a branch as well as parameters of
berries were ascertained.
Results. The plants of clone M3 affected with 0.1 % and 0.2 % solution finished
vegetation earlier in comparison to the control (September 28, September 25 and
October 7, respectively), i. e., vegetation period shortened for 13 and 10 days. It has
been determined by fixing the change in phenological phases that application of growth
regulators influenced the duration of the vegetation of female cultivar ‘Landė’, too.
The end of vegetation of plants treated with 0.2 and 0.1 % solutions and that of control
plants was fixed at the end of September or at the first decade of October. The growth
regulator shortened the vegetation period of cultivar ‘Landė’ from 9 to 15 days.
Shoots of clone M3 affected with solutions 0.1 % and 0.2 % of
2-chlorethylphosphonic acid solution were significantly shorter in comparison to the
control (23.3 ± 1.7 cm, 15.7 ± 1.1 cm and 32.3 ± 2.3 cm, respectively). Evaluation of
shoot length revealed the same tendency after 28 days. The shoots of control plants
demonstrated the most intensive growth; therefore their length at the end of vegetation
period on average was 62.1 cm (Fig. 1).
The solutions 0.1 % and 0.2 % of 2-chlorethylphosphonic acid suppressed growth
of cultivar ‘Landė’ during the first 14 days (Fig. 2). The length of shoots treated with
0.2% solution was significantly shorter (45.2 ± 2.8 cm) at the end of vegetation period,
too. The length of control plants shoots (63.0 ± 3.1 cm) and the same of plants treated
with 0.1 % solution (59.3 ± 2.5 cm) did not differ significantly (LSD 05
= 7.39).
58

Fig. 1. The influence of 2-chlorethylphosphonic acid solution on the length of
fruiting shoots of clone M3: A – before treatment, B – after 14 days,
C – after 28 days, D – at the end of vegetation period
1 pav. 2-chloretilfosfoninės rūgšties įtaka M3 klono derančiųjų ūglių ilgiui:
A – prieš purškimą, B – po 14 dienų, C – po 28 dienų, D – vegetacijos pabaigoje
Fig. 2. The influence of 2-chlorethylphosphonic acid solution on the length of
fruiting shoots of cultivar ‘Landė’: A – before treatment, B – after 14 days,
C – after 28 days, D – at the end of vegetation period
2 pav. 2-chloretilfosfoninės rūgšties įtaka veislės ‘Landė’ derančiųjų ūglių ilgiui:
A – prieš purškimą, B – po 14 dienų, C – po 28 dienų, D – vegetacijos pabaigoje
Plants of A. kolomikta treated with the growth regulator revealed strong
morphological changes. The 0.1 % and 0.2 % solutions induced fall of leaves from
1–4 internodes, moreover, the one-year-old shoots lost rambling abilities. The plants
treated with 0.2 % solution developed shrubby shape also. These changes reduced at
the end of vegetation period.
It has been determined that the number of fruiting shoots per metre length of
two-year-old branch of M3 control plants and the plants treated with 0.1 % solution
was statistically reliably larger as compared to that of plants treated with 0.2 %
solution (LSD 05
= 1.95) The number of fruiting shoots of cultivar ‘Landė’ treated with
2-chlorethylphosphonic acid did not differ significantly from the control (LSD 05
= 2.53)
(Fig. 3).
59

Fig. 3. The influence of 2-chlorethylphosphonic acid solution on the number of
fruiting shoots of clone M3 and cultivar ‘Landė’
3 pav. 2-chloretilfosfoninės rūgšties įtaka klono M3 ir veislės ‘Landė’
derančiųjų ūglių kiekiui
The spraying of cultivar ‘Landė’ plants significantly reduced the number of
flowers and berries in comparison to the control. The plants affected with 0.1 % and
0.2 % solution of 2-chlorethylphosphonic acid on average had 21.7 and 14.6 flowers
(control plants – 27.7 flowers) per metre length of branch, respectively. They set 14.9
and 10.2 berries (control plants – 19.3 berries), respectively (Fig. 4).
Fig. 4. The influence of 2-chlorethylphosphonic acid solution on the number of
flowers and berries of cultivar ‘Landė’
4 pav. 2-chloretilfosfoninės rūgšties įtaka veislės ‘Landė’ žiedų ir uogų kiekiui
Assessment of growth regulator related changes in the parameters of berries and
their mass confirmed that the average mass of berry enlarged by treatment of 0.1 %
solution in comparison to control plants was 2.5 ± 0.01 g and 2.0 ± 0.05 g, respectively.
The average berry mass of plants affected with 0.2 % solution was significantly less –
1.6 ± 0.01 g, LSD 05
= 0.102 (Fig. 5).
60

Fig. 5. The influence of 2-chlorethylphosphonic acid on the parameters
of berries and the average berry mass of cultivar ‘Landė’
5 pav. Veislės ‘Landė’ uogų matmenų ir vienos uogos vidutinės masės kitimas,
veikiant 2-chloretilfosfonine rūgštimi
Changes occurred in the parameters of berries also. Significant differences in
respect of berry length were determined (LSD 05
= 0.69). The width of berries of plants
was largest after treatment with 0.1 % solution, whereas the width of berries of plants
sprayed with 0.2 % solution and these of the control did not differ (LSD 05
= 0.79).
Discussion. In order to define the influence of 2-chlorethylphosphonic acid on
changes of different morphological characteristics, duration of phenological phases
were evaluated. The results corroborated comprehensive physiological impact on
male and female plants of A. kolomikta. This growth regulator changes intensity of
shoot growth, number of flowering shoot as well as of berries per metre length of a
branch. According to other authors, the number of fruiting shoots, the number and
average mass of berries per metre length of a branch were characterized as the fruiting
potential of Actinidia species (A. deliciosa, A. arguta) (Kulczewski, 2003; Samanci,
1997; Tiyayon, Strik, 2003).
Different trials were carried out using plant growth regulator treatments of Ethrel
(2-chlorethylphosphonic acid) in order to improve fruit size, yield and quality of
kiwifruit (A. deliciosa). The fruit size and quality was found better at a concentration
of 0.08 % than for the controls (Jindal et al., 2003). Studies of other authors revealed
effect of this growth regulator on fruit set and cropping of pears (Wells, McArtney,
1993) and rabbiteye blueberry (Kugishima et al., 2004).
Application of growth regulator determines shortening of vegetation period of
A. kolomikta. It could be used growing late cultivars, which berry ripening coincides
with early frosts in autumn. 2-chlorethylphosphonic acid retards shoot growth of
male ant female plants. It is very important to apply this growth regulator in the large
plantations with a purpose to reduce labour expenditures of pruning.
Conclusions. 1. The application of 2-chlorethylphosphonic acid significantly
decreases the growth of A. kolomikta shoots and causes the forwardness of the end
of vegetation period.
61

2. The important indices of fruiting potential such as number and mass of berries
per metre length of branch could be controlled using different solutions of
2-chlorethylphosphonic acid.
3. The solution 2-chlorethylphosphonic acid of 0.1 % significantly enlarges the
average berry mass.
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62

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14. Wells G. H., McArtney S. J. 1993. Fruit quality and cropping of pears II.
Response of ‘Nijisseiki’ Asian pears to the concentration of chlorethephon. Acta
Horticulturae, 329: 252–262.
15. Wiliamson J. G., Moust B. E. 1996. Possible use of ethephon for bloom delay in
blueberries // Proceedings of 8 th Southeast Blueberry Conference, 21–24.
16. Коропачинский И. Ю. (ред.) 1982. Интродукция древесных растений в
лесостепном Приобъе. Новосибирск.
17. Лапин П. И. (ред.) 1982. Исследование древесных растений при интродукции.
Москва.
SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
2- chloretilfosfoninės rūgšties poveikio Actinidia kolomikta augimui
ir vystymuisi tyrimai
L. Česonienė
Santrauka
Atliktų tyrimų tikslas buvo nustatyti 2-chloretilfosfoninės rūgšties poveikį Actinidia
kolomikta vyriško klono M3 ir veislės ‘Landė’ augalų augimui ir vystymuisi. Nustatyta, kad
paveikus augalus šiuo augimo reguliatoriumi, jų vegetacijos periodas sutrumpėjo 9–15 dienų.
Nupurkšti augalai pasižymėjo morfologiniais pakitimais. Paveikus augalus 0,2 % augimo
reguliatoriaus tirpalu, ūglių ilgis vegetacijos pabaigoje buvo statistiškai patikimai mažesnis.
Nupurkštiems 2-chloretilfosfoninės rūgšties tirpalu augalams buvo būdingas mažesnis žiedų ir
uogų kiekis. Veikiant skirtingoms augimo reguliatoriaus koncentracijoms, kito ir uogų dydžio
parametrai.
Reikšminiai žodžiai: augimo reguliatorius, derantis ūglis, klonas, veislė, vegetacijos
periodas.
63

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF HOR-
TICULTURE AND LITHUANIAN UNIVERSITY OF AGRICULTURE.
SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Photomorphogenic responses of garden cress to light in
altered gravity
Danguolė Raklevičienė, Danguolė Švegždienė, Regina Losinska
Institute of Botany, Laboratory of Plant Physiology, Sector of Gravitational
Physiology, Žaliųjų Ežerų 49, LT-80406 Vilnius, Lithuania
E-mail: danguole.rakleviciene@botanika.lt
The purpose of the present research was to determine growth and developmental
responses of garden cress (Lepidium sativum L.) seedlings to blue (450 nm,
4–5 µmol m -2 s -1 ) light and to its combined effect with red (660 nm, 13 µmol m -2 s -1 ) or far-red
(735 nm, 0.8–0.9 µmol m -2 s -1 ) illumination as well as to red with far-red light under usual and
altered (by a 50-rpm horizontal clinostat) gravity conditions. Lighting was provided by light
emitting diodes. Effects of light under altered gravity were evaluated after a 5-day cultivation
of seedlings on the clinostat or vertically at a stationary position both in 12 h per day light
photoperiod and without illumination. The plants were grown in special containers fitted to the
stationary control (1 g) and centrifuge-clinostat devices. Biometric analysis of organs showed that
inhibition of hypocotyl elongation and promotion of leaf expansion caused by applied lighting
were the same for both 1-g and clino-rotated plants. However, the rate of growth suppression
by blue light and by its combined action with far-red light was promoted in altered gravity. The
area of clino-rotated leaves in the combined red and far-red light was larger than that of 1-g ones
due to the more rapid radial expansion. Quantifiable assessment of photosynthetic pigments
in leaves showed that only in blue lighting the amount of chlorophyll b was enhanced under
clino-rotation as compared with 1 g. The obtained data show that plant reactions to the precise
spectral components and photon flux density of light can be modulated by gravity alterations
and support the view of the interaction between photo- and gravity-related responses.
Key words: clinostat, garden cress, gravity, growth, leaf, light, seedlings.
Introduction. Plants evolved and adjusted to constant, prolonged presence of
gravity and simultaneously changing action of solar lighting on Earth. Gravity is
important for the orientation of the plant. Primary shoots and roots of seedlings are
often used in studies of gravitropism, i.e. directed growth in response to gravity.
Organs of developing plants have a typical orientation relative to the gravity vector
(Kiss et al., 2002), which can be modified by light conditions (Hangarter, 1997).
Light- and gravity-related signalling pathways in plants interact and make it difficult
to determine whether plant growth is influenced by light, gravity or by the combined
action of these two stimuli. Existing interactions between responses to light and gravity
were shown using gravitropic or phototropic mutants (Hangarter, 1997; Kiss et al.,
2003; Lariquet, Fankhauser, 2004), altering gravity conditions on Earth by rotation
or reorientation of plants with regard to the gravity vector (Brown et al., 1996a; Vitha
et al., 2000) and performing investigations at microgravity in space (Stutte et al., 2006).
65

Gravity-related growth responses were shown in Arabidopsis (Merkys et al., 1984),
garden cress (Merkys, Laurinavičius, 1990), and other seedlings during experiments
carried out in microgravity in the dark or in white light of a high fluence rate. Light can
either enhance or reduce gravitropic responses of the plant (Hangarter 1997; Okada,
Shimura, 1992; Corell, Kiss, 2002). Photomorphogenic effects of light quality and
quantity play a crucial role in plant development and were not detailed under altered
gravity conditions.
Thus, taking into consideration the fact that spectral components (Hangarter,
1997), photon flux density (Galland, 2002) and action direction (Vitha et al., 2000;
Raklevičienė et al., 2005) of light are factors influencing gravity-related growth
responses of plants, we can presume that photophysiological effectiveness of a certain
light wavelength might be more obvious when gravitropic stimulation associated with
the vector of gravity is reduced by clino-rotation. Irradiation of clino-rotated plants
is one of the modes of determining the effects of separate components of the light
wavelength and fluence rate on plant growth.
The aim of the present work was to estimate the effects of blue (B) light and its
combined action with red (R) or far-red (FR) components of light spectrum on lightrelated
responses of axial organs and leaves of seedlings under gravity altered by
50-rpm horizontal clino-rotation (HC).
Object, methods and conditions. Seeds of garden cress (Lepidium sativum L.)
were planted on a transparent solid MS medium with one-half-strength salts (Murashige,
Skoog, 1962) and 0.2 % (w/v) gelrite (Sigma) and for 5 days were grown in containers
fitted to the device of a stationary vertical control (1 g) and to a horizontal clinostat
rotating at 50 rpm for creating altered gravity conditions. Seedlings were cultivated both
with and without illumination. Light emitting diodes (LEDs) were used for illumination
in the containers (cartridges with a light module ensured irradiation of the plant from
above). B (peak wavelength 450 nm, photon flux density 5 µmol m -2 s -1 ), R (660 nm,
13 µmol m -2 s -1 ) and FR (735 nm, 0.8-1 µmol m -2 s -1 ) LEDs were applied for irradiation
on the 50-rpm horizontal clinostat and in the vertical control device. The influence of
B light and the combined effect of B with R (B + R), B with FR (B + FR) and R with
FR (R + FR) light on the length of roots, hypocotyls, leafstalks and laminas as well as
on the amount of fresh biomass and leaves photosynthetic pigments was measured.
The photoperiod was 12 h per daylight. All experiments were performed at ambient
temperatures of 22 °C to 24 °C. Total intensities of lighting combinations B + R,
B + FR and R + FR were approximately 16–17.5 and 13.5 µmol m -2 s -1 , respectively.
The fluence rate of light was measured by a quantum sensor (PM-20, Sonopan, Poland).
At the end of each experiment, seedlings were photographed with the PENTAX *ist
D digital camera, leaves were scanned and images were analyzed using SigmaScan
Pro 5 (Jandel Scientific Software) on the PC platform. Spectrophotometrical analyses
of photosynthetic pigments in leaves were performed using the UV/VIS “Helios
Gamma” (190–1 100 nm) spectrophotometer. Concentrations of chlorophyll a (chl. a),
chlorophyll b (chl. b) and carotenoids were measured in three replicates after acetone
extraction and calculated according to Lichtenthaler (1987). Measurements of seedlings
grown in different lights were compared with the ones in the dark; also, measurements
of seedlings grown on the clinostat were compared with the ones at 1 g. Statistical
66

analysis was performed using Excel (version 7.0). The data presented in figures are
given as the mean ± standard error. Statistical significance was set at p < 0.05.
Results. In the dark, the length of hypocotyls and roots of seedlings
grown under 50 rpm clino-rotation did not significantly differ from the control ones
(Fig. 1 a, b).
Fig. 1. Length of hypocotyls (a) and roots (b)
of garden cress seedlings grown in the dark and in blue (B)
light or in blue light supplemented with red (B + R),
far red (B + FR) or red with far red (R + FR) spectral components of light in normal
(1 g) and altered gravity (HC) conditions
1 pav. Sėjamosios pipirnės hipokotilių ir šaknų ilgiai daigų, augintų tamsoje,
šviečiant mėlyna (M) ar kompleksu su raudona (M + R) ir tolimąją raudona
(M + TR), ar raudona su tolimąją raudona (R + TR) šviesa normalaus (1 g)
ir pakeisto svarumo sąlygomis
Monochromatic blue light inhibited the elongation of 1-g and clino-rotated
hypocotyls by 17 % and 29 %, respectively. Differences between clino-rotated and 1-g
hypocotyls were significant at p < 0.05. Simultaneous application of B + R spectral
components of light enhanced the suppression of hypocotyl growth approximately by
30 % independently on gravity conditions. However, in B + FR lighting, the clinorotated
hypocotyls were significantly shorter than the 1-g hypocotyls. The influence of
R + FR light was similar to that of B + R lighting. Thus, the applied B + FR combination
of lighting inhibited the hypocotyl elongation on clinostat more significantly than
at 1 g. Although the seedlings were cultivated on a transparent medium, roots were
unequally illuminated from above because of leaf shading. Differences between the
length of 1-g and clino-rotated roots were negligible in the dark, monochromatic B
and combined B + R or F + R illumination. Only in R + FR light the roots were shorter
in altered gravity.
Measurements of leafstalk and leaf lamina parameters in the dark and lights under
altered and normal gravity conditions are presented in Fig. 2.
Monochromatic B light as well as combined B + R or B + FR lighting promoted
the elongation of both leafstalks and laminas of leaves. It should be noted that the
combination of B + R enhanced the fluence rate of illumination. Despite comparatively
low fluence rate of FR light, its effect on the elongation of leaves was considerable
when FR was integrated with lights of other wavelengths, i. e. with 450 or 660 nm.
However, no differences between the lengths of 1-g and clino-rotated leaves were
determined in the applied lighting.
67

Fig. 2. Length of leafstalks and laminas of leaves in seedlings grown in different
lighting and gravity conditions
2 pav. Lapkočių ir lapalakščių ilgiai daigų, augintų skirtingomis apšviestumo ir gravitacinio
dirginimo sąlygomis
At the end of experiments, seedlings of garden cress had two leaves of different
sizes and the area of the leaf largely depended on the parameters of the lamina.
Measurements of the areas of leaves did not show statistically significant gravityrelated
differences in the dark and in the applied B light as well as in its combined
action with R (B + R) or FR (B + FR) light (Fig. 3).
Fig. 3. Area of leaves of garden cress seedlings grown in the dark and in combined
lighting under normal (1 g) and altered gravity (HC) conditions
3 pav. Lapų plotai sėjamosios pipirnės daigų, augintų skirtingomis apšviestumo ir normalaus
(1 g) bei pakeisto svarumo (HK) sąlygomis
68

Promotion of leaf expansion by B lighting conditions was similar to both 1-g and
clino-rotated plants. B + R and B + FR lightings increased considerably the area of
control and clino-rotated leaves. However, differences between the area of 1-g and
clino-rotated leaves were determined in combined R + FR lighting. In the dark, fresh
weight of leaves of average plant increased slightly, i.e. statistically insignificantly,
on clinostat as compared with control at 1 g (Fig. 4).
Fig. 4. Effect of illumination on fresh weight of leaves
in natural and altered gravity
4 pav. Šviesos poveikis lapų žaliajai masei natūraliomis ir
pakeisto svarumo sąlygomis
However, a statistically significant increment in leaf biomass was determined on
the clinostat in B light. Fresh weight of 1-g leaves was the same as in the dark, whereas
the integrated effect of the two components of light spectrum promoted similarly the
augmentation of leaf biomass in both gravity conditions. Nevertheless, a tendency of
a smaller amount of fresh biomass of clino-rotated leaves as compared with 1-g leaves
in B + R lighting was observed.
A negligible amount of photosynthetic pigments in etiolated 1-g and clino-rotated
leaves was determined. The applied monochromatic B light caused the appearance of
both chlorophylls and carotenoids in garden cress leaves (Fig. 5).
However, the amount of photosynthetic pigments significantly increased when R
and FR lights were applied simultaneously with B light. The increment of chlorophyll
a occurred independently of gravity conditions and strongly depended on R light (Fig.
5a). The influence of R light was predominant in the combined B+R lighting. Only
in B light the amount of chlorophyll b in clino-rotated leaves was larger than in 1-g
ones (Fig. 5b). In other lighting conditions, the amount of photosynthetic pigments
did not significantly depend on gravity conditions. The combined action of B + R
lighting resulted in an increment of carotenoids as compared with other conditions of
illumination independently on gravity conditions.
69

Fig. 5. Content of chlorophylls a and b (in graphs a and b, respectively),
their ratio (c) and content of carotenoids (d) in garden cress leaves in different
lighting and gravity conditions
5 pav. Chlorofilų a ir b kiekis (a, b) ir jų santykis (c) bei karotinoidų kiekis (d) sėjamosios
pipirnės lapuose esant skirtingoms šviesos ir gravitacinio dirginimo sąlygoms
Discussion. Previous studies demonstrated that gravity force affects various
processes in the life cycle of plants, i. e. seed germination, growth, flowering and seed
formation (Merkys, Laurinavičius 1983; Musgrave et al., 1997; Volkmann, Baluška,
2006). This revealed that terrestrial plants evolved under a 1 g gravitational environment
on Earth and acquired the ability to use gravistimuli to regulate their growth and
development. Thus, growth and morphogenetic responses of plants are considered
to be highly influenced by constant gravity. The elongation of the overground part of
seedlings was promoted by microgravity (Laurinavičius et al., 2001; Soga et al., 2002).
However, for further optimal development of plants, light with a strong effect on the
overground part is necessary. Inhibition of the elongation of the overground part as well
as an increase of leaves in light are estimated as positive reactions of the plant adapting
to environmental changes. Interactions between gravity and light-related responses
in plants have been investigated so far insufficiently and need precise approaches to
reveal masked physiological reactions.
Blue light rapidly and strongly inhibits hypocotyl elongation during the
photomorphogenic response known as de-etiolation, i. e. transformation of a darkgrown
seedling into a pigmented, photoautotrophic organism (Cosgrove, 1981; Folta,
Spalding, 2001). High-irradiance B light is especially effective in this respect as it
inhibits growth more quickly and to a greater extent than equal irradiances of red light.
Our earlier data on garden cress demonstrated that the effect of light wavelengths of
70

450, 660 and 735 nm applied in a comparatively low density of the photon flux had a
stronger inhibiting effect on the elongation of garden cress hypocotyls and leafstalks
when gravitropic stimulation was eliminated by clino-rotation as compared with natural
gravity (Raklevičienė et al., 2005, 2007). The presented data show that monochromatic
B light reduced the elongation of 1-g and clino-rotated hypocotyls by 16 % and 29 %,
combined B + R lighting – by 30 % and 45 %, B + FR – by 56 % and 61 %, R + FR –
by 33 % and 39 %, respectively. The impact of FR light showed a strong elongation
inhibiting effect. It is important because the photon-sensing system can be activated by
the FR (700–800 nm) spectral component of light and interacts with the gravity-sensing
system (Johnson et al., 1996). Our data on garden cress seedlings show a significant
growth inhibition caused by a simultaneous action of B + R lights and prove that under
changed gravity the inhibition is stronger as than under usual gravity conditions.
The size of leaves is determined at an early stage of plant development (Cookson
et al., 2005), but histological differentiation is definitely dependent on light spectral
quality (Lee et al., 2000) and could be related to gravity (Stutte et al., 2006; Raklevičienė
et al., 2007). In our experiments, the applied illumination enhanced the total length of
both 1-g and clino-rotated leaves. It should be noted that FR lighting combined with B
or R lighting increased the length of 1-g leafstalks most significantly (approximately
by 2.3 and 2.2 times, respectively). However, the total leaf length did not differ in both
gravitational environments. The area and fresh weight of garden cress leaves largely
depended on leaf lamina parameters, which usually increased in light due to the apicalbasal
elongation, radial expansion and tissue differentiation. In the dark, any differences
between 1-g and clino-rotated leaf areas and lamina lengths were determined. The area
and fresh weight of leaves increased significantly when plants were irradiated with
lights of two wavelengths simultaneously, i. e. FR + B or FR + R lighting. The latter
combination is physiologically and photochemically active in driving phytochrome
conversions each in the opposite direction. In our experiments, it promoted significantly
the radial expansion of clino-rotated leaf lamina and, therefore, we assumed that it was
involved in light and gravity interaction through phytochrome activity.
Also, our results related to the amount of photosynthetic pigments showed that
monochromatic R light or R + B light enhanced considerably the amount of chlorophylls
independently of gravity conditions. The influence of R light was predominating. Only
the B light provoked the gravity-dependent alteration in photosynthetic pigments
content: the amount of chlorophyll b in clino-rotated leaves increased approximately by
30 % in comparison with 1-g plants (Fig. 5b). The impact of light on the concentration
of carotenoids was the same for plants grown under both gravitational conditions.
A high chl. a /chl. b ratio, usually observed in chlorotic plants, indicates a reduced
antenna size relative to reaction centres of photosystems. It should be mentioned that
the excess energy in B light photon is largely converted into heat whose circulation
between leaves and air is retarded at a lower gravity level (Kitaya et al., 2003). Data
on the effect of gravity on photosynthesis is quite controversial (Brown et al. 1996b;
Tripathy et al., 1996; Stutte et al, 2005). Our findings are in partial agreement with
photosynthetic measurements that showed possibility of light-induced photosynthetic
71

esponses under altered gravity (Brown et al., 1996b; Stutte et al., 2005); however,
mechanisms for integration of red, far-red and blue light signalling in control of
photomorphogenesis remain largely unknown.
Conclusions. 1. Monochromatic blue light and its combined action with red
and far-red light in comparatively low light intensities caused a rate of inhibition
in clino-rotated hypocotyls more significant than in those grown in natural gravity
conditions.
2. Blue and red light-related elongation and radial expansion of leaves of garden
cress significantly increased when the action of these lights was combined with far-red
lighting. Simultaneous action of red and far-red lighting promoted a radial expansion
in clino-rotated leaves more significantly than in 1-g leaves.
3. Altered gravity did not provoke obvious changes in the amount of
photosynthetic pigments with the exception of chlorophyll b, the amount of which
increase in monochromatic blue light with photon flux density of approximately
4.5 µmol m -2 s -1 .
4. The obtained data support the opinion of the existing photo- and graviphysiological
interactions that can be modulated by precise parameters of light under
altered gravity conditions.
Acknowledgements. This work was supported by the Lithuanian State Science
and Studies Foundation.
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SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Fotomorfogenetinės sėjamosios pipirinės reakcijos į šviesа pakeisto
svarumo sąlygomis
D. Rachlevičienė, D. Švegždienė, R. Losinska
Santrauka
Darbo tikslas – nustatyti sėjamosios pipirnės (Lepidium sativum L.) daigų augimo ir
vystymosi reakcijas į mėlyną (450 nm, 4–5 µmol m -2 s -1 ) šviesą ir į kompleksinį jos poveikį su
raudona (660 nm, 13 µmol m -2 s -1 ) ar tolimąją raudona (735 nm, 0,8–0,9 µmol m -2 s -1 ) šviesa
bei į raudonos ir tolimosios raudonos šviesos poveikį natūralios gravitacijos ir horizontaliu
klinostatu (50 aps./min.) imituoto nesvarumo sąlygomis. Apšvietimui naudoti puslaidininkiniai
šviesos diodai. Šviesos poveikis daigams buvo įvertintas po 5 parų kultivavimo klinostatuojant
ir stabilioje vertikalioje padėtyje, esant 12 val. šviesos arba tamsoje. Daigai buvo auginami
specialiuose konteineriuose, prijungtuose prie stacionarios vertikalios kontrolės įrenginio ir
centrifugos-klinostato komplekso horizontalių aрių. Atlikus biometrinę daigų analizę buvo
nustatyta, kad naudoti apšvietimo moduliai slopino hipokotilių tįstamаjį augimą ir skatino
lapų augimą kaip įprasto, taip ir pakeisto svarumo sąlygomis. Tačiau, augimo tempo lėtinimas
apšvietus mėlyna ir mėlyna su tolimąja raudona buvo intensyvesnis pakeistame svarume.
Apšvietus raudona su tolimąją raudona, klinostatuotų lapų plotai dėl radialinio tįsimo buvo
didesni negu kontrolinių. Atlikus fotosintetinių pigmentų kiekybinį vertinimа buvo nustatyta,
kad tik mėlynoje šviesoje klinostatavimas padidino chlorofilo b kiekį lapuose. Rezultatai parodė,
kad augalų reakcijos į tam tikras šviesos spektrines komponentes ir fotonų srauto tankį gali
būti veikiamos gravitacijos ir patvirtina nuomonę apie sąveikas tarp reakcijų, indukuojamų
šviesa ir gravitacija.
Reikšminiai žodžiai: augimas, daigai, gravitacija, klinostatas, lapas, sėjamoji pipirnė,
šviesa.
74

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF HOR-
TICULTURE AND LITHUANIAN UNIVERSITY OF AGRICULTURE.
SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Gravisensing of garden cress roots under varying
g-magnitude
Danguolė Švegždienė, Dalia Koryznienė, Danguolė Raklevičienė
Institute of Botany, Laboratory of Plant Physiology, Sector of Gravitational
Physiology, Žaliųjų Ežerų 49, LT-08406 Vilnius, Lithuania
E-mail: danguole.svegzdiene@botanika.lt
The aim of present work was to characterize and compare the sensitivity of roots and
positioning of amyloplasts in gravity sensing cells, when the gravity (centrifugal) forces of
lower than 1 g magnitude were applied for gravitropic stimulation.
After 30 h of growth in 1-g conditions, etiolated seedlings of garden cress (Lepidium
sativum L.) were stimulated gravitropically on a centrifuge-clinostat. For sensitivity study of
roots, an exposition to the transverse action of 0.004, 0.019 or 1 g and following clino-rotation
has been applied. Quantitative analysis of curvature kinetics revealed a relation between the
magnitude of acting force and the value of threshold stimulus dose. It was evaluated that the
doses exceeding 2, 4 and 7 g × s could be effective for induction of root gravitropic reaction
by the forces of respective magnitude. Amyloplast positioning was analyzed in gravity sensing
cells of roots subjected to transverse directed forces of 0.001 g to 0.156 g for 4 h (resulting in 16,
65, 203, 563 or 2 252 g × s). Measurements were performed by light microscopy on semi-thin
median longitudinal sections of the root apices fixed in glutaraldehyde and embedded in Epon.
Significant and g-dependent sedimentation of amyloplasts was determined only in response to
the stimulus dose of 203 g × s and strongest ones. It exceeds considerably the earlier evaluated
threshold dose for root gravitropic stimulation by the force of the comparable magnitude. Our
data imply that there is no direct ratio between the gravitropic sensitivity of garden cress roots
to the action of the forces lower than 1-g magnitude and the respective alteration in amyloplast
positioning. It allows supposing that a slight, however, transient, sedimentation of statoliths
may trigger and transmit the gravitropic stimulus.
Key words: amyloplast, garden cress, gravity, root, sensitivity, stimulus.
Introduction. Gravisensing in roots occurs within specialized cap cells –
statocytes containing starch-filled amyloplasts (statoliths). On changing a magnitude
and/or direction of gravity force, statolith movement provides the information leading
to directional growth response of organ. A gravitropic response depends on a stimulus
dose (D) as a product of magnitude of gravity (g) or physiologically adequate centrifugal
force (Merkys et al., 1981) and the duration of its action (t), when they are stimulated at
90° angle. Sensitivity of root sensory system can be characterized by threshold stimulus
dose (D-threshold, maximal quantity of stimulation in g´s still unable to provoke
any response). Usually it is determined by stimulating the organ with corresponding
stimuli, registration of following responses and analysis of dose-response curves. Its
values vary in a wide range depending on the methods of stimulation and estimation
75

(Laurinavičius et al., 1998; Volkmann, Tewinkel, 1996; Perbal et al., 2002).
According to the hypotheses concerning actin-based gravisensing in roots, a
settling of amyloplasts constitutes the initial act of gravity sensing (Driss-Ecole et al.,
2003; Baluška, Hasenstein, 1997; Sievers et al., 1991). During the past decades, the
plant cytoskeleton has been identified as an important target for a manifold of signal
chains that are triggered by many environmental cues. Recent works give an opportunity
to better understanding of its role in gravisensing (Braun, Limbach, 2006; Kuya et al.,
2006; Palmieri, Kiss, 2005; Švegždienė et al., 2005, 2007). Despite a rapidly expanding
field of in vivo imaging of cytoskeletal elements and their links with amyloplasts, there
are only a few direct investigations on behaviour of these plastids in statocytes under
the stimulation by gravity force of lower than 1 g magnitude (Laurinavičius et al., 2001;
Volkmann, Tewinkel, 1996; Yoder et al., 2001). In most experiments carried out on
purpose to study the gravitropic sensitivity of plant organs and function of statocytes,
the 1-g force was applied for stimulation. Therefore, the aim of the present study was
to characterize and compare the sensitivity of cress root gravisensors and amyloplast
location within statocytes, using centrifugal forces of low magnitude for gravitropic
stimulation. Attention was focused on evaluating of the threshold dose of gravitropic
stimulus and its relation with the positioning of statoliths.
Object, methods and conditions. Garden cress (Lepidium sativum L.) was used
as plant material. Seeds were germinated and grown for 30 h in special cylinder-shaped
containers in darkness, at 23.0° ± 0.5 °C. A longitudinal axis of embryos and an original
direction of root growth were parallel to the vector of Earth gravity (1 g). For gravitropic
stimulation, the containers were placed on a centrifuge-clinostat with two-orthogonal
axes allowing independent or simultaneous rotation around the horizontal (clinostat)
and vertical (centrifuge) axes (Laurinavičius et al., 2001).
Responsiveness of roots to varying g-loads has been studied after an exposition to
clino-rotation (50 rpm) and centrifugal force of 0.004, 0.019 or 1 g till 60 min (resulting
in 2 to 600 g × s stimulus doses). Curvature from the original growth direction was
measured after the subsequent rotation on a clinostat for additional 60 min. Curvature
angles were plotted against logarithm of the resulting doses and a threshold dose was
calculated for the each applied centrifugal force.
To analyze a relationship between the location of amyloplasts and stimulus doses,
the seeds were germinated in open 10-ml thin glass funnels to retain their respective
orientation and to mark the direction of gravitropic stimulation. The funnels were fixed
in special plastic holders and placed in to containers for the vertical growth at 1 g. After
30 h, the containers were mounted on horizontal axes of centrifuge-clinostat and rotated
for 4 h by the speed of 10, 20, 50 or 100 rpm. In dependence on the distance from the
clinostat axes, the roots have been subjected to the action of horizontal clino-rotation
or centrifugal forces of 0.001, 0.004, 0.014, 0.039 or 0.156 g (resulting in the stimulus
doses of about 0 (simulated weightlessness), 16, 65, 203, 563 or 2 252 g × s). After
stimulation, the seedlings were fixed in 4 % (v/v) glutaraldehyde in 0.1 M sodium
phosphate buffer (pH 7.2) for 45 min without stopping the clino-rotation. The root
apices were then transferred into fresh portion of glutaraldehyde solution for 2 h at
+4 °C, postfixed in 1 % (w/v) OsO 4
, dehydrated and embedded in Epon by standard
procedures.
76

Statolith positioning was studied employing light microscopy on semi-thin median
longitudinal sections of root caps after staining by toluidine-blue. The plane of sections
was parallel to the direction of stimulation. Location of the individual amyloplast in
the cells of the 2 nd-4 th columella storey has been characterized as the horizontal
(x-position) and vertical (y-position) coordinates in the statocyte as in two-coordinate
system. x-positions represent the plastid distances from the morphological cell bottom,
which are expressed in percentages from the cell length. y-positions represent the
distances of plastid center from the right longitudinal cell walls in percentages of the
cell width. As a rule, 2–3 sections were analyzed of each test variant.
Digital images of roots and cap sections have been made by means of a
PENTAX*ist. D digital camera and analyzed by SigmaScan Pro5 (Jandel Scientific
Software). Statistical analyses were carried out using the standard package of MS
EXCEL 7 (Microsoft Corporation). Values are represented as a mean ± standard error
(SE). Statistical significance was determined using the Student’s t-test.
Results. R o o t s e n s i t i v i t y t o g r a v i t r o p i c s t i m u l a t i o n b y
the centrifugal forces of low magnitude. Figure 1 represents the
angles of root gravitropic curvature plotted against the common logarithm of stimulus
doses, which have been modelled by 0.004, 0.019 and 1 g centrifugal forces on the
centrifuge-clinostat.
Fig. 1. Dose-response curves of the gravitropic reaction of garden
cress roots to stimulation by the centrifugal force of different magnitude
1 pav. Dozės-atsako kreivės sėjamosios pipirnės šaknims gravitropiškai
reaguojant į dirginimą skirtingos amplitudės išcentrinėmis jėgomis
One can see that the responses are fitted correctly to the logarithmic model
(R = a + b × lg (D), where R – angle of curvature, D – stimulus dose, a
and b – constants) on account of the values of correlation coefficients (table). The
threshold stimulus doses were calculated by an extrapolation of the respective
regression line to zero response and equalled to 2, 4 and 7 g × s under stimulation by
the centrifugal force of 0.004, 0.019 and 1 g, respectively.
77

Table 1. Characteristics of experimental data fit by logarithmic model
R = a + b × lg (D)
1 lentelė. Aproksimuojančio eksperimentinius duomenis logaritminio modelio
R = a + b × lg (D) charakteristikos
Amyloplast positioning in dependence on the magnitude
of centrifugal force. Location of statoliths in the cells of the 2 nd –4 th columella
storey of garden cress roots has been analyzed after the 4-h horizontal clinorotation
or transverse stimulation by total stimulus doses of 16, 65, 203, 563 or 2 252 g × s
that have been modelled by centrifugal forces of 0.001, 0.004, 0.014, 0.039 or
0.156 g, respectively. Micrographs of these cells in the horizontally reoriented root
caps are presented in Fig. 2. The results of statistical analysis of statolith location are
presented in Fig. 3. In statocytes of vertically grown roots, amyloplasts were grouped
in the distal part. Their mean distance with respect to the morphological bottom
(x-position) and right longitudinal wall (y-position) amounted to 27.8 % and 50.0 %
from the total length and width of the cells, respectively. As shown in Fig. 2 and 3,
the amyloplasts moved significantly from this start position (differences statistically
significant at p ≤ 0.001) towards central part of the cells under the transverse stimulation
by all applied doses.
Fig. 2. Statocytes in garden cress roots after the 4-h stimulation
by ≈ 0 g (A, horizontal clinostat) or centrifugal forces of 0.001 (B), 0.039 (C)
and 0.156 g (D). Arrows indicate the direction of stimulation. Bar – 10 µm
2 pav. Statocitai horizontaliai paverstose sėjamosios pipirnės šaknyse po 4 val. dirginimo
≈ 0 g (A, horizontalus klinostatas), 0,001 (B), 0,039 (C) ir 0,156 g (D) išcentrinėmis jėgomis.
Rodyklės atitinka dirginimo kryptį. Mаstelis – 10 µm
After clinorotation and stimulation by the weakest 16 g × s dose (0.001 g for 4 h),
any appreciable difference in the statolith position has been detected as in longitudinal
as well transverse direction. On stimulation by the 65 g × s (0.004 g for 4 h), only a
tendency of slight plastid shift in parallel to the stimulus direction was obtained in
tested statocytes.
78

Fig. 3. Statolith location in statocytes of garden cress roots after the 4-h gravitropic
stimulation by centrifugal force of various magnitudes.
Start – position before stimulation, HC – horizontal clinostat
3 pav. Statolitų išsidėstymas sėjamosios pipirnės šaknyse po gravitropinio dirginimo
skirtingos amplitudės išcentrinėmis jėgomis. Startas – pozicija dirginimo pradžioje,
HK – horizontalus klinostatas
A significant transverse displacement of amyloplasts has been determined only
in response to the action of 203 g × s (0.014 g for 4 h). In this instance distance of
plastids from the right longitudinal cell wall decreased to 45.1 % versus 50.0 % in
root cells before stimulation (p ≤ 0.05). Under the action of the stronger stimulus
dose 563 g × 2 s (0.039 g for 4 h) the statoliths moved significantly in stimulus
direction, however, remained their longwise location at the cell center. Their mean
y-position decreased to 45 % from average statocyte width with respect to about 50 %
in control and clinorotated roots (p ≤ 0.05).
After the stimulation by 2 252 g × s (0.156 g for 4 h), the positioning of amyloplasts
has been changed considerably in comparison to previous tests. The mean values of
x- and y-position decreased to 38.6 % and 38.1 % of the total cell width and length,
respectively. This finding indicates that under these stimulation conditions the
sedimentation of statoliths occurred with simultaneous returning toward morphological
cell bottom.
Discussion. Plant roots are known to be sensitive to gravity alterations. In most
experiments for the study of plant gravisensing, the stimulus doses were modelled by
changing the action direction of Earth gravity (Hejnowicz et al., 1998; Perbal, Driss-
Ecole, 1994; Volkmann, Tewinkel, 1996). Earlier it was obtained a limited validity
of reciprocity rule (g × t = const) for garden cress roots, if the impact of lower than
1 g magnitude forces on gravitropic reaction has been tested (Laurinavičius et al.,
1998). The present study was devoted to detailing of that finding and its relation with
the motion of statoliths-amyloplasts in root gravisensing cells under such stimulation
conditions.
In an earlier study, we have shown that the gravitropic response of cress roots to
the smallest of tested forces of 0.004 g remained the same after 40 min of stimulation
while it grew until 60 min in response to 0.019 g or 1 g (Laurinavičius et al., 1998).
Therefore, these periods of g-exposure have been applied in our experiments for
79

oot sensitivity analysis. According to our data, the D-threshold values are equal to
2, 4 and 7 g × s under stimulation by the centrifugal force of 0.004, 0.019 and 1 g,
respectively. They are considerably lower than those of about 60 g × s evaluated for
lentil (Perbal, Driss-Ecole, 1994) and cress roots (Volkmann, Tewinkel, 1996) under
1-g stimulation. On the other hand, it is shown that the dose of 1 g × 2 s could be
effective for gravitropic stimulation using 1-g stimuli intermission (Hejnowicz et al.,
1998). Thus, the evaluated values of D-threshold allow confirming a preposition of
limited validity of the reciprocity rule under the action of gravity of lower than 1-g
magnitude.
Structural analysis of statocytes after exposure to the stimulus dose increasing up to
65 g × s (0.004 g × 4 h) demonstrated any significant displacement of statoliths in the
stimulus direction (Fig. 3), though much lower doses were effective for the induction
of root gravitropic responses (Fig. 1). Furthermore, the statoliths were displaced from
the distal to the central region of the cell (Fig. 2 and 3) like after transferring the 1-g
roots into microgravity or on the clinostat (Merkys et al., 1981; Driss-Ecole et al.,
2003; Gaina et al., 2003; Švegždienė et al., 2005). They sustained such lengthwise
location when a considerably stronger stimulus doses were applied.
Investigations on the involvement of the cytoskeleton in root gravisensing
demonstrate that actin filaments in any case take part in the positioning of amyloplasts
and their transport during stimulation (Hou et al., 2003; Driss-Ecole et al., 2003
Palmieri, Kiss, 2005). Therefore, the location of statoliths in the central part of the
statocyte after applying the doses modelled by centrifugal forces of low magnitude,
which are already capable to induce gravitropic curvature of roots itself, could be caused
by the elastic forces generated by actin filaments and acting in proximal direction.
Based on the presented data and the above discussion, we suggest that a slight, however,
transient, sedimentation of amyloplasts under the action of low magnitude acceleration
may trigger and transmit the gravitropic stimulus.
Conclusions. 1. Evaluation of threshold stimulus doses for root gravitropic
stimulation revealed a relation between the magnitude of acting force and the value
of D-threshold.
2. Any direct ratio is determined between the D-threshold and the responding
alteration in amyloplast positioning within gravisensing cells, when the force of lower
than 1-g magnitude is applied for root gravitropic stimulation.
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Gauta 2008 03 24
Parengta spausdinti 2008 04 21
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81

SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Gravitacijos jutimas sėjamosios pipirinės šaknyse
esant skirtingiems g-dydžiams
D. Švegždienė, D. Koryznienė, D. Raklevičienė
Santrauka
Darbas atliktas siekiant charakterizuoti ir palyginti šaknų jautrumа ir amiloplastų
išsidėstymą gravitaciją juntančiose lаstelėse, kai dirginama mažesnių už 1 g dydžių gravitacine
jėga. Etioliuoti sėjamosios pipirnės daigai, augę 30 val. 1-g sąlygomis, gravitropiniam dirginimui
buvo perkeliami į centrifugą-klinostatą. Atliekant šaknų jautrumo tyrimą, daigai dirginti 0,004,
0,019 ar 1 g dydžio jėgomis statmena kryptimi, po to klinostatuoti. Kiekybinė išlinkimų
kinetikos analizė atskleidė gravitacinio stimulo slenkstinės dozės šaknims priklausomybę nuo
jėgos dydžio. Šių dozių reikšmės lygios 2, 4 ir 7 g × s, kuomet šaknys veikiamos atitinkamai
0,004, 0,019 arba 1 g dydžio jėgomis. Amiloplastų išsidėstymo analizė gravitaciją juntančiose
lаstelėse atlikta paveikus šaknis 4 val. skersine kryptimi 0,001–0,156 g dydžio jėgomis (suminės
dozių reikšmės 16, 65, 203, 563 ar 2 252 g × 2 s). Esminė, nuo gravitacijos priklausoma
amiloplastų sedimentacija nustatyta tik paveikus 203 g × s ir didesnėmis dozėmis. Ji ženkliai
viršija slenkstinę 4 g × s dozę šaknims, gravitropiškai dirginamoms panaрaus dydžio jėga.
Daroma išvada, kad nėra tiesioginio ryšio tarp sėjamosios pipirnės šaknų gravitacinio jautrumo
ir amiloplastų viduląstelinio išsidėstymo, kai dirginama mažesnėmis už 1 g jėgomis. Duomenys
leidžia manyti, jog menki, tačiau trumpalaikiai ir kryptingi silpno gravitacinio stimulo sukelti
amiloplastų poslinkiai gali indukuoti atsakomаją reakciją.
Reikšminiai žodžiai: amiloplastas, gravitacija, jautrumas, stimulas, sėjamoji pipirnė,
šaknis.
82

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
The possibility to control the metabolism of green
vegetables and sprouts using light emitting diode
illumination
Akvilė Urbonavičiūtė 1 , Giedrė Samuolienė 1 , Aušra Brazaitytė 1 ,
Raimonda Ulinskaitė 1 , Julė Jankauskienė 1 ,
Duchovskis 1, 3 , Artūras Žukauskas 2, 3
1
Lithuanian Institute of Horticulture, Kauno 30, 54333 Babtai, Kaunas distr.,
Lithuania, e-mail: A.Urbonaviciute@lsdi.lt
2
Institute of Materials Science and Applied Research, Vilnius University,
Saulėtekio al. 9-III, LT-10222 Vilnius, Lithuania
3
JSC ‘Hortiled’
The influence of different solid-state lighting spectrum on the metabolism, as the
index of internal nutritional quality of green vegetables (lettuce, onion leaves, dill, parsley,
basil, marjoram, wheat grass, barley grass and leafy radish), was investigated. Plants were
illuminated with the basal red 640 nm light emitting diodes, supplemented with 450, 660
and 735 nm components for three to five days in different developmental phases. Reference
plants were grown under high-pressure sodium lamps. The content of carbohydrate, nitrate,
vitamin C, phenolic compounds and the antioxidant activity of the selected vegetable extracts
were evaluated. According to the obtained results, the red light effect on the metabolic system
depends on the plant specie. The lettuce, marjoram, wheat grass and leafy radish were found
to be the potentially suitable for cultivation under the light emitting diode lighting, due to the
positive increase in monosugar content, reduction of nitrates, bigger vitamin C content and
promoted antioxidant activity.
Key words: leafy vegetables, light quality, metabolism.
Introduction. Light is one of the most important environmental factors, acting on
plants not only as the sole source of energy, but also as the source of external information,
affecting their growth, development and metabolism. Plants are empowered with an
array of photoreceptors controlling diverse responses to light parameters, such as
spectrum, intensity, direction, duration: the red and far-red-absorbing phytochromes,
the blue and UV-A light absorbing cryptochromes, phototropins, and other implied
photoreceptors, absorbing in UV-A and green regions (Devlin et al., 2007). Spectral
light changes evoke different morphogenetic and photosynthetic responses that can
vary among different plant species. Such photo-response is of practical importance
in recent plant cultivation technologies, since feasibility to tailor illumination spectra
purposefully enables one to control plant growth, development, and nutritional quality.
The recent progress in solid-state lighting based on light-emitting diodes (LEDs)
facilitated and extended the research possibilities in this field and developed the outset
for new progressive vegetable-growing technologies.
83

It is known, that the red light is of major importance for plants (Kopcewicz,
Lewak, 1998) – for the development of the photosynthetic apparatus and morphogenetic
processes due to light-induced transformations in phytochrome system (Furuya, 1993).
Blue light, also essential for plants, affects the formation of chlorophyll, stomata
opening, and photomorphogenesis (Dougher, Bugbee, 1998; Shuerger et al., 1997; Heo
et al., 2002). Phytochromes, principally thought of as red/far-red reversible pigments,
yet they absorb well in the blue portion of the spectrum. Moreover, these receptors are
extremely sensitive to all light qualities across the spectrum and will initiate responses
to minor illumination form the light qualities across the spectrum (Folta, Maruhnich,
2007). Nevertheless, there are still no clear findings, how light components, such as
green, yellow or violet affect plant vitality. Herewith the main growth and development
processes, light affects the primary and secondary metabolism reactions, thus acting on
plant vital state and the nutritional quality of vegetable food. The knowledge, regarding
plant metabolism regulation by light spectral quality is still limited; although it is of
economical and ecological importance to explain this effect by scientific findings.
Especially for green vegetable cultivation, such as lettuce, onion leaves, dill, parsley
et al., which are intensively grown during the seasons of low solar irradiation. Moreover,
leafy vegetables and sprouts are the main source of bioactive compounds, positively
acting on human organism. Thus, elucidation of the illumination spectrum, which is
optimal in the view of vegetable growth rate, healthy development and well-balanced
metabolism, is of relevant value. The objective of this study was to evaluate the effect
of different light qualities, provided by light emitting diodes (LEDs) on the indices of
nutritional quality in green vegetables.
Object, methods and conditions. Lighting experiments were performed at the
Lithuanian Institute of Horticulture in 2006–2008. Different green vegetables were
cultivated under solid-state lighting units. Parsley (Petroselinum crispum (Miller)
Nyman ex A. W. Hill, cv. ‘Moss curled’), marjoram (Majorana hortensis Moench.),
onion leaves (Allium cepa L.), lettuces (Lactuca sativa cv. ‘Grand rapids’), dills
(Anethum graveolens L. cv. ‘Szmaragd’) and basil (Ocimum basilicum L.) were placed
under investigated lighting when plants matured, before gathering and illuminated for
three days. Therefore, vegetables were grown in the phytotron chambers in peat substrate
according to appropriate agrotechnical conditions. A photoperiod of 18 h was used and
the temperature of 21/15 °C (day/night) was maintained throughout the experiment.
Wheat grass (Triticum aestivum L. cv. ‘Рirvinta‘), barley grass (Hordeum vulgare L.
cv. ‘Aura’) and leafy radish (Raphanus sativus L. cv. ‘Tamina’) were grown under
light emitting diode (LED) lighting from the sowing time, as their growth period until
consumption is relatively short: plants were kept there for five days after germination.
Two treatments under the illumination units, containing different combinations of light
emitting diodes were performed as presented in Table. The treatment L1 contained
red basal component (640 nm, delivered by AlGaInP LEDs Luxeon TM type LXHL-
MD1D, Lumileds Lighting, USA)) and three supplemental components: blue (455 nm,
LuxeonTM type LXHL-LR5C, Lumileds Lighting, USA), red (662 nm, L660-66-60,
Epitex, Japan,) and far red (735 nm, L735-05-AU, Epitex, Japan). The treatment
L2 contained single basal 640 nm component. The total photon flux density in both
treatments was about 200 µmol m -2 s -1 . Reference plants were grown under illumination
by high-pressure sodium lamps (HPS, Son-T Agro Philips, USA).
84

Table. The light emitting diode combinations and flux densities
Lentelė. Kietakūnių šviestukų kombinacijos ir fotonų srauto tankiai
After the determined illumination period carbohydrate, vitamin C, nitrate, phenolic
compound content were determined and the antioxidant activity of selected plants
extracts was evaluated.
Samples for determination of carbohydrates were prepared by grinding 1 g of
leaf fresh matter and extracting it with 4 mL hot bidistiled water. After 24 h, the
extract was filtered through cellulose and membrane (pore diameter 0.2 µm) filters.
Chromatographic analysis was carried out using a Shimadzu 10A HPLC system
with refraction index detector (Shimadzu, Japan) and Adsorbosil NH 2
-column
(150 mm × 4.6 mm; Alltech, USA) with mobile phase of 75 % aqueous acetonitrile
at a flow rate of 1 mL min -1 .
Nitrate content in the fresh matter of the leafy vegetables was determined using the
potentiometric method described by Geniatakis et al. (2003). Oakton desktop ion meter
(Oakton, USA) and the nitrate selective electrode (Cole Parmer, USA) were used.
Vitamin C content was evaluated using spectrophotometric method, described by
Janghel et al. (2007). The extraction was performed by homogenising plant material
with 0.2 mol L -1 oxalic acid. The ability of extract to reduce methyl viologen in the
basic medium was determined and expressed as vitamin C content in the fresh material.
The Genesys 6 spectrophotometer was used for analysis (Thermospectronic, USA).
Total content of phenolic compounds was determined in the methanolic extracts
of fresh leaves by spectrophotometric Folin method (Ragaee et al., 2006). The final
concentration of phenolic compound is evaluated according to the gallic acid standard
calibration curves.
The antioxidant activity of the methanolic extracts of investigated leafy vegetables
was evaluated spectrophotometricaly as the ability to bind ABTS and DPPH radicals
(Ragaee et al., Kasparavičienė et al., 2004). The Genesys 6 spectrophotometer was
used for analysis (Thermospectronic, USA).
The error bars presented in figures and the tables are the standard deviations of the
three analytical measurements of the parameter. The data processing was performed
using MS Excel software.
Results. The content of primary and secondary plant metabolites, important as
the indices of human nutrition quality, is significantly affected by light spectral quality
and are dependant on plant species and developmental level.
P a r s l e y. The high flux of red light (Fig. 1) in the L2 treatment, enhanced
carbohydrate, especially sucrose, content in the parsley leaves. The reduction in the
flux of 640 nm component and the insertion of the blue (455 nm), red (662 nm) and
85

far red (735 nm) in the illumination spectra significantly reduced the accumulation of
monosugar, fructose and glucose in the plant material. Another positive effect is the
reduction in the nitrate content (Fig. 2). The higher the flux of red light, irrespectively
of the presence of other spectral components, the higher the decrease of the nitrate
content in parsley. It is consistent with the vitamin C concentration in plant fresh matter.
In the treatment L1 it is higher about 10 % as compared with the reference plants; and
in treatment L2 it is about 5 % higher, than in the treatment L1.
Fig. 1. Carbohydrate content in green vegetables, illuminated with different LED
combinations. R – control plants; L1 – illumination contain basal 640 nm and
supplemental 455, 662 and 735 nm components; L2 – single basal component
1 pav. Cukrų kiekis žalumyninėse daržovėse, švitintose skirtingomis LED kombinacijomis;
R – kontroliniai augalai, L1 – švitinti pagrindine 640 nm komponente ir papildomais 455,
662 ir 735 nm šviestukais; L2 – tik pagrindinė komponentė
M a r j o r a m. The investigated lighting was suitable for marjoram nutrition quality
improvement. In both treatments the significant increase in glucose (about 15 %), the
monosugar (Fig. 1), and the ~ 50 % reduction in nitrate content (Fig. 2) was observed.
Nevertheless, the selected solid-state lighting had no remarkable effect on vitamin C
accumulation in leaves (Fig. 3).
Onion leaves. The significant effect on photosynthesis product accumulation in
leaves was observed in L1 treatment, were the red and blue LED light combinations
were investigated. Such lighting promoted monosugar accumulation in leaves (Fig. 1).
Nevertheless, vitamin C accumulation in onion leaves was more stimulated by
illumination with solely 640 nm red light (Fig. 2). It was increased in this treatment
about 16 percents. Nitrate content in onion leaves was not significantly affected by
applied illumination.
Lettuce. The applied lighting source was found to be the most suitable for lettuce
photosynthetic system (Fig. 1). In the treatment L1, where the combination of all four
wavelengths was used, the fructose content was about 3 times higher and in treatment
L2, where the 640 nm red component was used, the increase was about 2.5 times, in
respect to reference, high pressure sodium lamp illumination. In L1 treatment, unlike in
other treatments, some sucrose was accumulated in the leaves. Nitrate metabolism was
86

also positively affected by LED lighting (Fig. 2). In this relatively short period 15 %
reduction in the lettuce, illuminated with all four components (the 640 nm, 662 nm,
445 nm and 731 nm combination) and 20 % reduction in the lettuce, illuminated with
the solely 640 nm light was observed. Vitamin C content was affected negatively –
50 % reduction was measured (Fig. 3).
Fig. 2. Nitrate content in green vegetables grown under different illumination.
R – control plants; L1 – illumination contain basal 640 nm and supplemental 450,
660 and 735 nm components; L2 – single basal component
2 pav. Nitratų kiekis žalumyninėse daržovėse, švitintose skirtingomis LED kombinacijomis;
R – kontroliniai augalai, L1 – švitinti pagrindine 640 nm komponente ir papildomais 455,
662 ir 735 nm šviestukais; L2 – tik pagrindinė komponentė
Dill. The metabolic system of this plant, from the nutritional viewpoint, reacted
negatively to the applied illumination. The fructose content decreased from 2.1 to
0.6–0.7 mg g -1 and glucose content decreased form 5.0 to 2.3–2.1 mg g -1 (Fig. 1).
Another unfavorable effect –50 % increase in nitrate content in treatment L1, and
about 30 % increase in treatment L2 (Fig. 2). Vitamin C concentration in leaves also
was affected negatively. It decreased about 5 times in the treatment L1 and about 30 %
in treatment L2 (Fig. 3).
Basil. The slight increase in monosugar content and traces of sucrose were
found in both treatments. In treatment L1, about 0.5 mg g -1 of maltose were detected
(Fig. 1). However, there were no remarkable effect on vitamin C concentration in
leaves (Fig. 3) and tenuous 5–10 % increase in nitrate ion concentration was found
in both LED treatments.
87

Fig. 3. Vitamin C content in green vegetables grown under different illumination.
R – control plants; L1 – illumination contain basal 640 nm and supplemental 450,
660 and 735 nm components; L2 – single basal component
3 pav. Vitamino C koncentracija žalumyninėse daržovėse, švitintose skirtingomis LED
kombinacijomis; R – kontroliniai augalai, L1 – švitinti pagrindine 640 nm komponente ir
papildomais 455, 662 ir 735 nm šviestukais; L2 – tik pagrindinė komponentė
W h e a t g r a s s. L1 treatment with all four investigated LED components was of
somewhat similar effect on sugar metabolism as compared to HPS lamps. Nevertheless,
the higher flux of red 640 nm component light reduced glucose concentration in
wheat grass in two times (Fig. 1). Photosynthetically active radiation promoted the
vital activity if the sprouts and the nitrate uptake from soil. In the treatment L1 the
~ 20 % increase in nitrate concentration was determined; and the high flux of red
light enhanced nitrate content by 65 % (Fig. 2). The inverse trend was observed in
vitamin C concentration (Fig. 3). In L1 treatment the slight increase in vitamin C
concentration was determined, and in the treatment L2 vitamin C concentration rose
almost three times. The light effect was significant for phenolic compound content
in sprouts (Fig. 4). Since the combination of different red and blue lights showed the
same effect for phenol accumulation in leaves, the wheat grass, grown under solely
red light accumulates about 10 % less of them. The antioxidant activity, according
to the ABTS radical scavenging activity (Fig. 5 A) was about 10 % higher in LED
treatments, as compared to HPS. Nevertheless, the DPPH radical scavenging activity
(Fig. 5 B) was opposite, about 20 % lower.
Barley grass. The lighting effect, similar to wheat grass was observed in barley
grass. Plants, grown under LED illumination for 5 days, accumulated only a half of
fructose, as compared to HPS and less of glucose: in the treatment L1 6.2 mg g -1 of
glucose were quantitated, in L2 – 3.5, when in reference plants – 7.5 mg g -1 of fructose
were detected (Fig. 1). Vitamin C content in the treatment L1 was about 20 % higher
than in reference plants, and in the L2 treatment – about two times higher (Fig. 3).
Despite the positive effect on vitamin C content in leaves, in the treatment L1 was
observed the 20 %, and in treatment L2 – ~ 10 % decrease in phenolic compound
88

concentration (Fig. 4), as compared to reference plants. Changes in the ABTS radical
scavenging activity (Fig. 5 A) were within the limits of the error; the DPPH radical
scavenging activity (Fig. 5 B) was found to be about 1.8 times higher in the treatment
L1.
Fig. 4. The content of phenolic compounds in green vegetables and sprouts grown
under different illumination; R – control plants,
L1 – illumination contain basal 640 nm and supplemental 450, 660 and
735 nm components, L2 – single basal component
4 pav. Fenoliniu junginių koncentracija žalumyninėse daržovėse ir želmenyse,
švitintose skirtingomis LED kombinacijomis; R – kontroliniai augalai,
L1 – švitinti pagrindine 640 nm komponente ir papildomais 455, 662 ir
735 nm šviestukais, L2 – tik pagrindinė 640 nm komponentė
Fig. 5. The antioxidant activity of green vegetables and sprouts grown under
different illumination; A – the ABTS radical scavenging activity;
B – the DPPH radical binding activity, R – control plants,
L1 – illumination contain basal 640 nm and supplemental 450, 660 and
735 nm components, L2 – single basal component
5 pav. Žalumyninių daržovių ir želmenų žaliavos antioksidacinis aktyvumas,
juo švitinant skirtingomis LED kombinacijomis;
A – ABTS radikalų surišimo aktyvumas, B – DPPH radikalų surišimo aktyvumas,
R – kontroliniai augalai, L1 – švitinti pagrindine 640 nm komponente ir
papildomais 455, 662 ir 735 nm šviestukais,
L2 – tik pagrindinė 640 nm komponentė
89

L e a f y r a d i s h. LED illumination significantly inhibited sugar accumulation
in leafy radish (Fig. 1); Glucose and sucrose concentration was more than two times
lower, although in L2 treatment (sole red light) sugar concentration was closer to
reference. Negative effect was for vitamin C content (Fig. 3): in plants, grown under
reference illumination, it was about 13 mg g -1 and in plants, grown under LED
illumination it was ~ 3 mg g -1 . The total content of phenolic compounds (Fig. 4) and
DPPH radical scavenging activity (Fig. 5 B) varied within the limits of the error in the
L1 and reference treatments. Although high flux of red light inhibited accumulation
of phenolic compounds in leaves and enhanced DPPH radical scavenging activity. In
the ABTS scavenging activity (Fig. 5 A) the opposite trend was observed.
Discussion. The selected solid-state lighting differentially affected the metabolic
system of the investigated green vegetables. The most sensitive response was in the
sugar, the main photosynthesis product, and accumulation in green leaves. Carbohydrate
metabolism lies at the very heart of the sensitive self-regulatory system of plant
development (Koch, 2004), therefore the light changes not only affect sugar, as the
index of nutritional quality, content, but also participate as the signaling molecule in
the regulation of important vital processes. Notwithstanding, a high total concentration
of carbohydrates is one of desirable parameters in view of food quality. The nutritional
quality also depends on the percentage of monosugars. However, the positive effect
both of the solely 640 nm red light illumination and in the combination with other red
light wavelengths and blue light was observed in lettuce, onion leaves and marjoram.
In parsley, positive effect was seen only growing them under high flux of red light
(L2 treatment). Negative reduction in sugar content was detected in dill, wheat grass
and barley grass. Although it is stated that red light positively affects the formation
and performance of photosynthesis system (Spalding, Folta, 2005), it is evident, that
this effect is specie-dependant. This presumption is confirmed analyzing the results
of the nitrate metabolism. Reduction of nitrates content is definitely of importance for
improvements of nutritional quality of vegetable food. Nitrate reductase activity and
the synthesis of this enzyme are also red-light sensitive and dependant on genetically
determined features (Appenroth et al., 2000; Lillo, 2004). The nitrate content
significantly decreases in lettuce and marjoram; due to nitrate reductase activity nitrates
are reduced to nitrite ions and incorporated to the content of ammonium and amino
acids. Sole 640 nm red light had the more pronounced effect on nitrate metabolism,
as compared to the investigated combination of other light wavelengths. However,
no significant light effect was detected in parsley and onion. Therefore in dill, basil
and wheat grass the high flux of red solid-state light stimulated vital activity of plants,
nitrate uptake and the increase in its concentration in leaves.
Biologically active compounds are quite sensitive to lighting conditions. One
of them, vitamin C, significantly increases in parsley, onion leaves, wheat grass
and barley grass. This could be associated with the stating, that vitamin C actively
accumulates in metabolically active tissues and acts as signaling molecule, coordinating
the performance of protective mechanism of antioxidant system (Pastori et al., 2003).
Significant light effects on the content of phenolic compounds and antioxidant activity,
as the rate of the radical scavenging, was observed only in leafy radish. The enhanced
antioxidant activity could be associated to the expression of antioxidant defence
90

genes, defending the plant cells against light-induces photooxidative damage (Wu
et al., 2007).
Conclusions. Light effect is a subsequence of the action of complex signal
transduction network, including different enzymes, primary and secondary metabolites
and messengers. Therefore, it could be employed as the tool for the purposeful plant
metabolism, herewith the nutritional quality of vegetable food regulation. However,
it is difficult to attain comprehensively positive effect, because different metabolic
systems differentially react to the lighting conditions. Moreover, there are contradiction
between the natural plant needs, metabolic homeostasis and the agronomic, nutritional
objectives. The lettuce, marjoram, wheat grass and leafy radish were found to be the
potentially suitable for growing under the light emitting diode lighting, due to the
positive increase in monosugar content, reduction of nitrates, higher vitamin C content
and promoted antioxidant activity. The light quality requirements of other plants are
different, although the combination of different red and far red lights with the blue
light had the superior effect, due to activation of more diverse array of light-sensitive
receptors.
Acknowledgements. This study was supported by the Lithuanian Science and
Study foundation under the high technology project PHYTOLED.
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Gauta 2008 04 16
Parengta spausdinti 2008 04 29
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SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Galimybė kontroliuoti žalumyninių daržovių ir želmenų
metabolizmą kietakūnės šviesos pagalba
A. Urbonavičiūtė, G. Samuolienė, A. Brazaitytė, R. Ulinskaitė,
J. Jankauskienė, P. Duchovskis, A. Žukauskas
Santrauka
Tirtas skirtingo kietakūnės šviesos spektro poveikis lapinių daržovių (salotų, svogūnų
laiškų, krapų, petražolių, bazilikų, mairūno, kviečių, miežių želmenų ir lapinių ridikėlių)
metabolizmo, apsprendžiančio daržovių maistinę kokybę, rodikliams. Augalai skirtinguose
augimo tarpsniuose švitinti 3–5 dienas pagrindine 640 nm bangos ilgio šviesa, papildyta 450,
660 ir 735 nm šviesą emituojančiais diodais. Palyginamieji augalai auginti po aukšto slėgio
natrio lempomis. Nustatytas angliavandenių, vitamino C, nitrato jonų ir fenolinių junginių kiekis
bei antioksidacinis aktyvumas. Pagal gautus rezultatus, tirto apšvietimo poveikis metabolizmui
labai priklauso nuo augalo rūšies bei išsivystymo laipsnio. Salotos, mairūnai, kviečių želmenys
ir lapiniai ridikėliai potencialiai tinkami jų maistinės kokybės gerinimui kietakūnės šviesos
pagalba, dėl рvitinant padidėjusio monocukrų, vitamino C kiekio, antioksidacinio potencialo
ir reikšmingos nitratų kiekio redukcijos žaliavoje.
Reikšminiai žodžiai: lapinės daržovės, šviesos kokybė, metabolizmas.
92

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Photosynthesis and some growth parameters of sweet
pepper grown under different light conditions
Gabriela Wyżgolik, Joanna Nawara, Maria Leja
Department of Plant Physiology, Faculty of Horticulture,
Agricultural University, 29 Listopada 54, 31-425 Krakуw, Poland
E-mail: gabriela.wyzgolik@bratek.ogr.ar.krakow.pl
The influence of various light intensity on growth, net photosynthesis rate and yielding of
bell pepper Capsicum annuum L. cultivar ‘Spartacus’, was investigated. Sweet pepper was grown
on rockwool in plastic tunnel divided into two parts covered by polyethylene films (Ginegar –
part I, Gemme 4 S – part II), which differentiated in light permeability, its dispersion and PAR
exertion. In part I light intensity was lower than in part II. Gas exchange was measured by LCi
(ADC England). WUE was expressed as net photosynthesis to transpiration ratio. Photosynthetic
pigments were determined according to photometric method given by Wellburn (1994). LAI and
LAR were calculated using leaf blade area, dry matter content and tunnel area per single plant.
Different light intensity influenced growth parameters and photosynthesis but not yield. Plants
grown under lower light intensity were significantly higher and had larger leaf blades, so as a
result value of LAI and LAR were higher. The concentration of chlorophylls and carotenoids was
also higher but photosynthesis intensity was lower. WUE was low 1.85 µmol (CO 2
) · mol -1 (H 2
O)
and stable while in plants grown in part II was higher but decreased gradually during the day
from 4.17 µmol (CO 2
) · mol -1 (H 2
O) to 2.46 µmol (CO 2
) · mol -1 (H 2
O).
Key words: LAI, light intensity, net photosynthesis, sweet pepper, WUE.
Introduction. Plant morphogenesis and photosynthesis are affected by many
factors. One of the basic is light. The energy of solar irradiation absorbed by green plants
ranges mainly from 400 to 700 nm (PAR) (Czarnowski, Cebula, 1994a). It is broadly
known that the increase of light intensity stimulates the process of photosynthesis.
Photosynthesis is one of the most important factors affecting biomass production
(Evans, 1975) closely associated with growth rate.
Plant growth analysis is an explanatory, holistic and integrative approach of
interpreting plant form and function. It uses simple primary data, such as weights,
areas, volumes and contents of plant components to investigate processes within and
involving the whole plant (Hunt et al., 2002).
Solar irradiation together with other environmental factors creates a specific
phytoclimate for cultivation in plastic tunnels (Czarnowski, Cebula, 1994b). The light
and thermal conditions can either limit or improve sweet pepper yielding (Somos,
1984). According to Stępowska and Kosson (2003), pepper plants need many sunny
days during growing period and the best effects are obtained in greenhouses with
climate control. In Poland sweet pepper is mostly cultivated in non-heated greenhouse,
hence, this vegetable grown in tunnels with climate control is relatively new crop
in our country with potential to expand production in future. Modern polyethylene
93

films consist of many layers for better temperature control but therefore changes light
permeability.
The aim of the present studies was to compare the effect of different light intensity
resulting from application of foils of various light transparency and diffusion on
photosynthesis intensity accompanied by certain growth parameters.
Object, methods and conditions. Sweet pepper (Capsicum annuum L. ‘Spartacus’,
De Reuiter Seeds) was grown on rockwool in a double-layered polyethylene tunnel
divided into two parts of different light intensities. Part I was covered by Ginegar foil,
part II by Gemme 4 S (light transparency 86 % and 90 %, light diffusion 58 % and
15 %, respectively). The PAR intensity measured by Hobo data logger in part I was
200–220 µmol m -2·s -1 and 650–800 µmol m -2 s -1 , in part II 300–320 µmol m -2 s -1 and
950–1 000 µmol m -2 s -1 for cloudy and sunny days, respectively. Nutrient levels were
provided in concentrations recommended for sweet pepper cultivation and supplied
by the automatic fertigation system. Shoots were pruned to form a plant structure of
2 main branches.
For analysing the plant growth thirty plants from each tunnel were used. Length of
branches was measured once a week, leaf area and dry weight of leaves and branches
were determined in 94 DAT (Day After Transplanting).
The leaf area index (LAI) was calculated using leaf blade area to tunnel area
per single plant. The leaf area ratio (LAR) was expressed as leaf area to dry matter
content per single plant.
Total and marketable yield was determined at each harvest. Fruits for marketable
yield were classified according to WE 1455/1999.
Net photosynthesis and transpiration ratio were determined using portable gas
exchange system (LCi, ADC England). Measurements were taken on cloudless days.
Water use efficiency (WUE) was expressed as net photosynthesis to transpiration ratio.
Photosynthetic pigments were determined according to photometric method given by
Wellburn (1994).
Results were statistically verified by the Fisher test at P = 0.05. Photosynthesis
intensity and WUE were analysed for its polynominal (quadratic and linear, respectively)
effects by regression analysis.
Results. In both parts of the tunnel during whole growing period plant height was
increasing but pepper grown under lower light intensity (in part I) was always higher.
The minimum difference was 3.5 cm. Leaf blades area was also larger by 35 % than in
plants grown in part II, so as a result value of LAI and LAR were significantly higher
by 36.5 % and by 35.7 %, respectively (Table 1).
Table 1. Growth parameters of sweet pepper grown in different light intensity
1 lentelė. Saldžiosios paprikos, augintos skirtingame šviesos intensyvume, augimo
parametrai
94

Higher but non-significant values of both total and marketable yields were obtained
in part I. However, in part II the participation of unmarketable fruits was lower than in
part I by 16.1 % and by 18.6 %, respectively. Light intensity in part II slightly reduced
level of all determined photosynthetic pigments. Net photosynthesis rate in part II
significantly exceeded that of part I (Table 2).
Table 2. Net photosynthesis, yield and pigments of sweet pepper grown in different
light intensity
2 lentelė. Grynoji fotosintezė, derlius ir pigmentai saldžiojoje paprikoje, augintoje
skirtingame šviesos intensyvume
Irrespectively to type of applied foil, photosynthesis intensity corresponded to
increasing PAR (Fig. 1).
Fig. 1. Net photosynthesis (P n
) of sweet pepper grown under
different light conditions
1 pav. Saldžiosis paprikos, augintos skirtingomis šviesos sąlygomis,
grynoji fotosintezė (P n
)
In the case of WUE value, the differences between parts of the tunnel were
distinct. In part I WUE was low but remained stable during the day while in part II
the relatively high WUE based on data obtained at morning hours gradually lowered.
The minimum WUE (2.46 µmol (CO 2
) · mol -1 (H 2
O)) observed in this part of tunnel
at 14:00 did not reach any values calculated for part I (Fig. 2).
95

Fig. 2. Water use efficiency WUE of sweet pepper grown under
different light conditions
2 pav. Saldžiosis paprikos, augintos skirtingose šviesos sąlygose,
vandens naudojimo efektyvumas (WUE)
Discussion. As described in introduction, light intensity strongly affects the
processes of photosynthesis and morphogenesis. In present investigations higher
radiation decreased elongation growth of plants grown in part II of the tunnel. Similarly
in study reported by Siwek and Libik (1996) sweet pepper ‘Spartacus’ grown in single
layered plastic tunnel was significantly lower than peppers grown in tunnel covered by
additional layers. It is widely known that light delays elongation growth. There are two
light-sensing systems involved in these responses, the blue light sensitive system and
the red light sensitive (phytochrome) system. Phytochrome R : FR ratio is essential for
physiological response (Kopcewicz, 2005). Significant reduction in stem elongation of
sweet pepper seedlings could be achieved by the exclusion FR light at the end of the
day by covering west and south facing walls of the chambers or by exposing plants to
photoselective films at the end of the day (Rajapakse, Li, 2004). In our study part I of
the tunnel (low light intensity) was additionally shaded by neighbouring buildings from
west, so it is possible that P FR
dominated in plants grown in part II delayed stem growth.
P FR
stimulates leaf growth (Kopcewicz, 2005). In our study less intensive elongation
growth was accompanied by reduction of leaf blade area. Similar relationships were
observed by Siwek and Libik (1996). In further investigations the spectral permeability
of Ginegar and Gemme 4 S films should be determined. Not only light intensity may
reduce rate growth but also deficit of water (Basiouny et al., 1994). In these studies the
limitation of leaf growth was probably caused by water stress. According to Xu et al.
(1994) severe water stress does not occur frequently in greenhouse crop production,
short-term mild water stress can occur. Decrease of WUE observed in part II indicated
mild mid-day water stress because photosynthesis remained stable during the day with
simultaneous increase of transpiration. Growth delay is the first reaction on even very
mild waters stress, while photosynthesis is reduced at stronger water stress (Kacperska,
2005, Xu et al., 1994). LAI value is characteristic for cultivars but can be modified by
cultivation conditions (Czarnowski, Cebula, 1994b). These authors determined similar
LAI value (1.96–2.06) for sweet pepper ‘Spartacus’ grown in single layered tunnel
as we did in plants grown in part II (1.97). In investigations of Lorenzo and Castilla
96

(1995) the higher value of LAI induced significantly higher total and commercial yields.
Our study corresponded with these findings. Differences in total and marketable yields
between part I and part II were non-significant but in part I higher LAI values was
accompanied by higher yields. It suggests that higher LAI (proportional to leaf blade
area) might compensate lower photosynthesis rate, which depends on light intensity.
On our study photosynthesis course was similar in both parts of tunnel, however, net
photosynthesis values were significantly lower in part I (21.05 µmol CO 2
m -2 s -1 ) as
compared to (26.35 µmol CO2 m -2 s -1 ) in part II.
The findings presented in this article suggest that different kinds of polyethylene
films strongly affected growth and photosynthesis of sweet pepper but had no any
affect on the yield.
Conclusions. 1. Lower light intensity increased stem elongation, leaf blade area
and leaf area index.
2. Lower PAR intensity limited photosynthesis rate.
3. In lower PAR intensity higher LAI value compensated net photosynthesis
reduction.
4. Polyethylene films of lower transmittance did not affected total and marketable
yields of sweet pepper ‘Spartacus’.
Acknowledgement. The study was financed by the State Committee for Scientific
Research, Poland under project No 2 P06R 021 30.
References
Gauta 2008 04 03
Parengta spausdinti 2008 04 29
1. Czarnowski M., Cebula S. 1994a. Solar spectral irradiance in cultivated plants
under cover in submontane region I. Cucumber plants in the greenhouse. Folia
Horticulturae, 4(2)2: 3–14.
2. Czarnowski M., Cebula S. 1994b. Solar spectral irradiance in cultivated plants
under cover in submontane region III. Sweet pepper plants in the plastic tunnels.
Folia Horticulturae, 4(2): 25–34.
3. Evans L. T. 1975. The physiological basis of crop yield. In: Evans L. T. (eds.)
Crop physiology Cambridge University Press, London-New York-Melbourne,
327–355.
4. Basiouny F. M., Basiouny K., Maloney M. 1994. Influence of water stress on
abscisic acid and ethylene production in tomato under different PAR levels.
Journal of Horticultural Science, 69(3): 535–541.
5. Hunt R., Causton D. R., Shipley B., Askew A. P. 2002. A Modern Tool for Classical
Plant Growth Analysis. Annals of Botany, 90: 485–488.
6. Kacperska A. 2005 Stres spowodowany niedoborem wody-stres wodny. In:
J. Kopcewicz, S. Lewak (eds.) Fizjologia roъlin. PWN, Warszawa, 626–629.
7. Kopcewicz J. 2005. Rozwój wegetatywny. In: J. Kopcewicz S. Lewak (eds.)
Fizjologia roślin. PWN, Warszawa, 510–512.
8. Lorenzo P., Castilla N. 1995. Bell pepper yield response to plant density and
radiation in unheated plastic greenhouse. Acta Horticulturae, 412: 330–334.
97

9. Rajapakse N. C., Li S. 2004. Exclusion of far red light by photoselective greenhouse
films reduces height of vegetable seedlings. Acta Horticulturae, 631: 193–199.
10. Siwek P., Libik A. 1996. Wpływ warunków mikroklimatu w tunelach foliowych z
podwójnym przykryciem na wzrost i plonowanie papryki. Nowe rośliny i techn.
w ogrodnictwie, 3: 63–68.
11. Somos A. 1984. The paprika. Akademiai Kiado, Budapest.
12. Stępowska A., Kosson R. 2003. The influence of substrate types in non-heated
plastic tunnel cultivation on sweet pepper yielding and postharvest quality of
fruits. Vegetable Crops Research Bulletin, 58: 95–102.
13. Xu H. L., Gauthier L., Gosselin A. 1994. Photosynthetic responses of greenhouse
tomato plants to high solution electrical conductivity and low soil water content.
Journal of Horticultural Science, 69(5): 821–832.
14. Wellburn A. R. 1994. The spectral determination of chlorophylls a and b, as well
as total carotenoids, using various solvents with spectrophotometers of different
resolution. Journal of Plant Physiology, 144: 307–313.
SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Saldžiosios paprikos fotosintezė ir kai kurie augimo parametrai
auginant skirtingomis apšvietimo sąlygomis
G. Wyżgolik, J. Nawara, M. Leja
Santrauka
Buvo tirta įvairaus šviesos intensyvumo poveikis saldžiosios paprikos Capsicum
annuum L. veislės ‘Spartacus’ augimui, neto fotosintezei ir derliui. Saldžioji paprika buvo
auginta mineralinėje vatoje plastiko tuneliuose, perskirtuose б dvi dalis ir uždengtuose
polietileno plėvele (Ginegar – I dalis, Gemme 4 S – II dalis), kuri skyrėsi šviesos pralaidumu,
jos išsisklaidymu ir FAR. I dalyje šviesos intensyvumas buvo mažesnis negu II. Dujų apykaita
buvo matuota LCi (ADC England). Vandens panaudojimo efektyvumas (WUE) išreikštas kaip
neto photosintezės ir transpiracijos santykis. Fotosintezės pigmentai buvo nustatyti fotometriniu
metodu pagal Wellburn (1994). Lapų ploto indeksas (LAI) ir lapų ploto santykis (LAR) nustatytas
naudojant lapų plotą, sausųjų medžiagų kiekį ir tunelio plotą vienam augalui. Skirtingas šviesos
intensyvumas darė įtaką augimo parametrams ir fotosintezei, bet ne derliui. Augalai augę
esant mažesniam šviesos intensyvumui buvo žymiai didesni ir turėjo didesnį lapų plotą, dėl
to LAI ir LAR reikšmės buvo didesnės. Chlorofilų ir karotinoidų koncentracija buvo didesnė,
tačiau fotosintezės intensyvumas buvo mažesnis. I dalyje augusių augalų WUE buvo žemas –
1,85 µmol (CO 2
) · mol -1 (H 2
O) – ir stabilus, o II dalyje augusių augalų buvo aukštesnis, tačiau
dienos metu sumažėjo nuo 4,17 µmol (CO 2
) · mol -1 (H 2
O) iki 2,46 µmol (CO 2
) · mol -1 (H 2
O).
Reikšminiai žodžiai: LAI, neto fotosintezė, saldžioji paprika, Šviesos intensyvumas,
WUE.
98

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Actualities in plant cold acclimation
Nijolė Anisimovienė, Jurga Jankauskienė, Leonida Novickienė
Laboratory of Plant Physiology, Institute of Botany, Žaliųjų Ežerų 49, LT-08406,
Vilnius, Lithuania, e-mail: nijole.anisimoviene@botanika.lt
The studies to reveal the possible involvement of phytohormone indole-3-acetic acid
(IAA) in mechanism of adaptive processes of cold acclimation of poorly wintering plants have
been carried out.
The composition of soluble and membranous structures proteins as well as IAA amount and
status in organs significant for wintering (root collum, terminal bud, developing inflorescence
and leaves) of oilseed rape during autumn growth and preparing for wintering period have
been analyzed. To clarify the manifestation of IAA in biochemical-physiological processes,
the model trials employing physiological analogues of auxin – compounds TA-12 and TA-14
were fulfilled.
Basing on the obtained results the supposition on influence of IAA on readjustment of
protein metabolism and composition in developing inflorescence and root collum organs cells
has been proposed. Under the influence of both compounds the number of individual proteins
increased. These changes involved not only the formation of specific thermostabile proteinsdehydrins,
but also proteins that may contribute to other biochemical-physiological processes of
the cells related to cold acclimation. According to preliminary results under the effect of tested
compounds the modifications of IAA fund composition takes place. The amount of reserve IAA
complexes was increased significantly.
Under these biochemical-physiological modifications the cold acclimation and wintering
of oilseed rape was partially improved.
Key words: indole-3-acetic acid, physiological analogues of auxin, cold acclimation,
proteins, rape.
Introduction. All biennial plants during autumn growth season have a specific
cold acclimation period related to plant readiness for survival the wintering period:
increasing in frost resistance, maintaining the dormancy – a temporary suspension
of visible growth of any plant structure containing meristem (Shuliakovskaja,
Anisimovienė, 1985; Anderson et al., 2001). At this period the growth is under the
arrest by external environmental and internal factors (Horwath et al., 2003).
Plants of natural flora exhibit differentiated programs that allow an adaptation
of growth and development processes under the shortening photoperiod and at low,
above zero, temperatures in autumn, therefore cold acclimated fully (Jeknič, Chen,
1999; Welling et al., 2002). Cold acclimated plants become not only freezing tolerant
but they can also retain specific levels of metabolic routes for wintering during eco-,
para- and endo-dormancy (Ivonis et al., 1984; Horwath et al., 2003). However, the
hardiness of some crops, such as winter rape, wheat or barley (Gavelienė et al., 2002;
99

Stupnikova et al., 2003; Borovskii et al., 2002), fruit plants – strawberry, blueberry
(Dhanaraj et al., 2005) is problematic.
In most cases while analyzing cold acclimation mechanism the attention is focused
on the effect of low temperature (mostly, 4°), changes of cold regulated COR genes
expression: their up- or down-regulation and proteins of these gene products formation
(Fowler, Thomashow, 2002). Basing on experimental data obtained during the past
two decades the following opinion has been proposed: significant role in plant cold
acclimation may be ascribed to the late embryogenesis abundant (LEA) genes (Svensson
et al., 2002; Welling et al., 2002; Stupnikova et al., 2003). The relationship between
formation and accumulation of dehydrin group proteins with shortening photoperiod,
low non-freezing temperatures, as well as drought, chilling and freeze stress have
been discovered (Jeknič, Chen, 1999; Welling et al., 2002; Fowler, Thomashow, 2002;
Borovskii et al., 2002; Stupnikova et al., 2003). The significant biochemical feature of
these proteins is thermostability (Svensson et al., 2002; Borovskii et al., 2002).
During autumnal growth and cold acclimation periods many plant growth and
developmental processes as well as various biochemical-physiological changes are
involved (Novickienė et al., 2004; Velička et al., 2005). Therefore, the change of
expression of genes regulating activity of these processes that may be related or are
under the control of plant hormone system action as well as the synthesis of proteins
that takes part in these processes realization are being or may be modified.
So far while analyzing the role of phytohormones in plant cold acclimation
processes the attention has been concentrated on abscisic acid (ABA) (Fowler,
Thomashow, 2002; Welling et al., 2002) The formation of several LEA group proteins
under the effect of ABA, cold, drought stress has been discovered (Welling et al.,
2002; Fowler, Thomashow, 2002). In spite of that plants cold acclimate at short day
and low temperature independently (Welling et al., 2002) and in their cells not only
ABA-dependent but also ABA-independent cold acclimation pathway is functioning
(Fowler, Thomashow, 2002); the role of other phytohormones in cessation of plant
growth, developmental and metabolic processes during cold acclimation is unclear.
There are only scarce publications on the role of phytohotmone indole-3-acetic acid
(IAA or auxin) fund transformation in plant autumn growth and its relationship to
cold acclimation and dormancy development in most cases in tissues of hardly cold
acclimating plants – pine, birch (Shuliakovskaja, Anisimovienė, 1985; Weilling
et al., 2002). Thus the matter to understand how, by witch molecular mechanism,
phytohormones can participate in plant autumn growth, developmental and cold
acclimation processes and help plants to adapt to changes in their environmental
factors as well as sustain dormancy during wintering period stays open. Our attention
is focused on understanding how the IAA may be implicated in regulatory network
of plant adaptive processes poor cold acclimating among them.
The aim of investigations is to evaluate possible implementation of auxin in
biochemical processes, namely changes in protein composition and the IAA amount
and status, in organs significant for wintering during autumnal plant growth and cold
acclimation period – under shortening photoperiod (day/night duration) and low
temperature at the second part of this period. The determination of the peculiarities
of protein fund and IAA status changes and their relationship, as well as the search of
100

the possibilities to modify cold acclimation of poorly wintering plants, namely oilseed
rape, is expected.
Object, methods and conditions. As a test object the organs significant for
wintering – terminal bud later on developing inflorescence and root collum as well
as leaves of oilseed rape (Brassica napus L.) varieties ‘Casino’ and ‘Valesca’, are
used. The period of studies comprised from 1st decade of September till 1st decade
of November, i. e. under the shortening photoperiod (day/night from 13/9 to 9/13 h)
and under the influence of low temperatures in the second half of it.
For investigation of the protein fund transformation the test materials (developing
inflorescence, root collum and leaves) fixed in N and stored at -70 ° have been used.
Extraction of soluble proteins (ESP) fraction was performed by 100 mM TRIS-HCl
buffer, pH 8.3 with additives (1 mM EDTA, 1 mM PMSF and 1 mM DDT) at the ratio
1 : 10 (w/v). For structural proteins (HSP) fraction isolation the same buffer with 1 %
non-ionic detergent Triton X-100 (without additives) was used. The protein fractions
were extracted at 4 °C and separated by centrifugation (10 000 g × 25 min.), washed
and stored at -70 °C until further analysis (Shuliakovskaja, Anisimovienė, 1985;
Anisimovienė et al., 2004). The isolation of specific thermostabile proteins-dehydrins
from soluble and structural proteins fractions samples was achieved by heating at 70 °C
and centrifugation at 30 000 g Ч 60 min. (Svensson et al., 2002).
In all stages of investigations protein content was monitored by the Bradford
method (1967). Purification of protein fractions was carried out applying procedures
of precipitation with ammonium sulphate (up to 85 % saturation) and multistage
chromatography on Sephadex G-25 and G-100 columns successively, XAD 7 and
dialyzed through 1 or 12.5 kDa semipermeable membranes (in detail described
Anisimovienė et al., 2004).
Protein composition was tested with non-denaturing PAGE (Laemmli, 1970):
170 µg of proteins were introduced into the tube or 35 µg on the line. Gels were
visualized with Coomassie brilliant blue R 250, in some cases with silver nitrate.
The proteins were characterized according to their electrophoretical mobility (EM)
and molecular mass (MM). In the case of native PAGE, the mM of proteins has been
evaluated in accordance with the localization of – 547, 272, 132, 66, 45, 29 and
14.2 kDa proteins-standards.
To characterize the possible implementation of auxin in plant cold acclimation
processes that extend during autumnal growth and cold acclimation period the
model trials employing synthetic physiological analogues of auxin – calcium
-4-(2-chloroehoxycarbonylmethyl)-1-naphthalenesulfonate (compound TA-12) and
ω–trialkyl-ammonioalkyl ester of 1-napthylethanoic acid (compound TA-14), having
features of this phytohormone, but more convenient for practical goals (Merkys et al.,
1993; Novickienė et al., 2004; Anisimovienė et al., 2006) have been used. The plants
were exposed to the effect of these compounds in optimal concentration (Novickienė
et al., 2004; Velička et al., 2005) as 2 mM and 4mM water solution respectively in a
4–5 true leave rosette phase. Later on the effect of compounds TA-12 and TA-14 on
composition of soluble and structural proteins fractions as well as the composition
of thermostabile proteins-dehydrins in both of them has been analyzed by the way
described earlier in this chapter.
The second part of all tested materials has been used for analysis of IAA level
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and status. The amount of IAA in free form, labile bound IAA conjugates with low
molecular masses compounds and hardly bound reserve IAA complexes with proteins
have been determined according to commonly used methods of physical and chemical
identification (Merkys et al., 1973; Anisimovienė, 1994; Ludwig-Muller et al., 1996;
Walz et al., 2002). Every indole compounds specimen extracted by 80 % ethanol was
introduced for analyzes and preliminary identification by liquid-liquid fractionation
according to pH, solid state-liquid partition on gel filtration columns (Sephadex
G-10, and other). Prepared ether soluble and n-butanol or ethylacetate soluble specimens
of indole compounds were analyzed on TLC in three different chromatography systems
(Anisimovienė, 1994; Anisimovienė et al., 2007). IAA in IAA-ester, IAA-amide and
IAA-protein forms have been elucidated after the ester or amide links rupture by 1, 7
and 5 N alkaline hydrolyses. Liberated from complexes IAA was extracted into ether
and chromatographied on TLC. IAA and its metabolites were characterized by R f
,
UV absorption spectra, reaction with Salkowski in comparison with synthetic indole
compound standards (Anisimovienė, 1994; Ludwig-Muller et al., 1996; Anisimovienė
et al., 2007).
Results. We consider that there are several expectancies for IAA involvement
in autumn growth and cold acclimation biochemical mechanism (biochemistry)
elucidation. One of them – the analysis of changes in protein fund composition of the
cell in the organs significant for autumn growth and cold acclimation and modeling
of these processes employing physiological analogues of auxin (Anisimovienė et al.,
2004; Velička et al., 2005). Another – clarification of the IAA turnover, level and
status in cells of these organs. IAA metabolic routes and proteins-enzymes that take
part in physiologically active IAA form homeostasis maintenance. IAA reserve forms
composition: labile bound IAA conjugates – IAA-esters and/or IAA-amides as well
as hardly bound IAA-protein complexes (Merkys et al., 1973; Anisimovienė, 1994;
Ludwig-Muller et al., 1996; Walz et al., 2002).
Therefore, the analysis of the proteins composition in autumn growth period (in the
mid- September) and peculiarities of their changes later – during cold acclimation period
(in the 3 rd decade of October) – was performed in organs significant for wintering
of rape, i. e. terminal buds or developing inflorescence and leaves cells. The proteins
functioning mainly in cytoplasm and appoplast (ESP fraction) as well as in structural
compartments of the cell – membranes and membranous surrounded compartments
(HSP fraction) have been analyzed separately. Complete isolation of this fraction was
achieved by 3 steps of extraction: 85, 15 and 5 % in succession. After repeated washing
of residues they have been used for HSP fraction isolation. Compete extraction of this
fraction was achieved by 2 steps: 75 and 25 . Thus for protein composition analysis
the specimens of both ESP and HSP fractions have been fully isolated: they have been
used clean and not contaminated by each other. Proteins bands detected on ESP or HSP
fraction electrophoregrams are characteristics for first or second fraction.
Comparative analysis of the results of protein composition in different organs of
winter rape at the autumn growth period (in the 1st decade of September) and later
on during their autumn growth and cold acclimation period (in mid or 3rd decade of
October) revealed common and specific features for organs and protein fraction.
At first it was revealed that during cold acclimation period number of individual
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protein in ESP and HSP fractions increased in cells of all investigated organs. In
developing inflorescence and root collum during 20–30 days of cold acclimation period
the number almost doubled. Most intensively their number increased in developing
inflorescence cells and ESP fraction than in root collum or HSP fraction. In both tested
organs having low or middle molecular masses: 14–45(66) kDa. Such transformations
are true for other varieties of winter rape (‘Accord’, ‘Kasimir’), although modifications
are exclusive for every variety (Anisimovienė et al., 2004; Velička et al., 2005). The
protein composition in leaves of ‘Casino’ and ‘Valesca’ are modified too (Fig. 1).
Fig. 1. The changes of easily soluble (ESP) and membranous structure (HSP)
protein composition during cold acclimation: 1 – Leaves; 2 – Developing
inflorescence; 3 – Root collum
1 pav. Lengvai tirpių (LTB) ir membraninių struktūrų (STB) baltymų sudėties pokyčiai
grūdinimosi periodu: 1 – lapai; 2 – besivystantis žiedynas; 3 – šaknies kaklelis
The increase in proteins’ number in organs is related not only to their synthesis
or accumulation of ones (Fig. 1), but also to the destruction or decrease in their
accumulation. Degree of formation or accumulation of newly formed proteins exceeds
their appearance than disappearance in both fractions of all tested organ cells.
This feature is also characteristic for protein composition changes during autumn
growth and cold acclimation period in hardly cold acclimating plant cells and is related
to preparing for wintering and dormancy-keeping (Ivonis et al., 1984; Jeknič, Chen,
1999). It is revealed that in cells of pine buds in the middle of September ten (10)
protein components are presented while the 30 days later – 17.
Analysis of electrophoregrams of ESP and HSP fractions derived from developing
inflorescence and root collum tissues cells of cold acclimating winter rape (in the middle
or 3rd decade of October) revealed that in their spectrums several (5–7) individual
103

proteins not characteristic to autumn growth period are emergent.
Results of model explorations show that auxin type compounds clearly influence
protein metabolism processes of winter rape developing and root collum tissues cells
during cold acclimation period. The appearance – increase in accumulation or synthesis
of certain proteins not characteristic to control plants occurs. Alongside the several
growth processes of these plants that are under influence or control of auxin (Qin et al.,
2005) are modified (Table).
Table. Effect of physiological analogues of auxin on biochemical-physiological
characteristics
Lentelė. Auksino fiziologinių analogų poveikis biocheminėms-fiziologinėms savybėms
Namely these changes result in the increase of tested plants’ cold acclimation and
keeping the dormancy during over-wintering: only 5 % of TA-14 and 15 % of TA-12
affected plants perished during 2005–2006 wintering, while the percent of perished
plants in control reached 25 %. They are in agreement with the data of investigators
(Gavelienė et al., 2002; Novickienė et al., 2004; Velička et al., 2005), which show that
auxin physiological analogous compounds TA-12 and TA-14 have the positive effect
on winter rape growth, developmental processes and wintering.
Having in mind that the level and status of phytohormones (IAA, ABA) in hardly
cold acclimated plants is innovated and they are coherent to prepare for wintering
(Ivonis et al., 1984; Shuliakovskaja, Anisimovienė, 1985; Welling et al., 2002) the
analysis of IAA level and status in organ tissues significant for rape wintering has been
started. The attention was focused on elucidation of the amount of physiologically
active IAA form (as IAA molecule) and amount of its reserve forms – complexes of
various chemical structure and composition (Ivonis et al., 1984; Anisimovienė, 1994;
Ludwig-Muller et al., 1996; Walz et al., 2002). The amount of IAA, labile bound
low molecular IAA conjugates – complexes with charboxydrates and amino acids
104

extractable by 70–80 % organic solvents and high molecular IAA-protein complexes
not extractable by these solvents have been analyzed.
According to the data obtained so for, the level of IAA as well as the ratio of free
IAA and IAA complexes (Fig. 2) in different organs of winter rape ‘Casino’, ‘Valesca’,
at the autumn growth stage is not identical. At this period the amount of free IAA is
significantly higher in the main IAA synthesizing organs – terminal bud (developing
inflorescence) and rosette leaves (Fig. 2) than in root collum.
Fig. 2. The changes of IAA and labile bound low molecular mass
IAA-conjugates amount during cold acclimation period: 1 – leaves;
2 – developing inflorescence; 3 – root collum
2 pav. IAA ir labiliai sujungtų su mažos molekulinės masės junginiais
IAA konjugatų kiekio pokyčiai grūdinimosi periodu: 1 – lapai;
2 – besivystantis žiedynas; 3 – šaknies kaklelis
After 30 days of cold acclimation period (in mid-October) in developing
inflorescence and root collum cells the ratio of free IAA and IAA conjugates increases.
But the amount free IAA, particularly in root collum is too steep in comparison to
other organs. In hardly cold acclimated plants, namely pine and birch, during the cold
acclimation level of free IAA sharply decreases (Ivonis et al., 1984).
Besides that, according the preliminary data of model trials, it was remarked
that under the influence of auxin type compounds, particularly compound TA-14, the
formation reserve of hardly hidrolysible IAA-protein complexes may be enhanced.
In developing inflorescence of ‘Valesca’ it reaches about 11 µg IAA/10 g of fresh
weight or 12 %. In root collum it may be enhanced about from 17 to 27 µg IAA/10 g
of fresh weight. It is possible to assume that formation of bound IAA forms in winter
rape as in hardly cold acclimated plants may be related with dormancy keeping. In
October all or almost all IAA in pine buds is in bound state, primarily as IAA-protein
complexes (Ivonis et al., 1984). Hereby the obtained results exhibit the possibility
of phytohormone IAA involvement into biochemical processes related with cold
105

acclimation of poorly wintering plants.
Discussion. Presented data of model trials suggest possible involvement of
IAA in physiology and biochemistry of plant cold acclimation. Externally applied
physiological analogous of auxin – compounds TA-12 and TA-14 have positive
effect on processes that are under IAA control. The necessity and role of auxin for
morphogenesis, development, division, elongation, initiation of leaves and floral is
known (Gavelienė et al., 2002; Cheng, Zhao, 2007; Qin et al., 2005; etc.). Besides,
under the influence of physiological analogues of auxin not only specific thermostabile
protein-dehydrins that determine plant cold acclimation frost-resistance are formed
newly. Basing on the obtained preliminary results it is possible to suppose that other
from newly formed proteins may be related with readjustment of IAA synthesis,
metabolism or other biochemical processes of hormonal system action due to dormancy.
Formation of labile bound IAA conjugates – IAA-esters and/or IAA-amides is related
with the temporary inactivation of IAA. Processes may be contributed by IAA-amino
synthetases encoded by early auxin up regulated GH3 family genes, IAA-amino
hydrolases such as ILR1, ILR3 and proteins related to IAA – glucosides formation or
re-hydrolyses (Staswick et al., 2005; Woodward, Bartel, 2005). Besides the proteins
IAA-enzymes, the proteins such as IAA-carriers, IAA-receptors may participate in
IAA amount and activity regulation (Woodward, Bartel, 2005; Staswick et al., 2005;
Anisimovienė et al., 2006; 2007).
Having in mind the observed possibility of physiological analogues of auxin to
increase the amount of IAA-protein complexes in winter rape organs significant for
cold acclimation it is possible to assume that IAA-protein complexes formed in winter
rape developing inflorescence and root collum tissues may perform function of the
hardly hydrolysible reserve complexes function and by the diminishing of the level of
physiologically active IAA form may to have influence the endo-dormancy processes
of winter rape. The obtained results uphold preposition that as in hardly acclimated
plant cells the soluble and structural protein composition as well as level of free IAA
and its status may be the important molecular-biochemical factors determining plant
cold acclimation and dormancy during over-wintering of poorly cold acclimated
plants, too.
Although, in spite of above mentioned, for eventual elucidation and explication of
possibility of IAA involvement in cold acclimation mechanism a lot of supplementary
experiments employing other methodical approaches and presumptions must to be
carried out.
Conclusions. The composition of soluble, mainly cytoplasmic, and membranous
structure proteins in the cell as well as IAA amount and status may be important
biochemical-physiological factors determining or influencing cold acclimation of
winter rape.
Not only on specific thermostabile protein-dehydrins formation but possible as
well as those that may be related to growth processes, phytohormone level and status
modification during cold acclimation may be formed under influence of physiological
analogues of auxin.
106

Externally supplied physiological analogues of auxin – compounds TA-12 and
TA-14 –increased oilseed rape wintering.
These biochemical modifications may be considered as the important interior
factors that improve winter rape cold acclimation.
Acknowledgement. The research was partially supported by Lithuanian State
Science and Studies Foundation.
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SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Augalų grūdinimosi problemos
N. Anisimovienė, J. Jankauskienė, L. Novickienė
Santrauka
Nustatyta lаstelės tirpių ir membraninių struktūrų baltymų sudėtis bei IAR kiekis ir
būklė žiemojimui svarbiuose organuose: šaknies kaklelyje, virрūniniame pumpure arba
besivystančiame žiedyne ir lapuose rudeninio augimo ir grūdinimosi – pasiruošimo žiemoti
metu modeliniu augalu panaudojant žieminį rapsą. Tirta fitohormono indolil-3-acto rūgšties
(IAR) dalyvavimo galimybė blogai žiemojančių augalų grūdinimosi – pasiruošimo žiemojimui
adaptaciniuose procesuose. IAR dalyvavimo šiuo metu vykstančiuose biocheminiuosefiziologiniuose
procesuose išryškinimui buvo atliekami modeliniai bandymai, panaudojant
fiziologinius auksino analogus – junginius TA-12 ir TA-14.
Modelinių eksperimentų duomenų pagrindu daroma prielaida apie galimą IAA poveikį
baltymų metabolizmo bei sudėties pertvarkai besivystančio žiedyno ir šaknies kaklelio lаstelėse.
Tirtų junginių poveikyje padidėja individualių baltymų skaičius. Šie pokyčiai apima ne tik
specifinius termostabilius baltymus-dehidrinus, bet ir kitus baltymus, kurie taip pat galbūt
gali dalyvauti kituose fiziologiniuose-biocheminiuose procesuose, susijusiuose su augalų
užsigrūdinimu ir žiemojimu.
Preliminariniais duomenimis šių junginių poveikyje modifikuojama IAA fondo cheminė
sudėtis, padidinama rezervinių IAA kompleksų koncentraciją. Šios biocheminės-fiziologinės
modifikacijos darė įtaką rapsų užsigrūdinimui ir žiemojimui.
Reikšminiai žodžiai: indolil-3-acto rūgštis, fiziologiniai auksino analogai, grūdinimasis,
baltymai, rapsai.
109

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Nutritional diagnosis of apple-tree growing in the
nitrogen fertilizer factory region
Jurga Sakalauskaitė 1 , Eugenija Kupčinskienė 2, 4 ,
Darius Kviklys 1 , Laisvunė Duchovskienė 1 ,
Akvilė Urbonavičiūtė 1 , Gintarė Šabajevienė 1 , Aida Stiklienė 2 ,
Juratė Bronė Šikšnianienė 1 , Ričardas Taraškevičius 3 ,
Alfredas Radzevičius 3 , Rimantė Zinkutė 3 , Pavelas Duchovskis 1
1
Lithuanian Institute of Horticulture, Kauno 30, 54333 Babtai, Kaunas distr.,
Lithuania, e-mail j.sakalauskaite@lsdi.lt
2
Lithuanian University of Agriculture, Department of Ecology, Studentų 11,
LT-5336 Kaunas, Akademija, Lithuania
3
Institute of Geology and Geography, Department of Environmental Geochemistry,
T. Рevčenkos 13, LT-03223 Vilnius, Lithuania
4
Kaunas University of Medicine, Department of Pharmaceutical Chemistry and
Pharmacognosy, Mickevičiaus 9, LT-44307 Kaunas, Lithuania
The aim of the study was to evaluate the changes in the content of macroelements (N, P,
Ca, Mg and Fe), microelements (Mn, Zn, Cu, B, Co, Mo,) and nonessential elements (Ti, V Cr,
Pb, Ba, Ni, Ag, Al, Sr, Sn) in the leaves of apple-trees in the nitrogen fertilizer factory area, the
main industrial pollution source in Lithuania. As a control, a garden in a ‘relatively clean’ district
(Babtai) was selected. A deficiency (< 2.1–2.4 %) of nitrogen in the leaves of apple-trees was
documented in the investigated sites. The greatest amount of P was determined in the leaves
collected from control trees, where deficiency of the nitrogen was the highest. According to our
results, we maintain that apple-trees growing in Babtai garden sustain deficiency of N, Ca, Fe,
Zn and greatly accumulate P and Ba. Apple-trees growing in the vicinity of nitrogen fertilizer
factory sustain the deficiency of N, Ca, Mn, Zn, B and Zn; and accumulate P, Fe and Mo greater
than optimum content. Under the influence of nitrogen fertilizer factory, accumulation of some
heavy metals (Fe, Mo, V, Ti, Pb) in the leaves of apple-tree may occur.
Key words: air pollution, Malus domestica, element, leaves, soil.
Introduction. Air pollutants are known to affect the nutrient metabolism of trees
in different ways. Low concentrations of some compounds, such as nitrogen oxides,
sulphur dioxide and hard particles, may serve as nutrient sources and might be used
by plants (Fangmeier et al., 1994; Rennenberg et al., 1996). Higher concentrations of
gaseous air pollutants may increase or decrease the uptake of nutrients by the roots,
and can intensify nutrient leaching from leaves, as a consequence of membrane damage
(Seufert, 1990). In addition to these direct effects, the deposition of air pollutants to
the soil is known to alter soil chemical characteristics, such as pH-value and buffering
capacity, thus affecting the availability of nutrients and increasing the leaching of
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mineral elements from the soil. It may result in deficiencies of the nutrients in the
plants, mainly of magnesium, calcium and potassium, as well as in severe nutritional
imbalances (Shaw, McLeod, 1995; Oren, 1996). Apart from these effects on the mineral
balance of the soil, acidification may provoke root damage and impair mineralization
(Seufert, 1990).
Plants are known to accumulate a wide range of pollutants (Salt et al., 1998),
and in the studies of the trees growing in the urban and industrial areas element
accumulation by higher plants, lichens and mosses has been documented (Bargagli
et al., 1998). Uptake of the elements in plants depends on many factors, among them
climate plays a fundamental role modifying nutrition. The chemical analysis of the
leaves has often been applied as a valid method for studying air pollution (Zwolinski
et al., 1998). While great attention has been paid to conifers decline under anthropogenic
influence, much less evidence is available about horticultural species reaction growing
in adverse gaseous and dusty environment. Excesses or deficiencies of nutrients are
a special concern in fruit trees where nutritional imbalances may affect the yield for
more than a single season (Pestana et al., 2004), since new growth depends on the
nutrients stored in the plant.
In present paper, the effect of emitted pollutants from nitrogen fertilizer factory
on nutritional status of the apple-tree, the dominant orchard tree species of Lithuania,
is presented.
Object, methods and conditions. The leaves of the apple-tree and soil samples
were collected in the area affected by the nitrogen fertilizer plant (NFF) JSC Achema
(ranges of the annual total emissions in 2001–2004 were 5.6–4.9 thousand t). In order to
test the influence of air pollution on the mineral nutrition of apple-tree cv. ‘Antonowka’
(Malus domestica L.), leaf sampling was performed in July. Leaves of the apple-trees
and soil samples were collected north-eastward from pollution source, in the direction
of prevailing wind at a distance of 2.5 km from the nitrogen fertilizer factory.
The garden of the intermediate location regarding industrial area (district Babtai,
Lithuania) and at the same time distant from the mentioned pollution source, was
taken as a reference site where soil samples were collected and leaves for the analyses
were collected from the apple-tree cv. ‘Antonowka’ (Malus domestica L.). In each
site the leaves were collected from three apple-trees and soil samples were collected
from location. Only fully expanded leaves were collected for elemental analysis.
Preparation of the leaf samples for this analysis consisted of heating, gradual primary
ashing with free access of oxygen, final ashing at 450 °C and homogenization. For the
determination of the total content of 18 elements: Ag, Al, B, Ba, Ca, Co, Cr, Cu, Fe,
Mn, Mo, Ni, P, Pb, Sr, Ti, V and Zn the samples of the leaves were analysed by atomic
emission spectrophotometry (DFS-13); at a laboratory of the Institute of Geology and
Geography, taking part in the international intercalibrations of Wageningen University.
Internationally certified reference materials were used when analyzing the samples of
the leaves. The content of N was determined by Kjeldal method (Allen, 1989).
All data were analyzed by ANOVA (ANOVA for MS Excel, version 3.43) and
Fisher’s LSD test procedure (α = 0.05) and correlation analyses.
Results. The soil pH of ‘relatively clean’ garden (district Babtai, Lithuania) was
neutral (~ 7.3), while in the garden soil near the nitrogen fertilizer factory was acid
(~ 6.2).
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When compared to the control garden, according to N content, no significant
differences were found for soil and for the apple-tree leave growing near the nitrogen
fertilizer factory (Table 1). The significant greater amount of P was determined in the
leaves from the control trees with the greater nitrogen deficiency and with significantly
(p < 0.01) lower content in the soil. Deficiency of Ca (optimum interval 1.1–2.0 %)
was determined in the leaves sampled in the control and the nitrogen fertilizer factory
gardens. The foliar Mg levels did not present greater variations among selected
gardens. The significantly lower amount of Mg was determined in the garden soil near
the nitrogen fertilizer factory, but this did not reached deficiency level on apple-tree
(0.22–0.35 %; Sadowski, 1990). Significantly lower amount of soil Fe was determined
in the nitrogen fertilizer factory site as compared to reference. Excess of this element
was determined in the leaves from the garden near the nitrogen fertilizer factory. The
greater soil Fe content, determined in the reference site does not decided the optimal
Fe content in the apple-tree leaves at this site.
Table 1. Amount of macroelements (mg kg -1 ) in the leaves of the apple-tree (Malus
domestica L.) and soil (mean value ± confidence interval, * indicates significant
difference from the control area, when a = 0.05). C – control (Babtai garden),
NFF – nitrogen fertilizer factory.
1 lentelė. Makroelementų kiekis obelų lapuose ir dirvožemyje (vidurkis ± pasikliautinas
intervalas, * žymi skirtumus nuo kontrolės, kai a = 0.05). C – kontrolė (Babtų sodas),
NFF – azoto trąšų gamykla (AB “Achema”).
The amount of Mn in the leaves of the apple-trees grown in the control garden
was significantly higher by a factor 5 as compared to the leaves collected from the
site with industrial emission (Table 2). The significant higher content of Mn was
determined in the garden soil near the nitrogen fertilizer factory, but Mn deficiency was
registered in the leaves of the apple tree (Sadowski, 1990; Mochecki et al., 1986). The
B content in the soil under industrial emissions was significant lower as compared to
control garden. The leaves of the apple-trees were B deficient (Wojcik, 2004), except
the apple-tree leaves of the control garden, where B corresponded optimal value for
the growth. Similar to B the soil and foliar Cu content presented significant decrease
in polluted garden as compared to the control garden. The greater amount of Zn was
determined in the apple-tree leaves from the control, while soil Zn content was similar
113

in the investigated sites. The foliar Co levels did not present greater variation, but
soil Co content in the industrial site was significantly lower as compared with control
garden soil. The optimum content of Mo was determined in the apple-tree leaves
samples from the control garden (Sadowski, 1990), while excess of this element was
observed in the leaves from the garden near the nitrogen fertilizer factory. Significant
lower V and Ti content was determined in the garden soil near the nitrogen fertilizer
factory, but foliar V and Ti level showed an increase by a factor 2 as compared to the
control garden.
Table 2. Amount of microelements (mg kg -1 ) in the leaves of the apple-tree (Malus
domestica L.) and soil (mean value ± confidence interval, * indicates significant
difference from the control area, when į = 0.05). C – control (Babtai garden),
OR – oil refinery, CF – cement factory, NFF – nitrogen fertilizer factory.
2 lentelė. Mikroelementų kiekis obelų lapuose ir dirvožemyje (vidurkis ± pasikliautinas
intervalas, * žymi skirtumus nuo kontrolės, kai į = 0.05). C – kontrolė (Babtų sodas),
NFF – azoto trąšų gamykla (AB “Achema”).
Such nonessential elements as Cr, Ni in the leaves of the apple-tree corresponded
to the concentrations normally found in plants (Barker, 1989; Markert, 1996). Foliar and
soil Pb content in the garden near the nitrogen fertilizer factory was significantly higher
as compared to control site. Foliar and soil Ba level of the apple-trees growing in the
nitrogen fertilizer factory site showed significant decrease as compared to the control
garden. The foliar Al, Sr and Sn levels did not present greater variations. The soil Al
content was significant lower and Sr content was significant greater near the nitrogen
fertilizer factory. The foliar Ag level showed significant lower content as compared
with control leaves, while soil Ag content did not presented greater variation.
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Table 3. Amount of nonessential elements (mg kg -1 ) in the leaves of the apple-tree
(Malus domestica L.) and soil (mean value ± confidence interval, * indicates
significant difference from reference area, when į = 0.05). C – control (Babtai
garden), OR – oil refinery, CF – cement factory, NFF – nitrogen fertilizer
factory.
3 lentelė. Neesminių elementų kiekis obelų lapuose ir dirvožemyje ((vidurkis ±
pasikliautinas intervalas, * žymi skirtumus nuo kontrolės, kai į = 0.05). C – kontrolė
(Babtų sodas), NFF – azoto Trąšų gamykla (AB “Achema”).
Discussion. Significant differences among the macro- and micronutrient levels
in the leaves from the trees growing in reference site and nitrogen fertilizer factory
area were determined (Tables 1, 2). The optimum content of nitrogen in the leaves
of the apple-tree is 2.1–2.4 % (Intensive technologies…, 2005). Despite abundant
emissions of the nitrogen oxides from factory the deficiency of the nitrogen in the
leaves of apple-trees was determined in each site of investigation. The present N
pollution dissatisfies the nitrogen demands of apple-trees. Ammonium emitted from
nitrogen fertilizer factory is not sufficient for optimal apple-tree nutrition. The N
deficiency in the soil induces the lack of N in the leaves of apple-tree. Deficiency of
N stimulates the assimilation of P (Sadowski, 1990). Similar situation was found in
our study: the greatest amount of P was determined in the leaves from the control trees
with nitrogen deficiency and with significant lower P content in the soil. The optimum
content of P in the leaves of the apple-trees is 0.15–0.16 % (Sadowski, 1990), so the P
content determined in the apple-tree leaves from control and polluted sites was higher
than optimum. The abundance of P suppresses the assimilation of Zn, Fe and other
microelements (Intensive technologies…, 2005).
Deficiency of Ca element was determined in the leaves sampled in the control
and the nitrogen fertilizer factory gardens (optimum interval 1.1–2.0 %; (Sadowski,
1990). The lack of Ca to fruit-trees occurs very rarely (Intensive technologies…, 2005).
Excess of Fe element was determined in the leaves from the garden near the nitrogen
fertilizer factory; meanwhile its deficiency was documented for the control garden.
Iron chlorosis (Fe deficiency) is an important nutritional disorder among the fruit trees.
It results from impaired acquisition and use of the metal by plants rather than from a
115

low-level Fe in the soil. The most common cause of Fe chlorosis is the bicarbonate
ion, which occurs in high levels in calcareous soils (Pestana et al., 2004). High levels
of P in the tissues of the plants are often associated with Fe deficiency. The deficiency
of Fe in apple-tree leaves from Babtai garden may influence the neutral pH level in
the soil (~ 7,3), and excess of P in plant tissues.
The highest Mn mean concentration was apparently observed in the leaves sampled
from the control garden. Amount of this element was higher by a factor 5 as compared
to the leaves collected from the sites with industrial emission. Under elevated industrial
emission Mn deficiency was registered in the leaves of the apple tree (Sadowski,
1990; Mochecki et al., 1986). According to Adriano (1986) the deficiency range of
Mn for the most plant species is < 20 mg kg -1 . Only in the leaves of the control garden
the concentration of B corresponded optimal value for the growth, in the nitrogen
fertilizer garden the leaves of the apple-trees were B deficient (Wojcik, 2004). The
B content in the soil was significant lower near investigated factory as compared to
control garden; so foliar B concentration was depended on soil B concentration. Boron
deficiency is particularly prevalent in the light textured soils, where water-soluble
B readily leaches down the soil profile and becomes unavailable for plants (Walsh,
Golden, 1953). According to Shear and Faust (1980) the deficiency range of Zn for
the apple-tree is less than 14 mg kg -1 . In all investigated sites Zn deficiency occurred.
Zinc deficiency may be caused by high amount of P (Loneragan et al., 1979). In our
study excess of P was determined in the apple-tree leaves sampled near the nitrogen
fertilizer factory and control garden (Table 1). Also Zn deficiency is associated with
high pH, calcareous soil in which Zn availability is greatly reduced (Neilsen, 2003).
Under deficiency of Zn the fruit-tree resistance to cold stress is reduced (Intensive
technologies..., 2005; Mochecki et al., 1986). On the basis of the V data obtained by
us as compared with Barker (1989) and Markert (1996) results, it can be deduced, that
V was present in leaves of apple-tree from the control garden in concentration lower
than is normally found in plants.
Only leaves collected in the garden near the nitrogen fertilizer factory had mean
Pb concentration higher than the background level (Barker, 1989; Markert, 1996).
The greater accumulation of Pb in the apple-tree leaves may influence the intensive
transport in Jonava region. Baker (1989) and Markert (1996) report that Ba requirement
in the plant tissue is 40 mg kg -1 . This means that the garden near the nitrogen fertilizer
factory was the place where Ba leaf concentration was optimum. Excess of Ba was
determined in the apple-tree leaves from the control garden.
According to Neilsen, Neilson (2003), 16 chemical elements (O, C, H, N, P, K,
Ca, Mg, S, Mn, Fe, B, Cl, Cu, Zn, Mo) are essential for the normal and healthy growth
of apple-trees. Also greater concentrations of N, P, S, K, Ca and Mg are necessary. On
basis of the results of other authors and data shown in Tables 1–3, we maintain that
apple-trees growing in Babtai garden, selected as control site, sustain deficiency of
essential elements such as N, Ca, Fe, Zn and greatly accumulate P and Ba. Apple-trees
growing in the vicinity of nitrogen fertilizer factory sustained the deficiency of N, Ca,
Mn, Zn, B and Zn; and accumulated P, Fe and Mo more than optimum content.
Natural differences in the soil fertility among the sites, direct absorption of gaseous
air pollutants by the leaves or deposition to the leaf surfaces and pollution-induced
116

alterations of soil chemistry are the possible reasons for the differences in the nutritional
status of adult trees growing at various sites in the vicinity of the industrial pollution
sources (Klumpp et al., 2002).
Conclusions. The deficiency of the nitrogen in the leaves of the apple-trees was
determined in each site of investigation. The N deficiency in the soil induces the lack
of N in the leaves of apple-tree. Nitrogen deficiency stimulated the assimilation of P in
the apple-tree leaves. Deficiency of such essential elements as Mn, Ca, Zn and B was
determined in the leaves of apple-trees growing near nitrogen fertilizer factory. Under
the influence of the nitrogen fertilizer factory, accumulation of some heavy metals (Fe,
Mo, V, Ti, Pb) in the leaves of the apple-tree may occur. It shows that present industrial
activities may change and worsen nutrition quality of the fruit trees.
Acknowledgement. This work was supported by Lithuanian State Science and
Studies Foundation under project FIBISTRESS.
Gauta 2008 03 27
Parengta spausdinti 2008 04 22
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13. Pestana M., Varennes A., Goss M. J., Javier Abadнa J., Faria E. A. 2004. Floral
analysis as a tool to diagnose iron chlorosis in orange trees. Plant and Soil,
259: 287–295.
14. Rennenberg H., Herschbach C., Polle A. 1996. Consequences of air pollution on
shoot-root interactions. Journal of Plant Physiology, 148: 296–301.
15. Sadowski A. 1990. The evaluation of fertilization demand of orchard plants.
Akademia, Warsawa, (in Polish).
16. Salt D. E., Smit R. D., Raskin I. 1998. Phytoremediation, Ann. Rev. Plant Physiol.
Plant Mol. Biol., 49: 643–668.
17. Seufert G. 1990. Flow rates of ions in water percolating through a model ecosystem
with forest trees. Environment. Pollution, 68: 231–252.
18. Shaw P. J. A., McLeod A. R. 1995. The effects of SO 2
and O 3
on the foliar nutrition
of Scots pine, Norway spruce and Sitka spruce in the Liphook open-air fumigation
experiment. Plant, Cell and Environment, 18: 237–245.
19. Shear C. B., Faust M. 1980. Nutritional ranges in deciduous tree fruits and nuts.
Hort. Rev., 2: 142–163.
20. Walsh T., Golden J. D. 1953. The boron status of Irish soils in relation to the
occurrence of boron deficiency in some crops in acid and alkaline soils. Int. Soc.
Soil Trans., 2: 167–71
21. Wojcik P. 2004. Impact of boron, biomass production and nutrition of aluminium
stressed apple rootstocks. Journal of Plant Nutrition, 27(11): 2 003–2 046.
22. Zwolinski J., Matuszcyk I., Zwoliсska B. 1998. Accumulation of sulphur and
metals in and on pine (Pinus sylvestris L.) and spruce (Picea abies (L.) Karst.)
needles in industrial regions in southern Poland. Folia For Polinica, 40: 47–57.
SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Obelų, augančių azoto trąšų gamyklos poveikyje, mitybinės būklės
įvertinimas
J. Sakalauskaitė, E. Kupčinskienė, D. Kviklys, L. Duchovskienė,
A. Urbonavičiūtė, G. Šabajevienė, A. Stiklienė, J. B. Šikšnianienė,
R. Taraškevičius, A. Radzevičius, R. Zinkutė, P. Duchovskis
Santrauka
Tyrimo tikslas buvo įvertinti makroelementų ((N, P, Ca, Mg and Fe), mikroelemetų (Mn,
Zn, Cu, B, Co, Mo) ir neesminių elementų kiekių pokyčius obelų lapuose prie azoto trąšų
gamyklos (AB “Achema”), vieno iš pagrindinių taršos šaltinių Lietuvoje. Kaip kontrolė buvo
118

pasirinktas sodas sąlyginai švariame rajone, Babtuose. Abejose tyrimų vietose buvo nustatytas
N trūkumas obelų lapuose (< 2,1–2,4 %, Sadowski, 1990). Didžiausias P kiekis nustatytas obelų
lapuose, rinktuose nuo kontrolinių medžių, kur N trūkumas buvo didžiausias. Pagal gautus
rezultatus, obelys, augančios Babtų soduose patiria N, Ca, Fe, ir Zn trūkumą ir yra linkusios
kaupti P ir Ba. Obelys, augančios prie azoto trąšų gamyklos, patiria N, Ca, Mn, B ir Zn trūkumą,
o P, Fe ir Mo kaupia daugiau nei optimalus kiekis. Azoto trąšų poveikyje, obelys buvo linkusios
kaupti sunkiuosius metalus, tokius kaip Fe, Mo, V, Ti, Pb. Tai rodo, kad dabartinė gamyklos
veikla gali keisti ar pabloginti obelų, augančių šiose vietose, mitybinę būklę.
Reikšminiai žodžiai: oro tarša, obelis (Malus domestica), mitybos elementai, lapai,
dirvožemis.
119

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Oxidative stress in the tobacco plants at hypothermia
Valeriy Popov, Olga Antipina, Tamara Trunova
Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya Street
35, 127276 Moscow, Russia, e-mail: trunova@ippras.ru
Tobacco plants (Nicotiana tabacum L.) transformed with the desC gene for acyl-lipid
∆9-desaturase from a thermophilic cyanobacterium Synechococcus vulcanus were cultivated
on the agarized Murashige and Skoog medium at 22 °C and 16 h photoperiod. Tobacco plants
transformed with an empty binary vector pGA 482 served as the reference. The investigations
showed that, in contrast to the reference plants, transgenic plants maintained a higher activity
of antioxidant enzymes during 2 h incubation at 2 °C, as a result, these plants resisted more
efficiently to the accumulation of reactive oxygen species and reduced the rate of the lipid
peroxidation. The activity of antioxidant enzymes in the transformed plants is apparently related
to the operation of the introduced desC gene for acyl-lipid ∆9-desaturase because the enhanced
activity of the latter enzyme increased the relative content of polyunsaturated fatty acid in
membrane lipids. This way promoted the liquid state of membranes during the chilling. These
changes helped to preserve the cellular homeostasis and thereby maintain the steady synthesis
of antioxidant enzymes at hypothermic conditions; as a result, cold resistance of transformed
tobacco plants increased.
Key words: Nicotiana tabacum, transgenic plants, acyl-lipid desaturase, antioxidant
enzymes, cold resistance.
Introduction. Chilling of heat-loving plants is known to induce oxidative
processes in their cells. These processes are initiated by reactive oxygen species
(ROS), which arise from disturbed operation of electron transport chains in plant cells
and bring about various manifestations of chilling damage (Hariadi and Parkin, 1993;
Prasad et al., 1994). It is assumed that at low temperatures, the antioxidant systems of
heat-loving plants fail to overcome the increasing level of ROS and peroxides arising
there from; such failure is an initial stage of injury (Hariadi and Parkin, 1993).
A b b r e v i a t i o n s / Sutrumpinimai: FA – fatty acid; MDA – malondialdehyde;
POL – peroxidation of lipids; ROS – reactive oxygen species; SOD – superoxide
dismutase.
ROS content in plant cells is under stringent multilevel control of the antioxidant
system comprising, in particular of, superoxide dismutase (SOD) and catalase
(Lukatkin, 2002). However, at low above-zero temperatures ROS are produced so
rapidly and in such amount that the mechanisms of antioxidant protection become
inefficient. The maximum of temporary ROS production is attained to the interval from
15 min to 2–3 h (Merzlyak, 1989).ROS formed in the cell in the course of chilling
stress activate lipid peroxidation (POL). O 2
-
may be a precursor of hydrogen peroxide.
After it’s reduction the hydroxyl radical (OH) of high oxidative ability is produced
121

(Merzlyak, 1989; Lukatkin, 2002) and induces a cascade of oxidation reactions
leading to malondialdehyde (MDA), one of POL products (Vladimirov and Archakov,
1972). Because MDA is formed as a result of peroxidation of polyunsaturated FA and
most lipids with double bonds are components of membranes (Mazliak, 1977), the
accumulation of POL products at low temperatures is thought to indicate the injury of
membrane structures (Baraboi, 1991). The content of unsaturated FA in the membranes
declines while the level of saturated FA remains the same; the former process elevates
the viscosity of membranes and results in the oxidation of thiol groups of the membrane
proteins bringing about an increase in proton permeability and a decrease in electrical
field strength of membranes. These changes are accompanied by the inactivation of
membrane-bound enzymes (Merzlyak, 1989), which may disturb cell metabolism and
cause plant death under hypothermia (Lukatkin, 2002). Therefore, the investigation
of the characteristics of ROS formation and accumulation of POL products is crucial
for clarifying the mechanisms of cold resistance in heat-loving plants.
It was shown earlier that in the tobacco plants with the transformed gene for acyllipid
∆9-desaturase from Synechococcus vulcanus induced the formation of a double
bond at ∆9 position of stearate with the production of oleic acid. The increase in the
relative content of polyunsaturated FA in the membrane lipids resulted in higher cold
resistance of transformed tobacco plants (Orlova et al., 2003). The processes of POL in
the transgenic plants were less active than in the control plants (Popov et al., 2005).
In this context, the aim of this work was to study the effect of the introduced desC
gene for acyl-lipid ∆9-desaturase from Synechococcus vulcanus on the development
of oxidative stress and operation of antioxidant enzymes at hypothermia in heat-loving
tobacco plants.
Object, methods and conditions. Investigations were carried out with tobacco
(Nicotiana tabacum L.) plants comprising the inserted desC gene for acyl-lipid
∆9-desaturase from a thermophilic cyanobacterium Synechococcus vulcanus under the
control of the 35S promoter. The expression of this gene was confirmed by molecular
methods (Orlova et al., 2003). The tobacco plants transformed with an empty binary
vector pGA 482 were used as the control. Aseptic tobacco plants were cultured on
the agarized Murashige and Skoog medium supplemented with ferulic acid (1 mg/l),
kanamycin (25 mg/l), and claforan (250 mg/l). The plants were cultivated at 22 °C
and a 16 h photoperiod. Cold treatment was conducted at 2 °C for 2 h using a climatic
chamber MIR-153 (Sanyo, Japan). These chilling parameters were selected in order
to ensure the maximum of ROS production attained in the period from 15 min to
2–3 h (Merzlyak, 1989).
Fresh plant material was homogenized in cold 0.06 M phosphate buffer (pH 7.4).
The homogenate was centrifuged at 12 000 g for 20 min, and the supernatant was used
to determine SOD and catalase activities.
Hydrogen peroxide (H 2
O 2
) was assayed using the method based on the production
of a colored compound (a complex of titanium peroxide) (Brennan and Frenkel, 1977).
The concentration of H 2
O 2
was expressed as nmol/g fr wt.
The rate of POL processes was assessed by the accumulation of MDA, one of
the products of oxidation. MDA content was determined by the color reaction with
thiobarbituric acid and subsequent measurement of optical density at 532 nm using an
122

SF-46 spectrophotometer (LOMO, Russia). MDA content was expressed as mmol/g
fr wt (Zhirov et al., 1982).
Activity of SOD (EC 1.15.1.1) was determined using the method based on its
ability to compete with nitro blue tetrazolium for superoxide radicals (Beauchamp and
Fridovich, 1971). SOD activity was expressed as units/mg protein.
Catalase (EC 1.11.1.6) was assayed according to Kumur and Knowles (Kumur
and Knowles, 1993). A decline in the optical density during 1 min after the addition
of 10 mM H 2
O 2
to the enzyme extract was recorded at 240 nm. Enzyme activity was
expressed as the unit of optical density per mg of protein per min. Protein was assayed
according to Bradford (Bradford, 1976). All experiments were repeated six times in
triplicate, and the results were analyzed statistically. The tables show the means and
their standard errors (Dospekhov, 1977).
Results. Under the cold stress, the production of hydrogen peroxide is of great
importance because it can promote the formation of hydroxyl radical, another and more
toxic species of reactive oxygen (Chen and Patterson, 1988). Table 1 shows that the
initial content of peroxides before chilling was about the same in both treatments. The
measurements made right after chilling showed that low temperature 2 °C induced
a significant rise in the peroxide content in both treatments. However, in the plants
transformed with the gene for acyl-lipid ∆9-desaturase, the peroxide level was much
lower than in the control plants.
Table 1. H 2
O 2
and MDA content of control and transgenic tobacco plants that
were incubated at low temperatures
1 lentelė. H 2
O 2
ir MDA kiekis kontroliniuose ir modifikuotuose tabako augaluose, kai
buvo auginami žemos temperatūros sąlygomis
In addition to the hydrogen peroxide assay, we determined the rate of POL assessed
by the MDA content as an index of the activity of oxidation processes set by ROS
and, therefore, reflects plant resistance to hypothermia. The results presented in Table
1 show that chilling for 2 h of the control plants at 2 °C produced almost two-fold
increase in the MDA level, suggesting the active POL processes under these conditions.
In the leaves of tobacco plants transformed with the gene for acyl-lipid ∆9-desaturase,
such chilling did not essentially change the MDA content.Thus, our results show that
chilling the control plants at low above-zero temperature induced H 2
O 2
accumulation
and also activated POL. At the same time, when the plants transformed with the gene
for acyl-lipid ∆9-desaturase were chilled, the level of H 2
O 2
increased to the level much
lower than in the control plants. The rates of POL in the transformed plants before
and after chilling were essentially the same. It follows that the transformed tobacco
plants are more resistant to the oxidative stress under low above-zero temperatures.
123

Probably, this phenomenon is accounted for by an elevated activity of antioxidant
enzymes participating in ROS elimination. A considerable increase in the contents of
H 2
O 2
and MDA in the control tobacco plants apparently points at the lower efficiency
of antioxidant system in these plants under hypothermic conditions. In order to verify
this assumption, we conducted experiments, which included the determination of
activities of antioxidant enzymes SOD and catalase. These enzymes play important
role in plant resistance to the oxidative stress under hypothermic conditions.
Table 2. SOD activity of control and transgenic tobacco plants that were incubated
at low temperatures
2 lentelė. SOD aktyvumas kontroliniuose ir modifikuotuose tabako augaluose, kai buvo
auginami žemos temperatūros sąlygomis
Table 2 shows that before chilling, the initial activity of SOD in both treatments
was essentially the same. In the control plants, a sharp decrease in SOD activity was
recorded 30 min after chilling as compared to the plants that were not chilled. The
most considerable decrease in the activity (four-fold) was observed in the control
tobacco plants 1 h after the chilling. After 2 h incubation at low temperature, the
activity of SOD somewhat increased; however, it remained below the initial level
(preceding the chilling treatment). In the plants transformed with the gene for acyl-lipid
∆9-desaturase, a decline in SOD activity 30 min after chilling was less considerable.
As compared to the control plants, SOD activity in the transformed plants remained
much higher during the whole period of chilling, and after 2 h chilling, it considerably
rose and even exceeded the initial level (before chilling). Thus, after 2 h chilling, the
control plants that are less resistant to hypothermia showed a considerable decrease
in their SOD activity, while in the more resistant transformed plants, the activity of
this enzyme significantly increased. This evidence agrees with the already published
data suggesting that in the course of chilling, the most susceptible plants manifested
the greatest suppression of SOD activity (Lukatkin, 2002).In our experiments, catalase
activity in the control and transformed tobacco plants was almost the same before
chilling; however, in the course of chilling at 2 °C, the activity of the enzyme in these
plants changed differently (Table 3). Within the first hour of chilling, catalase activity
somewhat rose in both treatments; however, this increase was more pronounced in the
transformed plants. The continuation of chilling treatment up to 2 h resulted in a decline
of catalase activity in the leaves of the control plants and induced its considerable rise
in the transformed plants.
124

Table 3. Catalase activity of control and transgenic tobacco plants that were
incubated at low temperatures
3 lentelė. Katalazės aktyvumas kontroliniuose ir modifikuotuose tabako augaluose, kai
buvo auginami žemos temperatūros sąlygomis
Discussion. One of the main antioxidant enzymes is SOD that utilizes superoxide
radical with the formation of H 2
O 2
(Beauchamp and Fridovich, 1971). Detoxification of
ROS produced under oxidative stress involves catalase, one more important antioxidant
enzyme contributing to the rapid utilization of H 2
O 2
(Brennan and Frenkel, 1977). It
was shown earlier that cold resistance of some plants closely correlates with the activity
of this enzyme. For instance, after the chilling of heat-loving plants, their catalase
activity considerably decreased, whereas in the plant species of higher cold resistance,
catalase activity under chilling increased or did not change (Gianinetti et al., 1993;
Okane et al., 1996).Thus, the activity of antioxidant enzymes can largely account for
our experimental data concerning the level of H 2
O 2
and MDA under hypothermia.
For instance, in the control plants 2 h chilling brought about a considerable decline
in the activity of SOD and almost did not affect the activity of catalase. This led to
a considerable accumulation of H 2
O 2
and therefore promoted the POL processes. At
the same time, such chilling significantly elevated the activities of all antioxidant
enzymes in the transformed plants and resulted in a moderate increase in the H 2
O 2
level, as compared to the control plants, and the stabilization of POL processes.The
published evidence indicates that one of the major differences between cold-susceptible
and cold-resistant plants is associated with the ability of resistant plants to reduce the
impairing effect of cold and suppress the formation of cold-induced free radicals by
activating the antioxidant enzyme system (Zhang et al., 1995). Our data persuasively
corroborate this hypothesis.
Conclusions. Summing up our data, we conclude that in contrast to the control
plants, the transgenic plants were able to maintain the activity of antioxidant enzymes
on a higher level throughout the whole period of chilling, which enabled them to
efficiently resist the accumulation of ROS and to decrease the rate of POL processes.
One would assume that the activity of antioxidant enzymes in the transgenic plants
depends on the operation of the introduced desC gene for acyl-lipid ∆9-desaturase;
its activity results in an increase in the relative content of polyunsaturated FA in the
membrane lipids ensuring the liquid state of membranes during chilling. In this way
cellular homeostasis is preserved and the steady synthesis of antioxidant enzymes is
maintained under hypothermia; as a result, cold resistance is enhanced in transformed
tobacco plants.
125

Acknowledgments. This work was supported by the Russian Foundation for
Basic Research, project no. 06-04-48291.
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Gauta 2008 03 07
Parengta spausdinti 2008 04 15
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Stress. Saransk.
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Lipids and Lipid Polymers in Higher Plants, Berlin: Springer-Verlag.
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membranes, Itogi Nauki i Tekhniki, Ser. Fiziol. Rast., 6: 5–167.
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antioxidant responses in arabidopsis Thaliana Callus. Planta, 198: 371–377.
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Trunova T. I., Tsydendambaev V. D., Los D. A. 2003. Transformation of tobacco
with a gene for the thermophilic acyl-lipid desaturase enhances the chilling
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126

15. Popov V. N., Orlova I. V., Kipaikina N. V., Serebriiskaya T. S., Merkulova N. V.,
Nosov A. M., Trunova T. I., Tsydendambaev V. D., Los D. A. 2005. The effect
of tobacco plant transformation with a gene for acyl-lipid ∆9-Desaturase from
Synechococcus vulcanus on plant chilling tolerance. Russ. J. Plant Physiol.,
52: 664–667.
16. Prasad T. K., Anderson M. D., Martin B. A., Stewart C. R. 1994. Evidence for
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17. Vladimirov Y. A., Archakov A. P. 1972. Lipid Peroxidation in biological
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18. Zhang J. X., Cui S. P., Li J. M., Wei J. K., Kirkham M. B. 1995. Protoplasmic
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SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Oksidacinis stresas tabako augaluose hipotermijos sąlygomis
V. Popov, O. Antipina, T. Trunova
Santrauka
Tabako augalai (Nicotiana tabacum L.), modifikuoti desC genu, buvo auginami agarizuotoje
Murashige ir Skoog terpėje 22 °C temperatūroje ir esant 16 h fotoperiodui, o augalai, pakeisti
tuščiu binariniu vektorium pGA482, buvo kaip kontrolė. Tyrimai parodė, kad modifikuoti
augalai išlaikė didesnį antioksidacinių fermentų aktyvumą per 2 val. inkubaciją esant 2 °C.
Todėl šie augalai efektyviau priešinosi ROS kaupimuisi ir sumažino lipidų peroksidacijos tempą.
Antioksidacinių fermentų aktyvumas modifikuotuose augaluose yra aiškiai susijęs su minėto
geno veikimu, kadangi šių fermentų aktyvumo padidėjimas padidino santykinį nesočiųjų riebiųjų
rūgščių kiekį membranų lipiduose. Šis kelias skatino skystа membranų būklę per šaldymą. Šie
pokyčiai padeda apsaugoti ląstelių homeostazę ir kartu palaiko pastoviа antioksidacinių fermentų
sintezę hipotermijos sąlygomis. Todėl transformuotų augalų atsparumas šalčiui padidėja.
Reikšminiai žodžiai: acyl-lipidų desaturazė, antioksidaciniai fermentai, atsparumas
šalčiui, modifikuoti augalai, Nicotiana tabacum.
127

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Reaction of model plant Crepis capillaris to
stress-inducing factors – ozone and UV-B
Vida Rančelienė, Regina Vyšniauskienė
Institute of Botany, Žaliųjų ežerų str. 49, Vilnius LT-08406, Lithuania
E-mail: vida.ranceliene@botanika.lt
Stress inducing effect of ozone and UV-B on plants is well known. However, despite the
different molecular mechanisms of their action, both factors have common mechanisms, such
as affected photosynthesis and synthesis of photosynthetic pigments, induction of oxidative
burst. Action of different doses of both factors was compared on the same plant material of
Crepis capillaris in experimentally changed temperature (21/14 °C, 25/16 °C) and CO 2
(350
and 700 ppm) conditions. The doses of UV-B were 0; 2; 4 kJm -2 and of O 3
– 20; 40; 80 ppb.
The most sensitive for UV-B was the leaf area, which decreased proportionally to the dose of
UV-B. C. capillaris has primarily developed mechanisms of adaptation to alterations of the
tested environmental conditions. That was shown by increased activity of SOD (superoxide
dismutase), relatively stable level of pigment synthesis and only slow alteration of protein content
in leaves. However, the observed effects depended on temperature and CO 2
conditions.
Key words: Crepis capillaris, complex action, elevated CO 2
, temperature, ozone,
UV-B.
Introduction. Rising concentrations of CO 2,
tropospheric ozone (O 3
) and surface
level of UV radiation as a result of increasing industrial activity (IPCC, 2001; Karnosky
et al., 2005; Hidema, Kumagai, 2006) alter plant performance in both natural and
managed ecosystems. Enriched CO 2
and O 3
typically have opposing effects on plant
productivity, resistance to pathogens and other physiological features, including activity
of photosynthesis (Allen et al., 1997; Krupa, 2003; Ashmore, 2005; Rämö et al., 2006;
Qaderi et al., 2007; Awmack et al., 2007; Plessl et al., 2007). However, joint action of
those factors is investigated insufficiently and depends upon plant species, genotype,
geographic location and many uncontrolled environmental conditions (Feder, Shrier,
1990; Allen et al., 1997; Krupa, 2003; Karnosky et al., 2005; Rämö et al., 2006; Koti
et al., 2006; Tegelberg et al., 2008).
Our many-year experience shows that model plant Crepis capillaris (L.) Wallr.
is sensitive enough to action of UV-B and O 3,
and that effects of both environmental
factors depend upon plant parameters. The most sensitive parameters are fresh weight
and leaf area (Rančelienė et al., 2005; 2006). The use of model plant allows escaping
uncontrolled environmental conditions and revealing affect pure effect of the interaction
of investigated factors.
The aim of our investigation was to analyze complex action of UV-B, CO 2
and O 3
on various features of model plant Crepis capillaris (L.) Wallr., representing different
129

aspects of plant response to the examined environmental factors: integrative trait – leaf
area; protein content in leaves, expressing synthetic capacity of plant and destructive
processes. On the other hand, concentration of chlorophylls and carotenoids in leaves,
partially expressing photosynthetic activity of leaves; activity of superoxide dismutase
in leaves, expressing alterations of the protective systems of plant.
Object, methods and conditions. Seeds of Crepis capillaris were harvested at the
Laboratory of Cell Engineering of the Institute of Botany (Vilnius). For experiments
with ozone, plants were grown for 21 days, for other experiments – 28 days in the same
conditions as for seed harvesting in the growth chamber in controlled conditions. Thus
conditions for preliminary preparation of plants were the same in all our experiments
with 12/12 h dark cycle (21 ± 2 °C) in pots filled with soil and watered daily with tap
water. In each pot five plants were grown. For illumination lamps OSRAM L36/77
Flora (PAR 53 µm m -2 s -1 ) were used.
During the second stage, the preliminary grown plants were transferred to the
growth chambers of the Lithuanian Institute of Horticulture (Babtai, Kaunas district).
Illumination conditions in Babtai were different from those in Vilnius: lamps SON-T
Agro (PAR – 100 µm m - І s - №) were used with 16/8 h photoperiod. Experiments were
conducted only after 2 days adaptation in a new regime.
Irradiation with UV-B was made with UV-B lamps TL 40W/12 RS (Philips).
Doses were measured with radiometer VLX-3 (Vilber-Lourmat, France) equipped with
a 312 nm probe. The UV-B doses were 0; 2; 4 kJ m - І d - №. The ozone concentrations
were maintained by ozone generator OSR-8 (Ozone Solutions, Inc.) 7 h a day, 5 days
a week. The ozone level was determined by portable ozone sensor OMC-1108 (Ozone
Solutions, Inc.) Ozone concentrations were 20, 40 and 80 ppb daily. Different CO 2
regime was created with an automatic gas system in a phytotron chamber and monitored
by a CO 2
controller (Regin, Sweden).
An integrated regime of both factors, CO 2
and temperature, on the investigated
plants was analyzed in two variants of treatment: at normal temperature and
CO 2
(and as control) conditions of CO 2
concentration were 350 ppm at 21 °C –
day /14 °C – night, and elevated temperature and CO 2
conditions were 700 ppm CO 2
at 25 °C – day /19 °C – night.
All experiments were made in three replications. The position of pots was
randomized. Plants were examined the next day after treatment. For comparison of
different features, tested in our experiments, all results are expressed in percentage
to control plants. The leaf area of three–five plants taken from different pots in each
sample was measured. Leaves were scanned and analyzed with the Sigma Scan Pro.
The same plants were used for determination of fresh and dry weight.
Concentrations of chlorophylls and carotenoids were determined in 100 % acetone
by method of Wettshtein (1957) with spectrophotometer at 662, 644 and 440.5 nm for
chlorophyll a, chlorophyll b and carotenoids, respectively.
For determination of SOD (SOD, EC 1.15. 1.1) activity and protein content in
leaves, leaf material was grounded with an extraction buffer in 0.05 M Na-K phosphate
buffer pH 7.8 at 4 °C. Supernatants were used as a crude extract for SOD assays by
Beyer (Beyer, Fridovich, 1987) and for soluble protein determination according to
Bradford (1976). Conditions are described in Rančelienė et al. (2005).
130

Data analysis was carried out employing the package of statistical analysis tools
of MS Excel 2003 (Microsoft Corporation) program. We considered treatment effects
at P = 0.05 level.
Results. An action of ozone and UV-B on Crepis capillaris plants is contradictory
and very clearly depends upon the plant parameter used for the evaluation of effect.
That peculiarity of UV-B or ozone action is revealed very clearly if both stress-inducing
factors are employed to affect several distinct features of the same plants. So, according
to our previous work on C. capillaris (Rančelienė et al., 2006), it was expected that the
leaf area is the most sensitive parameter for evaluation of UV-B and ozone action. At
the present series of investigation, it was true only for UV-B. The leaf area decreased
proportionally to the UV-B dose (Fig. 1).
Fig. 1. Effects of UV-B (A) and ozone (B) on leaf area of Crepis capillaris plants
depending on temperature and CO 2
concentration. Conditions of plant irradiation
with UV-B or ozone (O 3
) treatment: UVB and O 3
– at 21/14 °C day/night and
CO 2
– 350 ppm; UVB + t° – at elevated temperature 25/19 °C and CO 2
350 ppm;
UVB + t° + CO 2
and O 3
+ t° + CO 2
– at 25/19 °C and CO 2
– 700 ppm
1 pav. UV-B (A) ir ozono (B) poveikis Crepis capillaris augalų lapų plotui,
priklausomai nuo temperatūros ir CO 2
koncentracijos. Poveikio UV-B ir ozonu (O 3
) sąlygos:
UVB ir O 3
– temperatūra dieną/naktį 21/14 °C, CO 2
– 350 ppm;
UVB + t° – 25/19 °C, CO 2
– 350 ppm; UVB + t° + CO 2
ir
O 3
+ t° + CO 2
– 25/19 °C ir CO 2
– 700 ppm
However, action of ozone in the normal environment or in conditions of elevated
temperature and CO 2
on leaf area was insufficient or even slightly stimulating (Fig. 1).
The reason for such discrepancy becomes clear when action of UV-B is tested in
conditions of increased (25/16 °C) temperature. It was unexpected, but negative effect
of UV-B was convincingly restored namely if C. capillaris was treated with UV-B
in conditions of increased temperature. Concentration of CO 2
did not have impact
on restoration effect, because expression of observed effect was about the same,
independently of elevated CO 2
concentration as additional factor (Fig. 1). Slight positive
effect on leaf area was observed also for ozone in conditions of elevated temperature
and CO 2
in treated plants environment (Fig. 1).
Manipulation with temperature and CO 2
conditions allowed us to show negative
effect of UV-B on concentrations of carotenoids and chlorophyll a and b in leaves
(Fig. 2).
131

Fig. 2. Effects of UV-B (A) and ozone (B) on content of photosynthetic pigments
in leaves of Crepis capillaris depending on temperature and CO 2
concentration.
Conditions of plant irradiation with UV-B or ozone (O 3
) treatment: UVB and
O 3
– at 21/14 °C day/night and CO 2
– 350 ppm; UVB + t° – at elevated temperature
25/19 °C and CO 2
– 350 ppm; UVB + t° + CO 2
and
O 3
+ t° + CO 2
– at 25/19 °C and CO 2
– 700 ppm.
2 pav. UV-B (A) ir ozono (B) poveikis fotosintezės pigmentų kiekiui Crepis capillaris
augalų lapuose, priklausomai nuo temperatūros ir CO 2
koncentracijos.
Poveikio UV-B ir ozonu (O 3
) sąlygos: UVB ir O 3
– temperatūra dieną/naktį 21/14 °C,
CO 2
– 350 ppm; UVB + t° – 25/19 °C, CO 2
– 350 ppm;
UVB + t° + CO 2
ir O 3
+ t° + CO 2
– 25/19 °C ir CO 2
– 700 ppm.
132

However, clearer negative effect was observed under conditions of both modifying
factors, i. e. temperature and CO 2,
being elevated. Concentration of pigments in ozone
treated plants altered insufficiently.
Content of soluble proteins in leaves of C. capillarisafter treatment with different
doses of UV-B or ozone was about the same as in leaves of the control, untreated
plants (Fig. 3).
Fig. 3. Effects of UV-B (A) and ozone (B) on soluble protein content
in leaves of Crepis capillaris depending on temperature and CO 2
concentration.
Conditions of plant irradiation with UV-B or ozone (O 3
) treatment:
UVB and O 3
– at 21/14 °C day/night and CO 2
– 350 ppm;
UVB + t° – at elevated temperature 25/19 °C and CO 2
– 350 ppm;
UVB + t° + CO 2
and O 3
+ t° + CO 2
– at 25/19 °C and CO 2
– 700 ppm
3 pav. UV-B (A) ir ozono (B) poveikis tirpių baltymų kiekiui Crepis capillaris
augalų lapuose, priklausomai nuo temperatūros ir CO 2
koncentracijos.
Poveikio UV-B ir ozonu (O 3
) sąlygos: UVB ir O 3
– temperatūra dieną/naktį 21/14 °C, CO 2
–
350 ppm; UVB + t° –25/19 °C, CO 2
– 350 ppm;
UVB + t° + CO 2
ir O 3
+ t° + CO 2
– 25/19 °C ir CO 2
– 700 ppm.
Manipulation of temperature or temperature and CO 2
conditions did not alter
that relation.
Very appreciable effect of UV-B and especially ozone on activity of SOD in
leaves was observed (Fig. 4). After treatment with ozone, activity of SOD increased
about 3.5 times, and the effect was proportional to increasing concentration of ozone.
UV-B treatment also increased activity of SOD in leaves but not so strongly (about
1.5 times) and not so proportionally to UV-B dose.
It is worth noticing that under conditions of elevated temperature and CO 2
activity
of SOD in leaves of UV-B treated plants was lower, but differences between doses
of UV-B remained: the activity of SOD in leaves decreased proportionally to dose of
UV-B. However, opposite effect for UV-B and ozone in those conditions of treatment
became obvious (Fig. 4).
133

Fig. 4. Effects of UV-B (A) and ozone (B) on superoxide dismutase (SOD) activity
in leaves of Crepis capillaris depending on temperature and CO 2
concentration.
Conditions of plant irradiation with UV-B or ozone (O 3
)
treatment: UVB and O 3
– at 21/14 °C day/night and CO 2
– 350 ppm;
UVB + t° – at elevated temperature 25/19 °C and CO 2
– 350 ppm;
UVB + t° + CO 2
and O 3
+ t° + CO 2
– at 25/19 °C and CO 2
– 700 ppm.
4 pav. UV-B (A) ir ozono (B) poveikis superoksido dismutazės (SOD)
aktyvumui Crepis capillaris augalų lapuose, priklausomai nuo temperatūros ir
CO 2
koncentracijos. Poveikio UV-B ir ozonu (O 3
) sąlygos:
UVB ir O 3
– temperatūra dieną/naktį 21/14 °C, CO 2
– 350 ppm;
UVB + t° – 25/19 °C, CO 2
– 350 ppm;
UVB + t° + CO 2
ir O 3
+ t° + CO 2
– 25/19 °C ir CO 2
– 700 ppm.
Discussion. Results of the present work very convincingly show that action of
UV-B and ozone noticeably depends on environmental conditions of plant treatment.
According to many investigators (Krupa, 2003; Frohnmeyer, Staiger, 2003; Kakani
et al., 2003; Busotti et al., 2005; Brazaitytė et al., 2006), the most sensitive feature of
UV-B and ozone action is concentration of photosynthetic pigments in leaves. In our
previous works the most sensitive parameters to ozone or UV-B action were the leaf
area and plant fresh weight (Rančelienė et al., 2005; 2006). During the present work,
the leaf area decreased significantly only after irradiation with UV-B, but significant
negative effect was also observed on photosynthetic pigment concentration in plants
treated with UV-B in environment of elevated temperature and CO 2
.
On the other hand, this investigation confirms previous conclusion that C. capillaris
plants have well expressed means against UV-B and ozone action. One of such means
may be significantly increasing SOD activity in the leaves of treated plants. For ozone
treatment this effect was revealed independently of environmental conditions. Effects
of UV-B on SOD activity depended on temperature and CO 2
concentration. Under
elevated conditions of temperature and CO 2
, SOD activity in leaves was even slightly
lower.
Increasing SOD activity also confirms the conclusion of many investigators that
one of the main and common mechanisms of both stress-inducing factors, UV-B and
ozone, is oxidative burst (Krupa, 2003; Frohnmeyer, Staiger, 2003; Rančelienė et al.,
2005; Mittler, 2006).
134

Conclusions. 1. Effects of UV-B and ozone on Crepis capillaris plants depend
on plant feature, used for evaluation of the effect, and on environmental conditions of
treatment (temperature and concentration of CO 2
).
2. The most informative features are leaf area and activity of superoxide
dismutase.
3. Crepis capillaris plants have natural systems to avoid negative action of UV-B
and ozone.
Acknowledgements. This research was supported by the Lithuanian State Science
and Studies Foundation programme “APLIKOM”. The authors gratefully acknowledge
the Lithuanian Institute of Horticulture for possibility to perform our experiments in
growth chambers of the Institute.
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veiksnius – ozoną ir UV-B
V. Rančelienė, R. Vyšniauskienė
Santrauka
Stresą sukeliantis ozono ir UV-B poveikis augalams yra pripažintas reiškinys, tačiau
nepaisant skirtingų molekulinių veikimo mechanizmų, abiem stresą sukeliantiems veiksniams
būdingos ir bendros savybės: neigiamas poveikis fotosintezei, oksidacinis „sprogimas“ ir kt.
Šiame darbe abiejų veiksnių poveikis ištirtas priklausomai nuo temperatūros (21/14 °C) ir
(25/16 °C) ir CO 2
koncentracijos (350 ir 700 ppm). UV-B dozės buvo 0; 2; 4; kJ/m 2 , o O 3
– 20;
40; 80 ppb. Poveikis priklausė nuo UV-B tyrimo sąlygų ir nuo pasirinkto tyrimams požymio.
Vienas svarbiausių požymių, ypač UV-B, buvo lapų plotas. Fotosintezės pigmentų (chlorofilo
a ir b, karotinoidų) koncentracija lapuose pakito priklausomai nuo poveikio UV-B sąlygų.
Labai padidėjo superoksido dismutazės (SOD) aktyvumas. UV-B ir ozono poveikis pastebimai
priklausė nuo temperatūros ir CO 2
koncentracijos.
Reikšminiai žodžiai: Crepis capillaris, kompleksinis poveikis, ozonas, padidėjusi
temperatūra, CO 2
, UV-B.
137

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Alteration of source-sink relations in the leaves of
in vitro plants of two Solanum tuberosum L. genotypes
under hypothermia
Nina Astakhova, Alexander Deryabin, Maxim Sinkevich,
Stanislav Klimov, Tamara Trunova
Timirayzev Institute of Plant Physiology, Russian Academy of Sciences,
Botanicheskaya st. 35, Moscow, 127276 Russia, e-mail: trunova@ippras.ru
Growth, ultrastructural organization of photosynthetic apparatus and CO 2
-exchange
were investigated in the leaves of potato (Solanum tuberosum L.) plants cv. ‘Desiree’ of wild
type (control) and transformed with vector carrying yeast invertase gene under the control of
tuber-specific patatin promoter B33 class I, fused with proteinase II inhibitor leader peptide to
provide enzyme location in apoplast. Plants grown in vitro on the Murashige and Skoog medium
supplemented with 2 % sucrose. At optimal growth temperature (22 °C), the transformed plants
differed from the plants of wild type by retarded growth and a lower rate of photosynthesis
as calculated per plant. Photosynthesis and leaf dry weight ratio in transformed plants was
higher than in control plants. Under hypothermia (5 °C), dark respiration and especially
photosynthesis of transformed plants turned out to be more intense than in control plants. After
a prolonged exposure to low temperature (6 days at 5 °C), in the plants of both genotypes, the
ultrastructure of chloroplasts changed. Absolute areas of sections of chloroplasts increased in
transformed plants. By some ultrastructural characteristics: the number of granal thylakoids (per
a chloroplast section area), transformed plants turned out to be more cold resistant than control
plants. The obtained results are discussed in connection with changes in source-sink relations
in transformed potato plants. These changes modify the balance between photosynthesis and
retarded efflux of assimilates, causing an increase in the intracellular level of sugars and a rise
in the resistance to chilling.
Key words: Solanum tuberosum, chilling stress, chloroplast ultrastructure, photosynthesis,
respiration, yeast invertase gene.
Introduction. The potato plant transformed with vector carrying yeast invertase
gene under the control of tuber-specific patatin promoter B33 class I, fused with
proteinase II inhibitor leader peptide to provide enzyme location in apoplast is an
organism with modified source-sink relations (Stitt et al., 1990). Sink ability therein
is reduced because of an elevated activity of acid insoluble invertase cleaving
the sucrose to monosaccharides (glucose and fructose); as a result, the efflux of
assimilates from photosynthesizing tissues is suppressed, and they accumulate
in the leaves (Deryabin et al., 2003; 2007). In transformed plants, the threshold
concentration of sucrose necessary to initiate the formation of tubers was low
(1–2 %). The process of tuberization therein was intensified when the content of
sucrose in the growth medium increased up to 10 %. At the same time, in the plants
139

of wild type, the concentration of sucrose in the growth medium optimal for the onset
of tuberization was 8 %, and its further elevation sharply suppressed this process
(Aksenova et al., 2000). Expression of this gene, with the apoplastic localization of the
enzyme, produced larger tubers, though in lower numbers; in contrast, the transformed
plants with invertase localized in the cytosol produced smaller tubers in higher numbers
per plant (Sonnewald et al., 1997). Moreover, the leaves of transformed plants were
notable for a high activity of all the forms of invertase (especially, of the acid form),
an elevated content of sugars, were more resistant to chilling than the plants that only
contained a GUS marker gene controlled by the CaMV 35S promoter. We assumed
that the elevated cold resistance of transformed plants depends on a modified ratio
between sugars in the mesophyll cells induced by the foreign invertase (Deryabin
et al., 2003; 2005).
Exposure of cold-hardy species to low, non-freezing temperatures induces genetic,
morphological and physiological changes in plants, which result in the development
of cold hardiness and the acquisition of freezing tolerance. The ability of plants
to acquire freezing tolerance from cold acclimation has been shown to involve the
reprogramming of gene expression networks (Fowler, Thomashow, 2002; Kreps
et al., 2002). Photosynthetic cold acclimation has been reported to be an essential
component of the development of cold hardiness and freezing tolerance and requires
the complex interaction of low temperature, light and chloroplast redox poise (Gray
et al., 1997; Wanner, Junttila, 1999). It is well established that both short- (hours to
days) and long- (weeks to months) term exposure to low temperature results in similar
trends for accumulation of soluble sugars, modifications of thylakoid membrane lipid
composition, enhanced antioxidant and reactive oxygen species scavenging capacities
and increased non-photochemical quenching (Uemura, Steponkus, 1997; Savitch et al.,
2000; 2002).
Acclimation may be defined as changes that occur in plant in response to chilling
temperatures, which confer subsequent tolerance to the cold (Huner et al., 1993).
Since the chilling tolerance of plants depends on the resistance of their photosynthetic
apparatus (Levitt, 1980), it was important to study the effect of expression of a foreign
gene that caused changes in source-sink relations on the structure of chloroplasts
and on the rate of photosynthesis and dark respiration in potato plants. Preservation
of a high rate of photosynthesis at low positive temperatures is an important factor
of cold hardening and subsequent survival of plants during overwintering (Klimov,
1987). In chilling-sensitive plant species, photosynthesis is the first to be suppressed.
At the beginning, this suppression is reversible and becomes irreversible after longer
exposures (Klimov et al., 1999). For instance, in cucumber plants grown at 22 °C, a
prolonged exposure to low temperature of 10 °C (for 5 days) in the light irreversibly
inhibited photosynthesis, whereas in more chilling-resistant tomato, the suppression
of photosynthesis caused by the same exposure was reversible.
It was shown (Klimov, 2004) that the accumulation of sugars and their distribution
in the leaves and crowns of winter cereals during adaptation to cold depend on crucial
changes in their translocation and modification in CO 2
-exchange. For instance, low
140

hardening temperatures suppressed stronger respiration than photosynthesis.
Depending on specific genetic features and characteristics of temperature
stress, the plants during the adaptation period produce cells of specific ultrastructure
(Kratsch, Wise, 2000). It was shown that, in cells of frost-resistance plants exposed to
hypothermia, there was an increase in the number of cell elements, and an increase in
the number of plastoglobules (Jian, 1997). This prevented intracellular ice formation
and caused an increase in the plant resistance to the formation of extracellular ice.
Exposure of chilling-sensitive plants to low positive temperatures induces destructive
changes in the cell ultrastructure (Lukatkin, 2002). In the palisade cells of potato
leaves exposed to low temperatures (5 °C for 10 days followed by a gradual fall of
temperature to 0 and -2 °C), Balagurova et al. (1980) detected some ultrastructural
changes of chloroplasts: thylakoids became sinuous, and starch grains disappeared.
At the same time, these researchers revealed a greater tolerance of potato leaves to
hypothermia as compared with plants that were not subjected to chilling.
In relation to the foregoing, the aim of this work was to elucidate a possible
relationship between the greater chilling tolerance of transformed potato plants and the
structure and functions of their photosynthetic apparatus, in particular, with chloroplast
ultrastructure and the CO 2
-exchange.
Object, methods and condition. Investigations were conducted with the plants of
potato (Solanum tuberosum L., cv. ‘Desiree’) transformed with vector carrying yeast
invertase gene under the control of tuber-specific patatin promoter B33 class I, fused
with proteinase II inhibitor leader peptide to provide enzyme location in apoplast.
Wild-type potato plants, cv. ‘Desiree’, were used as control material. The plants were
taken from the collection of clones produced as a result of cooperation between the
researchers of Max Planck Institute of Molecular Plant Physiology (Golm, Germany)
and Chailakhyan Laboratory of Growth and Development (Timiryazev Institute of
Plant Physiology, Russian Academy of Sciences, Moscow, Russia).
The plants were micropropagated in vitro and grown in a controlled-climate
chamber at the Institute of Plant Physiology, Russian Academy of Sciences, at 22 °C
and 16 h illumination with the luminescent lamps producing white light (illuminance
of 4 klx) for 5 weeks in test-tube culture on Murashige and Skoog medium containing
2 % sucrose and the vitamins (mg/l): thiamine-0.5, pyridoxine-0.5, and meso-inositol-
60.0.
Plant length was measured weekly. In order to study the effect of hypothermia,
5-week-old plants were transferred for 6 days to a chamber with controlled temperature
of 5 °C and an illuminance of 4 klx. Judging by the results obtained by other researchers,
who worked with this potato cultivar (Svensson et al., 2002), the temperature regime
and the duration of chilling, we employed, were optimal for the investigations of this
kind.
In order to investigate the ultrastructure of the mesophyll cells, the leaves
from the middle part of 5-week-old plants were detached and fixed for 4 h with
2.5 % glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. After four washings in the
same buffer, the material was fixed with 1 % solution of OsO 4
and embedded in the
141

Epon 812 resin. Ultrathin sections of palisade parenchyma were prepared using an
LKB-2 ultra microtome (LKB, Sweden) and contrasted with a saturated solution of
uranyl acetate at 37 °C for 30 min followed by lead citrate at 20–22 °C for 15 min.
The sections were examined with a TEMSCAN 100CX2 electron microscope (Jeol,
Japan). Morphometric investigations of the cells and chloroplasts were made using a
MOP-VIDEOPLAN apparatus (Reichert, Austria). For electron-microscopic
examinations, the samples were taken from 5 leaves each of 4 plants of every type
of treatment. Morphometric measurements were based on the examination of 70
chloroplasts.
CO 2
-exchange of photosynthesis and dark respiration was measured using an
open type unit equipped with a URAS 2T infrared gas analyzer (Hartmann und Braun,
Germany) and attendant devices produced by the same manufacturer according to the
procedure described earlier (Klimov et al., 1999). The content of CO 2
in the air blown
through the leaf chamber was 0.0419 % by volume, and a sensitivity of the gas analyzer
was 0.005 % by volume over the whole scale. A difference between the content of
CO 2
at the inlet and outlet of the leaf chamber did not exceed 1–2 % of the content of
CO 2
in the air. In order to measure gas exchange, by 4 test tubes, each with one plant,
were accommodated in the exposure chamber placed within the working space of a
Gronland climate cabinet (ILKA, Germany). The rate of a temperature fall within the
climate cabinet was 1 °C min -1 , and the accuracy of its maintenance was ± 0.5 °C. Within
the leaf chamber, temperature was checked using a fixed mercury thermometer. As a
source of light, we used a Proton slide projector (Diaproektor, Russia) with a 300 W
incandescent lamp placed outside the climate cabinet. The plants in the leaf chamber
were illuminated through a special window closed as needed with a lightproof gate.
Because of a heat-insulating effect of double glass in the window of the climate cabinet,
the temperature in the leaf chamber both in the light and in the dark did not deviate
from the desired value. The light intensity on the plant level of 1 000 µmol (m 2 s) -1 was
saturating for photosynthesis of the investigated objects. Gas exchange was recorded
right after attaining a desired temperature in the leaf chamber. In order to reduce the
experimental error related to diurnal dynamics of photosynthesis, gas exchange in the
plants of both types was determined within the same time interval (from 9:30 to 14:00).
Duration of a single measurement (or one replicate) was 15–20 min.
Gas exchange was evaluated from the rate of net CO 2
assimilation determined
during the light-dark transition and the rate of dark respiration measured 10–15 min
after turning off the light. Net assimilation of CO 2
was a result of apparent assimilation
and light respiration evaluated from the release of CO 2
during the first 3–5 min after
turning off the light. Table 1 shows the means of three measurements and their standard
errors. Each measurement was taken using 4 plants with 3 replicates.
142

Table 1. CO 2
-exchange in control potato plants (C) and transformed plants (T)
at temperature 22 °C and after the exposure to cold (5 °C)
1 lentelė. Kontrolinių bulvių (C) ir pakeistų augalų (T) CO 2
apykaita 22 °C temperatūroje
po 6 dienų grūdinimo 5 °C temperatūroje.
The results were treated statistically using the Student’s t test for pair samples,
P = 0.05. Figures show the means of typical experiments comprising three replicates
and their standard errors. Differences reliable at a 95 % level of significance are
discussed.
Results. The insertion of yeast invertase gene in potato plants caused retardation of
their linear growth after 3 weeks of growth (Fig. 1). As compared with the plants of wild
type, relative growth rate of transformed plants declined their dry weight accumulation
more than twice (43 mg (g per day) -1 ) in comparison to 20 mg (g per day) -1 .
Fig. 1. Relative growth rate of control potato (1)
and transformed plants (2) at temperature of 22 °C
1 pav. Kontrolinių bulvių (1) ir pakeistų augalų (2)
santykinis augimo greitis 22 °C temperatūroje
143

The obtained results concerning the rate of photosynthetic gas exchange in potato
plants of investigated genotypes are shown in Table 1. It is apparent that the rate of
photosynthesis upon light saturation calculated on a leaf dry weight basis was higher
in transforming plants both at normal (22 °C) and low (5 °C) temperature. We consider
that a greater rate of photosynthesis observed in the transformed plants depends on a
more pronounced suppression of leaf dry weight accumulation, on which the calculation
of photosynthesis was based.
The photosynthetic apparatus of transforming plants was more cold-resistant.
Whereas in the plants of wild type, the fall of temperature from 22 to 5 °C caused a
full cessation of photosynthesis, in transformed plants, photosynthesis declined only
by 86 %. The rate of dark respiration was also higher in transformed plants at both
temperatures. The same as photosynthesis, dark respiration of transforming plants
was more resistant to the lowering of temperature and declined by 64 % as compared
with 70 % in control plants. However, these differences are not great and fall within
the experimental error.
Insertion of the gene for yeast invertase also brought about a change in the structure
of chloroplasts in transforming plants (Fig. 2).
Fig. 2. Section area of chloroplasts in control potato (1)
and transformed plants (2) at temperature of 22 °C and after
6 days of chilling at 5 °C
2 pav. Kontrolinių bulvių (1) ir pakeistų augalų (2)
chloroplastų plotas 22 °C temperatūroje
po 6 dienų grūdinimo 5 °C temperatūroje
The area of chloroplast sections in transforming plants turned out to be by 34 %
greater than in control material, which agrees with a higher rate of photosynthesis in
transformed plants (calculated on a leaf dry weight basis). As a result of expression
of the gene for yeast invertase, the number of grana and thylakoids in transforming
plants increased by 1.5 times (Table 2). We did not find differences in the number of
plastoglobules per chloroplast and the number of thylakoids per granum between the
plants of investigated genotypes.
144

Table 2. Structural organization of chloroplasts in control potato plants (C) and
transformed plants (T) at temperature 22 °C and after 6 days of chilling at 5 °C
2 lentelė. Kontrolinių bulvių (C) ir pakeistų augalų (T) chloroplastų struktūra 22 °C
temperatūroje po 6 dienų grūdinimo 5 °C temperatūroje.
Long (6 days) exposure with low temperatures (5 °C) caused differences between
the genotypes in the rates of growth and photosynthesis and induced morphometric
changes in the chloroplasts and their structural elements. In the plants of investigated
genotypes, the area of chloroplast section increased; in transforming plants, it was by
55 % greater than in control material (Table 2).
Discussion. It is known that long-term potato plant micropropagation in vitro
accelerates the processes of autoselection aiming at the selection of plants with better
growth characteristics under the given conditions, in particular at heterotrophic nutrition.
As a result, the selection by heterotrophy leads to a decrease in the photosynthetic
activity of these plants (Tsoglin, Gabel’, 1994). The measurements taken by these
researchers showed that the amount of carbon dioxide diffusing from the ambient air
through the cotton wool plug ensured only 5–6 % of photosynthetic demands of the
test-tube plants, and due to nutrient media rich in organic compounds, their growth
was predominantly heterotrophic. This suggests that photosynthesis of potato plants
in vitro is largely restricted by the shortage of carbon dioxide. The researchers sentenced
that the plants possessed properly formed photosynthetic machinery and, taking into
consideration the low level of gas exchange between the environment and the medium
within the test-tube, that the main photosynthetic function of the plants comes to
assimilation of CO 2
released as a result of heterotrophic growth, and also that oxygen
emanated by the plants contributes to heterotrophic growth (Tsoglin et al., 1991).
At 22 °C, the rates of photosynthesis and dark respiration the same as the ratio
of photosynthesis to respiration calculated on a whole plant basis in transforming
plants were lower than in control material. However, at low temperature (5 °C), these
145

characteristics in transformed plants were greater than in control material. Upon a fall
of temperature from 22 to 5 °C, the ratio of photosynthesis to respiration in control
plants approached zero; at the same time, in transforming plants, it decreased by 60 %
(Table 1). Because the value of this ratio at low temperatures is one of the important
indicators of cold and frost resistance in plants (Klimov et al., 1999), these results
corroborate a higher cold resistance of transforming plants as compared with the plants
of wild type we revealed earlier (Deryabin et al., 2004).
It should be noted that swelling of chloroplasts in the cold is one of the typical
responses of the cellular ultrastructure observed not only in plants sensitive to cold
(Klimov et al., 1999; Kratsch, Wise, 2000) but also in frost-resistant plants in the course
of hardening (Stefanowska et al., 2002). In plants of both genotypes, long exposure to
low temperatures caused a decrease in number of plastoglobules in the chloroplasts;
in transforming plants, this reduction was more pronounced. Moreover, in contrast
to control plants, hypothermia resulted in a decrease in the number of thylakoids in
the chloroplasts.
One of the symptoms of adaptation to cold on the level of chloroplast organization
is a reduction in the number of granal thylakoids per chloroplast area unit (Trunova,
Astakhova, 1999). By this parameter, the transformed plants also turned out to be
more cold resistant than control material. Whereas in the plants of wild type, the
number of granal thylakoids remained the same, in transforming plants, it decreased
1.5 times. Thus, the transformed plant of potato with inserted gene for yeast invertase
is a plant with modified source-sink relations (Stitt et al., 1990). Elevated activity of
acid-insoluble invertase in transforming plants, we have discovered earlier, suppressed
the efflux of assimilates from the photosynthesizing cells and tissues (Deryabin et al.,
2003).
Conclusions. Our investigations have shown that a disturbance of the balance
between photosynthesis and assimilate efflux manifests itself in a decrease in the
relative growth rate of the transformed plants. At the same time, they preserve the
higher rate of photosynthesis in the leaf tissues under hypothermia as compared with
control plants and that was accompanied by partial reduction of chloroplast structural
elements.
Acknowledgements. This work was supported by the Russian Foundation for
Basic Research, project No. 07-04-00601.
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148

SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Asimiliacinių ir sandėlinių audinių ryšio kitimas dviejuose in vitro
Solanom tuberosum L. genotipų lapuose hipotermijos metu
N. Astakhova, A. Deryabin, M. Sinkevich, S. Klimov, T. Trunova
Santrauka
Augimas, fotosintetinio aparato pertvarkymas ir CO 2
kitimas buvo nagrinėtas bulvių
(Solanom tuberosum L.) veislės ‘Desiree’ lapuose. Bandymai atlikti su laukinio tipo (kontrolė) ir
pakeistom bulvėm su mielių invertazės geno vektoriumi nešėju, kontroliuojamu šakniagumbiams
specifiniu I klasės B33 patatino promotoriu, su proteinazės II inhibitoriaus pirminiu peptidu,
siekiant aptikti fermento vietа apoplaste.
Augalai auginti in vitro Murashige ir Skoog terpėje praturtintoje 2 % sacharoze.
Optimalioje augimo temperatūroje (22 °C), pakeisti augalai skyrėsi nuo laukinio tipo augalų
lėtesniu augimu bei silpnesne fotosinteze. Pakeistuose augaluose fotosintezės ir sausosios
masės santykis buvo didesnis nei kontroliniuose augaluose. Paaiškėjo, kad po hipotermijos
poveikio (5 °C), pakeistų augalų kvėpavimas tamsoje ir ypač fotosintezė vyko intensyviau nei
kontroliniuose augaluose. Po ilgesnio poveikio žema temperatūra (6 dienas 5 °C), abiejų genotipų
augaluose, chloroplastų ultrastruktūra pakito. Absoliutūs chloroplastų plotai didėjo pakeistuose
augaluose. Pagal kai kurias struktūrines charakteristikas, tokias kaip bendras tilakoidų skaičius
(chloroplastų ploto vienete), paaiškėjo, kad pakeisti augalai buvo labiau atsparūs šalčiui, nei
kontroliniai augalai. Gauti rezultatai aptariami siejant su asimiliacinių bei sandėlinių audinių
kitimų santykiu pakeistuose bulvių augaluose. Šie kitimai pakeičia balansа tarp fotosintezės
ir vėluojančio asimiliuotų transporto, sukeliančio viduląstelinių cukrų kiekio padidėjimą bei
atsparumą šaldymui.
Reikšminiai žodžiai: chloroplastų ultrastruktūra, fotosintezė, kvėpavimas, mielių
invertazės genas, Solanum tuberosum, šaldymo stresas.
149

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF HOR-
TICULTURE AND LITHUANIAN UNIVERSITY OF AGRICULTURE.
SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Radish response to distinct ozone exposure and to its
interaction with elevated CO 2
concentration and
temperature
Jurga Sakalauskaitė, Aušra Brazaitytė, Akvilė Urbonavičiūtė,
Giedrė Samuolienė, Gintarė Šabajevienė, Sandra Sakalauskienė,
Pavelas Duchovskis
Lithuanian Institute of Horticulture, Kauno 30, 54333 Babtai, Kaunas distr.,
Lithuania, e-mail: j.sakalauskaite@lsdi.lt
The objective of the investigation was to evaluate the consequences of ozone (O 3
)
stress and interaction of ozone with elevated carbon dioxide (CO 2
) and temperature on some
physiological aspects of growth and photosynthesis in radish plants. Two different experiments
with 12-days-old radish plants were carried out under phytotron conditions. During the first
experiment, plants were exposed to 40, 80 and 160 µg m -3 O 3
concentrations, ambient CO 2
;
day/night temperature was 21/14 °C. During the second experiment the same O 3
concentrations
were kept, CO 2
concentration was 700 ppm; day/night temperature was 25/16 °C.
The primary effect of ozone on plants was reduction in growth. Dry weight accumulation,
leave area and rhizocarp diameter were reduced significantly under ozone stress. In combined
treatment, elevated CO 2
and temperature protected the adverse effect of O 3
on radish plants.
It was established the accumulation of biomass and induced development of radish rhizocarp
under increased O 3
concentration in the interaction with elevated CO 2
and temperature.
Ozone does not have significant negative impact on photosynthesis pigment synthesis.
Intensified photosynthesis pigment synthesis was determined in the radish leaves, which
developed under ozone exposure. Integrated impact of ozone and elevated CO 2,
and temperature
induced chlorophyll a, b and carotenoid synthesis in old and newly developed radish leaves.
Elevated CO 2
concentration and temperature caused elimination of toxic effect of O 3
on
radish plants.
Key words: radish (Raphanus sativus L.), ozone (O 3
), carbon dioxide (CO 2
), temperature,
biometry, photosynthesis.
Introduction. Increasing concentrations of atmospheric CO 2
from preindustrial
levels have been accompanied by an increase in the frequency and duration of
tropospheric O 3
episodes. Atmospheric concentrations of both trace gases are expected
to increase in the 21st century as global industrialization and emissions of CO 2
and
O 3
precursors continue to grow (Prather et al., 2001; Prentice et al., 2001). Because
increases in atmospheric CO 2
and O 3
concentrations have demonstrable effects on crop
growth, yield, and water use, there is significant concern about the possible effects of
these gases on agricultural production and practices (Fuhrer, Booker, 2003; Morgan
et al., 2003; Fiscus et al., 2005; Ainsworth, Long, 2005).
151

Tropospheric O 3
has long been known to be phytotoxic and causes the greatest
amount of damage on vegetation of any gaseous pollutant by reducing growth and
productivity of many plant species through reductions in photosynthesis, accelerated
leaf senescence and decreased root growth (Bortier et al., 2000; Rudorff et al., 2000).
Atmospheric CO 2
enrichment typically increases net photosynthesis, biomass, leaf
area, and less consistently, yields, in a number of C 3
crop species (Ainsworth, Long,
2005). It is hypothesized that plants growing in elevated CO 2
may be protected from
O 3
damage by reducing O 3
uptake because elevated CO 2
often reduces stomatal
conductance (Mousseau, Saugier, 1992). Elevated CO 2
may also provide substrates
for detoxification and repair processes against O 3
damage (Rudorff et al., 2000). How
these two greenhouse gases together affect plant growth and metabolism, has been
studied by some laboratories, but the results are often contradictory.
Photosynthetic system of plants responds to effect of various stresses sensitively
and measurements of photosynthetic pigments in plants affected by stresses can be one
of the suitable methods for establishing oxidative stresses (Sircelj, Batic, 1998). The
content of chlorophylls and carotenoids, the main pigments of leaf, provides valuable
information about plant physiological status.
The objective of this investigation was to evaluate the consequences of ozone stress
and interaction of ozone with elevated CO 2
and temperature on some physiological
aspects of growth and photosynthesis in radish plants.
Object, methods and conditions. Investigations were carried out in Laboratory of
plant physiology at Lithuanian Institute of Horticulture under controlled environment
chambers, to avoid impact of other environment factors. Radish (Raphanus sativus L.
cv. ‘Žara’) was used in vegetative experiments.
Radish plants were sowed in peat substrate, 25–30 seeds per 5 L pot. Until
germination and one week after, plants were grown in greenhouse, where temeprature
was 25 ± 3 °C and the light source was natural solar radiation. Then plants were
transferred to phytotron chambers with 16 h photoperiod. Light was provided by
SON-T- Agro (Philips, USA) lamps.
Two different experiments with 12-days-old radish plants were carried out. During
the first experiment, plants were exposed to 40, 80 and 160 µg m -3 O 3
concentrations,
ambient CO 2
(~ 350 ppm); day/night temperature was 21/14 °C. During the second
experiment the same O 3
concentrations were kept, CO 2
concentration was 700 ppm;
day/night temperature was 25/16 °C.
Photosynthesis pigment content and biometric measurements were performed
after 7 days of each exposure. Rhizocarp diameter, height and assimilation area of five
randomly chosen plants of each variant was measured. Portable leaf area meter CI-
202 (CID Inc., USA) was used for assimilation area measurements. Plant tissues were
oven-dried at 105 °C for 24 h to determine dry weight. Photosynthesis productivity
was calculated according to the formula:
152

where P pr
– photosynthesis productivity (g m -2 day -1 ); M 2
– M 1
– increase of dry
weight per given time period (g); L 1
, L 2
– leaf area at the beginning and at the end of
given time period, respectively (m 2 ); T – term of period (days).
Chlorophyll a, b and carotenoid content in green matter was determined in 100 %
acetone extracts using spectrophotometrical Wettstein method (Гавриленко, 2003)
with a Genesys 6 spectrophotometer (ThermoSpectronis, USA).
All data were analyzed by ANOVA (ANOVA for MS Excel, version 3.43) and
Fisher’s LSD test procedure (p ≤ 0.05).
Results. Ozone had adverse impact on radish overground part growth, rhizocarp
development, leaves area and dry matter accumulation (Table 1). Significant decrease
in radish overground part growth, as compared with control plants, was determined at
the end of exposure to 80 and 160 µg m -3 ozone concentrations. Radishes treated with
higher O 3
concentrations (80 ir 160 µg m -3 ) developed significantly lower leaves area
as compared with control plants. Ozone inhibited the development of radish rhizocarp
consequently twice as less value for rhizocarp diameter was determined under exposure
to 80 and 160 µg m -3 ozone levels.
Table 1. Biometric indices and photosynthesis productivity of radish under
different ozone (O 3
) concentrations. Significant differences from control treatment
are denoted by bold numbers at p ≤ 0.05 and italic numbers at p ≤ 0.01 (mean ± SE,
n = 5).
1 lentelė. Skirtingos koncentracijos ozono (O 3
) poveikis ridikėlių biometriniams rodikliams
ir fotosintezės produktyvumui. Reikšmingi skirtumai nuo kontrolinių augalų pažymėti
paryškintais skaičiais, kai p ≤ 0,05, ir pasvirais skaičiais, kai p ≤ 0,01 (vidurkis ± standartinė
paklaida, n = 5).
Significantly lower amount of dry matter was accumulated in radish treated
with higher ozone concentrations; in the leaves, dry matter was accumulated like in
control plants, but significantly lower amount of dry matter was accumulated in radish
rhizocarp.
Significant decrease in photosynthesis productivity was observed under all ozone
levels. Control plants produced on average 1.07 mg cm -2 of dry matter per day, while
plants exposed to 80 and 160 µg m -3 ozone concentrations produced only 0.57 and
0.42 mg cm -2 of dry matter per day, accordingly.
In combined treatment, elevated CO 2
(700 ppm) and temperature (25/16 °C day/
night) protected the adverse effect of O 3
on radish plants (Table 2).
153

Table 2. Biometric indices and photosynthesis productivity of radish under
different ozone (O 3
) concentrations, increased temperature and carbon dioxide
(CO 2
) concentration. T – 25/16 °C day/night; CO 2
– 700 ppm. Significant
differences from control treatment are denoted by bold numbers at p ≤ 0.05 and
italic numbers at p ≤ 0.01 (mean ± SE, n = 5).
2 lentelė. Skirtingos koncentracijos ozono (O 3
) bei padidėjusios temperatūros ir anglies
dioksido (CO 2
) koncentracijos kompleksinis poveikis ridikėlių biometriniams rodikliams
ir fotosintezės produktyvumui. T – 25/16 °C dieną/naktį; CO 2
– 700 ppm. Reikšmingi
skirtumai nuo kontrolinių augalų pažymėti paryškintais skaičiais, kai p ≤ 0,05 ir pasvirais
skaičiais, kai p ≤ 0,01 (vidurkis ± standartinė paklaida, n = 5).
It was determined the similar over-ground part height and leave area of all radishes.
While rhizocarp diameter under higher ozone concentration was significantly greater
as compared with control plants. Increased O 3
concentration in the interaction with
elevated CO 2
and temperature induced the accumulation of dry matter, particularly in
radish rhizocarp. Photosynthesis productivity of radish grown under increased O 3
, CO 2
concentrations and temperature was significantly greater as compared with control
plants. Control radish produced on average 0.48 mg cm -2 of dry matter per day, while
plants exposed to 80 and 160 µg m -3 ozone concentrations, elevated CO 2
and temperature
produced 0.94 and 0.77 mg cm -2 of dry matter per day, accordingly.
No significant differences of chlorophyll a, b and carotenoids concentration were
determined in damaged leaves within any level of ozone exposure (Fig. 1). Only
chlorophyll a and b ratio was significantly lower under the highest ozone concentration.
Rapid regeneration of new leaves was the typical reaction of radish under ozone stress.
Significant increase in photosynthesis pigment content was determined in regenerated
leaves immediately after the end of exposure to ozone. Chlorophyll a and b ratio was
similar in newly developed leaves of all radishes.
Integrated impact of ozone and elevated CO 2,
and temperature induced chlorophyll
a, b and carotenoids synthesis in old and newly developed radish leaves. Significant
increase in chlorophyll a and b content, as compared to control plants was determined in
old leaves immediately after the end of exposure to 160 µg m -3 ozone levels. Chlorophyll
a and b ratio was similar in old and newly developed radish leaves.
154

Fig. 1. Photosynthesis pigment synthesis under different ozone (O 3
) concentrations.
Significant differences from control treatment are denoted by an asterisk (*) at
p ≤ 0.05 (mean ± SE, n = 3).
1 pav. Skirtingos koncentracijos ozono (O 3
) įtaka fotosintezės pigmentų sintezei ridikėlių
lapuose. Reikšmingi skirtumai nuo kontrolinių augalų pažymėti žvaigždute (*) p ≤ 0,05
(vidurkis ± standartinė paklaida, n = 3).
Fig. 2. Photosynthesis pigment synthesis under different ozone (O 3
) concentrations,
increased temperature and carbon dioxide (CO 2
) concentration. T – 25/16 °C day/
night; CO 2
– 700 ppm.. Significant differences from control treatment are denoted
by an asterisk (*) at p ≤ 0.05 (mean ± SE, n = 3).
2 pav. Skirtingos koncentracijos ozono (O 3
) bei padidėjusios temperatūros ir anglies
dioksido (CO 2
) koncentracijos kompleksinis poveikis fotosintezės pigmentų sintezei
ridikėlių lapuose. T – 25/16 °C dieną/naktį; CO 2
– 700 ppm.
Reikšmingi skirtumai nuo kontrolinių augalų pažymėti žvaigždute (*) p ≤ 0,05
(vidurkis ± standartinė paklaida, n = 3).
155

Discussion. Ozone enters plants through leaf stomata, oxidizes plant tissues
and causes alteration in biochemical and physiological processes. Long-term chronic
exposure to ozone can lead to a reduction in growth and crop yield, resulting from
the inhibition of photosynthesis, premature senescence, altered biomass partitioning
and changes to reproductive processes (Black et al., 2000; Saitanis, Karandinos,
2002). According to the results of our experiments, used ozone concentrations (80
and 160 µg m -3 ) adversely affected radish growth. Ozone caused radish foliar injury at
the beginning of the experiment; induced leaf desiccation, necrosis and shedding, as a
result leaf area of radishes treated with higher ozone concentrations was significantly
lower at the end of experiment as compared with control plants. Literature indicates
that ozone accelerates leaf senescence and premature leaf loss. These processes are
determined by increase of free radicals in plants cells (Farage et al., 1991; Brunshon-
Harti et al., 1995). Thought the typical reaction of plants to ozone stress was the rapid
regeneration of new leaves, photosynthesis assimilates were accumulated and used to
repair processes of injured photosynthesis apparatus, instead of transporting to other
plant organs.
Radish rhizocarp diameter was reduced roughly by a factor 2 in comparison to
control plants at the end of ozone exposure. Ozone may cause greater disruption of
processes below ground than above, and these changes may occur before changes are
observed above ground (Hofstra et al., 1981). Thought roots are not injured by ozone
directly, they are influenced through indirect mechanism; distribution of photosynthesis
assimilates changes under ozone exposure, later and sink activity.
Ozone stress reduced the radish photosynthesis productivity. Plant productivity
reduces because ozone induce stomatal conductance as a defence mechanism for
further injury (Heath et al., 1994). Although decreased stomatal conductance reduces
further ozone uptake and damage, CO 2
fixation decline, possibly leading to increased
photorespiration and production of phosphoglycolate (Andersen, 2003). Photosynthesis
productivity of radishes treated with ozone, decreased because of intensified plant
respiration and increased energy demand for repair processes. Besides, a part of
assimilated carbon is used for the synthesis of antioxidants and other secondary
compounds, which are necessary for quenching free radicals (H 2
O 2
, O 2
H, O 2,
OHį)
originating from reactions of ozone with water and other solutes in the apoplasm.
Many authors have shown that ozone decreased chlorophylls content in plant
leaves (Sircelj, Batic, 1998; Hassan et al., 1999; Saitanis at al., 2001). However, no
significant negative impact of ozone on chlorophylls and carotenoids synthesis in radish
leaves was observed. Only chlorophyll a and b ratio was significantly lower in the old
radish leaves under highest ozone concentration. Insignificant decline in chlorophyll a
and insignificant increase in chlorophyll b in old radish leaves determined chlorophyll
a and b ratio reduction. Moreover, photosynthetic pigments content in newly developed
leaves have even significantly increased under ozone exposure. It shows that ozone
induced synthesis of photosynthetic pigments in newly developed radish leaves.
Increased CO 2
concentration (700 ppm) and temperature (25/16 °C day/night)
reduced adverse affect of ozone on radish growth. At the end of the experiment, radishes,
treated with higher ozone concentration in consort with elevated temperature and CO 2
concentration, cropped significantly larger rhizocarps, accumulated significantly greater
156

amount of dry matter, intensified photosynthesis productivity. Photosynthesis pigment
synthesis (chlorophyll a, b and carotenoid) intensified along with increasing ozone
concentration. There is growing evidence that rising atmospheric CO 2
concentration
will reduce or prevent reductions in the growth and productivity of C 3
crops attributable
to ozone (O 3
) pollution, but the mechanism of such an effect remain unclear. Firstly, in
many crop plants, stomatal conductance (gs) declines as atmospheric concentrations
of CO 2
increase. The resulting decrease in gs lowers the amount of O 3
absorbed by the
leaf and subsequent O 3
injury. A second explanation for the protective effect of elevated
CO 2
against O 3
injury involves increased photosynthate availability that could be used
for repair and detoxification processes (Polle, Pell, 1999). Plants grown in elevated CO 2
might also be able to increase or maintain pools of antioxidants that confer resistance to
O 3
injury (Polle, Pell, 1999). A third proposal suggests that elevated CO 2
concentrations
shift the intercellular CO 2
concentration (Ci) away from Rubisco-limited assimilation
toward ribulose-1,5-bisphosphate (RuBP)-regeneration-limited assimilation (McKee
et al., 2000). This possibly lowers the suppressive effect of O 3
on photosynthesis by
moving the photosynthetically limiting assimilation process away from an O 3
-sensitive
region (Rubisco activity) to the region where photosynthetic electron transport for
RuBP-regeneration limits assimilation (McKee et al., 2000). Photosynthetic electron
transport processes are less inhibited by O 3
compared with carboxylation activity
(McKee et al., 2000; Long, Naidu, 2002; Fiscus et al., 2005).
The present study revealed, that increasing concentrations of tropospheric ozone
would suppress, to varying degree, the radish growth, and photosynthesis productivity.
Rising atmospheric carbon dioxide along with temperature are likely to afford
protection against the adverse effects of ozone on radish growth and photosynthesis
due to reduced ozone uptake, increased carbon assimilation and other factors.
Conclusions. In general, investigated ozone concentrations inhibited the radish
overground part growth, rhizocarp development, injured photosynthesis apparatus
induced leaves desiccation and shedding, diminished radish photosynthesis productivity.
Ozone concentrations somewhat reduced photosynthetic pigment concentration in old
radish leaves, while the synthesis of given pigments was stimulated in newly developed
leaves under ozone exposure. Radishes, exposed to the same ozone concentrations but
along with doubled carbon dioxide (700 ppm) and increased temperature (25/16 °C
day/night), cropped significantly larger rhizocarp, accumulated greater amount of dry
biomass, intensified photosynthesis productivity. Interactive effect of ozone, elevated
carbon dioxide and temperature stimulated photosynthesis pigment synthesis in old
and newly developed radish leaves.
Acknowledgements. The work was supported by Lithuanian State Foundation
of Science and Studies under project APLIKOM.
Gauta 2008 04 04
Parengta spausdinti 2008 04 24
157

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SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Kompleksinis ozono ir didėjančios anglies dioksido koncentracijos
bei temperatūros poveikis valgomajam ridikėliui
J. Sakalauskaitė, A. Brazaitytė, A. Urbonavičiūtė, G. Samuolienė,
G. Šabajevienė, S. Sakalauskienė, P. Duchovskis
Santrauka
Kompleksinis ozono (O 3
), didėjančios anglies dioksido (CO 2
) koncentracijos ir
kylančios temperatūros poveikio ridikėlių biometriniams rodikliams ir fotosintezės
pigmentams tyrimas atliktas Lietuvos sodininkystės ir daržininkystės instituto,
Augalų fiziologijos laboratorijos fitotroniniame komplekse. Vykdyti du skirtingi eksperimentai.
Pirmo eksperimento metu ridikėliai veikti tokiomis ozono koncentracijomis: 40, 80 ir 160 µg m -3 ,
anglies dioksido koncentracija buvo apie 350 ppm (dabartinė aplinkos), temperatūra – dieną
159

21 °C, naktį 14 °C. Antro eksperimento metu ridikėliai veikti tokiomis pačiomis ozono
koncentracijomis, tik anglies dioksido koncentracija buvo padidinta iki 700 ppm ir temperatūra
pakelta iki 25/16 °C dieną/ naktį.
Bandymo metu naudotos ozono koncentracijos lėtino ridikėlių antžeminės dalies augimą,
šakniavaisių vystymаsi, buvo kaupiama mažiau sausųjų medžiagų, pažeidė fotosintezės aparatą,
skatino lapų džiūvimą ir numetimą. Kompleksinio poveikio metu, padidėjusi anglies dioksido
koncentracija ir aukštesnė temperatūra sumažino neigiamą ozono poveikį ridikėliams. Ridikėliai,
augę aukštesnės ozono, anglies dioksido koncentracijos ir temperatūros aplinkoje, sukaupė iš
esmės daugiau sausųjų medžiagų, formavo didesnius šakniavaisius.
Ozonas neturėjo neigiamo poveikio fotosintezės pigmentų sintezei, o ozono aplinkoje
susiformavę ridikėlių lapai iš esmės daugiau sintetino chlorofilo a, b ir karotinoidų. Kompleksinis
ozono, anglies dioksido ir temperatūros poveikis paskatino visų fotosintezės pigmentų sintezę
senuose ir naujai ozono aplinkoje susiformavusiuose lapuose.
Reikšminiai žodžiai: valgomasis ridikėlis (Raphanus sativus L.), ozonas (O 3
), anglies
dioksidas (CO 2
), temperatūra, biometriniai rodikliai, fotosintezė.
160

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Response reactions to the complex influence of radionuclides
and heavy metals
Jūratė Darginavičienė 1 , Virgilija Gavelienė 1 , Donatas Butkus 2 ,
Benedikta Lukšienė 3 , Sigita Jurkonienė 1
1
Institute of Botany, Žaliųjų ežerų 49, LT-08406 Vilnius, Lithuania
2
Vilnius Gediminas Technical University, Saulėtekio 11, LT-01513 Vilnius,
Lithuania
3
Institute of Physics, Savanorių 231, LT-01513 Vilnius, Lithuania
E-mail: jurate.darginavicienė@botanika.lt
The aim of the work – to evaluate peculiarities of complex influence, frequently taking
place in natural environment, of 90 Sr or 137 Cs with heavy metals and components of mineral
nutrition on the growth of spring wheat seedlings.
10 days-old green wheat seedlings (Triticum aestivum L., ‘Nandu’) were grown in sand
and nutritional medium with additions of 90 Sr, 137 Cs and heavy metals (mixture: Co(NO 3
) 2
,
Cd 2
(NO 3
), ZnCl 2
, CuCl 2
, CrO 3
, Pb(NO 3
) 2
and Ni(NO 3
) 2
). Obtained data show that growth of
wheat seedlings shoots in nutritional medium with 90 Sr in complex with NO 3
"
significantly
activates RNA-polymerase II (RNP-II) in the model system of isolated nuclei. The nutritional
medium with 90 Sr and heavy metals (HM) at the low concentrations of radionuclide activated
RNP-II, at high concentrations – inhibited. 90 Sr at high concentration (14.6 Bq ml -1 ) increased
the growth inhibiting influence of HM. 137 Cs on the background of NO 3
"
activated the growth
(lenght and weight) of wheat seedlings shoots, but on the background of HM growth activating
influence of 137 Cs weakened or disappeared. The influence of 90 Sr and 137 Cs on growth, weight
and activity of RNP- II in the model system of the isolated nuclei depended on the nutritional
medium and changed from activation to inhibition of the processes. Inhibition was characteristic
for complex influence of 90 Sr or 137 Cs with heavy metals.
Key words: complex influence, heavy metals, 90 Sr, 137 Cs.
Introduction. Constant control of radionuclide levels in soil, air and living
organisms is obligatory in the world subjected to everyday anthropogenic influences.
Alongside with changes evoked by radioactive radiation in plants, the quantitative
changes of cell metabolism disturbing the growth, development of plants and lowering
their productivity are highly significant. There is the opinion that nowadays rapid change
of environmental conditions override adaptive potential of plants (Schudzentubel, Polle,
2002). Air and soil pollution, acid precipitation, salinity, increasing UV radiation arise
in plants stress conditions and force the search for mechanisms of plant tolerance and
adaptivity. In order to devise new strategies for improved tolerance, it is important
to understand the basic principles as to how pollutants are taken up and act at the
cellular and tissue levels. The main literature data is concentrated on the investigations
of radionuclides uptake to the plant and changes in gene expression influenced by
161

adioactive contamination of soil (White, Broadley, 2000). Model experiments with
radionuclides are obscure. More information is available concerning mechanisms
of action of heavy metals (Suzuki et al., 2002) but not their complex action with
radionuclides.
The aim of presented work was to evaluate peculiarities of complex influence of
90
Sr and 137 Cs with heavy metals and components of mineral nutrition on the growth
of spring wheat seedlings.
Object, methods and conditions. Test object – 10-days-old spring wheat seedlings
(Triticum aestivum L., ‘Nandu’) grown under permanent light, in sand. Radionuclides,
heavy metals and nutritional medium were added to calcined sand.
Nutritional medium (mg l -1 ): CuSO 4
· 5H 2
O – 0.125; MgCl 2
– 125; CaCl 2
– 112.5;
KCl – 125; MnSO 4
– 0.125; FeCl 2
– 12.5; NH 4
Cl – 4.25; NaH 2
PO 4
· H 2
O – 45.
The solution of heavy metals (HM/SM) was prepared from salts: Co(NO 3
) 2
· 6H 2
O;
Cd 2
(NO 3
) · 4H 2
O; ZnCl 2
; CuCl 2
· 2H 2
O; CrO 3
; Pb(NO 3
) 2
and Ni(NO 3
) 2
· 6H 2
O.
Concentration of heavy metals was determined using the Atomic Absorbtion Flame
spectrophotometer (Hitachi, Model 150-70) with error of 1 % (Lukšienė et al.,
2002).
So cold “nitrate control” was used in the experiments. This means that additional
quantity of NaNO 3
, which corresponded the quantity of heavy metals in the form
of salts, was added to control variants. 90 Sr and 137 Cs used activities were prepared
from solutions: 1.46 · 10 2 , 14.6 · 10 2 , 146 · 10 2 and 1.48 · 10 2 , 14.8 · 10 2 , 148 · 10 2 Bq l -1
respectively. Growth was measured on 100 seedlings: length and weight of seedling
was determined. Nuclei from wheat seedlings were isolated with modifications to
wheat (Merkys, 1988). The system of isolated nuclei for the provocation of activity
of RNA-polymerase II (RNP-II) contained Tris-HCl, pH 7.6, triphosphates UTP, GTP,
CTP and 14 C-ATP (0.1 mM; 3.1 MBq g -1 , Hartmann Analytic). The work of enzyme
was stopped after 40 min. incubation at +37°C by cold trichloroacetic acid. Pellets were
gathered and cleaned on membrane filters (2.5 Ø, Pragopor) by trichloroacetic acid
and ethanol. Radioactivity was checked by scintilliation counter LS 1801 (Beckman,
USA). Specificity of mark incorporation was tested by į-amanythine. Summary protein
was determined according to Bradford (1976).
Tables and figures show arithmetical values of 2–3 experiments with no less
3–4 replications in each and their standard deviations. Differences reliable at P ≥ 0.95
are discussed.
Results. Significance of nitrate level for growth and RNP-
I I a c t i v i t y. Experiments with wheat seedlings grown on nutritional medium with
-
different concentrations of NO 3
within the range of 5–100 mg 100 ml -1 revealed that
growth of shoots of seedlings and their weight increase proportionally to concentration
-
of NO 3
(Fig. 1, A).
162

Fig. 1. Influence of NO 3
į on growth and RNP-II activity
of wheat seedling shoots.
A – Growth changes: white columns – length of shoot,
dark columns – weight of shoot in percents from control
(100 % = correspondingly – 6.1 cm and 2.5 g 100 units -1 ).
B – Changes of RNP-II activity. 1 – Controls; 2 – 5; 3 – 20;
4 – 100 mg NO 3
į100 ml -1 H 2
O.
1 pav. NO 3
į įtaka kviečių daigų augimui ir RNP-II aktyvumui.
A – augimo pokyčiai: balti stulpeliai – antžeminės dalies ilgis,
tamsūs – antžeminės dalies svoriai, iрreikрti procentais nuo kontrolės
(100 % = atitinkamai – 6,1 cm ir 2,5 g 100 vienetų -1 ).
B – RNP-II aktyvumo pokyčiai. 1 – kontrolė, 2 – 5; 3 – 20;
4 – 100 mg NO 3
į 100 ml -1 H 2
O.
So the highest used dose of NO 3
į promoted more than 200 % increase in length
and weight of seedling shoots. The same tendency we saw in the changes of RNP-
II activity. Nuclei from the cells of shoots grown in medium with 100 mg 100 ml -1
NO 3
į exhibited RNP-II activity more than nine times higher than the control without
NO 3
į (Fig. 1, B). So, these data show some significant moments: first of them – the
growth (changes of length and weight) of shoots reflects the changes undergoing in
nuclei (changes in activity of RNP-II). Second moment is methodical – if the nitrate
salts of heavy metals are used, experiment variants obligatory must contain “nitrate
control”.
Impact of complex treatment by 90 Sr or 137 Cs and HM on growth and RNP-II
activity of spring wheat seedling shoots. The influence of 90 Sr on growth and RNP-II
activity in model systems of isolated nuclei was evaluated on the different background
of NO 3
į and HM. The obtained data have shown that 90 Sr impact in complex with NO 3
į
and in complex with HM is different (Table 1).
163

Table 1. Impact of 90 Sr on length and weight of wheat seedling shoots
1 lentelė. 90 Sr poveikis kviečių daigų antžeminės dalies ilgiui ir svoriui
NO 3
į10 and 15 mg 100 ml -1 with different concentrations of 90 Sr induced slight
inhibition of growth parameters, but HM at 10 and 15 mg 100 ml -1 concentrations
inhibited the growth of shoots up to 51 and 62 % correspondingly. All concentrations
of 90 Sr (1.46 – 146 Bq 100 ml -1 ) on the background of NO 3
į (10 mg 100 ml -1 ) activated
RNP-II in the model system of isolated nuclei Fig. 2, A). Simultaneously the activity
of RNP-II in nuclei of seedlings under the influence of 90 Sr changed in dependence of
the concentration of radionuclide (Fig. 2, B) and from activation (at 1.46 Bq 100 ml -1 )
shifted to inhibition (146 Bq 100 ml -1 ).
Fig. 2. Impact of 90 Sr on the activity of RNP-II of wheat seedlings shoots.
A – Control and all variants contain NO 3
į (10 mg 100 ml -1 H 2
O).
B – Control and all variants contain HM (10 mg 100 ml -1 H 2
O).
1 – Controls; 2 – 90 Sr 1.46 Bq 100 ml -1 ; 3 – 90 Sr 14.6 Bq 100 ml -1 ;
4 – 90 Sr 146 Bq 100 ml -1 .
2 pav. 90 Sr poveikis kviečių daigų antžeminės dalies RNP-II aktyvumui.
A – kontrolėje ir visuose variantuose po 10 mg 100 ml -1 H 2
O NO 3
į
B – kontrolėje ir visuose variantuose po 10 mg 100 ml -1 H 2
O sunkiųjų metalų.
1 – kontrolė, 2 – 90 Sr 1,46 Bq 100 ml -1 ; 3 – 90 Sr 14,6 Bq 100 ml -1 ;
4 – 90 Sr 146 Bq 100 ml -1 .
137
Cs on the background of 10 mg 100 ml -1 NO 3
į increased the growth parameters
of shoots (Table 2). At both used concentrations 1.48 Bq 100 ml -1 and 148 Bq 100 ml -1 137 Cs
approximately twice increased the length and weight of shoots. This comparison was
164

made between “nitrate control” and variants enriched by 137 Cs. Control and experimental
variants were supplied with equal concentration of NO 3
į, so the supposition could
be done that growth activation were induced exactly by 137 Cs. Nevertheless in the
case of HM presence in the medium, the shoot become slightly (10 %) shorter, but
its weight slightly (up to 15 %) increased. 137 Cs in the complex with HM also arose
growth activation, but it was not as great as in complex with NO 3
į. The enlargement
in weight was 15"23 % in dependence of 137 Cs doses.
Table 2. The impact of 137 Cs on length and weight of wheat seedling shoots. NO 3
į
and HM both (10 mg 100 ml -1 ).
2 lentelė. 137 Cs poveikis kviečių daigų antžeminės dalies ilgiui ir svoriui. NO 3
į ir SM,
10 mg 100 ml -1 .
The analysis of activity of RNP-II of nuclei isolated from cells of shoots grown
in medium with 10 mg 100 ml -1 NO 3
į have shown 137 Cs induced enlargement by more
than 20 % (Fig. 3, A). 137 Cs on the background of HM significantly, more than twice,
inhibited the plant growth (Fig. 3, B). So, HM additions induced the different changes
of 137 Cs than the ones occurring on the background of NO 3-
.
Fig. 3. Impact of 137 Cs on the activity of RNP-II of wheat seedling shoots.
A – Control and all variants contain NO 3
į (10 mg 100 ml -1 H 2
O).
B – Control and all variants contain HM (10 mg 100 ml -1 H 2
O). 1 – Controls;
2 – 137 Cs 1.48 Bq 100 ml -1 ; 3 – 137 Cs 148 Bq 100 ml -1 .
3 pav. 137 Cs poveikis kviečių daigų antžeminės dalies RNP-II aktyvumui.
A – kontrolėje ir visuose variantuose po 10 mg 100 ml -1 H 2
O NO 3
į.
B – kontrolėje ir visuose variantuose po 10 mg 100 ml -1 H 2
O sunkiųjų metalų.
1 – kontrolė; 2 – 137 Cs 1,48 Bq 100 ml -1 ; 3 – 137 Cs 148 Bq 100 ml -1 .
165

These data confirm that changes in RNP-II activity depend on NO 3
į content.
HM plus 137 Cs severely inhibited RNP-II activity which resulted in further growth
inhibition, but influence of 137 Cs in the nuclei was more intensive compared with the
plant growth.
Discussion. Contamination of soils and water with radionuclides and heavy
metals creates major environmental problems, leading to considerable losses in
plant productivity and hazardous human health effects. The harmful influence of
radionuclides, especially of long life 90 Sr and 137 Cs, for human and plants is well known.
The same could be said about heavy metals. Exposure to toxic metals can intensify in
plant the production of reactive oxygen species, which are continuously produced in
both unstressed and stressed plant cells (Gratao et al., 2005). Some of reactive oxygen
species are highly toxic.
On the other hand the contaminating materials are frequently not solitary and
their complex influence on plant growth is very significant. For this reason, we tried to
study the effects of heavy metals mixture on the impact of radionuclides contaminating
the soil on plant growth. The work was performed with spring wheat seedling shoots.
Roots were not investigated because they were in direct contact with contaminating
elements and there were difficulties with selection of only adsorbed to root surfaces
cationes and the ones directly participating in root metabolism.
Plant cells contain 5"50 mM nitrates in their cytosol and vacuole (Pouliguin
et al., 1991). Nitrates are obligatory for growth of plants (Ramage, Williams, 2002)
and response reactions of plants to nitrates are connected with the work of systems
in nuclei and phytohormones (Forde, 2002). For this reason the obligatory “nitrate
conrol” in our work was used.
The obtained data revealed differences in plant response reactions to 90 Sr and 137 Cs
in complex with the mixture of heavy metals or without them. The influence of 90 Sr
was in most cases inhibitory, but the action of 137 Cs was more complicated. Cs belongs
to the group of elements similar to potassium. When external to plant concentration
of 137 Cs is lower than 200 µm it could be nontoxic for plants (White, Broadley, 2000),
but this is mostly determined by other ions present in plant growth substrates. In our
experiment we used different ions such as K + , NH 4
, Na + , Mg 2+ which can influence
the uptake of caesium to plant. For this reason special question was – what is the
influence of used heavy metals for uptake of radionuclides under conditions of our
experiments. The obtained data showed that neither addition of heavy metals nor
altering of concentration of nitrate ions do not leaded to changes of accumulation or
distribution of 137 Cs in spring wheat seedlings (Butkus et al., in press).
So the changes in radionuclides action were induced as a result of complex
processes in plant body and affected by heavy metals. On the level of nuclei the changes
initiated by 137 Cs on the background of different nitrate content differ from the ones
induced by 137 Cs in complex with heavy metals. In the first case the activation of RNP-II
and consequently growth of seedling takes place, in the second case – approximately
double inhibition of RNP-II activity.
166

Conclusions. 1. Growth of wheat seedlings shoots in nutritional medium with
90
Sr in complex with NO 3
"
significantly activates RNP-II in the model system of
isolated nuclei. The nutritional medium with 90 Sr and HM at the low concentrations
of radionuclide activated RNP-II, at high concentrations – inhibited. 90 Sr at high
concentration (14.6 Bq ml -1 ) increase the growth inhibiting influence of HM.
2. 137 Cs on the background of NO 3
"
activate the growth (lenght and weight) of
wheat seedlings shoots. On the background of HM growth activating influence of
137
Cs weakens or disappears.
3. Activity of RNP-II under the influence of 137 Cs on the background of NO 3
"
increases, but on the background of HM inhibition (50%) is exhibited.
4. Impact of radionuclides 90 Sr and 137 Cs on the growth and activity of RNP-II
depends from composition of nutritional medium and changes from activation to clear
inhibition. The last is exhibited as complex influence of 90 Sr or 137 Cs and HM.
Acknowledgements. The work was supported by Lithuanian State Science
and Studies Foundation. The help of assistant Liudmila Chramova is gratefully
acknowledged.
References
Gauta 2008 03 31
Parengta spausdinti 2008 04 24
1. Bradford M. M. 1976. A rapid and sensitive method for quantification of
mikrogram quantities of protein utilizing the principe of protein-dye binding.
Analytical Biochemistry, 72(1–2): 249-254.
2. Butkus D., Lukšienė B., Darginavičienė J., Maksimov G., Konstantinova M.,
Gavelinė V., Druteikienė R. 2008. Peculiarities of 137 Cs accumulation in spring
wheat. Nucleonica. Spaudoje.
3. Forde B. G. 2002. The role of long-distance signalling in plant responses to nitrate
and other nutrients. Journal of Experimental Botany, 53(366): 39–43.
4. Gratao P. L., Polle A., Lea P. J., Azevedo R. A. 2005. Making the life of heavy
metal-stressed plants a little easier. Functional Plant Biology, 32: 481–494.
5. Lukšienė B., Tautkus S., Druteikienė R., Gvozdaitė R. 2002. Impact of geothemical
surroundings on the distribution of 239, 240 Pu in soil at the Baltic seaside. Ekologija,
4: 34–38.
6. Merkys A. J., Darginavičienė J. V., Žemėnas J. A. 1988. IAA complexes in
plasmalemma and their supposed function. Proceedings of International conference
Physiology and Biochemistry of Auxins in Plants. Praha, Chechoslovakia, 175–
179.
7. Pouliquin P., Grouzis J. P., Gibrat R. 1999. Electrophysiological study with oxonol
VI of passive NO 3
-
transport by isolated plant root plasma membrane. Biophysical
Journal, 76: 360–373.
8. Ramage C. M., Williams R. R. 2002. Mineral nutrition and plant morphogenesis.
In vivo Cell Developmental Biology-Plant, 38: 116–124.
167

9. Schudzendubel A., Polle A. 2002. Plant responses to abiotic stresses: heavy metalinduced
oxidative stress and protection by mycorrhization. Journal of Experimental
Botany, 53(372): 1 351–1 365.
10. Suzuki N., Yamaguchi Y., Koizumi N., Hiroshi S. 2002. Functional characterization
of a heavy metals binding protein Coll19 from Arabidopsis. The Plant Journal,
32: 165–173.
11. White P. J., Broadley M. R. 2000. Mechanism of caesium uptake by plants. New
Phytologist. Review, 147: 241–256.
SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Atsako reakcijos į kompleksinį radionuklidų ir sunkiųjų metalų
poveikį
J. Darginavičienė, V. Gavelienė, D. Butkus, B. Lukšienė, S. Jurkonienė
Santrauka
Darbo tikslas – įvertinti 90 Sr ar 137 Cs kompleksinio poveikio su mineralinės mitybos
elementais ir sunkiaisiais metalais ypatybes vasarinių kviečių augimui.
10 parų žali kviečių (Triticum aestivum L. ‘Nandu’) daigai buvo auginami smėlyje
mitybinėje terpėje keičiant azoto kiekį, su radionuklidų 90 Sr ar 137 Cs ir sunkiųjų metalų priedais
mišinyje: Co(NO 3
) 2
, Cd(NO 3
) 2
, ZnCl 2
, CuCl 2
, CrO 3
, Pb(NO 3
) 2
ir Ni(NO 3
) 2
.
Pagal gautuosius rezultatus kviečių daigų augimas ir RNR-polimerazės II aktyvumas
izoliuotų branduolių sistemoje priklauso nuo mitybinės terpės sudėties ir gali kisti nuo
aktyvacijos iki inhibicijos. Pastaroji yra būdinga 90 Sr arba 137 Cs kompleksiniam poveikiui su
sunkiaisiais metalais.
Reikšminiai žodžiai: kompleksinis poveikis, sunkieji metalai, 90 Sr, 137 Cs.
168

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Investigation of water regime and drought resistance
of various kinds of plants applying phytomonitoring
methods
Oleg Ilnitsky, Ivan Paliy, Tatiana Bystrova, Sergey Radchenko,
Nikolay Radchenko
Nikita Botanical garden, st. Yalta, Ukraine, e-mail: ilnitsky@yalta.crimea.ua
Agrophysics SRI, st. St.-Petersbur, Russia, e-mail: radchenko@peterlink.ru
There were carried out investigations of optical features of leaves in the near infra-red
radiation (970 nm) of plant intact organs of the number of agricultural and decorative species with
the water regime changes. The laser photometer “Perfot-93” and micrometer type “Turgomer-1”
served as the instrument of the study. The investigations were carried out with different plant
species and in different regions (Krasnodarsky region, Leningrad district-Russia, Southern Coast
of Crimea-Ukraine). The high correlation dependence (0.97–0.98) of optical parameters of leaves
on their thickness was determined. The differences in sensitivity of the optical characteristics
to the changes of water regime, connected with eco-physiological peculiarities of plants were
noticed. There exists a principle opportunity to use the methods of parallel control of optical
characteristics of leaves in the near infra-red radiation and water status of plants as an instrument
for investigation of ecological-physiological character and as an element of precise agriculture
technology for the control of water regime in sowings.
Key words: water regime, drought resistance of plants, optical properties, thickness of
a leaf.
Introduction. Researches of optical properties of plant leaves in a visible part of
spetrum are connected usually with a condition of pigmentary system and structure
of leaves (Brandt, Tageeva, 1967; Penuelas et al., 1993; Penuelas et al., 1997; Surin
et al., 1997; Radchenko et al., 1998; Dallon, 2005). The studying of the water status
of plants was operated more often in a range of lengths of waves more than 1 000
nanometers. For definition of water stress of plants by the analysis of measurements
of reflection on several key lengths of the waves called “strips of water” were used
Interference and laser spectrometers. The most known “strips of water” are 1400 and
1900 nanometers. It is shown that reflection on these lengths of waves corresponds to
the maintenance of water in fabrics of plants (Penuelas et al., 1993; Penuelas et al.,
1997). However, these spectrometers are rather expensive. Besides, it is difficult to
measure water stress on these lengths of waves on distance because of a high level of
absorption by fume of water of a terrestrial atmosphere.
Today there are some inexpensive spectrometers on silicon diodes, suitable for
exact measurements of lengths of waves up to 1 000 nanometers, including remote
measurements. The strip of 970 nanometers historically was considered too small for
169

exact measurement of water stress, however it has been shown (Penuelas et al., 1997),
that it can be used for indication of water stress of the close crops where the index of
a leave surface varies not too much.
The purpose of the present work was the research of opportunities to use the
characteristics of reflection and absorption of intact leaves of plants for an estimation
of the water status and drought resistance of various kinds of plants by means of laser
spectrophotometer with simultaneous registration of leaf plate thickness.
Object, methods and conditions. We studied a water mode and drought resistance
of various kinds of plants by measurement of leaves optical properties (Lisker, 1998)
with laser spectrophotometer “Perfot-93” (the working of Leningrad optic-mechanical
association, Russia) and measurements of their thickness with device “Turgoromer-1”
(Kuchnirenko, 1975). “Perfot-93” allows registering simultaneously values of
integrated factors of radiation reflection, absorption and passage and it was used for
discrete measurements. Changes of leaves optical characteristics were compared with
condition of water mode intact plants – increase of water deficiency, reaction on having
watered. The thickness of leaves in all experiments was used as a characteristic of
their level of water concentration.
As objects of researches, there were used various kinds of cultural plants, both the
ones, which are grown up in conditions of vegetative experiment (Table 1, 2) and the
decorative and fruit plants growing in field conditions (Tables 3, 4, 5). Measurements
were carried out in triple frequency in the morning (at 6–7 o’clock) and in the middle
of day (at 13–14 o’clock) synchronously on not separated leaves of the plants grown
in identical conditions. In the morning leaves have the greatest turgor (for a night it
is restored), and in the middle of day at the maximal intensity of external conditions
it was minimal.
For development of universal methods the researches were conducted on various
kinds of plants and in different regions (Krasnodar territory and Leningrad region –
Russia, Southern coast Crimea-Ukraine).
The climatic conditions in these regions are various: the southern coast of Crimea –
dry subtropics, Krasnodar territory – southern black-earth Russia, St.-Petersburg and
Leningrad region – northwestern part of Russia.
In practice the procedure of measurement of optical parameters of a leaf consists
in registration of the falling, reflected and passing radiation, and the absorption factor
is calculated according to the formula:
A = 100 - (T + R), (1)
where А – absorption (factor of absorption, %);
T – passing, %;
R – reflection, %.
It is known, that this formula (1) is applicable in a visible part of spectrum (Leman,
1961), but in near infra-red area of spectrum this formula is not used. We had been
offered the formula (2), which allows identifying results of an irradiation of a leaf
from various surfaces.
A’ = A / (A + T), (2)
The term “specific absorption”, is a quotient from division of average value of
absorption into average value of thickness of a leaf and characterizes an inclination
170

of corresponding lines of trends of A. This dependence simply becomes:
A = C + Kd, (3)
where C – a constant, d – thickness of a leaf, K = A / d – specific absorption.
The factor K in essence is a certain characteristic of sensitivity for leaves of
the given cultures with dimension of % of absorption / % of the change of leaf
thickness.
Mathematical data processing was made applying various methods of statistical
analysis (program STATISTICA).
Results. The results of laser testing of the plants water status together with
monitoring of leaf thickness showed high correlation of these two characteristics and
high enough sensitivity surpassing the known one on literary facts. Some results of
experiments in conditions of vegetative experience are shown in Table 1.
Table 1. Ranges and sensitivity of leaf near infrared radiation reflection change,
when changing plant water status*
1 lentelė. Lapų infraraudononosios spinduliuotės atspindžio pokyčio diapazonas ir
jautrumas, keičiantis vandens režimui augaluose*
The range of changes of the characteristic of reflection DR for the wheat cultivar
‘Saratovskaya-29’ made 4% on average background R = 39.5 %, i. e. depending on the
condition of water regime reflection change ≈ 10 %, for the cultivar ‘Leningradka’ –
accordingly DR = 3.2 % and 8 %. The average changes of thickness of leaves during
experiment between waterings made for ‘Saratov’ 18 %, for ‘Leningradka’ – 25 %.
Various cultures differently reacted to change of reflection in reply to changes
of water concentration (changes of leaf thickness, %) and various ranges of lifetime
changes of reflection. It depends on ecologic-physiological properties of plants, and
obviously can serve as their certain ecological characteristic.
171

It is interesting to note, that according to the degree of drought resistance
decrease experimental plants settle down in a following number: Rose > ‘Saratovskaya-
29’ > ‘Leningradka’ > Tomato.
Table 2. The estimated factors of absorption A’ = A / (A + T) and of leaf near
infrared radiation absorption sensitivity change, when changing of plant water
status*
2 lentelė. Absorbcijos A’ = A / (A + T) faktoriaus įvertinimas ir lapų infraraudonosios
radiacijos absorbcijos jautrumo kitimas, keičiantis vandens režimui augaluose*
In Table 2 values of specific absorption of tomato and rose leaf near infrared
radiations are shown. They differ essentially, that testifies the distinction in features
of their water regime.
As our researches have shown, the maximal thickness of leaves is observed in
the morning (at 6–7 o’clock), and minimal – at 14–15 o’clock. Distinctions of these
parameters are explained with features of water mode of various kinds and sorts of
plants and can serve as their ecological characteristic (Radchenko, et. al., 1998).
By analogy to existing in the scientific literature ten-mark estimations of drought
resistance (Eremeyev, 1964) we have tried to estimate some kinds our offered method.
In Table 3 the results of such measurements for 22 kinds of plants are presented.
Researches were made in Nikita botanical garden (Ukraine, Crimea, Yalta).
Table 3. Interrelation between drought resistance of various kinds of plants and
change of leaf thickness *
3 lentelė. Įvairių augalų rūšių atsparumo sausrai santykis su lapų storiu *
172

Table 3 continued
3 lentelės tęsinys
From Table 3 it is seen, that investigated kinds settle down in a number of
ecological groups, on a degree of drought resistance and have received good enough
concurrence (R 2 = 0.92) with results known earlier for the given kinds (Eremeyev,
1964). We carried out similar investigations on southern coast of the Crimea in the
park of settlement Katseveli.
In Table 4 the results of investigations defying the factors of absorption and
sensitivity of leaf near infrared radiation absorption change for various kinds of
plants are shown. A number of sensitivity and, hence, drought resistances, looks as
follows:
Laurus nobilis ← Rubus occidentalis ← Olea europea ← Pistasea mutica ←
Lonicra Caprifolium ← Prunus divaricata ← Rosa ← Malus domestika ← Lonicera
Caprifolum
Apparently from the analysis of results (Table 4) drought resistance of investigated
various species of plants shows following results: the highest drought resistance
evergreen species xerophyte and on half xerophyte have the highest resistance,
mezophyte have lower one.
Less resistable species - Malus domestica ← Rose ← Malus domestica ← Lonicera
Caprifolium.
To confirm efficiency of the offered methods similar researches have been
made in various regions of Russia (Krasnodar territory, St.-Petersburg and in
Leningrad region). Results of such researches are shown in Table 5. As well as above,
on 10-point system series of drought resistance of investigated kinds of plants have
been calculated. The results of these researches are also proved to be true according
to the data of the scientific literature (Sytnik et al., 1994; Udovenko, 1988).
173

Table 4. Factors of absorption and sensitivity of leaf near infrared
radiation absorption change when changing of plant water status (Kazeveli,
21–22-06-2007)*
4 lentelė. Absorbcijos faktoriai ir lapų infraraudonosios radiacijos absorbcijos jautrumo
kitimas, keičiantis vandens režimui augaluose (Kazeveli 21–22-06-2007)*
Table 5. Daily changes of leaf thickness of various plants* (measurements of all
series were made within two days with intervals of 3–5 hours).
5 lentelė. Įvairių augalų lapo storio dienos pokyčiai* (kiekvienos serijos matavimai atlikti
per dvi dienas 3–5 val. intervalu)
174

Table 5 continued
5 lentelės tęsinys
Discussion. In a near infrared range of radiation, which was investigated,
discrepancy of energy reflection by various leaf surfaces (bottom and top) was found
out. This difference made approximately 10%. Therefore, it is known (Leman, 1961)
that the formula (1) is not to apply in an investigated range. Our investigations allowed
offering the formula (2), which allows identifying results of leaf irradiation from
various surfaces.
According to the earlier poorly investigated near infrared range, it was established,
that the change of absorption factor A (%) depending on leaf water concentration can
reach 25–30 %. The same high sensitivity to water posses the thickness of a sheet
plate of 15–35 %, while the change of thickness of the sprout (stalk) on the same plant
makes 0.2–2.5 % possesses also.
In the scientific literature there was described the possibility to use reflection
methods to estimate drought resistance of various kinds of plants (Eremeyev, 1964;
Kuchnirenko, 1975; Udovenko, 1988; Penuelas et al., 1993; Penuelas et al., 1997;
Dallon, 2005). The results received by us on various kinds of plants – wheat (two
grades), tomato, soy, fruit (14 kinds and grades), decorative, etc., and also in various
geographical regions (southern coast of Crimea – dry subtropics, Krasnodar territory –
southern black-earth Russia, St.-Petersburg and Leningrad region – the northwestern
part of Russia) prove to be true according to these sources.
The investigations also have shown, that there is high correlation dependence
(0.7–0.98) between optical properties of leaves and their thickness.
In this connection, the offered method of ranging of kinds of plants and cultures
with a degree of drought resistance is represented quite comprehensible. The minimal
change of thickness of a sheet plate within day serves as a criterion.
Conclusions: 1. The investigations that have been carried out in various
geographical regions and on various kinds of plants have allowed to develop universal
methods of studying of plant water mode and drought resistance.
2. There exists gradual dependence of absorption of near infrared radiation on
thickness of a leaf. In a range of little changes of leaf thickness the dependence has
linear character.
175

3. The function of leaf thickness can serve as the characteristic of optical
properties at linear approximation of radiation absorption as the steepness of a line
of this function trend.
4. There were noticed the distinctions in sensitivity of optical characteristic
changes of the water mode, caused by eco-physiological features of plants.
5. There is a basic opportunity to use a method of the parallel control of leaf near
infrared radiation optical properties and plant water status as the tool of researches of
eco-physiological character and as element of technology of exact agriculture for the
control of crop water mode. The investigations, which were carried out, undoubtedly
seem perspective both in the theoretical aspect and in practical application.
References
Gauta 2008 03 27
Parengta spausdinti 2008 04 25
1. Brandt A. V., Tageeva S. V. 1967. Optical parameters of vegetative organisms.
Moscow, Science.
2. Curran P. J., Dungan J. L., Peterson D. L. 2001. Estimating the foliar biochemical
concentration of leaves with reflectance spectrometry: testing the Kokaly and
Clark methodologies. International Journal of Remote Sensing, 76: 349–359.
3. Dallon D. 2005. Measurement of water stress: Comparison of reflectance at 970
and 1450 nm. In: Utah State University. Crop Phys. Lab., 1–5.
4. Еremeyev G. N. 1964. Laboratory-field method of an estimation of drought
resistance of fruit and other plants and the results of its application. In: Coll.
Scientific works. Moscow, Kolos. 456–472.
5. Kushnirenko M. D. 1975. Physiology of water exchange and drought resistance
of fruit plants. Kishinev, Chtiinza.
6. Leman B. M. 1961. The course of the light culture of plants. Moscow, Science.
7. Lisker I. S. 1998. Laser-optical methods, devices and systems of the automated
research of plants and seeds. In: Agrophysics methods and devices (in 3 volumes).
Plants and environment of their dwelling. Spb. AFI. 3: 299–311.
8. Lisker I. S., Radchenko S. S. 1999. Laser-optical and hydromechanical methods
of diagnostics of stresses in plants in ontogeny. In: Field experiments – for steady
land tenure. The works of 3-rd International colloquium. Spb. 1: 51–52.
9. Penuelas J., Pinol J., Ogaya R., Filella I. 1997. Estimation of plant water
concentration by the reflectance water index WI (R900/R970). International
Journal of Remote Sensing, 18: 2 869–2 875.
10. Penuelas J., Filella Bell C., Serrano L., Save R. 1993. The reflectance at the
950–970 nm region as an indicator of plant water status. International Journal of
Remote Sensing, 14: 1 887–1 905.
11. Radchenko S. S. Ivanov V. M., Marichev G. A., Tchernyaev E. V. 1998.
The method of monitoring of thickness of a leaf plate. In: Agrophysics methods
and devices (in 3 Vol.). Plants and environment of their dwelling. SPb. AFI.
3: 159–166.
176

12. Sytnik K. M., Brajon A. V., Gorodetskij A. V., Brajon A. P. 1994. The ecological
dictionary. Kiev, Naukova dumka.
13. Surin V. G. 1997. Precision field spectrometry: possibilities and prospects. Earth
Obs. Rem. Sens. 14: 973–984.
14. Udovenko G. V. 1998. General requirement to methods and principles of
diagnostics of stability of plants to stresses. In: Diagnostics of stability of plants
to stressful influences. Leningrad: VIR.: 5–10.
SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Įvairių rūšių augalų vandens režimo ir atsparumo sausrai
tyrimai, naudojant fitomonitoringo metodus
O. Ilnitsky, I. Paliy, T. Bystrova, S. Radchenko, N. Radchenko
Santrauka
Buvo atlikti grupės žemės ūkio ir dekoratyvinių augalų lapų optinių savybių tyrimai
infraraudonojo spinduliavimo (970 nm) srityje, keičiantis vandens režimui. Tyrimams naudoti
lazerinis fotometras „Perfot-93“, mikrometras „Turgomer-1”. Tyrimai buvo atlikti su skirtingomis
augalų rūšimis ir skirtinguose regionuose (Rusijos Krasnodaro krašte bei Leningrado srityje
ir Pietiniame Krymo krante, Ukrainoje). Buvo nustatyta aukšta koreliacija (0,97–0,98) tarp
lapų optinių savybių ir jų storio. Nustatyti optinių charakteristikų jautrumo skirtumai, susiję
su vandens režimo pokyčiais ir nulemti ekofiziologinių augalų ypatybių. Egzistuoja galimybė
naudoti lapų optinių charakteristikų infraraudonojo spinduliavimo srityje ir vandens būklės
augale paralelinės kontrolės metodа kaip instrumentа tiriant ekofiziologines savybes ir kaip
precizinės žemdirbystės elementа kontroliuojant pasėlių vandens režimа.
Reikšminiai žodžiai: atsparumas sausrai, lapų storis, optinės lapų savybės, vandens
režimas.
177

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Effects of UVB radiation on photosynthesis pigment
system and growth of pea (Pisum sativum L.)
Rima Juozaitytė, Asta Ramaškevičienė, Algirdas Sliesaravičius,
Natalija Burbulis, Ramunė Kuprienė, Vytautas Liakas,
Aušra Blinstrubienė
Lithuanian University of Agriculture, LT-53067 Noreikiрkės, Studentų 11, Kaunas
distr., Lithuania, e-mail: rima.juozaityte@lzuu.lt
The object of the work was to determine the effects of UV-B radiation on growth of
peas (Pisum sativum L.) and photosynthesis pigment system. Research was carried out
in phytotron complex at the Lithuanian Institute of Horticulture and Laboratory of Agro
Biotechnology at Lithuanian University of Agriculture. Plants were grown in 5 L vegetative
pots in neutral peat substrate (pH 6.0–6.5), 25 plants per pot. A photoperiod of 16 h and
temperature of 21 °C/17 °C (day/night) was maintained throughout the experiment. Highpressure
sodium lamps ‘Son-T Agro’ (Philips) were used for illumination. UV-B radiation
was generated using UV-B lamps (TL 40W/12 RS UV-B Medical, Philips). Plants were
treated with UV-B radiation for 5 days. There were investigated UV-B radiation doses of
0 (control), 1, 3, 5, 7 and 9 kJ m -2 each day. Shoot height, dry weight, stomata density and
leaf area were measured at 1, 3, 5 day of experiment immediately. Content of photosynthesis
pigments were determined spectrophotometrically in 100 % acetone extracts. After the
first day of UV-B radiation there was established exposition toxic UV-B effect on aboveground
length, dry biomass and number of leaves. It was established that increasing
UV-B radiation doses and expositions, there was tendency of pigment content decrease.
Key words: growth, peas, photosynthetic pigments, UV-B radiation.
Introduction. Stratospheric ozone layer absorbs the most of space UV radiation
(100 to 400 nm) including solar and other sources, thus protecting plants and other
living organisms from harmful UV radiation effect. The thickness of ozone layer
is rapidly decreasing and intensity of UV radiation is increasing since seventies
of the last century (Krizek, 1998). Shortest UV waves are most detrimental to the
living organisms. Usually, UV radiation is divided to UV-A (315–400 nm), UV-B
(280–315 nm) and UV-C (100–280 nm) spectral ranges. UV-C radiation is completely
absorbed by ozone layer while UV-A radiation is unaffected, though, such radiation
does no harm to plants. However, the UV-B radiation intensity is mostly affected by
the thickness of stratospheric ozone layer and exactly this type of radiation is most
harmful to plants. Recently, research on UV-A radiation also showed some adverse
effect on plants (Helsper et al., 2003; Krizek, 2004).
Many authors have determined the wide range of UV-B radiation effects on plants.
Higher than normal levels of UV-B causes various damages to plant such as DNA and
179

membrane injuries, photosynthetic or hormone systems’ disorders (Rozema et al., 1997;
Jansen et al., 1998; Hollosy, 2002). Molecular-level changes affect other processes
including gene activity, metabolism, photosynthesis intensity and, consequently, the
growth of the whole plant. Some genes are identified as “UV-B dependent genes” and
are responsible for synthesis of UV screening compounds, DNA repairs and activation
of ant oxidative enzymes (Brosche, Strid, 2003).
Continuous measurements of UV radiation level were initiated in Lithuania only
since 2000. Lithuanian Hydrometeorology Service does such measurements at Kaunas
meteorology and Palanga aeronautical meteorology stations. Obtained data shows that
UV-B radiation intensity depends on season, daily time and meteorology. Average daily
dose during fine weather reaches 2.5 kJ m -2 (Jonavičienė, 2005).
Objectives to determine UV-B radiation effect on pea (Pisum saivum L.) growth
and photosynthetic pigment system were raised.
Object, methods and conditions. Research was carried out at Lithuanian
University of Agriculture, Laboratory of Agro biotechnology while vegetative research
took place in phytotron complex at Lithuanian Institute of Horticulture. Research
object – pea (Pisum sativum L.), species ‘Ilgiai’ (leafy).
Pea was grown in 5 L vegetative pots in neutral peat substrate (pH 6.0–6.5).
Research was done in three replications, each of 25 plants. Pea was germinated
and grown for one week in a greenhouse and then carried to growth chambers were
photoperiod of 16 hours and temperatures at 21/17 °C (day/night) were maintained.
UV-B daily doses of 0 (reference), 1, 3, 5, 7 and 9 kJ m -2 were investigated. UV-B
radiation was generated by TL 40 W/12 RS UV-B Medical lamps (Philips, Austria).
Irradiation experiment lasted 5 days.
Shoot height, dry mass, number of stoma and leaf area (Win Dias meter) were
measured after 1, 3 and 5 days of exposure. Photosynthetic pigments were analyzed by
spectrophotometer in 100 % acetone extract according to Vetstein method (Wettstein,
1957). Average values and SDs were calculated by MS Excel.
Results. Shoot height has decreased even after the first day of exposure to
UV-B (Fig. 1 A). Lowermost shoots were observed after the fifth day of exposure
to 7 and 9 kJ m -2 daily doses, which reached only 58 and 59 % of reference height,
respectively.
Shoot dry mass followed the pattern of shoot height and decreased even after the
first day of exposure (Fig. 1 B). It reached only 55 % of reference value after the fifth
day of exposure under 9 kJm -2 of UV-B.
Adverse effect of UV-B radiation on leaf area was also determined (Fig. 2). Leaf
area decreased even after the first day of exposure. Most reduced leaf area values were
observed after the fifth day of exposure to 7 and 9 kJ m -2 and reached only 52 and
63 % of reference values, respectively.
Number of stoma has increased up to 23 % after the first day of exposure to
1 kJ m -2 (Fig. 3). Other dosage resulted in zero effect. However, as the exposure time
increased, the significant decrease of stoma counts was observed.
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Fig. 1. Effect of UV-B radiation on pea (Pisum sativum L.) growth parameters: A –
height growth, B – above-ground dry biomass
1 pav. UV-B spinduliuotės poveikis sėjamojo žirnio (Pisum sativum L.) augimo
parametrams: A – aukščio didėjimui, B – antžeminės dalies sausai biomasei
Fig. 2. Effect of UV-B radiation on pea (Pisum sativum L.) leaf area
2 pav. UV-B spinduliuoutės poveikis sėjamojo žirnio (Pisum sativum L.) lapų plotui
Fig. 3. Effect of UV-B radiation on stomata content in pea
(Pisum sativum L.) leaf area
3 pav. UV-B spinduliuotės poveikis sėjamojo žirnio (Pisum sativum L.)
žiotelių skaičiui lape
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First day of exposure to UV-B radiation had no effect on chlorophyll a amounts
in pea leaves (Table 1). However, a decrease in chlorophyll a was observed after the
third and fifth day of exposure.
1 Table. Effect of UV-B radiation on chlorophyll a content
1 lentelė. UV-B spinduliuotės poveikis chlorofilo a kiekiui
Amount of chlorophyll b remained steady even after the third day of exposure
(Table 2). The most significant decrease in chlorophyll b amount was observed after
the fifth day of exposure to 5 kJ m -2 daily doses.
2 Table. Effect of UV-B radiation on chlorophyll b content
2 lentelė. UV-B spinduliuotės poveikis chlorofilo b kiekiui
The first day of exposure had no effect on chlorophyll a and b ratio (Table 3).
However, a and b ratio have decreased as the time of exposure have increased.
Table 3. Effect of UV-B radiation on chlorophyll a/b ratio
3 lentelė. UV-B spinduliuotės poveikis chlorofilo a/b santykui
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4 Table. Effect of UV-B radiation on carotenoid content
4 lentelė. UV-B spinduliuotės poveikis karotinoidų kiekiui
Carotenoid content remained steady or even decreased even after the first day of
exposure (Table 4). However, carotenoid content have decreased after the fifth day of
exposure to UV-B radiation.
Discussion. This research revealed that pea growth pattern is very sensitive to
UV-B radiation. The adverse effect of UV-B radiation was observed after the first day
of exposure. Other scientist also shows significant UV-B radiation effect on barley
growth parameters including stem height, sprout count, leaf area and biomass (Correia
et al., 1999, Nasser, 2001). Plant sensitivity to UV exposure might be determined by
direct damage to cell structural and functional elements or by ineffective acclimatization
process (Smith et al., 2000).
As aforementioned, shoot height, dry biomass and leaf area decreased even
after the first day of exposure. UV-B radiation may induce leaf differentiation and
senescence processes via modification of leaf structure (Kakani et al., 2003, 2004).
Plants are capable to accommodate to certain UV-B radiation as well as to light
intensity, though tolerance range are determined by plant species, age, duration of
exposure and other factors. If the UV-B dosage exceeds the limits of tolerance, plant
leaf anatomy is changed and biomass is decreased (Coleman, Day, 2004, Kakani
et al., 2003, Zhao et al., 2003). Nevertheless, other authors (Liu et al., 1995; Stephen
et al., 1999; Schmitz-Hoerner and Weissenbock, 2003; Valkama et al., 2003) show
that biomass or photosynthetic pigment content does not change under the exposure
to UV-B radiation or such variation is insignificant. Hakala et al. (2002) determined
sensitivity of various agricultural plant species including barley, wheat, oat, clover,
timothy, fescue and potato to exposure to UV-B radiation (as if ozone layer would be
decreased by 30 %) and found no significant variation of biomass accumulation or
yield. Thus, it was shown that C 4
type of plants are resistant to UV-B radiation.
Stomata formation and permeability is determined by leaf age and other factors
including UV-B radiation intensity (Day, Neale, 2002). In our study, stomata counts
on pea (Pisum sativum L.) leaves remained unchanged only after the first day of
exposure to UV-B radiation. Similar results are presented by Day and Neale (2002)
where stomata counts and permeability were changing if exposure period and intensity
is increasing.
Photosynthesis is very important process in plants as it determines biomass
increase, thus, is a subject for UV-B exposure research. Photosynthesis process is based
on chlorophylls’ system and, if such system is altered by UV-B radiation, biomass
183

increase is hindered; hence, such feature might be used to determine UV-B sensitive
plants. Accordingly, plants, which are able to keep chlorophylls’ system unchanged,
are far more resistant to UV-B radiation (Smith et al., 2000). During our study, amount
of chlorophyll a remained unchanged after the first day of exposure, but significantly
decreased if the exposure period and intensity increased. Amount of chlorophyll b was
more stable, but decreased at the end of experiment. Some authors have stated, that
content of chlorophyll a and carotenoids remains unchanged under the exposure to
UV-B, while amount chlorophyll b decreases (Barsig and Malz, 2000). However, our
study revealed that chlorophyll a was more sensitive to UV-B radiation than chlorophyll
b. Such variation could be based on the injury of thylakoid lumen, where the center of
light harvesting system – chlorophyll a – is being damaged and disintegrates (Rengel
et al., 1989). Decrease of chlorophylls’ a to b ratio under exposure to UV-B radiation
was also shown by other authors (Smith et al. 2000).
Carotenoids called xanthophylls are the main protective agents dissipating excess
energy and protecting photoreaction center from auto-oxidation (Yamamoto, Bassi,
1996). Our study revealed that content of carotenoids remained unchanged during
first three days of exposure, but varied at the fifth day of exposure accordingly to
radiation intensity.
In general, pea growth and biomass accumulation was hindered even after the
first day of exposure and the plant response intensity was adequate to the duration and
intensity of exposure to UV-B radiation. Thus, it could be concluded that pea plants
are very sensitive and hardly adapt to the increased UV-B radiation.
Conclusions. 1. An adverse UV-B effect on pea (Pisum sativum L.) growth was
observed even after the first day of exposure.
2. Decrease of amounts of photosynthetic pigments in pea (Pisum sativum L.)
leaves indicates plant sensitivity to UV-B radiation.
Acknowledgement. The research was funded by Lithuanian State and Studies
Foundation under the priority research project “Complex effect of anthropogenic
climate and environmental changes on vegetation of forests and agro ecosystems”.
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SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
UV-B spinduliuotės poveikis sėjamojo žirnio (Pisum sativum L.)
augimui ir fotosintezės pigmentų sistemai
R. Juozaitytė, A. Ramaškevičienė, A. Sliesaravičius, N. Burbulis,
R. Kuprienė, V. Liakas, A. Blinstrubienė
Santrauka
Darbo tikslas – nustatyti UV-B spinduliuotės poveikį sėjamojo žirnio (Pisum sativum L.)
augimui ir fotosintezės pigmentų sistemai.
Bandymas vykdytas kontroliuojamose sąlygose Lietuvos sodininkystės ir daržininkystės
instituto fitotrone bei Lietuvos žemės ūkio universiteto Agrobiotechnologijos laboratorijoje.
Augalai auginti neutralaus rūgštumo substrate (Ph 6–6,5), 5 L talpos vegetaciniuose induose,
po 25 augalus. Fotoperiodas – 16 val., temperatūra – 21 °C/17 °C (dieną/naktį), šviesos
šaltinis – „SON-T Agro“ (PHILIPS) lempos). UV-B spinduliuotę skleidė UV-B lempos
(TL 40W/12 RS UV-B Medical, Philips). Augalai UV-B spinduliuote švitinti 5 dienas. Tirtos
tokios UV-B spinduliuotės dozės: 0 (kontrolė), 1, 3, 5, 7 ir 9 kJ m -2 per dieną.
Po 1, 3, 5 dienų UV-B spinduliuotės poveikio nustatytas antžeminės dalies aukštis, sausoji
biomasė, žiotelių skaičius, lapų plotas (WinDias matuoklis). Fotosintetiniai pigmentai nustatyti
spektrofotometriniu būdu 100 % acetono ištraukoje pagal Vetšteiną.
Atlikus tyrimą jau po 1 dienos poveikio nustatytas toksiškas UV-B poveikis antžeminės
dalies ilgio, sausos biomasės ir lapų ploto augimui. Didėjant UV-B spinduliuotės dozėms ir
poveikio laikui, nustatytas tendencingas fotosintetinių pigmentų kiekio mažėjimas.
Reikšminiai žodžiai: augimas, fotosintetiniai pigmentai, UV-B spinduliuotė, žirniai.
186

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Ozone influence on photosynthesis pigment system and
growth of Soya (Glycine max (L.) Merr.) under
warming climate conditions
Asta Ramaškevičienė, Rima Juozaitytė, Algirdas Sliesaravičius,
Egidija Venskutonienė, Liuda Žilėnaitė
Lithuanian University of Agriculture, LT-53067 Akademija, Kaunas distr.,
Lithuania, e-mail: astar@vic.lt, astara@delfi.lt
The aim of this work: to estimate ozone influence on Soya in warming climate. Experiments
were performed at the Laboratory Agrobiotechnology of the Lithuanian University of Agriculture
and at phytotron complex of the Lithuanian Institute of Horticulture. Climate conditions were
modelled as follows: current climate – temperature day/night 21/14 °C CO 2
concentration –
350 ppm (700 mg m -3 ), warming climate – temperature day/night 25/16 °C and photoperiod –
14 h. The investigated concentrations of ozone were as follows: 20 ppb (40 µg m -3 ) – control,
40 ppb (80 µg m -3 ) and 80 ppb (160 µg m -3 ) (7 h each day, 5 days per week). The plants were
grown in 5 litre pots in peat substrate (6–6.5 pH), 25 units per pot. Experiments were provided in
3 replications. Before germination and one week after, seedlings were grown in the greenhouse
and then moved to phytotron. At the end of the experiment the aboveground part of Soya shoots
was cut and its length and dry biomass were measured. Photosynthesis pigment content was
determined in green leaves.
In current climate conditions ozone inhibited the length of shoots and biomass of Soya
accumulation. Nevertheless, at warming climate conditions all ozone concentrations stimulated
biomass accumulation and length growing, respectively 50 % and 66 %. Ozone influence on
photosynthetic pigments in leaves of Soya was toxic in both climate variants, but at warming
climate ozone influence was lesser. Ozone influence on carotenoids in the leaves of Soya at
current climate conditions was toxic, the level of this pigment was lower by 18–29 %, but at
warming climate conditions the level of carotenoids were similar to control.
Key words: ozone, Soya, plants, photosynthesis pigments.
Introduction. Recent climate changes, depletion of the stratosphere ozone layer
and the increase of the ultraviolet radiation intensity, acid rains and the rise of ground
level ozone concentration have been considered major anthropogenic processes causing
the negative changes of state, productivity and biological diversity of plants. These
issues are crucially significant since the state of Lithuania as a geographical centre of
Europe is unfavourable from the viewpoint of the far transfers of polluted air and the
processes of regional environment pollution. Moreover, the global climate changes
may also have a severe negative impact on forest and agro-ecosystems vegetation in
our geographical latitude.
Greenhouse gas emissions and their concentrations have risen considerably over
the last 150 years due to human activities. The concentration of carbon dioxide in the
187

atmosphere has increased by 30 % and it is assumed that the percentage will have
doubled by the end of the century. After the amount of CO 2
has doubled, the average
temperature may increase from 2 to 5 °C (Ward, Strain, 1999).
Despite the successful joint international attempts to considerably reduce the
emission of sulphur into the air and sulphur drops on the earth surface (Jasinevičienė,
Šopauskienė, 1999), they still greatly exceed the limits foreseen for natural ecosystems.
The efforts made to control the emergence of nitrogen compounds in the environment
have been by far less successful. Excess of nitrogen causes serious problems of land
and water ecosystems eutrofication. Besides, nitrogen dioxide is the main primary cause
for ground level ozone and it adds to the increase of ground level ozone concentrations
that have been observed over the last decades (Girgždienė, Girgždys, 2003; Fichman,
1991).
65 µg m -3 is suggested by the World Health Organization as a marginal concentration
of ozone which, if exceeded, causes damage to ozone-sensitive plants (Morison,
Lawlor, 1999). In Lithuania such ground level ozone concentration is present over
one third of the vegetation period. Plants take in ozone together with air through leaf
stomata. When ozone resolves into the free radicals and other formations, membranes
of cells experience the effect of fall: their permeability changes, the radicals directly
affect membranes and plasma, flows of information, trans-membranous potential falls
into disarray. Damages at the molecular level inevitably alter the activity of genes,
metabolism, intensity of photosynthesis, and the latter, in turn, influences the whole
plant growth. Most frequently observed damages by ozone are as follows: slowed
down growth, increased sensitivity of plants to diseases and pests, accelerated ageing
processes, spotty, yellowing leaves, the loss of leaves (Agrawal et al., 1993).
It is assumed that yields of cereals in Lithuania have decreased by 5–10 % due
to ozone effect. Loses of wood in Lithuanian forests can also be similar (Ozolinčius,
Stakėnas, 2001). Loses during dry seasons that have been more frequent over the recent
years might even increase and lead to the growing risk of agricultural business. Shortterm
(strong) or recurring impact of ozone alters the composition of plants organic
matter, their mass and translocation.
The main problem when researching the impact of warming climate on plants is
that all the factors (temperature, CO 2
gas, ozone, growing intensity of the ultraviolet
radiation, etc.) have the joint effect on plants the same way as on other living organisms,
therefore, the research of their complex impact and the prediction of further possible
changes of state, productivity and biological diversity of plants are not only vitally
important but also very complicated. The effect of climate changes on crop yield differs
greatly. This depends on plant species, soil characteristics, pests and diseases, direct
impact of CO 2
on plants, correlation between CO 2
and weather temperature, water
regime, mineral nutrients, weather quality and adaptation processes (IPCC, 2001).
Two types of stressors interaction – synergy and cross-adaptation – were
established when researching the interrelation of external factors of the environment.
In the first case external factors reinforce each other’s impact and under the influence
of one stressor plants become more sensitive to the impact of other stressors (Butler
et al., 1999; Das et al., 1997; Sliesaravičius et al., 2002; Duchovskis et al., 2004).
However, in cases when mechanisms of plant adaptation to unfavourable external
188

factors are similar, plats become more resistant to the impact of other stressors if they
have adapted to one unfavourable factor (Larcher, 1995; Alexieva et al., 2003).
Research carried out by diverse authors proves that the increased concentration of
CO 2
in the air partially neutralizes the negative effect of ozone on plants (Fuhrer, 2003).
Despite the fact that the increased concentration of CO 2
reduces the negative effect of
O 3
as the visible damage of leaves is much less, however, it is not eliminated entirely.
The protective effect of CO 2
under the ozone stress is due to the decreased permeability
of stomata because the ability of O 3
to get into the leaves is lessened; moreover, the
impact of oxidation stress caused by O 3
on photosynthesis is also relieved. Actually, it
is assumed that the protection due to the increased quantity of CO 2
is determined by the
restricted ability of ozone to get into the leaves. The increased concentration of CO 2
decreased transpiration in Pinus ponderosa seedlings by 5 %. It has been established
that the flows of ozone into plasmolema are more dependent on the permeability of
stomata rather than the quantity of O 3
detoxicating combinations (Fuhrer, 2003).
Photosynthesis is one of the major physiological processes whose intensity changes
according to the changing conditions of the environment. Pigments are the part of the
system of photosynthesis and their changes can be used when studying the impact of
ozone on plants (Calatayud, Barreno, 2004). The majority of authors indicate that the
increased quantity of CO 2
reduces the negative impact of ozone on the intensity of plant
photosynthesis (Heagle et al., 2000), however, the increased temperature frequently
eliminates the stimulating effect of CO 2
(Fuhrer, 2003).
The research aim was to investigate the changes in resistance of
Soya (Glycine max (L.) Merr.) under warming climate conditions and increasing
concentration of ground level ozone.
Object, methods and conditions. The research was carried out at the Laboratory
of Agro-biotechnologies of the Faculty of Agronomy of the Lithuanian University
of Agriculture and the Phytotron of the Lithuanian Institute of Horticulture in
2005–2006 using the ‘Progress’ species of Soya (Glycine max (L.) Merill.). The
plants were grown in five-litre vegetative pots in turf substratum of neutral acidity
(pH 6–6.5). Each experiment was repeated three times each time growing 25 plants.
Plants were kept in the greenhouse until germination and one week after, and
then transferred to the phytotron. The photoperiod lasted for 14 hours. The plants
acclimatized for two days before they were exposed to the treatment. Three
variants of ozone concentration were researched: 40 µg m -3 – control; 80 µg m -3 ;
160 µg m -3 . Present climate conditions: day/night temperature – 21/14 °C; CO 2
concentration – 700 mg m -3 . Conditions of warming climate: day/night temperature –
25/16 –C; CO 2
concentration – 1 400 mg m -3 . Ozone was generated using the generator
OSR-8 (Ozone Solutions, Inc.) 7 hours a day 5 days a week. The duration of exposition
in phytocameras was 9 days. Ozone concentration was measured by the portable ozone
monitor OMC-1108 (Ozone Solutions, Inc.). The measurements were accomplished as
follows: the height of each plant was measured before the treatment; at the end of the
experiment the height of the plant was measured, the above-ground dry biomass and the
quantity of photosynthetic pigments were established. The pigments of photosynthesis
in green leaves were established by spectro-photometric method in 100 % extract
according to Wetstein (Wettstein, 1957).
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Statistical indicators of the research data were measured by the method of
dispersion analysis. Statistical reliability of the experiment data was evaluated after
measuring the standard square bias and the lowest absolute margin of the essential
difference (R 05
). Statistical programs “MINITAB” ANOVA single-factorial statistical
analysis was employed for the statistical assessment of the data.
Results. Having researched the effect of ozone gas under present climate
conditions it has been established that the growth of the above-ground part was
more suppressed by the extensively studied concentration of O 3
. Height increase
in this variant was 16 % less compared to the control (Fig. 1). After analyzing the
dry mass of the above-ground part of Soya grown in cameras with different ozone
concentration it has been established that both studied concentrations of ozone (80 µg
m -3 and 160 µg m -3 ) suppressed the accumulation of dry mass (Fig. 1). Depending on
the concentration of O 3
the dry mass was 8 and 7 % less in comparison to the control
plants. The differences compared to the control were essential.
Fig. 1. The impact of ozone on the height and dry biomass of
Soya above-ground part under present climate conditions
1 pav. Ozono poveikis gauruotosios sojos antžeminės dalies aukščiui
bei sausai biomasei dabartinio klimato sąlygomis
Having accomplished the experiment of ozone impact on Soya under the conditions
of warming climate and having analyzed the results, quite different tendencies were
observed with reference to the experiment under present climate conditions. The growth
of the above-ground part of Soya was stimulated in the studied concentrations of ozone
under greater average day temperature and double concentration of CO 2
gas (Fig. 2).
The increase of the above-ground part height of Soya that grew under the influence
of 80 and 160 µg m -3 ozone concentrations was larger by 35 and 9 % respectively
compared to the impact of 40 µg m -3 ozone concentration. The increase of the aboveground
part height of Soya under the impact of 80 µg m -3 ozone concentration was
essential compared to the control. Analogous results were obtained after analyzing
the data of Soya above-ground part dry mass (Fig. 2). 80 µg m -3 ozone concentration
mostly stimulated the dry mass accumulation: the dry Soya mass was 1.5 times greater
compared to the treatment with 40 µg m -3 ozone concentration. The greatest studied
ozone concentration had a slightly less stimulation on the accumulation of the above-
190

ground part dry mass. The mass of these plants was 40 % greater compared to the
treatment of 40 µg m -3 ozone concentration. However, differences compared to the
control were essential.
Fig. 2. The impact of ozone on the height and dry biomass of Soya
above-ground part under warming climate conditions
2 pav. Ozono poveikis gauruotosios sojos antžeminės dalies aukščiui
bei sausai biomasei atšilusio klimato sąlygomis
Having analyzed the effect of diverse ozone concentrations under present climate
conditions on the synthesis of Soya photosynthetic pigments it has been established that
the O 3
gas concentrations greatly suppressed the synthesis of chlorophyll a (Fig. 3A).
The amount of chlorophyll a in the leaves of Soya grown under the influence of 80
and 160 mg m -3 ozone concentrations was less by 26 and 40 % respectively compared
to the control. Differences in data were essential. Having analyzed the amount of
chlorophyll a in Soya grown in different ozone concentrations under warming climate
conditions the decrease in the amount of chlorophyll (25–9 %) has also been observed,
however, essential differences have been detected only in the variant with 80 mg m -3
ozone concentration.
Ozone gas also considerably suppressed the synthesis of chlorophyll b under
present climate conditions (Fig. 3B). Under the influence of the two studied ozone
concentrations the amount of chlorophyll b in the leaves of Soya was less by 31 and
38 % respectively compared to the control plants. The results under warming climate
conditions were similar to those of chlorophyll a; the synthesis of chlorophyll b was
more suppressed in cases of Soya grown under the treatment of 80 mg m -3 ozone
concentration. The amount of chlorophyll b in the leaves of Soya in this ozone
concentration decreased by 21% compared to the 40 мg m -3 concentration.
The changes of chlorophyll a and b ratio in the leaves of the studied plants under
the conditions of changing ozone concentration were considerably less evident than
the chlorophyll a and b amount changes (Fig. 3A, B, D).
191

Fig. 3. The effect of ozone on the photosynthetic pigments of Soya:
A – chlorophyll a, B –chlorophyll b, C – carotenoids, D – chlorophylls a/b
3 pav. Ozono poveikis gauruotosios sojos fotosintetiniams pigmentams:
A – chlorofilas a, B – chlorofilas b, C – karotinoidai, D – chlorofilai a/b
The ratio of chlorophyll a and b of Soya treated by 80 µg m -3 ozone concentration
under present climate conditions increased by nearly 8 %, whereas in the variant
with 160 µg m -3 concentration this ratio was just slightly less than that of the control
plants. However, the above said differences are not statistically reliable. The studied
ozone concentrations did not have substantial influence on the ratio of chlorophyll a
and b under warming climate conditions. The ratio of chlorophyll in all variants of
the experiment was similar, that is, it approximated 2.6. This shows that the increased
CO 2
gas concentration and higher temperature were the factors reducing stress for
Soya caused by ozone gas.
Ozone gas under present climate conditions intensely suppressed carotenoid
synthesis in the leaves of Soya (Fig. 3C). The quantity of carotenoids in the leaves
of Soya treated by 80 and 160 µg m -3 ozone concentrations was less by 18 and 29 %
respectively compared to the control. The decrease in the quantity of carotenoids
proves that plants are unable to resist the effect of ozone and experience extreme stress.
However, it has been established that under warming climate conditions there have
been no significant differences compared to the control. Stable retention of carotenoid
quantity in leaves shows that the system of resistance to stress is fairly strong.
Discussion. When ozone gets into the leaves of a plant, its above-ground part, it
has a toxic impact on the whole plant. After the photosynthesis in leaves is disrupted,
the plant uses energy and organic compounds to eliminate the outcomes of leaf cells
damage and relieve stress, thus, their dislocation into the leaves is disturbed. It is
192

evident from the comparison of the results of the two experiments using ozone that
both studied ozone gas concentrations under present climate conditions suppressed the
growth of Soya seedlings. Suppression was greater at 160 µg m -3 ozone concentration
treatment. The stage of plant sensitivity when it is possible to observe external changes
(dots, spots or crosses (usually white)) on the leaves is different. The most sensitive
tobacco-plant of Bel-W3 species reacts after 1 hour in 40 ppb ozone (Saitanis et al.,
2001). Other sensitive plants have a similar reaction only under approximately 100 ppb
ozone concentration. Our experiments under warming climate conditions have not
established toxic impact of ozone on the growth of Soya. Though the increasing ozone
concentration under present climate conditions suppressed the growth of the aboveground
part height of Soya, however, such growth was stimulated under warming
climate conditions. Having compared the results of dry mass they were also analogous,
however differences were even more distinct.
Literary sources maintain that if plants are grown in the usual CO 2
concentration
but higher temperature, the total amount of assimilated carbon reduces (Fuhrer, 2003).
Thus, if the rise in temperature results in the increase of CO 2
concentration, the intensity
of photosynthesis also enhances (Owensby et al., 1993). According to Kimball et al.
(2002) experiments, the increased amount of CO 2
stimulated the accumulation of C 3
grass biomass by 12 %, the yield of wheat and rice was 10–15 % bigger and the yield
of potatoes increased by 28 % compared to the usual CO 2
concentration. However
for C 4
plants the yield supplement due to the increased CO 2
concentration seems to
be considerably less. Positive effect of greater CO 2
concentration in the air has also
been observed in the experiment when 45-day-old sugar-maple seedlings were treated
by O 3
. Under high O 3
concentration together with the present CO 2
concentration the
speed of assimilation and the amount of Rubisco decreased, and the total biomass
declined by 45 %. It has also been detected that glucose 6-phosphate dehydrogenase
(G6PDH) and activity of anaplerotic CO 2
fixation by phosphoenolpyruvate carboxylase
(PEPC) increased in these seedlings. This evidences that mechanisms of detoxication
and oxidative damage amendment became more active. Therefore under high O 3
concentration together with the higher CO 2
concentration the total biomass decreased
by only 21 %, and G6PDH and PEPC stimulation was much more evident than in
case of high O 3
and the present CO 2
concentration (Gaucher et al., 2003; Fumagalli
et al., 2001).
The quantity of photosynthetic pigments, their proportion in Soya leaves is a
significant index for the mechanism of photosynthesis. According to literary sources,
the amount of chlorophyll a in the leaves of trees has a positive correlation with the
effectiveness of reaction centers of photosystems, and the amount of chlorophyll a with
the content and size of the photosystem II complexes (Кершанская, 2000). Having
analyzed the impact of ozone under present climate conditions the decrease in the
quantity of photosynthetic pigments – chlorophylls a and b and carotenoids that was
analogous to morphometric parameters was observed. However, under warming climate
conditions the toxic effect of ozone was much less, though stimulating effects have not
been detected. This evidences that the photosynthetic system of a plant is much more
sensitive than the height and mass of the above-ground part. It can be hypothesized that
if the impact of ozone was prolonged, both height and mass would reduce. The duration
193

of ozone impact is very significant for plant photosynthesis (Adedipe et al., 1973). This
is important to investigate due to the fact that concentration of ozone in nature is very
dynamic: it depends on daytime, wind direction, traffic intensity and work of factories.
Literary data shows that long-term ozone concentration gas, though 5–6 times less, has
the same effect on photosynthesis as short-term high concentration fumigation It has been
established that the stage of plant photosynthesis suppression approximates to 100 ppb
an hour, however, in some cases the process of photosynthesis is suppressed by only
200–500 ppb ozone concentration gas. In case of long-term ozone treatment (longer
than one day) the stage is much less, it only reaches 35–45 ppb (Lin et al., 2001).
Under warming climate conditions only slight changes in the quantity of
carotenoids upon ozone treatment were detected, it was close to the control. The main
function of carotenoids is the protection of photosystems from the photooxidative
damage. Xantophyll cycle carotenoids play the main role in photo-protection by
dissolving the excess of awakening energy (Fuhrer, Bodur, 2003). Thus, the longer
the ability of a plant to sustain a stable or slightly greater number of carotenoids, the
more it is resistant to the stressor.
Conclusions. Under present climate conditions ozone gas suppressed the growth
of Soya (Glycine max (L.) Merill.), however, under warming climate conditions ozone
stimulated the growth of Soya.
Ozone impact on the quantity of photosynthetic pigments in the leaves of Soya
under present and warming climate conditions was negative; however, ozone impact
under warming climate conditions was less toxic.
Soya has the potential to adapt to the studied warming climate conditions (CO 2
concentration is 1 400 mg m -3 , day/night temperature is 25/16 °C) even if the ground
level ozone concentration increases up to 160 mg m -3 .
Acknowledgment. Research was supported by Lithuanian State Science and
Studies foundation.
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Pažemio ozono įtaka sojos fotosintetiniams pigmentams bei
augimui (Glicine max (L.) Merr.) рylančio klimato sąlygomis
A. Ramaškevičienė, R. Juozaitytė, A. Sliesaravičius, E. Venskutonienė,
L. Žilėnaitė
Santrauka
Bandymai vykdyti LSDI fitokamerų komplekse ir LŽŪU AF agrobiotechnologijos
laboratorijoje. Darbo tikslas – išsiaiškinti gauruotosios sojos (Glycine max (L.) Merr.) atsparumo
pokyčius šylant klimatui bei didėjant pažemio ozono koncentracijai. Tikslui pasiekti buvo
numatyti šie uždaviniai: 1) nustatyti priežemio ozono skirtingų koncentracijų įtaką sojų daigų
augimui dabartinio bei pakitusio klimato sąlygomis; 2) ištirti ozono bei CO 2
dujų poveikį sojų
fotosintetinių pigmentų gamybai dabartinio bei pakitusio klimato sąlygomis. Tiriant ozono
poveikį gauruotajai sojai (Glycine max (L.) Merr.) dabartinio klimato sąlygomis temperatūra
dieną/naktį buvo 21/14 °C, atšilusio klimato sąlygomis temperatūra buvo 25/16 °C. Fotoperiodo
trukmė – 14 val. Tirti trys ozono koncentracijos ore variantai: 20 ppb (40 µg m -3 ) – kontrolė,
196

40 ppb µg m -3 ) ir 80 ppb (160 µg m - 3 ). Ozonas buvo generuojamas 7 val. per dieną, 5 dienas
per savaitę. Ekspozicijos fitokamerose trukmė – 9 dienos. Augalai buvo auginami neutralaus
rūgštumo substrate (6–6.5 pH) po 25 vnt. 5 litrų vegetaciniuose induose trimis pakartojimais.
Iki sudygimo ir vieną savaitę sudygus soja buvo auginama šiltnamyje, o po to perkelta į
fitokameras. Bandymų pabaigoje buvo nustatyta sojos antžeminės dalies aukštis, sausa biomasė
ir fotosintetinių pigmentų kiekis.
Dabartinio klimato sąlygomis ozono dujos slopino sojų antžeminės dalies ilgio ir sausos
biomasė augimą. Atšilusio klimato sąlygomis gauruotosios sojos augimas ir sausos biomasės
kaupimas buvo stimuliuojamas, priklausomai nuo ozono koncentracijos, atitinkamai 50 % ir
66 %. Išanalizavus ozono poveikį fotosintetiniams pigmentams, nustatyta, kad chlorofilo kiekis
sojų lapuose dabartinio klimato sąlygomis tendencingai mažėjo, didėjant priežemio ozono
koncentracijoms. Tačiau atšilusio klimato sąlygomis, chlorofilo kiekis, palyginti su kontrole,
sumažėjo nežymiai. Karotinoidų kiekis sojų lapuose, veikiant ozonui, dabartinio klimato
sąlygomis mažėjo18–29 %, o atšilusio klimato sąlygomis išliko panašus į kontrolinių augalų.
Reikšminiai žodžiai: ozonas, soja, augalai, fotosintetiniai pigmentai.
197

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Complex influence of different humidity and
temperature regime on pea photosynthetic indices in
VI–VII organogenesis stages
Sandra Sakalauskienė 1 , Gintarė Šabajevienė 1 ,
Sigitas Lazauskas 3 , Aušra Brazaitytė 1 , Giedrė Samuolienė 1, 2 ,
Akvilė Urbonavičiūtė 1, 2 , Jurga Sakalauskaitė 1 ,
Raimonda Ulinskaitė 1 , Pavelas Duchovskis 1, 2
1
Lithuanian Institute of Horticulture, Kauno 30, 54333 Babtai, Kaunas distr.,
Lithuania, e-mail: s.sakalauskiene@lsdi.lt
2
Lithuanian University of Agriculture, Studentų g. 11, LT-53361, Akademija,
Kaunas distr., Lithuania
3
Lithuanian Institute of Agriculture, LT-58343 Akademija, Kėdainiai distr.,
Lithuania, e-mail: sigislaz@lzi.lt
The aim of the study was to investigate the influence of different humidity and temperature
regime on pea photosynthetic indices. Vegetative experiments were carried out in the phytotronic
complex of Plant Physiology Laboratory at the Lithuanian Institute of Horticulture in 2007.
There was investigated pea cultivar ‘Pinochio’ (Pisum sativum L.). Peas were grown under
conditions of different temperature (21/16 °C and 30/23 °C day/night) and humidity (40–45 %
and < 10 % normal/dryish) regime. Different combinations of temperature and humidity regime
significantly influenced plant physiological processes. There was established the smallest clear
photosynthesis productivity, relational growth speed, fresh and dry weight of the pea, which
grew in dry substratum at the temperature of 30 °C. High temperature and the lack of humidity
also inhibited the synthesis of chlorophylls a and b. At the temperature of 30 °C under both
humidity regimes the accumulation of carotenoids in pea became more intensive.
Key words: lack of humidity, photosynthetic pigments, photosynthesis indices,
temperature.
Introduction. More and more attention to the influence of climatic changes on
plants is paid in the world (Bray et al., 2000; Fuhrer, 2003). Plant injures and the
decrease of their productivity most often depends on unfavourable factors, which exist
naturally in the environment. Plant physiological processes are directly connected with
the regime of temperature and humidity and their change. These factors are among the
most important ones – the plant growth and development processes and productivity
depends on them (Alexieva et al., 2003). Higher temperature distinguishes itself
with stress effect. Temperature, which is even a few degrees higher than the usual
one, influences the function of many ferments and causes protein expression of heat
stress (Jenkins et al., 1997). The ratio of photosynthesis and respiration determine
plant growth. When temperature increases, the intensity of respiration increases also,
199

therefore energetic plant resources are lost (Kirnak et al., 2001). Higher temperature
quickens plant growth and development, shortens the duration of different development
stages, therefore the final biomass production may decrease. The first and the most
sensitive plant response to water deficit is the decrease of turgor and slowing down of
growth processes (Kirnak et al., 2001). The lack of water worsens carbon assimilation,
which depends on opening and shutting of jaws. Moreover, the lack of water and high
temperature can stimulate the formation of free radicals and active oxygen derivatives,
which disturb the processes of plant metabolism (Alexieva et al., 2003; Chaves et al.,
2003). In order to support the growth and productivity, plant must adjust itself to
stress conditions and develop the specific tolerance mechanisms (Wang et al., 2003).
Plant reaction to the changes of temperature and humidity depends on plant type,
cultivar, genetic properties, age and the level of development (Yamaguchi-Shinozaki
et al., 2002). Plants of the different level of development react on the stress factors
differently (Lawlor, 2002; Lawlor and Cornic, 2002). It was observed that plants,
which adjusted to one stressor, became more resistant to the complex influence of
stressors also (Duchovskis et al., 2003). Nevertheless, the complex influence of the
limited duration change of temperature and humidity regime on the productivity of
agricultural plants is investigated insufficiently.
Aim of investigation – to analyze the influence of the complex humidity and
temperature regime on pea photosynthetic indices in VI–VII organogenesis stages.
Object, methods and conditions. Vegetative experiments of the complex
influence of humidity and temperature regime were carried out in the phytotronic
complex of Plant Physiology Laboratory at the Lithuanian Institute of Horticulture.
There was investigated pea cultivar ‘Pinochio’ (Pisum sativum L.). Plants were
grown in vegetative pots of 5 l. Substratum was prepared from peat of neutral acidity
(6–6.5 pH) and sand (3 : 1). There were grown twenty pea plants per each vegetative
pot. From germination up to VI–VII organogenesis stage plants were grown in the
greenhouse, and then transferred to phytocameras, where 16 h photoperiod was created
and for the illumination Son-T-Agro (PHILIPS) lamps were used. Organogenesis stages
were established according to F. Kuperman (Kуперман et al., 1982). Four variants of
experiment were carried out in five replications each. Under two temperature regimes
(21/16 °C and 30/23 °C day/night) there was investigated the effect of humidity and
drought (< 10 %) of normal (40–45 %) substratum. Humidity of substratum was
measured with Delta-T Devices soil humidity measurer HH2. Duration of the effect
was 10 days. Right away after the effect plants were transferred back to the greenhouse,
where uniform conditions to all plants were created and their regeneration was
observed for 7 days. During investigations, biometrical measurements were carried
out. The amount of photosynthesis pigments in fresh weight was established preparing
100 % acetone extracts and analyzing them by spectrophotometrical Wettstein’s
method (Гавриленко et al., 2003). There was used spectrophotometer Genesys 6
(ThermoSpectronic, JAV).
Assimilation area was measured with leaf area measurer CI-202 (CID Inc., USA).
Plant dry weight was established drying at the temperature of 105 °C.
Pure photosynthesis productivity (F pr
) was calculated according to the formula:
F pr
= 2(M 2
- M 1
) / (L 1
+ L 2
)T (1)
200

Here (M 2
- M 1
) – increase of dry weight during certain period;
L 1
and L 2
– leaf area at the beginning and at the end of the period;
T – duration of period in 24 h (Bluzmanas et al., 1991).
Relational speed of growth (R) was calculated according to the formula:
R = W 2
- W 1
/ t 2
- t 1
(2)
Here W 1
and W 2
– dry weight at the beginning and at the end of the period;
t 1
and t 2
– the beginning and the end of the period in 24 h (Coombs et al.,
1985).
Standard deviation of the investigation data average was calculated using program
MS Excel.
Results. Different combinations of temperature and humidity regime significantly
influenced pea photosynthesis system (Fig. 1). The least amount of chlorophyll a was
established in pea, grown in dry substratum at the temperature of 30 °C.
Fig. 1. Photosynthetic pigment content in different temperature and humidity
combinations. N – substratum of normal humidity (40–45 %), S – dryish substratum
(< 10 %). A – chlorophyll a; b – chlorophyll b; c – carotenoids.
1 pav. Žirnių fotosintezės pigmentų kiekis skirtinguose temperatūros ir drėgmės režimo
deriniuose. N – normalaus drėgnumo substratas (40–45 %); S – sausokas (< 10 %).
A – chlorofilas a; b – chlorofilas b; c – karotinoidai.
The smallest amount of chlorophyll b accumulated pea, which suffered the lack of
humidity under both temperature regimes. The biggest amount of carotenoids was
established in pea grown at the temperature of 30 °C. After 7 days of regeneration
period the amount of chlorophylls a, b and carotenoids strongly decreased. The smallest
amount of photosynthetic pigments was in pea, which had suffered humidity lack and
201

high temperature of 30 °C (Fig. 1). Both the lack of humidity and high temperature
negatively influenced pea assimilation area, fresh and dry weight (Fig. 2, 3, 4). After
10 days of humidity and high temperature of 30°C there was established the smallest
pea assimilation area, fresh and dry weight. After 7 days of regeneration period in all
the combinations of temperature and humidity regime, assimilation area, fresh and
dry weight strongly increased (Fig. 2, 3, 4).
Fig. 2. Pea assimilation area in different temperature and humidity combinations.
N – substratum of normal humidity (40–45 %),
S – dryish substratum (< 10 %).
2 pav. Žirnių asimiliacinis plotas skirtinguose temperatūros ir drėgmės režimo deriniuose.
N – normalaus drėgnumo substratas (40–45 %);
S – sausokas (< 10 %).
Fig. 3. Pea fresh weight in different temperature and humidity combinations.
N – substratum of normal humidity (40–45 %),
S – dryish substratum (< 10 %).
3 pav. Žirnių žalioji masė skirtinguose temperatūros ir drėgmės režimo deriniuose.
N – normalaus drėgnumo substratas (40–45 %);
S – sausokas (< 10 %).
202

Fig. 4. Pea dry weight in different temperature and humidity combinations.
N – substratum of normal humidity (40–45 %),
S – dryish substratum (< 10 %).
4 pav. Žirnių sausoji masė skirtinguose temperatūros ir drėgmės režimo deriniuose.
N – normalaus drėgnumo substratas (40–45 %);
S – sausokas (< 10 %).
Independently on substratum humidity after 10 days of effect, peas, which were
grown at the temperature of 30 °C, produced essentially smaller above-ground part
(Fig. 5). After regeneration period pea height almost didn’t change (Fig. 5).
Fig. 5. The height of pea above-ground part in different temperature
and humidity combinations. N – substratum of normal humidity (40–45 %),
S – dryish substratum (< 10 %).
5 pav. Žirnių antžeminės dalies aukštis skirtinguose temperatūros ir
drėgmės režimo deriniuose. N – normalaus drėgnumo substratas (40–45 %);
S – sausokas (< 10 %).
Temperature and humidity regime influenced pea pure photosynthesis productivity
and relational growth speed (Fig. 6). After 10 days of effect the biggest pure
photosynthesis productivity and relational growth speed was of the pea, which grew
under optimal conditions (at the air temperature of 21 °C and substratum humidity
203

40–45 %). The smallest pure photosynthesis productivity and relational growth speed
was established in pea, which suffered the lack of humidity and high temperature of
30 °C (Fig. 6).
Fig. 6. Pea photosynthesis productivity (a) and relational growth speed
(b) after 10 days effect in different temperature and humidity combinations.
N – substratum of normal humidity (40–45 %),
S – dryish substratum (< 10 %).
6 pav. Žirnių fotosintezės produktyvumas (a) ir santykinis augimo greitis
(b) po 10 dienų poveikio skirtinguose temperatūros ir drėgmės režimo deriniuose.
N – normalaus drėgnumo substratas (40–45 %); S – sausokas (< 10 %).
Discussion. The effect of biotic and abiotic factors significantly influences
plant physiological processes. Some of them are trophic, causes plant nutrition,
others have side effect, which often is harmful. When factors are unfavourable or
the level of trophic factors unsuitable, this causes unusual reaction in plants – stress.
Stress determines changes in the normal plant physiological processes. Draught and
extreme temperatures are among environmental factors, which most often inhibit
plant productivity (Chaves et al., 2003; 2004; Flexas et al., 2004). Photosynthesis
is one of the main physiological processes, which determine plant productivity. The
efficient work of photosynthesis apparatus is guaranteed only by optimal amount and
ratio of photosynthetic pigments. Our results show that, when pea suffered the lack
of humidity, the amount of chlorophylls a, b significantly decreased and at the high
temperature the negative influence revealed itself even more. At the high temperature
the amount of carotenoids very increased. Carotenoids participate as antioxidants,
which preserve plant cells from oxidation stress (Alexieva et al., 2003), therefore it is
possible to think that pea developed defence mechanisms against draught and stress,
caused by high temperature. Plants have unique mechanisms to react to the continually
changing environmental conditions: they feel the environment and accommodate their
physiological and metabolic processes for the preserving of homeostasis. Reaction
to stressors is determined by plant genome and the interaction of the changed
environmental conditions (Pastori, Foyer, 2002).
One of the most important photosynthesis indices is pure photosynthesis
productivity. It is expressed by dry matter amount, which is produced during 24 h
by plant per leaf assimilation area unit (Bluzmanas et al., 1991). Pure photosynthesis
productivity best of all reflects the effect of environmental factors on plant growth
204

and development (Ничипорович, 1987; Третьяков, 1998). The obtained results of the
investigations showed that the lack of humidity and high temperature decreased pure
photosynthesis productivity and relational speed of growth. This means that plant for
the preserving biological vitality used more energy and assimilates than created them
during photosynthesis process.
Many authors indicate, that stress caused by draught negatively influence the
activity of assimilation apparatus, accumulation of dry matter, violate metabolism
(Kage et al., 2004; Grzesiak et al., 1999).
One of the elements of productivity is to keep high photosynthetic potential. Our
investigations showed that pea sensitively reacted on the stress caused by draught
and high temperature. The lack of humidity inhibited the accumulation of fresh and
dry weight and the growth of assimilation area. Peas under the optimal conditions
regenerated themselves.
Conclusions. 1. Pea reaction to the stress of the lack of humidity and high
temperature manifested itself in the decrease of chlorophylls a and b synthesis and
increase of carotenoids.
2. The lack of humidity at the temperature 30 °C decreased pea photosynthesis
productivity, relational growth speed and dry weight.
3. High temperature of 30 °C inhibited the growth of the above-ground part.
Acknowledgement. Authors are grateful to Lithuanian State Fund of Science and
Studies and Lithuanian Agricultural Ministry for financial support to this project.
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8. Flexas J., Bota J., Cifre J., Escalona J. M., Galmes J., Gulias J., Lefi E., Martinez-
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206

SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Skirtingo drėgmės ir temperatūros režimo kompleksinis poveikis
žirnių fotosintetiniams rodikliams VI–VII organogenezės etapuose
S. Sakalauskienė, G. Šabajevienė, S. Lazauskas, A. Brazaitytė,
G. Samuolienė, A. Urbonavičiūtė, J. Sakalauskaitė, R. Ulinskaitė,
P. Duchovskis
Santrauka
Darbo tikslas – ištirti kompleksinį temperatūros ir drėgmės režimo poveikį žirnių
fotosintetiniams rodikliams. Vegetaciniai bandymai atlikti 2007 metais Lietuvos sodininkystės
ir daržininkystės instituto Augalų fiziologijos laboratorijos fitotroniniame komplekse. Tirtas
sėjamasis žirnis (Pisum sativum L.) ‘Pinochio’. Žirniai auginti skirtingos temperatūros (21/16 °C
ir 30/23 °C dieną/naktį) ir drėgmės (40–45 % ir < 10 % normalus/sausokas) režimo sąlygomis.
Skirtingi temperatūros ir drėgmės režimo deriniai turėjo reikšmingos įtakos augalų fiziologiniams
procesams. Žirnių, augusių sausame substrate, esant 30 °C temperatūrai, grynasis fotosintezės
produktyvumas, santykinis augimo greitis, žalioji ir sausoji masė buvo mažiausi. Aukšta
temperatūra ir drėgmės deficitas taip pat slopino chlorofilų a ir b sintezę. 30 °C temperatūroje,
esant abiem drėgmės režimams, žirniuose suintensyvėjo karotinoidų kaupimasis.
Reikšminiai žodžiai: drėgmės deficitas, fotosintetiniai pigmentai, fotosintezės rodikliai,
temperatūra.
207

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Changes in the activity of antioxidant enzyme
superoxide dismutase in Crepis capillaris plants
after the impact of UV-B and ozone
Regina Vyšniauskienė, Vida Rančelienė
Institute of Botany, Žaliųjų ežerų str. 49, Vilnius LT-08406, Lithuania
E-mail: regina.vysniauskiene@botanika.lt
The aim of the work was to determine the changes of antioxidant enzyme superoxide
dismutase (SOD) activity in model plant Crepis capillaris after the impact of UV-B and ozone.
Comparison of the SOD activity in Crepis capillaris leaves after the effect of small adaptational
doses of UV-B and ozone with the control revealed that after UV-B (3 kJm -2 ) the SOD activity
increases 1.4 times and after the impact of ozone the SOD increases by 1.94 times. After larger
UV-B (9 kJm -2 ) doses the SOD activity increases even 2 times. Still three times higher ozone
dose already inhibits the SOD activity but does not reach the level of the control.
Investigations of the impact of adaptation on repeated impact of UV-B and ozone showed
that SOD activity to Crepis capillaris plants is similar and increases by 1.74 and 1.98 times
comparing with the control. Even when plants are adapted to one factor and influenced by the
other (cross adaptation), the SOD activity remains similar. The research showed that adaptation
by small UV-B and ozone doses to one and the other factor influences the increase of SOD activity
in plants. The increased SOD activity after UV-B irradiation and ozone should be considered
as adaptational response of a plant to oxidative stress caused by unfavourable factors.
Key words: Crepis capillaris, ozone, UVB, adaptation, superoxide dismutase,
EC 1.15. 1.1.
Introduction. Unfavourable environmental factors, like UVB and ozone, are
usually related with each other. Incident UVB radiation (in particular, the waveband
297–310 nm) is increasing due to the reduction in the stratospheric ozone concentration
(Caldwell et al., 1989). Environmental stress factors are known to cause oxidative
stress. Stress caused by these abiotic factors induces the excess of free radicals. Part
of free radicals cause changes in macromolecules, such as DNA and proteins. The
other part acts as a signal triggering the protective antioxidant mechanisms of plants
(Ballarй, 2003). Enzyme system is one of such mechanisms. Such antioxidative
enzymes as superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) detoxify
the excess of free radicals (Mazza et al., 1999). Selection of test-plants characterizing
the contamination is essential while investigating the impact of natural factors upon
ambient environment. Harmful factors are usually investigated separately, and complex
mechanism of the impact of both factors is still under investigated.
Aim of the work was to determine changes in the activity of antioxidative enzyme
superoxide dismutase (SOD) in leaves of Crepis capillaris after complex impact of
209

UVB and ozone and to answer the question whether enzyme of antioxidative stress
SOD participates in protective mechanisms of plant.
Object, methods and conditions. Crepis capillaris (L.) Wallr. plants grown for 2
weeks in a phytotron at the Laboratory of Cell Engineering of the Institute of Botany
and later transferred into the phytotron of the Lithuanian Institute of Horticulture
were used. Growth regime – 16/8 hour photoperiod; 21/16 °C day/night temperature.
The plants were divided into groups of unequal size: control (K) – unaffected plants;
plants affected by ozone or UVB, i.e. affected for 5 days by low adaptive doses: or
3 kJ m -2 d -1 (UVB), or 120 µg m -3 ozone (O 3
), respectively. During the second stage of
the research the first portion of plants was divided into 3 groups: control – unaffected
plants (K + K) affected plants by threefold dose (without adaptation): UVB (9 kJ m -2 d -1 )
or ozone (360 µg m -3 ), marked (K + UV) and (K + O 3
), respectively. Second group of
plants adapted by UVB or ozone was also affected by the same threefold doses only in
various combinations: combining the first impact with the second one: UVB + UVB;
O 3
+ O 3
; UVB + O 3
; O 3
+ UVB. During the second stage of the research exposition
to UVB or ozone was 7 days.
Activity of superoxide dismutase (SOD, EC 1.15. 1.1). Leaf material (1g) was
grounded with extraction buffer (2 ml), consisting of 1mM EDTA, 0.1 % Triton
X-100 in 0.05M Na-K phosphate buffer pH 7.8, with mortar and pestle at 4 °C. The
homogenates were centrifuged for 15 min at 12,000 Ч g (4 °C), and supernatants were
used as crude extract for soluble protein quantification according to Bradford (1976)
with bovine serum albumin as the standard.
Total SOD activity was assayed by the inhibition of the photochemical reduction
of nitro blue tetrazolium (NBT) according to modified method of Beyer and Fridovich
(1987). The reaction mixture (2.2 ml) consisted of 50 mM (Na-K) phosphate (pH 7.8)
and 20 µl of leaves extracts. Each extract was assayed twice with three replications
and measured by spectrophotometer at 560 nm.
The statistical data analysis was carried out by packet of statistical analysis tools
of MS Exel 2002 (Microsoft Corporation) program.
Results. It has been determined that in the course of adaptation, after 5 days
treatment (i. e. first measurement) by low UVB (3 kJ m -2 d -1 ) or ozone (120 µg m -3 )
doses the concentration of soluble proteins decreased comparing with the control (K).
The decrease was particularly significant after ozone treatment (table).
Table. Protein content changes after UVB and ozone treatment in leaves of Crepis
capillaris plants. Protein, mg g -1 F. w.; F. w. – fresh weight.
Lentelė. Baltymo kiekio pokyčiai po UV ir ozono poveikių Crepis capillaris augalų
lapuose, mg g -1
210

After of 7 days direct treatment of plants with threefold UVB (9 kJm -2 ) and ozone
(360 µg m -3 ) doses increased the content of proteins in comparison with the control
(K + K) (i. e. second measurement). It is rather difficult to compare these data because
control plants differ by their age. Differences between the controls K and K + K
could be predetermined by the fact that in leaves of control (K + K) plants, which are
older, than in the control (K). Investigation of the impact of adaptation upon repeated
treatment with UVB and ozone (table) revealed no significant impact of adaptation
with UVB or ozone on the protein content.
However, comparison of the SOD activity in Crepis capillaris leaves after the
impact of small adaptation doses of UVB and ozone with the control revealed that
after UVB treatment (3 kJm -2 ) the SOD activity increases to 1.4 times and after the
impact of ozone the SOD increases to 1.94 times. After higher UVB (9 kJ m -1 ) dose
SOD activity doubles, but after threefold O 3
dose SOD activity is lower but does not
reach the level of the control.
Fig. Complex action of UVB and ozone on SOD activity in Crepis capillaris
plants adaptation. Exposition of 5 days in 3 kJ m -2 d -1 UVB or 120 µg m -3 O 3
environment. Treatment without adaptation – exposition of 7 days in 9 kJ m -2 d -1
to UV or 360 µg m -3 O 3
; K and K + K – control plants, grown in the same
conditions without any treatment.
Pav. Kompleksinis UVB ir ozono poveikis Crepis capillaris SOD aktyvumui.
Adaptacija. 5 dienų ekspozicija 3 kJ m -2 d -1 UVB ar 120 µg m -3 ozono aplinkoje. Poveikis be
adaptacijos – septynių dienų ekspozicija 9 kJ m -2 d -1 UV ar 360 µg m -3 O 3
aplinkoje;
K ir K + K – kontroliniai augalai, auginti tomis pačiomis sąlygomis be poveikių.
Investigations of the influence of adaptation upon repeated UVB and ozone
treatment (UV + UV and O 3
+ O 3
) showed that SOD activity in Crepis capillaris
plants is similar and increases up to 1.74 and 1.98 times comparing with the control.
Even after adaptation of a plant to one factor and later under treatment with the other
211

(UV + O 3
; O 3
+ UV) SOD activity remains similar (Fig.). The research showed that
adaptation by low UVB and ozone doses towards one of the other factor influences
the increase of SOD activity in plants.
Discussion. Abiotic stress condition weakens plants; they become more susceptible
to pathogens, and this leads to extensive losses to of agricultural crop worldwide.
Usually the impact of environmental factors is not individual but combined. Therefore,
under conditions of climate changes plants have to adapt to simultaneous impact of
several factors: drought and heat; cold stress and drought combined with high light
conditions. For example, during heat and high ozone stresses plants open leaf stomata
for transpiration to avoid overheating, but thus the way for more abundant passing of
ozone into leaf intercellular ducts is opened, otherwise stomata would close. Therefore
in case of complex stress plants might require conflicting or antagonistic responses
(Pasqualini et al., 2003; Mittler, 2006).
The response type depends upon the plant genotype. Cultivated plants are more
susceptible to stresses than wild plants, which are better adapted to climate changes.
Even the slightest changes of climate factors could be relevant for agricultural plants.
Previous investigations have shown that even the lowest UVB (2 kJ m -1 ) and ozone
doses reduce the leaf size of a model plant Crepis capillaris; it is particularly true in
case of ozone (Rančelienė et al., 2006). Ozone, as a strong oxidant, or its secondary
derivatives, such as ROS (Reactive Oxygen Species) frequently cause leaf necroses.
It is known that viruses also cause necroses of plant leaves. In case of hypersensitivity
reaction necrotic spots, localizing the virus spread, form on the injured areas of leaves.
There is an opinion that ozone also triggers a hypersensitive response (Koch et al.,
2000). Therefore, necroses indicate the death of some cells irrespective of the stress
factor causing them: viruses, bacteria, UVB, ozone, cold and drought. So the damages
indicate the so-called programmed cell death (Pasqualini et al., 2002). But response
and defense genes are simultaneously induced, and they determine triggering of plant
defence mechanism to antioxidative response (Yun-Hee Kim et al., 2007). Antioxidative
enzyme systems, especially SOD enzyme, actively participates in this process. Our
research showed that after preadaptation by low doses the repeated treatment by other
factor doubly increases the activity of antioxidative SOD enzyme. It demonstrates that
plants adapted to one factor are frequently more tolerant to other factors. It confirms
the results of our earlier researches performed with cold-resistant interspecific potato
hybrids treated with UVB (Vyšniauskienė et al., 2006). However, results of other 7-year
long researches performed with plants growing in tundra showed that under conditions
of stratospheric ozone depletion and by enhanced UVB, the supplementation of UVB
in field produced no negative effect upon growth parameters (Rozema et al., 2006). It
demonstrates that in the course of time plants become UVB-tolerant.
Conclusions. 1. Increased activity of plant enzymes, such as SOD, after the UVB
and ozone irradiation was assessed as adaptational response of plant towards oxidative
stress caused by harmful factors. 2. Plants adapted to one factor are frequently more
tolerant to the impact of other factors.
212

Acknowledgements. This research was supported by the Lithuanian State Science
and Studies Foundation programme “APLIKOM”. The authors gratefully acknowledge
to the Lithuanian Institute of Horticulture for availability to perform our experiments
in growth chambers of Institute.
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Acevedo A., Scopel A. L., Ballare C. L. 1999. The effects of solar UV-B radiation
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213

12. Yun-Hee Kim, Soon Lim, Sim-Hee Han, Jae-Cheon Lee, Wan-Keun Song,
Jae-Wook Bang, Suk-Yoon Kwon, Haeng-Soon Lee, Sang-Soo Kwak.
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SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Antioksidacinio fermento superoksido dismutazės aktyvumo
pokyčiai Crepis capillaris augaluose po UVB ir ozono poveikio
R. Vyšniauskienė, V. Rančelienė
Santrauka
Darbo tikslas buvo nustatyti antioksidantinio fermento superoksido dismutazės (SOD)
aktyvumo pokyčius modelinio augalo Crepis capillaris augalams po UVB ir ozono poveikių.
Lyginant SOD aktyvumą Crepis capillaris lapuose po poveikio mažomis –adaptuojančiomis
UVB ir ozono dozėmis su kontrole, gauta, kad po UVB (3 kJ m -1 ) SOD aktyvumas pakyla 1,4
kartus, o po ozono poveikio SOD padidėja 1,94 karto. Po didesnės UVB (9 kJ m -1 ) dozės SOD
aktyvumas pakyla net 2 kartus, tačiau po trigubos O 3
dozės jau SOD aktyvumas mažesnis, tačiau
nesiekia kontrolės lygio. Tiriant adaptacijos poveikį pakartotiniam UVB ir ozono paveikiui
gavome, kad SOD aktyvumas Crepis capillaris augalams yra panašus ir lyginant su kontrole
padidėja 1,74 ir 1,98 karto. Tačiau ir adaptavus augalus vienam veiksniui, o poveikus augalus
kitu veiksniu (kryžminė adaptacija), SOD aktyvumas išlieka panašus. Tyrimai parodė, kad
mažų UVB ir ozono dozių adaptacija tiek tam pačiam, tiek ir kitam veiksniui, turi poveikį SOD
aktyvumo padidėjimui augaluose. Padidėjęs SOD aktyvumas po UVB spinduliuotės ir ozono
vertintinas kaip augalo adaptacinis atsakas į nepalankių veiksnių sukeltа oksidacinį stresą.
Reikšminiai žodžiai: Crepis capillaris, ozonas, UVB, adaptacija, superoksido dismutazė,
EC 1.15. 1.1.
214

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Photosystem II thermostability of apple tree leaves:
effect of rootstock, crown shape and leaf topology
Peter Ferus 1 , Marián Brestič 1 , Katarína Olšovská 1 ,
Anna Kubová 2
1
Department of Plant Physiology, Slovak Agricultural University in Nitra,
Tr. A. Hlinku 2, 949 76 Nitra, Slovakia
2
Experimental Orchard, Slovak Agricultural University in Nitra, Tr. A. Hlinku 2,
949 76 Nitra, Slovakia, e-mail: Marian.Brestic@uniag.sk
Apple tree leaves usually experience extremely high summer temperatures, which might
cause disturbances to their photosynthesis and negatively influence fruit loading and quality.
In this respect, in apple trees of cv. ‘Idared’ we evaluated the effect of rootstock (very dwarfing
M.9 and vigorous MM.104), crown shape (modified Slender spindle and modified Schlцsser
palmette) and leaf topology (leaves from the top of annual shoots, from the middle of the annual
shoots and from short sprouts on the tree trunk) on the photosystem II (PS II) thermostability at
the end of summer in 2006. For this purpose we analysed chlorophyll a fluorescence induction
curves after exposure of leaf samples to 30 minutes of 42 °C. Neither rootstock types nor crown
shapes caused any changes in the leaf PS II thermostability; however, significant differences in
these characteristics were found in relation to leaf position in the apple tree crown. In comparison
to leaves from annual shoots, which exhibited only moderate thermotolerance, a considerable
increase was observed in leaves from short sprouts on the tree trunk. Measured high capacity
of PS II thermotolerance is discussed in respect to plant polarity principles.
Key words: photosystem II thermotolerance, rootstock, crown shape, leaf topology, apple
tree (Malus domestica Borkh.).
Introduction. Leaves are the main photosynthetizing plant organs providing
assimilates for plant growth and fruit development. Leaves of apple tree (Malus
domestica Borkh.) are developed from the leaf buds or combined floral-leaf buds.
Passing exogenous dormancy, they expand to annual shoots with numerous leaf
insertions. Intensive growth of annual shoots is timed particularly during spring and
autumn. In summer their growth is usually decreased or almost stopped.
During vegetation period, developing leaves in the tree crown experience different
endogenous and exogenous conditions determining their morphological, anatomical
and biochemical characteristics, and hence their photosynthetic activity (Pearcy et al.,
2005). In southern Slovakia, summer air temperatures usually reach 40 °C, which
can have detrimental effects on photosynthesis (Salvucci and Crafs-Brandner, 2004)
and, in a consequence, on fruit loading and quality. The final negative effect depends
on leaf protective capacity on one side, and stress intensity and duration on the other
side (Larcher, 2003).
215

Supraoptimal air temperature usually limits leaf photosynthesis through stomatal
limitation of CO 2
uptake (Rahman, 2005; Cui et al., 2006), Rubisco deactivation (Law
and Crafs-Brandner, 1999), thylakoid membrane injury (Liu and Huang, 2000), and
photochemical PSII efficiency decline as a result of antenna detachment or impairment
of electron transport chain components (Srivastava et al., 1997; Strasser, 2004). In the
past two decades, a number of mechanisms alleviating these negative constraints was
identified, including thermal dissipation (Bukhov et al., 1998); switch from the noncyclic
to cyclic electron transport (Bukhov et al., 1999); enhanced photorespiration
(Sharkey, 2005); electron flux to oxygen (Mehler reaction) associated with up-regulated
antioxidant system (Sairam et al., 2000) and chlororespiration (Wang et al., 2006);
changes in the membrane composition in favour to saturated fatty acids (Larkindale and
Huang, 2004) and accumulation of chemical (Kuznetsov et al., 1999) and molecular
chaperones (Iba, 2002; Schroda, 2004).
Apple trees are composed of scion engrafted into a rootstock, which markedly
regulates their growth (Webster, 1995). Tree crowns are formed into particular shapes
in order to intercept more light and optimise fruit growth and ripening process. Both
rootstock and crown shape influence hormonal composition of the scion (Webster,
1995; Tworkoski et al., 2006), and therefore can have significant effect on many
physiological processes in leaves as well. Moreover, light conditions regulate leaves
expansion and evoke different acclimation syndromes (Kull, 2002).
Very little is known about how these circumstances modify photosynthetic
characteristics of apple tree leaves and what effect they have on photosynthetic
processes in combination with elevated air temperature. In this work we focused on
how rootstock differing in growth intensity, different crown shapes and leaf topology
influence photosystem II (PS II) thermostability of leaves exposed to conditions of
summer heat.
Object, methods and conditions. Plant material and cultivation.
Apple trees (Malus domestica Borkh.) of cv. ‘Idared’, were cultivated in Experimental
orchard of Slovak Agricultural University in Nitra, Slovakia, which is located on Nitra
river alluvium (loamy-clayed soil substrate). The scion cultivar was engrafted into
rootstock MM.104 (vigorous) and formed to modified Slender spindle or modified
Schlцsser palmette (20 years old trees), while apple trees engrafted into rootstock
M.9 (very dwarfing) were formed to Slender spindle (8-years-old trees). Rows were
oriented in north-southern direction, spaced 5 metres from each other, with in-row plant
distance of 5 metres (older trees) and 1 meter (younger trees). Trees were approximately
3 (older trees) and 2 metres high (younger trees), and their crown diameter/width was
2.5 m (older trees, modified Slender spindle), 1.25 m (older trees, modified Schlцsser
palmette) and 1 m (younger trees, Slender spindle), respectively.
In autumn, phosphoric and potassium fertilization combined with cultivation
was applied. During the vegetation period, trees were fertilized by nitrogen and for
prevention treated against powdery mildew (Podosphaera leucotricha), apple scab
(Venturia inaequalis) and herbivores. Weeds were regulated mechanically.
In the fruit growing and ripening growth stage (beginning of September), young
216

5th leaves of east-oriented annual shoots, leaves of middle part of the same shoots and
leaves of south-oriented short sprouts on the tree trunks were collected in the mornings
of three consecutive sunny days, transferred to laboratory, acclimated to darkness for
30 minutes and subjected to photosystem II (PS II) thermostability test.
Photosystem II thermostability test. Samples of leaf halves were enclosed in the
test tubes and submerged into water bath of 42 °C for 30 minutes in the darkness.
Before and after exposure of samples to high temperature, chlorophyll a fluorescence
induction kinetics of the samples (12 leaves per treatment, 5 replicates per a leaf) were
determined by Handy-PEA, Hansatech Instruments Ltd., UK. Their normalizations
enabled to determine relative variable fluorescence at J and I step (V j
and V i
), ratio of
fluorescence at K and J step of the induction curves (F k
/F j
) and maximal photochemical
efficiency of PS II (F v
/F m
). After that, the JIP test (Biolyzer 4HP v. 3.06 software,
Ronald Rodriguez) was applied to the fluorescence induction curves, which revealed
further characteristics of the PS II bioenergetic state (Strasser et al., 2000; Strasser
et al., 2004), such as maximal quantum yield of primary photochemistry (φ Po
), exciton
transfer efficiency to electron transport chain (ψ o
), electron transport yield (φ Eo
) and
thermal dissipation yield (φ Do
).
Determination of photosynthetic pigments concentration. For this purpose, second
halves of leaf samples were utilized. Segments of the samples were homogenised in
the presence of sea sand, MgCO 3
and 100 % acetone. After acetone vaporisation the
homogenates were transferred into 80 % acetone and after centrifugation for 2 minutes
at 2 500 rpm light absorption of the solutions was read at 470, 647 and 663 nm using
spectrophotometer Jenway, UK. The pigment concentrations per leaf area unit were
calculated according to Lichtenthaler (1987).
During the summer months, maximal daily air temperatures and daily rainfall in
the experimental orchard was recorded by automatic agrometeorological station.
Statistical data analysis (ANOVA) was accomplished using application
Statgraphics Plus v. 4.0.
Results. Photosynthetic apparatus of apple trees before high temperature treatment.
Chlorophyll a fluorescence induction curves of leaves from modified Slender spindle
apple trees engrafted into vigorous rootstock MM.104 (Fig. 1) showed very similar
course at the beginning (O step). At J step they started to differ significantly with much
more steeper fluorescence rise to I step in the leaves of south-oriented short sprouts
on the trunk (inner-crown leaves) followed by slighter rise to P step. Fluorescence
transients from J to P step in the 5th and middle leaves of east-oriented annual shoots
were almost parallel. However, the latter gained significantly lower values.
217

Fig. 1. Chlorophyll a fluorescence induction curves (OJIPs) in control
(full symbols) and high temperature (30 min of 42 °C in the darkness) treated
(empty symbols) leaves from apple trees of cultivar ‘Idared’ formed to modified
Slender spindle and engrafted into vigorous rootstock MM.104: 5th leaves of
east-oriented annual shoots (circles), leaves of the middle part of the same
shoots (squares) and leaves of the south-oriented short sprouts on the tree trunks
(triangles).
1 pav. Chlorofilo a fluorescencijos indukcijos kreivės nepaveiktuose
(užtušuoti simboliai) ir aukšta temperatūra (42 °C 30 min tamsoje) paveiktuose
(tuščiaviduriai simboliai) modifikuotos laibosios verpstės formos ‘Idared’ veislės obelų su
MM.104 poskiepiu lapuose. Imti penkti lapai nuo į rytus orientuoto metūglio (rutuliukai),
lapai nuo vidurinės šakų dalies (kvadratėliai) ir lapai nuo į šiaurę orientuotų vaisinių šakučių
( trikampėliai).
Parameters derived from fluorescence induction curves, such as basal fluorescence
(F o
) and relative variable fluorescence at the J and I step of the induction curves (V j
and
V i
) (Table 1) also confirm this tendency. On the other hand, maximal photochemical
efficiency of PS II (F v
/F m
) did not show statistically significant difference between
the leaves. Fluorescence at K versus J step of the induction curves (F k
/F j
) was slightly
enhanced in the inner-crown leaves. JIP test applied to the fluorescence induction
curves revealed significantly higher exciton transfer efficiency to electron transport
chain (ψ o
) and electron transport yield (φ Eo
) in the inner-crown leaves, but they had no
effect on maximal quantum yield of primary photochemistry (φ Po
) (Fig. 2). Thermal
dissipation yield (φ Do
) also showed balanced values in each leaf position.
218

Table 1. Parameters derived from chlorophyll a fluorescence induction curves
measured in 5th leaves of east-oriented annual shoots, leaves of middle part
of the same shoots and leaves of south-oriented short sprouts on the apple tree
trunks (inner-crown), of cultivar ‘Idared’ before the high temperature treatment
(30 min at 42 °C in the darkness): F o
– basal fluorescence, V j
and V i
– relative
variable fluorescence at J and I step of the fluorescence induction curve, F k
/F j
–
ratio of chlorophyll a fluorescence at K versus J step of the induction curve, and
F v
/F m
– maximal photochemical efficiency of PS II. Letters indicate statistically
significant difference at P = 0.01.
1 lentelė. Išvestiniai parametrai iš chlorofilo a fluorescencijos indukcijos kreivių,
matuotų ‘Idared’ veislės obelų penktuose lapuose nuo į rytus orientuoto metūglio,
lapuose nuo vidurinės šakų dalies ir lapuose nuo į pietus orientuotų vaisinių šakučių.
Analizės atliktos prieš aukštos temperatūros poveikį (42 °C 30 min tamsoje). F o
– bazinė
fluorescencija, V j
ir V i
– J ir I fluorescencijos indukcijos kreivės žingsniuose santykinai
kintanti fluorescencija, F k
/F j
– chlorofilo a fluorescencijos santykis tarp K ir J žingsnių,
F v
/F m –
maksimalus fotocheminis PS II efektyvumas. Raidės žymi statistiškai patikimus
skirtumus, kai P = 0,01.
219

Fig. 2. Parameters derived from chlorophyll a fluorescence induction curves
measured in the 5th leaves of east-oriented annual shoots, leaves of middle part
of the same shoots and leaves of south-oriented short sprouts on the tree trunks
(inner-crown) from apple trees, of cultivar ‘Idared’, formed to modified Slender
spindle and engrafted into vigorous rootstock MM.104, before and after the high
temperature treatment (30 min of 42 °C in the darkness): φ Po
– maximal quantum
yield of primary photochemistry, ψ o
– exciton transfer efficiency to electron
transport chain, φ Eo
– electron transport yield, φ Do
– thermal dissipation yield.
Letters indicate statistically significant difference at P = 0.01: italic – before, and
normal – after the high temperature treatment.
2 pav. Išvestiniai parametrai iš chlorofilo a fluorescencijos indukcijos kreivių, matuotų
modifikuotos laibosios verpstės formos ‘Idared’ veislės obelų su MM.104 poskiepiu
penktuose lapuose nuo į rytus orientuoto metūglio, lapuose nuo vidurinės šakų dalies ir
lapuose nuo į pietus orientuotų vaisinių šakučių.
Analizės atliktos prieš ir po temperatūros poveikio (42 °C 30 min tamsoje). φ Po
– maksimali
pirminės fotochemijos kvantų išeiga, ψ o
– eksitono perdavimo į elektronų transporto
grandinę efektyvumas, φ Eo
– elektronų transporto išeiga, φ Do
– šilumos sklaidos išeiga.
Raidės žymi statistiškai patikimus skirtumus kai P = 0,01, pasvirasis šifras – prieš,
normalus – po temperatūros poveikio.
220

In Slender spindle apple trees engrafted into very dwarfing rootstock M.9, the
middle leaves on annual shoots and the inner-crown leaves displayed lower fluorescence
values at J step of the fluorescence induction curves, followed by generally slowlier
fluorescence increase to P step in comparison to leaves of apple trees engrafted into
rootstock MM.104 (Fig. 3).
Fig. 3. Chlorophyll a fluorescence induction curves (OJIPs) in control
(full symbols) and high temperature (30 min of 42 °C in the darkness) treated
(empty symbols) leaves from apple trees of cultivar ‘Idared’, formed to Slender
spindle and engrafted into very dwarfing rootstock M.9: 5th leaves of east-oriented
annual shoots (circles), leaves of the middle part of the same shoost (squares) and
leaves of the south-oriented short sprouts on the tree trunks (triangles).
3 pav. Chlorofilo a fluorescencijos indukcijos kreivės nepaveiktuose (užtušuoti simboliai)
ir aukšta temperatūra (42 °C 30 min. tamsoje) paveiktuose (tuščiaviduriai simboliai)
modifikuotos laibosios verpstės formos ‘Idared’ veislės obelų su M.9 poskiepiu lapuose. Imti
penkti lapai nuo į rytus orientuoto metūglio (rutuliukai),
lapai nuo vidurinės šakų dalies (kvadratėliai) ir lapai nuo į pietus orientuotų vaisinių šakučių
(trikampėliai).
In these leaves F o
reached markedly lower values than in the 5th leaves of annual
shoots (Table 1). The 5th leaves also dominated in V j
, while F k
/F j
did not change with
leaf position. F v
/F m
was the highest in the inner-crown leaves. Significant differences
among the JIP test parameters were only obtained in the 5th leaves of annual shoots
and inner-crown leaves (Fig. 4). The inner-crown leaves reached higher φ Po
, ψ o
, φ Eo
,
and lower φ Do
than the 5th ones.
221

Fig. 4. Parameters derived from chlorophyll a fluorescence induction
curves measured in the 5th leaves of east-oriented annual shoots,
leaves of middle part of the same shoots and leaves of south-oriented short sprouts
on the tree trunks (inner-crown) from apple trees of cultivar ‘Idared’, formed to
Slender spindle and engrafted into very dwarfing rootstock M.9,
before and after the high temperature treatment (30 min of 42 °C in the darkness):
φ Po
– maximal quantum yield of primary photochemistry,
ψ o
– exciton transfer efficiency to electron transport chain,
φ Eo
– electron transport yield,
φ Do
– thermal dissipation yield.
Letters indicate statistically significant difference at P = 0.01:
italic – before, and normal – after the high temperature treatment.
4 pav. Išvestiniai parametrai iš chlorofilo a fluorescencijos indukcijos kreivių,
matuotų modifikuotos laibosios verpstės formos ‘Idared’ veislės obelų su M.9 poskiepiu
penktuose lapuose nuo į rytus orientuoto metūglio,
lapuose nuo vidurinės šakų dalies ir lapuose nuo į pietus orientuotų vaisinių
šakučių. Analizės atliktos prieš ir po temperatūros poveikio (42 °C 30 min tamsoje).
φ Po
– maksimali pirminės fotochemijos kvantų išeiga,
ψ o
– eksitono perdavimo į elektronų transporto grandinę efektyvumas,
φ Eo
– elektronų transporto išeiga,
φ Do
– šilumos sklaidos išeiga. Raidės žymi statistiškai patikimus skirtumus, kai P = 0,01,
pasvirasis šifras – prieš, normalus – po temperatūros poveikio.
222

Except of markedly lower fluorescence values at O and J step of the fluorescence
induction curves in the middle leaves of annual shoots, leaves form Schlösser palmette
apple trees engrafted into vigorous rootstock MM.104 (Fig. 5) exhibited very similar
fluorescence transients to those from modified Slender spindle apple trees engrafted
into the same rootstock.
Fig. 5. Chlorophyll a fluorescence induction curves (OJIPs) in control
(full symbols) and high temperature (30 min of 42 °C in the darkness) treated
(empty symbols) leaves from apple trees of cultivar ‘Idared’, formed to modified
Schlösser palmette and engrafted into vigorous rootstock MM.104:
5th leaves of east-oriented annual shoots (circles), leaves of the middle part
of the same shoot (squares) and leaves of the south-oriented short
sprouts on the tree trunks (triangles).
5 pav. Chlorofilo a fluorescencijos indukcijos kreivės nepaveiktuose
(užtušuoti simboliai) ir aukšta temperatūra (42 °C 30 min. tamsoje) paveiktuose
(tuščiaviduriai simboliai) modifikuotos Schlösser palmetės formos ‘Idared’ veislės
obelų su MM.104 poskiepiu lapuose. Imti penkti lapai nuo į rytus orientuoto
metūglio (rutuliukai), lapai nuo vidurinės šakų dalies (kvadratėliai) ir
lapai nuo į šiaurę orientuotų vaisinių šakučių (trikampėliai).
Consequently, F o
was reduced in the middle leaves of annual shoots, and V j
(Table 1) with JIP test parameters y o
and f Eo
revealed no difference between leaf
positions (Fig. 6).
Photosynthetic pigments concentration and chl./car. ratio exhibited no significant
difference between leaf positions in the modified Slender spindle apple trees engrafted
into rootstock MM.104 (Table 2). However, chlorophyll a/b ratio of inner-crown leaves
was the lowest among all leaf positions.
Despite of significant reduction of chlorophyll a concentration in the inner-crown
leaves of the Slender spindle apple trees engrafted into rootstock M.9 (this rootstock –
crown shape combination exhibited the highest chlorophyll a concentrations), leaf
position did not influence the chlorophyll a/b ratio. Chlorophyll b and carotenoids
concentrations and chl./car. ratio were also very similar.
Among rootstock – crown shape combinations, markedly lower chlorophyll a,
b and carotenoids concentrations accompanied by decrease of chlorophyll a/b ratio
223

in the inner-crown leaves were only observed in modified Schlösser palmette apple
trees on the rootstock MM.104. Besides, 5th and the middle leaves on annual shoots
of these apple trees dominated in carotenoid concentration.
Fig. 6. Parameters derived from chlorophyll a fluorescence induction curves
measured in the 5th leaves of east-oriented annual shoots, leaves of middle part
of the same shoots and leaves of south-oriented short sprouts on the tree trunks
(inner-crown) from apple trees, of cultivar ‘Idared’, formed to modified Schlösser
palmette and engrafted into vigorous rootstock MM.104, before and after the high
temperature treatment (30 min of 42 °C in the darkness):
φ Po
– maximal quantum yield of primary photochemistry,
ψ o
– exciton transfer efficiency to electron transport chain,
φ Eo
– electron transport yield, φ Do
– thermal dissipation yield.
Letters indicate statistically significant difference at P = 0.01:
italic – before, and normal font – after the high temperature treatment.
6 pav. Išvestiniai parametrai iš chlorofilo a fluorescencijos indukcijos kreivių,
matuotų modifikuotos Schlösser palmetės formos ‘Idared’ veislės obelų su MM.104
poskiepiu penktuose lapuose nuo į rytus orientuoto metūglio,
lapuose nuo vidurinės šakų dalies ir lapuose nuo į pietus orientuotų vaisinių šakučių.
Analizės atliktos prieš ir po temperatūros poveikio (42 °C 30 min tamsoje).
φ Po
– maksimali pirminės fotochemijos kvantų išeiga,
ψ o
– eksitono perdavimo į elektronų transporto grandinę efektyvumas,
φ Eo
– elektronų transporto išeiga, φ Do
– šilumos sklaidos išeiga.
Raidės žymi statistiškai patikimus skirtumus, kai P = 0,01,
pasvirasis šifras – prieš, normalus – po temperatūros poveikio.
224

Table 2. Photosynthetic pigment concentration (mg m -2 ) in the 5th leaves on
east-oriented annual shoots, leaves of middle part of the same shoots and leaves
of south-oriented short sprouts on the tree trunks (inner-crown) from apple trees,
of cultivar ‘Idared’, with different rootstock and crown shape. Letters indicate
statistically significant difference at P = 0.01.
2 lentelė. Photosintezės pigmentų koncentracija (mg m -2 ) skirtingos formos ‘Idared’ veislės
obelų su skirtingais poskiepiais penktuose lapuose nuo į rytus orientuoto metūglio, lapuose
nuo vidurinės šakų dalies ir lapuose nuo į pietus orientuotų vaisinių šakučių. Raidės žymi
statistiškai patikimus skirtumus kai P = 0,01.
P h o t o s y s t e m I I a c t i v i t y i n a p p l e t r e e l e a v e s t r e a t e d
w i t h h i g h t e m p e r a t u r e. Thirty minutes treatment by temperature of 42 °C in
the darkness caused a significant increase of F o
, F k
/F j
and φ Do
, and decrease of V i
, F v
/F m
and φ Po
in all rootstock-crown shape-leaf position combinations (Table 3). Inner-crown
leaves of apple trees on rootstock MM.104 also showed lower V j
and higher ψ o
.
In comparison to control measurements, fluorescence induction curves of leaves
from modified Slender spindle apple trees engrafted into rootstock MM.104 exhibited
higher fluorescence at the O step, reduced fluorescence at the J and particularly I step,
followed by markedly lower fluorescence values at the P step (Fig. 1). Parallel to these
changes, relations between fluorescence transients of leaves from different positions
stayed relatively stable.
225

Table 3. Evaluation of statistical difference of parameters derived from chlorophyll
a fluorescence induction curves between control and high temperature (30 min at
42 °C in the darkness) treated apple tree leaves. F o
– basal fluorescence, V j
and
V i
– relative variable fluorescence at J and I step of the fluorescence induction
curve, F k
/F j
– ratio of fluorescence at K versus J step of the fluorescence induction
curve, F v
/F m
– maximal photochemical efficiency of PS II, φ Po
– maximal
quantum yield of primary photochemistry, ψ o
– exciton transfer efficiency to
electron transport chain, φ Eo
– electron transport yield, φ Do
– thermal dissipation
yield. Double asterisk indicates on statistically significant difference at P = 0.01,
asterisks indicates on statistically significant difference at P = 0.05 and n. s. –
non-significant difference.
3 lentelė. Išvestinių parametrų iš chlorofilo a fluorescencijos indukcijos kreivių statistinių
skirtumų tarp nepaveiktų ir aukšta temperatūra (42 °C 30 min. tamsoje) paveiktų vaismedžių
įvertinimas. F o
– bazinė fluorescencija, V j
ir V i
– J ir I fluorescencijos indukcijos kreivės
žingsniuose santykinai kintanti fluorescencija, F k
/F j
– chlorofilo a fluorescencijos santykis
tarp K ir J žingsnių, F v
/F m
– maksimalus fotocheminis PS II efektyvumas. φ Po
– maksimali
pirminės fotochemijos kvantų išeiga, ψ o
– eksitono perdavimo į elektronų transporto
grandinę efektyvumas, φ Eo
– elektronų transporto išeiga, φ Do
– šilumos sklaidos išeiga.
Dvi žvaigždutės žymi statistiškai patikimus skirtumus, kai P = 0,01, žvaigždutė žymi
statistiškai patikimus skirtumus kai P = 0,05.
From them the lowest F o
and V j
values, and the highest F v
/F m
of inner-crown
leaves were derived (Table 4). Leaf positions did not influence values of V i
and F k
/F j
.
These results were reflected in JIP test parameters: the inner-crown leaves showed the
highest ψ o
, φ Eo
and φ Po
, but the lowest φ Do
(Fig. 2).
226

Table 4. Parameters derived from chlorophyll a fluorescence induction curves
measured in 5th leaves of east-oriented annual shoots, leaves of middle part of the
same shoots and leaves of south-oriented short sprouts on the apple tree trunks
(inner-crown), after the high temperature treatment (30 min at 42 °C in the dark):
F o
– basal fluorescence, V j
and V i
– relative variable fluorescence at J and I step of
the fluorescence induction curve, F k
/F j
– ratio of chlorophyll a fluorescence at K
versus J step of the induction curve, and F v
/F m
– maximal photochemical efficiency
of PS II. Letters indicate statistically significant difference at P = 0.01.
4 lentelė. Išvestiniai parametrai iš chlorofilo a fluorescencijos indukcijos kreivių,
matuotų ‘Idared’ veislės obelų penktuose lapuose nuo į rytus orientuoto metūglio,
lapuose nuo vidurinės šakų dalies ir lapuose nuo į pietus orientuotų vaisinių šakučių.
Analizės atliktos po aukštos temperatūros poveikio (42 °C 30 min. tamsoje). F o
– bazinė
fluorescencija, V j
ir V i
– J ir I fluorescencijos indukcijos kreivės žingsniuose santykinai
kintanti fluorescencija, F k
/F j
– chlorofilo a fluorescencijos santykis tarp K ir J žingsnių,
F v
/F m
– maksimalus fotocheminis PS II efektyvumas. Raidės žymi statistiškai patikimus
skirtumus kai P = 0,01.
Fluorescence induction curves in the middle leaves of annual shoots and innercrown
leaves from Slender spindle apple trees engrafted into rootstock M.9 did not
exhibit lower fluorescence values at the J step than before high temperature treatment
(Fig. 3). Except of this difference, fluorescence transients of these as well as the 5 th
leaves resembled those from modified Slender spindle apple trees engrafted into
rootstock MM.104. Also the parameters derived from fluorescence induction curves
showed the same tendency as in these apple trees (Table 4, Fig. 4).
Comparison of fluorescence induction curves in leaves from modified Schlösser
palmette apple trees engrafted into rootstock MM.104 before and after high temperature
227

treatment indicates the similar characteristics to those measured in the Slender spindle
apple trees on the same rootstock (Fig. 5). On the other hand, inner-crown leaves except
of the lowest F o
and V j
, and the highest F v
/F m
showed also the highest V i
(Table 4).
However, φ Po
, ψ o
, φ Eo
and φ Do
in these apple trees were also similar to the Slender
spindle apple trees (Fig. 6).
The JIP test parameters obtained from all the combinations of rootstock – crown
shape – leaf position after high temperature treatment showed similar φ Po
, ψ o
, φ Eo
and φ Do
in the 5th and the middle leaves of annual shoots (Fig. 7). However, despite
of almost equal ψ o
and φ Eo
, significantly higher φ Do
in the inner-crown leaves from
modified Schlцsser palmette apple trees engrafted into rootstock MM.104 led to
significantly lower φ Po
than in the Slender spindle apple trees engrafted into rootstock
M.9.
Fig. 7. Comparison of parameters derived from chlorophyll a fluorescence nduction
curves (φ Po
– maximal quantum yield of primary photochemistry, ψ o
– exciton
transfer efficiency to electron transport chain, φ Eo
– electron transport yield, φ Do
–
thermal dissipation yield) between treatments (rootstocks and crown shapes) in 5th
leaves of east-oriented annual shoots, leaves of middle part of the same shoots and
leaves of south-oriented short sprouts on the tree trunks (inner-crown) from apple
trees, of cultivar ‘Idared’, after the high temperature treatment (30 min at 42 °C in
the dark). Letters indicate statistically significant difference at P = 0.01.
7 pav. Išvestinių parametrų iš chlorofilo a fluorescencijos indukcijos kreivių
(φ Po
– maksimali pirminės fotochemijos kvantų išeiga, ψ o
– eksitono perdavimo į elektronų
transporto grandinę efektyvumas, φ Eo
– elektronų transporto išeiga, φ Do
– šilumos sklaidos
išeiga), matuotų modifikuotos laibosios verpstės formos ‘Idared’ veislės obelų penktuose
lapuose nuo į rytus orientuoto metūglio, lapuose nuo vidurinės šakų dalies ir lapuose nuo į
pietus orientuotų vaisinių šakučių. Analizės atliktos po poveikio aukšta temperatūra (30 min.
42 °C tamspje). Raidės žymi statistiškai patikimus skirtumus, kai P = 0,01.
228

Discussion. Leaf photosynthetic characteristics are influenced by internal and
external factors and reflect their time-space fluctuations. Despite of homogenous
rainfall distribution during summer months (Fig. 8), maximal daily air temperatures
often exceeded 35 °C, inducing enhanced thermotolerance in apple trees leaves. How
was this acclimatory process influenced by type of rootstock, crown shape and leaf
topology within the crown
Fig. 8. Course of maximal daily air temperatures and daily rainfall in summer
months in 2006, in the experimental orchard of Slovak Agricultural University
(Nitra, Slovakia)
8 pav. Maksimalių kasdieninių oro temperatūrų ir kritulių eiga 2006 metų vasaros mėnesiais
Slovakijos žemės ūkio universiteto eksperimentiniuose soduose, Nitra, Slovakija
Rootstock mainly influences: (1) the amount and/or ratio of promoting and
inhibiting endogenous hormones circulating within tree plant, particularly between
the root system and above-ground tree parts; (2) the movement of assimilates (e. g.,
sugars and amino acids) or mineral elements between the scion and rootstock; and
(3) the amount of water taken up and moved through the rootstock to scion (Webster,
1995).
Therefore, we could expect rootstock influence on photosynthetic apparatus as
well. Rootstocks with enhanced resistance to Erwinia amylovora, provide higher
resistance to scion (Jensen et al., 2003). Would it not be possible also in relation to
abiotic stressors
However, no difference in any of the JIP test parameter (φ Po
, ψ o
, φ Eo
and φ Do
) in
leaves with different crown localization, from apple trees engrafted into either vigorous
rootstock MM.104 or very dwarfing rootstock M.9 (Fig. 7) implies to the fact that
rootstock does not change the PS II thermostability of apple tree leaves. On the contrary,
according to Kamboj and Quinlan (1998) and also Kamboj et al. (1999), roots of M.9
accept less auxin and produce less cytokinin and more abscisic acid than MM.104,
which is supposed to stress-harden (Wang et al., 2003) the scion in larger extent.
229

Crown shape optimises light utilization efficiency, but also harmonizes the ratio
of fruiting and growing parts of trees. Different branch organisation may also change
hormonal composition (auxin-cytokinin ratio) in shoot tips, as mentioned by Tworkoski
et al. (2006) and thus also stability of leaf photosynthesis, because cytokinines reduce
its susceptibility to heat stress (Liu and Huang, 2002; Gupta et al., 2000). Nevertheless,
different crown forms (modified Slender spindle or modified Schlösser palmette) caused
no significant change in the PS II thermostability of leaves from any crown position
(Fig. 7), suggesting a similar hormonal balance.
Concentrating on PS II reactions to high temperature treatment in leaves from
different tree crown positions we detected a pronounced unification in fluorescence
induction transients (Fig. 1, 3, 5) and parameters derived from them (Table 4, Fig. 2,
4, 6). The highest electron transport rate to photosystem I (lower disturbances at
the acceptor side of PS II reaction centre, different impairment at the donor side
are excluded because of balanced F k
/F j
ratio), enhanced communication between
antenna complex and reaction centres of the PS II and the reduced thermal dissipation
of excitation energy led to the smallest decrease of photochemical PS II efficiency
(Strasser, 2004) and thus the highest PS II thermostability in the inner-crown leaves.
Response of the 5th and the middle leaves from the annual shoots was very similar.
Reduced chlorophyll a/b ratio in the inner-crown leaves from the older apple trees
on rootstock MM.104 (Table 2) points to their shaded character (Kull, 2002). Leaves
from modified Schlцsser palmette apple trees in comparison to modified Slender spindle
were likely more exposed to higher light intensities, therefore a slight movement of
photosynthetic pigment characteristics was expected to sunny type. However, this was
not observed. Because of massive light transmission into the crown of younger apple
trees on rootstock M.9, there was no partition into sun and shade type in leaves.
Higher PS II thermotolerance in the inner-crown leaves is in contrast to their
shade character, because shade leaves usually contain less xanthophyll cycle pigments
(Demmig-Adams and Adams, 1992), less enzymatic and non-enzymatic radical
scavengers (Logan et al., 1998) and have reduced photorespiratory activity (Muraoka
et al., 2000), thus their protection capacity is reduced.
Also finding of Niinemets et al. (1999) that increased optimal temperature
for maximal photosynthetic electron transport in poplar leaves is correlated with
integrated quantum flux density, can not be taken into account because of lower PS II
thermostability of sun leaves from annual shoots in comparison to shaded inner-crown
leaves, no difference between shaded leaves in apple trees with different crown shape,
and difference between leaf positions in apple trees engrafted into rootstock M.9. All
these results confirm our suggestion that there is no relation between light acclimation
syndrome and PS II thermotolerance in the apple tree crowns. So, is the leaf age
responsible for the PS II thermostability differences between leaf positions
Juvenile elm leaves exhibited lower thermotolerance in respect to electron transfer
from PS II antenna complex to reaction centre and electron transport to PS I (mainly
limited by oxygen evolving complex (OEC) impairment), compared to expanded
ones (Jiang et al., 2006). Balanced F k
/F j
in every leaf position in apple trees suggests
on reaching comparable OEC stability and hence leaf maturity, and further PS II
thermostability rise of the inner-crown leaves provides enhanced communication
230

etween anthenna complex and reaction centre of PS II and probably higher capacity
of protective mechanisms, utilizing electrons from electron transport chain. Secondly,
middle leaves of annual shoots, which are older than 5th leaves, did not exhibit enhanced
PS II thermostability. Also different period of elevated temperatures, which are required
for photosynthetic apparatus acclimation (Verdaguer et al., 2003), can be excluded,
because of limited growth of annual shoots during summer.
Therefore, we suppose that the effect of plant polarity and associated changes
in hormonal balance could be responsible for the difference in PS II thermostability
in these three leaf positions. Larger proximity to root apices and larger distance from
shoot apices shift the hormonal composition in short sprouts on the trunk in favour to
cytokinins content, which may reduce leaf susceptibility to heat stress (Liu and Huang,
2002; Gupta et al., 2000). Their effect is probably realized through enhanced antioxidant
enzymes level (Liu and Huang, 2002) or photorespiration (Tian et al., 2006).
Conclusions. Fruit trees experiencing extremely high summer temperatures
usually exhibit disturbances to photosynthetic process. Testing roles of rootstock vigour
(vigorous MM.104 and dwarfing M.9), crown shape and branch organization (Slender
spindle and Schlцsser palmette) as well as leaf position in apple trees (cv. ‘Idared’)
on photosystem II (PS II) thermostability (parameters derived from rapid chlorophyll
a fluorescence kinetics) points to no influence of first two factors. On the other hand,
the leaf topology seems to be the most important regulatory feature suggesting on
influence of trunk/root apex distance associated with cytokinin concentration.
Acknowledgement. This study was supported by the scientific-technical project
of the Grant Agency for Applied Research of the Slovak Ministry of Education
(AV/1109/2004).
Gauta 2008 04 04
Parengta spausdinti 2008 04 25
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SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Vaismedžio vainiko formos, poskiepio ir topologijos įtaka obelų
lapų II fotosistemos termostabilumui
P. Ferus, M. Brestič, K. Olšovská, A. Kubová
Santrauka
Vasarа obelys dažnai patiria neigiamą aukštų temperatūrų poveikį, kuris gali lemti
fotosintezės sutrikimus ir neigiamai paveikti vaisių derlių ir kokybę. Atsižvelgiant į tai, buvo
įvertinta poskiepio (žemaūgis M.9 ir augus MM.104), vainiko formos (modifikuota laiboji
verpstė ir modifikuota) ir lapų topologijos (lapai imti nuo metinio ūglio virрūnės, vidurio ir
nuo vaisinių šakučių) įtaka ‘Idared’ veislės obelų II fotosistemos (PS II) termostabilumui 2006
metų vasaros pabaigoje. Tirtos chlorofilo a fluorescencijos indukcijos kreivės, 30 minučių
paveikus tiriamus lapus 42 °C temperatūra. Nei poskiepio tipas nei vaismedžio forma nesukėlė
lapo PS II termostabilumo pokyčių, tačiau esminiai skirtumai pastebėti tarp skirtingos lapų
pozicijos vaismedžio vainike. Lyginant su lapais nuo metūglio kurie pademonstravo tik nedidelę
termotoleranciją, žymus augimas nustatytas lapuose nuo vaisinių šakučių. Išmatuotas PS II
termotolerancijos pajėgumas aptartas atsižvelgiant į augalų poliškumo principus.
Reikšminiai žodžiai: II fotosistemos termotolerancija, lapo topologija, obelis (Malus
domestica Borkh.), poskiepis, vainiko forma.
234

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Quality changes in black currant berries during
ripening
Marina Rubinskienė, Pranas Viškelis, Vidmantas Stanys,
Tadeušas Šikšnianas, Audrius Sasnauskas
Lithuanian Institute of Horticulture, Kauno 30, 54333 Babtai, Kaunas distr.,
Lithuania, e-mail: m.rubinskiene@lsdi.lt
Black currant ‘Pilėnai’, ‘Vyčiai’, ‘Kriviai’ and ‘Gagatai’ berries of different stages of
maturity were studied at the Lithuanian Institute of Horticulture. At technical maturity, black
currant ‘Pilėnai’ and ‘Vyčiai’ berries were distinguished for having the greatest masses: 1.54 g
and 1.48 g, respectively. When berries overripened, their mass decreased from between 2.9 %
(‘Kriviai’) to as much as 38.3 % (‘Gagatai’). During berry ripening, the skin of black currants
decreases in firmness. Overripe berries of cvs. ‘Pilėnai’ and ‘Vyčiai’ softened the most: 3.8
times and 3.4 times, respectively. At technical maturity, berries of cvs. ‘Pilėnai’ and ‘Kriviai’
were distinguished for having the most firm skin: 63.09 N cm -2 and 53.9 N cm -2 , respectively.
The highest levels of ascorbic acid were found in berries at the beginning of ripening. Overripe
berries had the lowest levels of ascorbic acid. Among various cultivars, ‘Pilėnai’ and ‘Gagatai’
berries of differing maturity had the highest levels of ascorbic acid, 152–114 mg 100 g -1 and
147–103 mg 100 g -1 , respectively. With the exception of berries of cvs. ‘Vyčiai’ and ‘Pilėnai’,
larger amounts of pigments accumulated in overripe berries. Among all cultivars, mature
and overripe berries of cvs. ‘Kriviai’ and ‘Gagatai’ had the highest levels of anthocyanins, at
387.61–450.63 mg 100 g -1 and 349.79-393.91 mg 100 g -1 , respectively. The greatest amounts
of dry soluble solids were found in overripe berries. The highest levels were present in the
mature and overripe berries of cv. ‘Kriviai’ (14.55–16.95 %). The amount of titratable acidity
in black currant berries decreased during ripening. Significantly greater amounts of acids were
found in ‘Pilėnai’ berries of various stages of maturity (3.27–2.41 %).
Key words: chemical composition, skin firmness, berry weight, cultivar.
Introduction. Black currants (Ribes nigrum L.) are orchard plants widely
grown in Europe, especially in Russia, Poland and Germany. Currant growing in
Lithuania was described in studies by J. Strumila in 1820. The Lithuanian Institute
of Horticulture has carried out black currant cultivar selection and evaluation of the
quality of berries and their products for many years. Most often, the nutritional quality
of black currant berries is determined by the amount of ascorbic acid and PP-active
substances. Agroclimatic conditions in Lithuania, Latvia, Poland and Byelorussia are
favourable for a large accumulation of vitamin C. Black currant cultivars created and
grown in these countries exceed Scandinavian cultivars for the amount of ascorbic
acid in their berries (Žurawicz et al., 2000; Kampuse et al., 2002; Rubinskienė et al.,
2006). Anthocyanin concentrations in berries are mostly determined by the cultivar’s
genotype. High concentrations of pigments are found in the berries from Scandinavian
235

cultivars (350–450 mg 100 g -1 ) (Nes, 1993). According to our research, berries from
Lithuanian cultivars accumulate between 274.9 and 499.1 mg 100 g -1 of anthocyanins
(Rubinskienė, Viškelis, 2002).
Depending on the cultivar, the time-to-ripening for black currant berries can
vary by a month or more. In Middle Lithuania, berries of early cultivars ripen by
July 5, moderately early berries ripen between July 6–15, and late cultivars begin to
ripen during the third week of July. Different agroclimatic conditions influence berry
quality and the time-to-ripening for black currants of various cultivars (Bičkauskienė,
1973; Rubinskienė, 2004). Berry quality (firmness, weight) determines the black
currant storage potential, transportability and trade appearance. The biochemical
composition of the berry shows the specific qualities of various cultivars and the goal
for production. Therefore it is very important to choose the optimal harvest time for
different cultivars.
The aim of this article is to evaluate berry quality and to investigate the
accumulation of bioactive substances and other chemical compounds in black currant
berries during ripening, identifying the most suitable harvest time.
Object, methods and conditions. For the first time, berries of different stages
of maturity of the Lithuanian cultivars ‘Pilėnai’, ‘Gagatai’, ‘Vyčiai’ and ‘Kriviai’
were investigated. They were rated according to a scale composed by the authors:
pink (beginning of ripening), dark brown (50 % ripened), black (technical maturity)
and overripe berries. Dry soluble solids in black currant berries were established by
digital refractometer ATAGO; ascorbic acid content was assessed by titration with
2.6-dichlorphenolindophenol sodium salt solution; titratable acidity (expressed as
citric acid) was assessed by titration with a 0.1 N NaOH solution (Ермаков et al.,
1987). The total amount of anthocyanins expressed by cyd-3-rut was established
spectrophotometrically at a wave length of 544 nm (Wrolstad, 1976). Berry skin
firmness was established with a penetrometer ИДП-500, with a probe diameter of
1 mm.
Meteorological conditions have a strong influence on both the biochemical
composition of berries and the yield quality. According to data from the Lithuanian
Hydrometeorological Station, during the berry ripening period in June 2007, the highest
air temperature was 24–27 °C, 1.5–2.8 °C higher than the multiannual average. During
that month, there were 298 sunny hours in Middle Lithuania (33 hours longer than
average). Most of the precipitation fell during the last days of the month. The conditions
were favourable for both early and late cultivars. Air temperature in July was similar to
the multiannual average. July 17 th was the hottest day, and the air temperature reached
30–34 °C. Rainy weather prevailed; precipitation was 1.5 to 2.5 times the average
rainfall. There were 20–50 fewer hours of sunshine than the multiannual average.
Results. According to our data, the weight of the average black currant berry
depended on the time of ripening and the properties of the cultivar. With the exception
of the cultivar ‘Kriviai’, berry weight increased during ripening until the berries reached
technical maturity (Fig. 1). Black currant cultivars ‘Pilėnai’ and ‘Vyčiai’ produced
the biggest berries, with average weights of 1.54 g and 1.48 g, respectively. From the
beginning of ripening, their weights increased 26 % and 45 %. The average weight of
black currant ‘Gagatai’ berry at technical maturity increased 34.3 % from the beginning
236

of ripening, reaching 1.37 g. It was observed that when berries overripened, their
weight decreased. Less weight loss was observed in the berries of ‘Kriviai’ (2.9 %) and
‘Vyčiai’ (7.4 %); the greatest weight loss was seen in ‘Gagatai’ (38.3 %). The weight
of overripe berries of the ‘Pilėnai’ cultivar decreased by 14.9 %.
Fig. 1. Change in black currant berry weight during ripening
1 pav. Įvairių juodųjų serbentų veislių uogų masės kitimas nokimo metu
During black currant ripening, the firmness of the berry skin changed.
This index depended on both the cultivar’s properties and berry ripeness. The
firmest skins of all cultivars were in berries that were just beginning to ripen. The
firmness of black currant berry skins ranged from 241.94 N cm -2 (‘Pilėnai’) to
150.06 N cm -2 (‘Vyčiai’) (Fig. 2).
Fig. 2. Change in black currant berry skin firmness during ripening
2 pav. Įvairių juodųjų serbentų veislių uogų odelės tvirtumo kitimas nokimo metu
During ripening, the skin firmness of 50 % ripened berries from cultivars ‘Vyčiai’
and ‘Kriviai’ decreased by 26.9 % and 36.6 %, respectively; firmness of 50 % ripened
237

erries from the ‘Pilėnai’ and ‘Gagatai’ cultivars decreased by 45.8 % and 56.2 %. A
notable softening of the skin was observed in black currant berries of technical maturity.
The cultivar ‘Vyčiai’ decreased 69.8 %; skin firmness of the ‘Kriviai’, ‘Gagatai’ and
‘Pilėnai’ cultivars decreased by 56.9 %, 55.8 % and 51.9 %, respectively. At the stage
of technical maturity, berries of cultivars ‘Pilėnai’ and ‘Kriviai’ were noted for their
firm skin. When berries overripened, the skin of all black currant berries softened.
This effect was the greatest for the cultivars ‘Pilėnai’ (3.8 times) and ‘Vyčiai’ (3.4
times) (Fig. 2).
In analyzing the change in ascorbic acid during berry ripening, we observed that
changes in ascorbic acid levels in the berries of various cultivars depended on both
the physiological properties of cultivars and berry ripeness. In all of the investigated
cultivars, greater amounts of ascorbic acid were present in the berries at the beginning
of ripening (Fig. 3). Significantly larger amounts of ascorbic acid were observed in
the pink berries of ‘Pilėnai’ (152.0 mg 100 g -1 ) and ‘Gagatai’ (147.0 mg 100 g -1 ).
During ripening in the berries of all cultivars, we observed a decrease in the amount
of ascorbic acid. When berries of the above-mentioned cultivars reached technical
maturity, ascorbic acid concentrations decreased by 20.7 % and 18.7 %, respectively.
The amounts of ascorbic acid in the berries of cultivars ‘Kriviai’ and ‘Vyčiai’ decreased
by only 0.85 % and 7.8 %, respectively. The ascorbic acid content of these cultivars
further decreased as they became overripe: ‘Kriviai’ berries decreased by 26.2 %, and
‘Vyčiai’ berries decreased by 23.5 %. The amount of ascorbic acid in the berries of the
‘Pilėnai’ and ‘Gagatai’ cultivars decreased from 13.8 % to 5.4 % (Fig. 3).
Fig. 3. Change in ascorbic acid (AA) and anthocyanin
(A) content in black currant berries during ripening
3 pav. Askorbo rūgšties (AR) ir antocianinų
(A) kitimo dinamika juodųjų serbentų uogose nokimo metu
During ripening, the amount of pigment in the berries noticeably increased,
and at technical maturity, there was significantly more pigment in cultivars ‘Kriviai’
(387.61 mg 100 g -1 ) and ‘Gagatai’ (349.79 mg 100 g -1 ) (Fig. 3). The berries of cultivars
‘Vyčiai’ and ‘Pilėnai’ accumulated from 246.85 to 267.87 mg 100 g -1 of anthocyanins.
It was observed that when berries were overripe, the dynamics of the pigments differed
238

among cultivars. In the berries of cultivars ‘Kriviai’ and ‘Gagatai,’ the quantity of
anthocyanins increased 16.2–12.6 % (up to 450.63 mg 100 g -1 and 393.91 mg 100 g -1 ,
respectively) to produce the highest values overall. In overripe berries of cultivars
‘Vyčių’ and ‘Pilėnų,’ the concentration of pigments decreased: in berries of ‘Vyčiai’
by 3 %, and in berries of ‘Pilėnai,’ by 34.5 % (Fig. 3).
The quantity of dry soluble solids in berries depended on the properties of the
cultivars and the extent of ripeness. During ripening, the amount of dry soluble solids
in berries of all cultivars increased. The largest amount of was established in overripe
berries of ‘Kriviai’ (16.95 %); the lowest amount was in berries of ‘Pilėnai’ (14.7 %)
(Table). The amounts accumulated in berries of cultivars ‘Vyčiai’ and ‘Gagatai’ differed
only slightly.
Table. Change of biochemical composition in black currant berries during
ripening
Lentelė. Biocheminės sudėties kitimas juodųjų serbentų uogose nokimo metu
239

Larger amounts of titratable acidity were established in berries of the investigated
cultivars at the beginning of ripening. Overripe berries have the lowest amount of
organic acids. The berries of cultivar ‘Pilėnai’ were distinguished by having the largest
amount of acids (2.41 %); the lowest amounts were in the berries of the cultivar
‘Gagatai’ (2.12 %) (table).
Overripe berries of the cultivar ‘Kriviai’ were notable for having the largest
amount of dry soluble solids – 20.45 %. Large amounts (19.3 %) also accumulated in
berries of the cultivar ‘Gagatai’ (Table). The lowest amount was found in the berries
of cultivar ‘Vyčiai’. When evaluating the properties of cultivars, we observed that
during the ripening of cultivar ‘Pilėnai’ the amount of dry soluble solids increased
most of all, by 15.5 %.
Discussion. Set berries are green. Their unpleasant taste is due to organic
acids and fermentation substances. Since they contain large amounts of insoluble
protopectins and starch, the berries are firm. We noticed that berry firmness depended
on the biological properties of cultivars. The black currant cultivars ‘Pilėnai’ and
‘Gagatai’ were distinguished for berry firmness. At the second berry ripening stage,
when morphological and biochemical changes took place, their concentration of
pectins decreased (during overripening, by as much as 51–57 %) and berries softened
(Максименко, 1997). We observed that among the investigated black currant cultivars,
the changes in berry skin firmness took place unevenly during ripening. At the stage
of 50 % ripeness, skin firmness of ‘Pilėnai’ and ‘Gagatai’ berries decreased by 45.8 %
and 56.2 % (Fig. 2). When technical maturity was reached, the softening of ‘Pilėnai’,
‘Gagatai’ and ‘Vyčiai’ berry skins occurred more quickly than in the cultivar ‘Kriviai’.
At this maturity stage, the cultivars ‘Pilėnai’ and ‘Kriviai’ were distinguished for berry
skin firmness. According to our data, when berries were overripe, skin softening took
place quickly.
Studies of the biochemical composition of black currant berries of various ripeness
are carried out in Lithuania and as well as in other countries. We observed that the
amount of accumulated substances and changes in those levels in berries depends more
on the biological properties of the cultivar than on the growth conditions (Brennan,
1996; Viola et al., 2000; Rubinskienė et al., 2006).
The data of the ascorbic acid change confirmed the earlier results obtained by us
and by other investigators. Larger amounts of vitamin C are present at the beginning
of berry ripening (Fig. 3) (Максименко, 1999; Rubinskienė, Viškelis, 2002).
The quantity of anthocyanins in berries was due to cultivar properties and to
the time of ripening. As noted in earlier years of investigation, according chemical
analyses, the berries of cultivars ‘Kriviai’ and ‘Gagatai’ were distinguished for having
the greatest accumulation of anthocyanins (Fig. 3) (Viškelis, Rubinskienė, Jasutienė,
2001). We observed that overripe berries accumulate greater amounts of anthocyanins
(Rubinskienė, 2004). The data showed that pigment concentration did not increase
in berries of all the cultivars. The hot weather of July accelerated berry ripening, and
abundant precipitation negatively influenced berry quality of the later black currant
cultivars. We observed that overripe berries of the cultivars ‘Pilėnai’ and ‘Vyčiai’ were
very soft (Fig. 2) and were of sour taste. We think that the process of fermentation
(which took place in overripe berries) influenced anthocyanin degradation, and this
was the reason why the concentration of these pigments decreased (Fig. 3).
240

Our results on the dynamics of the changes in titratable acidity and dry soluble
solids in berries during ripening do not contradict previously published data. Greater
amounts of dry soluble solids are found in overripe berries, and greater amounts of
acids are found at the beginning of ripening (Максименко, 1997; Rubinskienė et al.,
2006). At the second stage of ripening, when the amounts of anthocyanins and dry
soluble solids significantly increase, titratable acidity starts to decrease in berries
(Fig. 3, Table).
Conclusions. 1. At the stage of technical maturity, berries of the cultivars ‘Pilėnai’
and ‘Vyčiai’ were distinguished for the largest weights: 1.54 g and 1.48 g, respectively.
When berries became overripe, their weight decreased by between 2.9 % (‘Kriviai’)
to 38.3 % (‘Gagatai’).
2. During black currant ripening, skin firmness decreases. Overripe berries of
cultivars ‘Pilėnai’ and ‘Vyčiai’ soften most of all: 3.8 and 3.4 times. At the stage
of technical maturity, the berries of cultivars ‘Pilėnai’ (63.09 N cm -2 ) and ‘Kriviai’
(53.9 N cm -2 ) had firm skin.
3. Greater amounts of ascorbic acid were present in berries at the beginning of
ripening. Overripe berries were the least vitaminous. At various stages of maturity,
berries of the cultivars ‘Pilėnai’ and ‘Gagatai’ were notable for ascorbic acid content:
152–114 mg 100 g -1 and 147–103 mg 100 g -1 , respectively. With the exception of the
berries of cultivars ‘Vyčiai’ and ‘Pilėnai’, greater amounts of pigment accumulate in
overripe berries. Berries of technical maturity and overripe berries of cultivars ‘Kriviai’
and ‘Gagatai’ were observed to have significantly greater quantities of anthocyanin:
387.61–450.63 mg 100 g -1 and 349.79–393.91 mg 100 g -1 , respectively. Larger amounts
of dry soluble solids were found in overripe berries. Larger quantities were present
in berries of technical maturity and in overripe berries of cultivar ‘Kriviai’ (14.55–
16.95 %). During ripening of the berries of black currants, the amount of titratable
acidity decreases. Significantly greater amounts of acids were found in the berries of
cultivar ‘Pilėnai’ of varying maturity (3.27–2.41 %).
Acknowledgement. This work was partly supported by Lithuanian State Science
and Studies
References
Gauta 2008 03 24
Parengta spausdinti 2008 04 14
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breeding. Vol. 2. Small fruit and vine crops. New York, 191–295.
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241

4. Nes A. 1993. Evaluation of blackcurrant cultivars in Norway. Sixth International
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juodųjų serbentų uogose: daktaro disert. santr. Kaunas.
6. Rubinskienė M., Viškelis P. 2002. Accumulation of ascorbic acid and anthocyanins
in berries of Ribes nigrum. Botanica Lithuanica, 8(2): 139–144.
7. Rubinskienė M., Viškelis P., Jasutienė I., Duchovskis P., Bobinas Č. 2006. Change
of biologically active constituents in black currants during ripening. J. Fruit Ornam.
Plant Res., 14(2): 237–246.
8. Viola R., Brennan R., Davies H., Sommerville L. 2000. L-ascorbic acid
accumulation in berries of Ribes nigrum L. J. of Horticulture Science &
Biotechnology, 75: 409–412.
9. Viškelis P., Rubinskienė M., Jasutienė I. 2001. Influence of black currant pigments
on berry technological properties. Sodininkystė ir daržininkystė, 20(3)–1: 229–
239.
10. Wrolstad R. E. 1976. Color and pigment analyzes in fruit products. Station Bulletin
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11. Žurawicz E., Pluta S., Danek J. 2000. Small fruit breeding at the research institute
of pomology and floriculture in Skierniewice, Poland. Proceeding of the Eucarpia
symposium on fruit breeding and genetics. Acta Horticulturae, 538: 457–461.
12. Ермаков А. И., Арасимович В. В., Ярош Н. П., Перуанский Ю. В.,
Луковникова Г. А., Иконникова М. И. 1987. Методы биохимического
исследования растений (Под ред. А. И. Ермакова), Агропромиздат,
Ленинград.
13. Максименко М. 1997. Аккумуляция питательных веществ в ягодах смородины
черной в процессе созревания. Состояние и проблемы садоводства России,
2: 207–210.
14. Максименко М. 1999. Динамика витамина С и его форм в процессе
созревания и технологической обработки смородины черной. Плодоводство,
12: 155–159.
SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Juodųjų serbentų uogų kokybės pokyčiai nokimo metu
M. Rubinskienė, P. Viškelis, V. Stanys, T. Šikšnianas, A. Sasnauskas
Santrauka
Lietuvos sodininkystės ir daržininkystės institute tirtos įvairios brandos juodųjų serbentų
‘Pilėnai’, ‘Vyčiai’, ‘Kriviai’ ir ‘Gagatai’ uogos. Didžiausia mase pasižymėjo techninę brandą
pasiekusios ‘Pilėnų’ (1,54 g) ir ‘Vyčių’ (1,48 g) juodųjų serbentų uogos. Uogoms pernokstant
jų masė sumažėja nuo 2,9 % (‘Kriviai’) iki 38,3 % (‘Gagatai’). Juodųjų serbentų nokimo metu
uogų odelės tvirtumas mažėja. Ženkliausiai suminkštėja persirpusios ‘Pilėnų’ (3,8 karto) ir
‘Vyčių’ (3,4 karto) uogos. Techninėje brandoje tvirta odele išsiskiria ‘Pilėnų’ (63,09 N cm -2 ) ir
‘Krivių’ (53,9 N cm -2 ) uogos. Didesni askorbo rūgšties kiekiai nustatyti uogose nokimo pradžioje.
242

Mažiausiu vitaminingumu pasižymi persirpusios uogos. Tarp veislių askorbo rūgšties kiekiu
išsiskiria įvairios brandos ‘Pilėnų’ (152–114 mg 100 g -1 ) ir ‘Gagatų’ (147–103 mg 100 g -1 )
uogos. Išskyrus ‘Vyčių’ ir ‘Pilėnų’ serbentus, gausesni pigmentų kiekiai susikaupia persirpusiose
uogose. Tarp veislių patikimai didesniu antocianinų kiekiu pasižymi techninės brandos ir
persirpusios ‘Krivių’ (387,61–450,63 mg 100 g -1 ) bei ‘Gagatų’(349,79–393,91 mg 100 g -1 )
uogos. Didesni tirpių sausųjų medžiagų kiekiai yra persirpusiose uogose. Daugiau jų nustatyta
techninės brandos ir persirpusiose ‘Krivių’ (14,55–16,95 %) uogose. Nokimo metu juodųjų
serbentų uogose sumažėja titruojamojo rūgštingumo kiekis. Patikimai didesni rūgščių kiekiai
rasti įvairios brandos ‘Pilėnų’ uogose (3,27–2,41 %).
Reikšminiai žodžiai: cheminė sudėtis, odelės tvirtumas, uogos masė, veislė.
243

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
The influence of fertilizers with nitrification inhibitor on
edible carrot photosynthesis parameters and productivity
Ona Bundinienė, Pavelas Duchovskis, Aušra Brazaitytė
Lithuanian Institute of Horticulture, Kauno 30, 54333 Babtai, Kaunas distr.,
Lithuania, e-mail: o.bundiniene@lsdi.lt
In 2005–2006 in the experimental field of the Lithuanian Institute of Horticulture,
in the IDg8-k / Calc(ar)i- Epihypogleyc Luvisols – LVg-p-w-cc it was investigated
the influence of fertilizers with nitrification inhibitor DMPP on the photosynthesis
parameters of carrot cultivar ‘Samson’ (Daucus sativus Röhl.) and their relation with
productivity. Sowing rate was 0.8 mln. unt. ha -1 of shoot seeds, sowing scheme –
62 + 8 cm. It was established that fertilizers with nitrification inhibitor DMPP (Entec-
Avant 12 7 16 + microelements + DMPP) increased assimilation area of carrot leaves
and crop and photosynthesis potential. The biggest total (70.1 t ha -1 ) and standard (49.7 t ha -1 )
carrot root-crop yield and the best standard yield output (70.7 %) were obtained applying
fertilizers with nitrification inhibitor. Standard carrot yield increased, when plant (r = 0.88)
and crop (r = 0.92) assimilation area and photosynthesis potential (correspondingly plant’s
photosynthesis potential r = 0.74, crop’s r = 0.91) increased, and the increasement of the net
photosynthesis productivity had negative (though not big) influence (plant’s photosynthesis
potential r = -0.4, crop’s r = -0.23) on productivity.
Key words: assimilation area, photosynthesis potential, photosynthesis, fertilizers with
nitrification inhibitor, edible carrot.
Introduction. The size of yield and crop productivity depend on plant
photosynthesis and breathing intensity, assimilation area, photosynthesis potential,
agroclimatic conditions and other factors (Третьяков; 1998, Romaneckas et al.,
2001). The relations of photosynthesis and yield are very complicate and often reveal
contradictions between plant needs and the purposes of a man as producer. Nevertheless,
all the means of photosynthesis increasement (optimization of light, humidity, crop
density, decrease of weediness and especially improvement of nutrition conditions,
i. e. balanced fertilization with mineral fertilizers) guarantee the increase of plant
production (Гриценко, Долгодворов, 1986; Чихов, 1997; Duchovskis, 2000; Velička
et al., 2007). When fertilizing with the optimal selected norms, nutrients are assimilated
more productively. Nitrogen is the most important among them. Nitrogen, especially
its nitric form, is very agile and easily washable element, therefore under unfavourable
meteorological conditions, its big losses are possible (Liet. dirvož. ..., 1998). The new
fertilizer with nitrification inhibitor (DMPP – 3.4 dimetilpyrazilphospfate, market
title – ENTEC) inhibits ammonium nitrogen turning into nitric nitrogen and in such
a way decreases the losses of its washing out (Hähndel, Zerulla, 2001), but does not
influence soil biological activity (Zerulla et al., 2001). Under the different temperature
245

and moisture conditions, more than 80 % of DMPP remained within the 0-to 5-mm
region around the granule, from 5 % to 15 % of DMPP were found in the 5- to 20-
mm region (Azam et al., 2001). Results show that DMPP may increase the mean crop
yield (grain yield by 0.24–0.29 t ha -1 , tuber yield potatoes by 1.9 t ha -1 , sugar beets
+ 0.24 t ha -1 and corrected sugar yield, biomass of carrots by 4.9 t ha -1 , onions – 1.9 t ha -1 ,
radish – 4.6 t ha -1 , lettuce – 1.4 t ha -1 , lambs lettuce – 1.9 t ha -1 , leek – 1.7 t ha -1 and
improve crop quality (e. g., reduced nitrate concentration in leafy vegetables). The
positive effects were especially pronounced at sites with a high precipitation rate or
intensive irrigation, and on light sandy soil (Pasda et al., 2001).
Different plants because of their uneven biological and morphological properties
distinguished themselves with uneven parameters of photosynthesis indices, which
optimization directly determines economical productivity (Suojala, 2000; Duchowski,
Brazaitytė, 2001). The increase of plant biomass during organogenesis directly depends
on the absorbed amount of FAR, which most often is determined by leaf area, their
position in space and the angle of turning to the sun (Guiducci et al., 1992; Duchowski,
Brazaitytė, 2001). The bigger coefficient of FAR using, the bigger is biomass yield
(Гриценко, Долгодворов, 1986). The parameters of crop photosynthesis are strongly
influenced by meteorological conditions and anthropogenic environment factors,
which dynamic depends on geographic latitude, local agroclimatic conditions and
the degree of environmental pollution (Sinclair, Horie, 1989; Gibberd et al., 2000;
Šiaudinis, Lazauskas, 2006).
The aim of the study is to establish the influence of fertilizer with nitrification
inhibitor DMPP (market title – Entec-Avant) on the parameters of photosynthesis of
edible carrot (Daucus sativus Röhl.) and their influence on productivity in comparison
with the effect of the other investigated fertilizers.
Object, method and conditions. In 2005–2006 experiments were carried out in the
Lithuanian Institute of Horticulture, in the IDg8-k / Calc(ar)i- Epihypogleyc Luvisols –
LVg-p-w-cc (Buivydaitė et al., 2001). The ploughing layer had little humus (1.53 %),
much phosphorus (341.5 mg kg -1 ), average amount of potassium (161 mg kg -1 ), also –
calcium and magnesium (respectively – 10 850 and 1 995 mg kg -1 ). In spring in the
ploughing and under ploughing layers it was found 58.7 kg ha -1 of mineral nitrogen
(little amount). Soil – pH – 7.55 (alkaline) (Liet. dirvož. ....., 1998).
Carrots of cultivar ‘Samson’ were grown on furrowed surface, sowing approximately
800 thousand germinable seeds per hectare. Sowing scheme – 62 + 8 cm (in the belt of
8 cm width seeds were sown in all directions). Carrot preplant was cabbage. The jobs
of vegetable supervision were carried out according to the technologies, accepted at the
LIH. Plants were fertilized with the rates and fertilizers indicated in the experimental
scheme only before sowing. Experiments were carried out according to the scheme:
1. Without fertilizers (N 0
P 0
K 0
) – control / Be trąšų – kontrolė
2. Monomial fertilizer, according to the data agrochemical characteristics /
Vienanarės trąšos, remiantis agrocheminių dirvožemio tyrimų duomenimis
3. Cropcare 10 10 20, using 600 kg ha -1 fertilizer / Cropcare 10 10 20, išberiant
600 kg ha -1 trąšos (N 60
P 60
K 120
)
246

4. F e r t i l i z e r w i t h n i t r i f i c a t i o n i n h i b i t o r E n t e c - Av a n t
N 12
P 7
K 16 +
microelements + DMPP, using 500 kg ha -1 fertilizer / trаša su nitrifikacijos
inhibitoriumi Entec-Avant N 12
P 7
K 16
+ mikroelementai + DMPP, išberiant 500 kg ha -1 trąšos
(N 60
P 35
K 80
).
In the second variant carrots before the sowing were fertilized with ammonium
saltpetre (34 % N), granuled superphosphate (20 % P 2
O 5
) and potassium magnesia
(30 % K 2
O); in the third variant – with the complex fertilizer with microelements
Cropcare 10 10 20 + MgO(4.15 %) + S(11 %) + microelements (B, Cu, Fe, Mn, Mo,
Zn, Se). In the fourth variant there was used fertilizer with nitrification inhibitor DMPP
(market title Entec-Avant). The fertilizer contains 12 % N, 7 % P 2
O 5
, 16 % K 2
O, 4 %
MgO, 5 % S, 0.02 % B, 0.01 % Zn, 0.8 % DMPP.
Initial area of field – 15 m 2 (length – 5 m, width – 3 m), record field 4.2 m 2 (length –
3 m, width – 1.4 m). Experiment variants were carried out in four replications.
Leaf assimilation area was measured for three times per vegetation: I – at 6–10
leaves stage (early growth, or 57–62 days after sowing, 2005-07-12, 2006-06-30);
II – the end of intensive leaf growth period (28–32 days after the first measurement
or 81–90 days after sowing 2005-08-09, 2006-07-26) and III – the end of intensive
root-crop growth period (28–32 days after the second measurement or 109–139 days
after sowing, 2005-09-08, 2006-08-26). 5 plants from each variant were taken and
the measurements were carried out. Analogically there were calculated the other
photosynthesis parameters (net plant and crop photosynthesis productivity, plant and
crop photosynthesis potential).
Leaf assimilation area was measured with the automat measurer (WinDias). Dry
matter were established gravimetrically, after drying at the temperature of 105 ± 2 °C
up to the unchangeable mass (Manuals, 1986). Crop assimilation area was calculated
by multiplying plant leaf assimilation area and crop density. Net photosynthesis
productivity was calculated according to the formula 1 (Третьяков, 1998):
F p
= M 2
-M 1
/ 1 / 2
(L 1
+ L 2
)T (1)
Here:
F p
– photosynthesis productivity, mg cm -2 per 24 h;
M 2
– M 1
– increase of dry weight during the period of investigation (mg);
L 1
+ L 2
– leaf area in the beginning of the period and at the end (cm 2 );
T – duration of the period, in 24 h.
Plant photosynthesis potential was calculated by summing leaf area of every 24 h
and crop area – taking into account crop density. The investigations of photosynthesis
parameters were carried out in the Laboratory of Plant Physiology at the Lithuanian
Institute of Horticulture.
There were established in soil samples: pH KCl
– by potentiometrical
(ISO 10390:2005), humus (%) – by dry burning (ISO 10694:1995), agile P 2
O 5
and
K 2
O (mg kg -1 )– by Egner-Rimo-Domingo (A-L, Gost 26208-84), mineral nitrogen
(mg kg -1 ) – by yonmetrical, calcium and magnesium (mg kg -1 ) – by atomic absorption
spectrometrical (SVP D-06) methods. Analyses were carried out in the Center of
Agrochemical investigations at the LIH.
Vegetables were gathered at the stage of technical maturity.
Data significance was evaluated by the method of one-factor dispersion analysis,
applying the program ANOVA, the relation among the different parameters – by
247

correlation regression analysis, the character and force of the interrelation – by
statistical analysis of the coefficients of the ways, applying the program STAT_ENG
(Tarakanovas, Raudonius, 2003).
M e t e o r o l o g i c a l c o n d i t i o n s. In 2005 May, July and August were
cooler and more humid, especially August, than the multiannual averages (Table).
In 2006 air temperature in the first two months was similar to the multiannual, but
during both months there fell averagely 42 % of multiannual precipitation rate, and
August was warmer, but very rainy (there was precipitation for 2.3 times more than the
multiannual average). July in both years of experiment was hot (air temperature 1.5 °C
was higher than the multiannual average) and very dry, especially in 2005, when only
5.3 % of multiannual month rate fell. September in both years of experiment also was
warmer, but in 2005 – dry (precipitation comprised 67 % of multiannual precipitation
rate), and 2006 – rainy (it rained for 2.8 times more than multiannual parameters).
Plant vegetation period in 2005 was warm and equaled to the multiannual average.
Plant vegetation period in 2006 was warmer and more humid than the multiannual
average. The average air temperature was 1.3 °C higher than multiannual average, and
precipitation was 8.5 mm more than multiannual average (Table).
Table. Meteorological conditions during plant vegetation
Lentelė. Meteorologinės sąlygos vegetacijos metu
Kaunas Meteorological Station, 2005–2006
Kauno meteorologinė stotis, 2005–2006 m.
Results. The formation of edible carrot plant assimilation area in different growth
periods took place unevenly, but fertilizing with mineral fertilizers increased. During
the period from sowing up to I measurement (early growth, the end of June – the
beginning of July, or 57–62 days after sowing) carrot leaf assimilation area, growing
them without fertilizers, was 82.1 cm 2 and under the influence of fertilizers it increased
10.5–50.7 cm 2 (Fig. 1). The biggest plant leaf assimilation area was fertilizing with
the fertilizer with nitrification inhibitor (Entec 12 7 16, 500 kg ha -1 ). At the end of
the intensive leaf growing period (II measurement, the end of July – the beginning of
Angust, or 81–91 day after sowing), leaf assimilation area of carrots grown without
248

fertilizers in comparison with the I measurement increased 2.4 and this of fertilized
ones – 2.5–3.3 times.
Fig. 1. Plant and crop assimilation area in carrot crop
of different fertilizing, 2005–2006.
1 pav. Augalo ir pasėlio asimiliacijos plotas skirtingo
tręšimo morkų pasėlyje, 2005–2006 m.
Fertilized plants, in spite of not fully suitable meteorological conditions, grew
better. Fertilizers increased carrot leaf assimilation area 1.6–1.7 times. The leaves of
carrots fertilized with monomial fertilizers and fertilizers with nitrification inhibitor
grew most quickly, but there weren’t significant differences between leaf assimilation
areas of plants fertilized with different fertilizers. At the end of intensive root-crop
growth period (III measurement, the end of August – the first half of September, or
109–139 days from sowing) leaf assimilation area in comparison with II measurement
decreased and this decrease in fertilized carrots was bigger (97.4–110.5 cm 2 ) than
in these grown without fertilizers (7.4 cm 2 ) (Fig. 1). Probably under the sufficient
amount of humidity plants recovered after hot and dry weather in July of both years
of investigation (especially in 2005) grew intensively and used soil nutrients, and
the fertilized plants intensively accumulated nutrients in root-crop and some leaves
already were wilted.
When plant leaf assimilation area increased, crop assimilation area also increased.
The biggest crop assimilation area (on the average 17.3 m 2 ha -1 ) was at the end of
intensive leaf growth period (II measurement), and measuring at the end of root-crop
growth (III measurement) this area was on the average 2.2 m 2 ha -1 smaller. During all
the measurements, fertilizing with all the investigated fertilizers crop assimilation area
was bigger than this of carrots grown without fertilizers (Fig. 1).
The biggest amount of dry matter was accumulated at the end of intensive rootcrop
growth period (III measurement). During all the measurements, the amount of
dry matter fertilizing carrots was bigger than this of carrots grown without fertilizers,
249

ut the essential differences weren’t found.
Meteorological conditions (humidity and temperature) of the different experimental
years influenced the increase of plant assimilation area: when temperature increased,
assimilation area decreased (2) and when the amount of precipitation increased,
assimilation area increased also (3):
Y = 1307.51 – 85.57 x, r = -0.94 ± 0.14, t = 4.41, F t
= 4.77** (2)
Y = 885.77 – 13.98 x + 0.003 x 2 , R = 0.823, F t
= 23.04** (3)
Used symbols / Sutartiniai ženklai:
r – coefficient of correlation / koreliacijos koeficientas
R – proportion of correlation / koreliacinis santykis
t – statistic derived in student t-test / studento t-testo kriterijus (skirtumo patikimumo
kriterijus)
F t
– variant ratio (F test
) / Fiрerio kriterijus
R 05
– LSD – least significant difference / mažiausias esminis skirtumas
* – data significant at P ≤ 0.05 probability level / patikimumas, esant 95 % tikimybės
lygiui
** – at P ≤ 0.01 probability level / patikimumas, esant 99 % tikimybės lygiui.
Duncan’s multiple range test, means followed by the same letter are not different
significantly at p < 0,05 / Tarp vidurkių pažymėtų ta pačia raide, pagal Dunkano kriterijų,
skirtumai neesminiai 95 % tikimybės lygiui
When assimilation area increased in the periods of early growth (from sowing
up to 6–10 leaves) and intensive leaf growth periods, the amounts of dry matter also
increased (r = 0.99 ir r = 0.97), and before the yield gathering, when leaf assimilation
area decreased, the amounts of dry matter also decreased (r = -0.64), but increased
carrot yield: assimilation area influenced the increase of total yield 31 % (r = 0.82),
the increase of the standard one – 32 % (r = 0.88).
The influence of environmental factors and technological means on photosynthesis
best of all is reflected by plant net photosynthesis productivity, which is expressed by
the ratio of dry weight increase during certain time and leaf assimilation area during
the same period. Plant photosynthesis productivity in the early carrot growth period
(from sowing up to 6–10 leaves) on the average was 0.37 mg cm -2 in 24 h, crop
photosynthesis productivity – 30.0 mg m -2 in 24 h (Fig. 2). The biggest carrot plant
photosynthesis productivity was fertilizing with monomial fertilizers, but essential
differences among plants fertilized with different fertilizers weren’t found. The biggest
crop net photosynthesis productivity was applying fertilizer with nitrification inhibitor
(Fig. 2).
During the period from sowing up till the end of intensive leaf growth (81–90 days
after sowing) plant photosynthesis productivity on the average was 0.55 mg cm 2 in
24 h and the biggest one – applying fertilizer with nitrification inhibitor, but essential
differences among plants fertilized with different fertilizers weren’t found. The net
photosynthesis productivity increase, in comparison with I measurement, increased
0.09–0.25 mg cm -2 in 24 h and it was the biggest one applying fertilizer with nitrification
inhibitor. Net photosynthesis productivity during the mentioned period on the average
was 60.5 mg m -2 in 24 h and it was the biggest fertilizing with monomial fertilizers
(Fig. 2).
250

Fig. 2. Parameters of the net photosynthesis productivity in the carrot crop of
different fertilizing, 2005–2006
2 pav. Grynojo fotosintezės produktyvumo rodikliai skirtingo tręšimo morkų pasėlyje,
2005–2006 m.
Plant and crop net photosynthesis productivity increased most intensively from the
end of the intensive leaf growth up till the end of intensive root-crop growth (between
II and III measurements, or 81–90, 109–139 days after sowing). Plant photosynthesis
productivity in comparison with II measurement at the end of July – in the beginning of
August on the average increased 0.62 mg cm -2 in 24 h or 2.2 times, crop photosynthesis
productivity increased 27.6 mg m -2 in 24 h or 1.8 times. The biggest increase of plant
net photosynthesis productivity was applying fertilizer with nitrification inhibitor
(1.17 mg cm -2 in 24 h), and this one of crop’s – applying Cropcare 10 10 20 (29.4 mg m -2
in 24 h). Crop net photosynthesis productivity applying fertilizer with nitrification
inhibitor increased 18.2 mg m -2 in 24 h (Fig. 2).
During the early (6–10 leaves) period of edible carrot growth (I measurement),
when plant assimilation area increased, plant net photosynthesis productivity increased
also (4). When the measurements were carries out at the end of intensive leaf growth
(II measurement), there weren’t dependency, and at the end of intensive root-crop
growth (III measurement), i. e., before yield gathering, photosynthesis productivity
even decreased, because when plants get older and leaves wilt, leaf assimilation area
decreased also (5):
Y = -135.16 + 64.24 x, r = 0.90 ± 0.18, t= 3.35, F t
= 25.15** (4)
Y = 281.551 - 5.67 x, r =-0.74 ± 0.28, t = 1.60, F t
= 7.2* (5)
Plant photosynthesis potential depended on leaf assimilation area and plant age,
and crop photosynthesis potential was influenced also by crop density. Fertilization with
all the investigated fertilizers increased plant and crop photosynthesis potential (Fig. 3).
In the early carrot growth period (from sowing up till 6–10 leaves) the biggest plant
and crop photosynthesis potentials were applying fertilizer with nitrification inhibitor.
In comparison with the mentioned parameters of carrot grown without fertilizers, plant
251

photosynthesis potential increased 1.6, crop’s – even 7.5 times. Most intensively plant
and crop photosynthesis potentials increased in the period of intensive carrot leaves
growth (from I up till II measurement). Plant photosynthesis potential in comparison
with I measurement on the average increased 4.2, crop’s – 1.6 times. Most intensively
plant photosynthesis potentials increased fertilizing carrots with Cropcare 10 10 20,
crop’s – fertilizing with monomial fertilizers. During the intensive root-crop growth, the
increased of plant and crop photosynthesis potential in comparison with II measurement
was insignificant (Fig. 3).
Fig. 3. Parameters of the photosynthesis potential in the carrot crop of different
fertilizing, 2005–2006
3 pav. Fotosintezės potencialo rodikliai skirtingo tręšimo morkų pasėlyje, 2005–2006 m.
Plant photosynthesis potential in the early period of plant growth (from sowing up
till 6–10 th leaf) and during intensive leaf growth directly and strongly depended on leaf
assimilation area (correspondingly r = 0.99; r = 0.96). In the last period, i. e., during
intensive leaf growth, dependency was averagely strong (r = 0.60). Plant assimilation
area influenced crop photosynthesis potential both in the early (I measurement, r = 0.88)
period and during the intensive root-crop growth (III measurement r = 0.90), but didn’t
have influence in the period of intensive leaf growth (II measurement) (Fig. 3).
Carrot root-crop total yield because of fertilizers increased on the average
8.2 t ha -1 , marketable – 4.9 t ha -1 (Fig. 4). The biggest total and marketable carrot
root-crop yields were obtained applying fertilizer with nitrification inhibitor (Entec-
Avant 12 7 16 + microelements + DMPP). Total yield in comparison with the yield
of carrots grown without fertilizers increased 10.3 t ha -1 , or 17.2 %; the marketable
one – 7.5 t ha -1 , or 17.8 %, but the output of marketable yield in comparison with this of
carrots grown without fertilizers increased only slightly, and because of the fertilizing
with Cropcare 10 10 20 and monomial fertilizers – even decreased (Fig. 4). Probably
rather rich in nutrients soil guaranteed suitable nutrition conditions, ant the application
of fertilizers increased only non-marketable yield.
252

Fig. 4. Influence fertilizer with nitrification inhibitor on the productivity of carrot,
2005–2006
4 pav. Trąšų su amonio stabilizatoriumi įtaka morkų produktyvumui, 2005–2006 m.
The path coefficient analysis showed that there were close relation between carrot
productivity (total and marketable yield) and photosynthesis parameters at the end of
root-crop growth (III measurement) (Fig. 5).
Fig. 5. Influence of different photosynthesis parameters on carrot productivity: a)
total productivity, b) standard productivity, 2005–2006
5 pav. Fotosintezės rodiklių įtaka morkų produktyvumui: a) suminis derlius, b) standartinis
derlius, 2005–2006 m.
253

There was investigated the influence of plant and carrot crop assimilation area, net
photosynthesis productivity and photosynthesis potential on total and marketable yield.
When plant and crop assimilation area (correspondingly plants r = 0.82 and r = 0.88,
crops r = 0.81 and r = 0.92) and photosynthesis potential (correspondingly plants
photosynthesis potential r = 0.71 and r = 0.74, crops – r = 0.79 and r = 0.91) increased,
total and marketable carrot yield increased also. The increase of photosynthesis
productivity had small or very small but negative influence (correspondingly plant’s
photosynthesis productivity r = -0.44 and r = -0.54, crops – r = -0.11 and r = -0.23).
Photosynthesis parameters of the first and two measurements had smaller influence
on the yield.
Discussion. Plant genetic properties most of all determined the formation of one
plant leaf assimilation area. The increased of red beet ‘Pablo’ F 1
leaf assimilation
area were observed at the end of July, and later on leaf area decreased (Tarvydienė
et al., 2004). In the beginning of plant vegetation, assimilation area increases slowly,
its activity isn’t effective, and at the end of vegetation crop photosynthesis decreases
because the leaves get old and wilt (Ничипорович, 1987). Our investigations with
carrots showed that the biggest leaf assimilation area increase is in the period of
intensive leaf growth, i. e., at the end of July – in the beginning of August. At the end
of intensive leaf growth (July – the beginning of August, 81–91 days after sowing),
leaf assimilation area of carrots grown without fertilizers increased in comparison
with I measurement 2.4 times, and this of fertilized carrots – 2.5–3.3 times. Fertilizers
increased carrot leaf assimilation area 1.6–1.7 times. The leaves of carrots, fertilized
with monomial fertilizers and fertilizers with nitrification inhibitor, grew most quickly,
but there weren’t essential differences among leaf assimilation areas fertilized with
different fertilizers. When plant leaf assimilation area increased, crop assimilation
area increased also.
According to the data of LAA investigations, nitrogen had the biggest positive
influence on the increase of dry weight and leaf parameters, while dry periods had the
biggest negative influence (Šiaudinis, Lazauskas, 2006). The data of our investigations
showed that the increase of temperature negatively influenced assimilation area, and
the increase of precipitation – positively.
Assimilation area very influences productivity (Romaneckas, 2001). During
intensive plant vegetation, in July– August, when leaf assimilation area is the biggest,
plants accumulate be biggest amount of photosyntheticaly active radiation (FAR);
photosynthesis and metabolism of organic substances is faster (Petronienė, 2000,
Tarvydienė, 2004). Assimilation area in July– August determined 52–72 % of red beet
root-crop yield (Petronienė, 2000). During our investigations, before harvesting, when
assimilation area decreased, the amounts of dry matter decreased also (r = -0.64), but
red beet yield increased: assimilation area influenced the increase of total yield 31 %
(r = 0.82), the increase of marketable yield – 32 % (r = 0.88).
One of the most important photosynthesis parameters, net photosynthesis
productivity, formed similarly as assimilation area, but its dynamic was more determined
by meteorological conditions and plant genetype. At the first ontogenesis stages net
photosynthesis productivity increased together with the increase of leaf assimilation
area (Tarvydienė et al., 2004). During our investigations, in the early carrot growth
254

period (from sowing up till 6–10 leaves, I measurement, the end of July–beginning of
June), when plant assimilation area increased, plant net photosynthesis productivity
increased also (r = 0.90), and during root-crop formation photosynthesis productivity
even decreased (r = -0.74).
Plant photosynthesis potential directly depended on leaf assimilation area and
crop density (Velička, 2007). Fertilizers changed plant photosynthesis potential
(Tarvydienė et al., 2004). During our investigations all the applied fertilizers increased
plant and crop photosynthesis potential (Fig. 3). Plant photosynthesis potential during
the early period of plant growth (I measurement, from sowing up till 6–10 leaves)
and intensive leaf growth period (II measurement) directly and strongly depended on
leaf assimilation area (correspondingly r = 0.99; r = 0.96). During the intensive rootcrop
growth, the dependency was averagely strong (r = 0.60). Plant assimilation area
influenced crop photosynthesis potential in early (r = 0.88) and intensive root-crop
growth periods (r = 0.90), but didn’t have influence in intensive leaf growth period
(III measurement).
All the photosynthesis parameters investigated during III measurement, influenced
carrot root-crop productivity (Fig. 5). Total carrot root-crop yield increased, when plant
and crop assimilation area (correspondingly r = 0.82 and r = 0.81) and photosynthesis
potential (correspondingly r = 0.71 and 0.79) increased, and photosynthesis productivity
had slightly negative influence (correspondingly r = -0.44 and r = -0.11). The
increase of plant and crop assimilation area (correspondingly r = 0.88 and r = 0.92)
and photosynthesis potential (correspondingly r = 0.74 and r = 0.91) had strong
positive influence on the increase of marketable yield; the increase of plant and crop
photosynthesis productivity had small negative influence (correspondingly r = -0.54
and r = -0.23).
Conclusions. 1. Fertilizers with nitrification inhibitor DMPP (Entec-Avant
12 7 16 + microelements + DMPP) increased carrot leaves and crop assimilation
area.
2. Fertilizers with nitrification inhibitor increased plant and crop net photosynthesis
productivity and plant and crop photosynthesis potential.
3. The biggest total (70.1 t ha -1 ) and standard (49.7 t ha -1 ) carrot root-crop yields
and the best output (70.7 %) of standard yield were obtained applying fertilizers with
nitrification inhibitor DMPP.
4. When plant and crop assimilation area and photosynthesis potential increased,
the productivity of carrot also increased, but the increase of photosynthesis productivity
had small or very small, but negative influence.
Acknowledgements. This study is supported by Lithuanian State Sciences and
Studies Foundation.
Gauta 2008 04 15
Parengta spausdinti 2008 04 25
255

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SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Trąšų su nitrifikacijos inhibitoriumi įtaka valgomosios morkos
fotosintezės rodikliams ir produktyvumui
O. Bundinienė, P. Duchovskis, A. Brazaitytė
Santrauka
2005–2006 m. Lietuvos Sodininkysts ir daržininkystės instituto bandymų lauke,
priesmėlio ant lengvo priemolio karbonatingajame sekliai glėjiškame išplautžemyje
(IDg8-k / Calc(ar)i- Epihypogleyc Luvisols – LVg-p-w-cc)) tirta trąšų su nitrifikacijos
inhibitoriumi DMPP įtaka ‘Samson’ veislės morkos (Daucus sativus Röhl.) fotosintezės
rodikliams ir jų sаryšis su produktyvumu. Sėjos norma – 0,8 mln. vnt. ha -1 daigių sėklų, sėjos
schema – 62 + 8 cm. Nustatyta, kad trąšos su nitrifikacijos inhibitoriumi DMPP (Entec-Avant
12 7 16 + mikroelementai + DMPP) padidino morkos lapų ir pasėlio asimiliacijos plotą bei
fotosintezės potencialą. Didžiausias suminis (70,1 t ha -1 ) ir standartinis (49,7 t ha -1 ) morkų
šakniavaisių derliai bei geriausia standartinio derliaus išeiga (70,7 %) gauti tręšiant trąša su
nitrifikacijos inhibitoriumi. Standartinis morkų derlius didėjo augant augalo (r = 0,88) ir pasėlio
(r = 0,92) asimiliacijos plotui, bei fotosintezės potencialui (atitinkamai augalo fotosintezės
potencialas r = 0,74, pasėlio – r = 0,91), o grynojo fotosintezės produktyvumo didėjimas turėjo
nors ir nedidelę, tačiau neigiamа įtaką (augalo fotosintezės produktyvumas r = -0,54, pasėlio –
r = -0,23) produktyvumui.
Reikšminiai žodžiai: asimiliacijos plotas, fotosintezės potencialas, fotosintezė, trąša su
nitrifikacijos inhibitoriumi, valgomoji morka.
257

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
The effect of additional fertilization with liquid complex
fertilizers and growth regulators on potato productivity
Elena Jakienė 1 , Virginijus Venskutonis 1 , Vytautas Mickevičius 2
1
Lithuanian University of Agriculture, Studentų 11, Akademija, LT-53067,
Kaunas distr., Lithuania, e-mail: Elena.Jakiene@lzuu.lt
2
Kaunas University of Technology, Radvilėnų pl. 19, LT-50270, Kaunas, Lithuania,
e-mail: Vytautas.Mickevičius@ctf.ktu.lt
Field tests were carried out at the Research Station of LUA during the period of
2004–2006. The effect of complex fertilizers and growth regulators on potato growth, tuber
development and productivity was investigated.
It was determined that the greatest addition to the potato yield (8–9 t ha -1 ) was produced and
approximately 60 % of bulky potatoes per one plant grew after additional foliar fertilization at the
end of bud stage with liquid complex fertilizers Atgaiva-2 or Atgaiva-P from “ARVI fertis”.
After spraying the potatoes with solutions of growth regulators Penergetic-P lapams
or Stilitas-
123, bulky potatoes constituted 55–58 % of yield structure. Under the influence of the above
mentioned growth regulators, potato yield significantly increased by 5–6 t ha -1 or 16–19 % in
comparison with the control variants.
It is advisable to fertilize table potatoes additionally with the blend of complex fertilizer
Atgaiva-2 and growth regulator Humicop. After additional foliar fertilization with this blend,
bulky potatoes constituted even 78 % of yield structure, while small potatoes were almost not
found (only 0.5 %) at all. Under the influence of the aforesaid additional fertilization, potato
yield reliably increased by 4.7 t ha -1 or 14.8 % in comparison with the control variants.
Key words: potatoes, liquid complex fertilizers, growth regulators, productivity.
Introduction. In the current market, consumers demand both rich and top quality
potato harvest. Productive and high quality potato seed is the first essential provision to
get rich and steady potato yield (Department of Statistics of the Republic of Lithuania).
In addition to agrotechnical measures, significance of exogenous growth regulators with
regard to development of vegetal productivity has been continuously growing. Growth
regulators are synthetic compounds, physiological analogues of natural phytohormones
that control the processes of growth and development as well as strengthen an immune
system of the plant. These substances cannot be replaced by watering or fertilixing
plants with microelement fertilizers (Венскутонене et al., 2004). Root development,
stem growth, blooming time or plant maturity can be stimulated subject to the usage
conditions of growth regulators, concentration thereof and physiological state of the
plant itself. In this way, growth regulators may alter the speed of ontogenesis, however,
the direction of ontogenesis remains unchanged since it is determined by genetic
information. Physiological activity of phytoregulators is expressed by their ability to
affect a particular component of a phytohormone system: to increase the amount of a
259

particular phytohormone through introduction of a physiological analogue of a plant
into its organism, to stimulate or inhibit biosynthesis of phytohormones, to block the
movement of phytohormones inside the plant, etc. (Меркис et al., 1994).
The activity of growth regulators mostly depends on the time of their application,
i. e. on the stage of plant growth and development, on the ability of plant tissues to
receive an external signal and integrate it both into the ways of a receptor system
of phytohormones and further transduction ways in order to induce the growth and
development of a separate part (Darginavičienė et al., 2002). Appropriate application
of growth regulators in potato growing technologies enable to control the time of
stolone formation, to effect their growth intensity, the process of tuber formation and
transportation of assimilants to tubers, as well as to increase starchiness of potato
tubers and productivity thereof (Венскутонене et al., 2004).
After spraying potato tubers with growth regulator stilite solutions, they sprout
faster and grow more intensely, the greater number of eyes form sprouts, the number
of stems of one plant increases. After moistening the seed tubers with stilite solutions
of 90 mg l -1 concentration, the significant yield growth amounts to 1.6–2.3 t ha -1 . The
tests carried out on the basis of many years have demonstrated that after spraying
potato plants with stilite solutions during the bud stage, productivity increases by
approximately 2–2.5 t ha -1 in comparison with the control tubers not treated with
growth regulators. Growth regulators may be sprayed onto the crops together with
liquid complex fertilizers. This provides new possibilities for control of potato growth
and productivity thereof (Jakienė, 2006; Makaravičiūtė, 2003).
The aim of the field tests is to determine the effect of growth regulators and
liquid complex fertilizers on formation of potato tubers and yield thereof.
Object, methods and conditions. Field tests were performed in the deep glay
carbonate leached soil of light clay loam (Balhihylogleyi – Calc(ar)ic Luvisol) at
the Research Station of LUA during the period of 2004–2006. The soil is neutral
(pH Hcl
7.1), of medium humusness (2.5 per cent), phosphorous and of medium
calcareousness.
In spring, when the soil surface dried, the soil was heavy harrowed. Later, the field
was cultivated and harrowed twice. Before planting potatoes, the field was fertilized
with complex fertilizer NPK 17:17:17. Early potatoes of cultivar ‘Karlen’ were planted
during the first ten days of May. Sprouts appeared on the soil surface during the first
week of June. After the complete sprouting of potatoes, test sectors were determined.
The area of the test sector was 3 m 2 . The test was carried out in three repetitions;
distribution of test cultivars was systemic.
When potato plants were 5–8 cm high, the test plants were sprayed with herbicide
Zenkor (0.4 l ha -1 ). When Colorado beetles appeared, Decis was used (0.15 l ha -1 ). Later,
potatoes were sprayed with Fastak (0.1 l ha -1 ). During the bud stage, test sectors were
sprayed according to the schedule provided in tables with liquid complex fertilizers
Atgaiva-2 and Atgaiva-P, solutions of growth regulators Stilitas-123, Penergetic-p lapams
,
Humicop and of the blend of the latter regulator and complex fertilizer.
In order to grow and develop, potatoes demand a lot of nutrients. Besides nitrogen,
260

phosphorus and kalium fertilizers that are required in terms of complexity, potatoes
need such microelements as magnesium (Mg), sulphur (S), boron (B), etc. The tested
liquid complex fertilizer Atgaiva-2 is in compliance with these requirements as besides
the main NPK it is enriched with the above-mentioned microelements. This fertilizer
is also very suitable for plants affected by stress (e. g. spring frost).
Complex liquid fertilizer Atgaiva-P is enriched with growth regulator Penergetic;
it is recommended for potatoes at the beginning of the bud stage in order to control
the processes of tuber formation.
At the Department of Organic Chemistry, Kaunas University of Technology,
growth regulator’s Stilitas-123 structural formula whereof is similar to that of
endogenous phytohormones are synthesized. Usually, these are formations of α, β, γ
amino acids with the connected different radicals of water-soluble salines. After entering
the plant, these synthetic growth regulators stimulate activity of natural endogenous
phytohormones, thus changing intensity of physiologic processes of the plant and
energising metabolism thereof. More active metabolism stimulates the physiological
processes that naturally take place in the plant when growth regulators are applied.
Even very small concentrations of these compounds (10 g of the regulator in 100 l -1
H 2
O) result in more intensive growth of plants, faster formation of the maximum
assimilation surface of leaves, more intense processes of photosynthesis, and more
rapid transportation of assimilants from leaves to the tuber, therefore plant productivity
increases (Jakienė et al., 2008). The tested growth regulator Stilitas-123: N was replaced
with β alanine sodium saline.
Potatoes are planted into lighter humous soils. Insufficient amount of humus
leads to worse physical and mechanical performances of soils (Ražukas, 2002;
Simanavičienė, 1999). The substance Humicop applied for the test does not increase
the amount of humus in soil, however, humat and fulvous acids contained by Humicop
activate and enhance development of soil microflora, improve assimilation of NPK
that is specially important for potato roots of weak permeability.
By supplementing the liquid complex fertilizer Atgaiva-2 with growth regulator
Humicop, the plants assimilate nutrients better, they grow and develop more intensely
(Jakienė, 2006).
Solution concentrations used for field tests are the following:
Atgaiva-2 65 l ha -1
Atgaiva-P 25 l ha -1
Humicop 60 l ha -1
Stilitas-123 30 g ha -1
Penergetic-P lapams
100 ml ha -1
One hectare was sprayed with 300 l -1 of operating solution.
The sectors of control tubers were sprayed with water.
The potatoes started to bloom at the beginning of July. The potatoes were harvested
in the second ten days of September. During the harvesting, potatoes of every plant were
grouped according to fractions and weighed. Statistic analysis of the obtained test data
was carried out by applying computer software ANOVA from package SELEKCIJA
(Tarakanovas et al., 2003).
261

Results. The obtained test results have shown that what concerns cultivation
of early potatoes, it is advisable to fertilize them additionally through leaves during
the bud stage with liquid complex fertilizers and growth regulators. Additional foliar
fertilization with the blend of solutions of complex fertilizer Atgaiva-2 and growth
regulator Humicop led to particular increase in potato productivity.
Table 1. The effect of additional foliar fertilization on the bigness of potato
tubers
1 lentelė. Papildomo tręšimo per lapus įtaka bulvių gumbų stambumui
Research Station of LUA, Average data of the period 2004–2006
LŽŪU Bandymų stotis, vidutiniai 2004–2006 m. duomenys
As the data in Table 1 demonstrate, the biggest potato tubers were provided by
the test variants additionally fertilized through leaves with the blend of solutions of
complex fertilizer Atgaiva-2 and growth regulator Humicop. Additional fertilization
with this blend resulted in as much as 78.8 % of bulky tubers in one plant and almost
no small potatoes were found. Medium sized potatoes amounted to 19.7 % of all
potato tubers of one plant. Just 1.5 % of small potatoes were present. This additional
fertilization resulted in less tubers of one plant but they were all bulky and weighed
over 80 g each.
58–60 % of bulky tubers have also grown after additional fertilization with
liquid fertilizer Atgaiva-2, Atgaiva-P and after applying growth regulator Stilitas-123.
Amount of medium size potatoes found in test variants was 18–25 %. After additional
fertilization of potatoes with complex fertilizer Atgaiva-P and solution of Stilitas-123
(15.3–17.1 % accordingly), amount of small tubers was less since growth regulator
Penergetic contained by this fertilizer and growth regulator Stilitas-123 stimulated more
intense growth of potato tubers. After applying liquid complex fertilizer Atgaiva-2,
the amount of small potatoes found was 21.9 % (Table 1).
After spraying the potato plants in the bud stage with the solutions of growth
regulators Penergetic-P lapams
or Humicop , over 50 % of bulky potatoes per plant was
found, whereas the rest of the tubers contained approximately the equal number of
medium sized and small potatoes.
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The greatest number of tubers, however, the smallest ones has grown in the control
test sections where the plants were not additionally fertilized through leaves.
Table 2. The effect of additional foliar fertilization on the potato yield
2 lentelė. Papildomo tręšimo per lapus įtaka bulvių derliui
Research Station of LUA, average data of the period 2004–2006
LŽŪU Bandymų stotis, vidutiniai 2004–2006 m. duomenys
The greatest yield was achieved after additional foliar fertilization with liquid
complex fertilizer Atgaiva-P and Atgaiva-2 (Table 2). Under the influence of the
aforesaid additional fertilization, the yield significantly increased by 8–9 t ha -1 or
26–28 % in comparison with the control variants. Additional foliar fertilization of
potatoes with the solutions of growth regulators Penergetic and Stilitas-123 as well
as with the blend of solutions of complex fertilizer Atgaiva-2 and growth regulator
Humicop also provided good results. The above mentioned test variants produced a
significantly greater (by 4.76–6.28 t ha -1 or 14.8–19.5 %) potato yield.
In the test variants, where for additional fertilization growth regulator Humicop
solely was applied, the potato yield increased just slightly (0.92 t ha -1 or 2.8 %) and
not significantly.
Taking into account the purpose of the grown potatoes, it advisable to spray
potatoes grown for industrial processing with the solutions of growth regulators
Stilitas-123 or Penergetic-P lapams
after additional foliage fertilization thereof with
liquid complex fertilizer Atgaiva-P at the beginning of the bud stage. Under the
influence of this additional fertilization, the potato yield increases by approximately
5.34–9.12 t ha -1 or 16.6–28.3 %. In the yield structure, medium sized potatoes and
bulky potatoes make up 25 and 55–59 %, respectively.
In the yield structure of potatoes grown for food, bulky potatoes should dominate.
In this case, it is advisable to apply the blend of solutions of complex fertilizer Atgaiva-2
and growth regulator Humicop for additional foliar fertilization. In the test variants,
where this blend of fertilizer and growth regulator was applied, 78 percent of bulky
potatoes in one plant were found, whereas almost no small potatoes were present.
Nevertheless, the number of tubers per plant was by half smaller compared to the
number of tubers grown in control test sectors. Under the influence of this additional
fertilization, the potato yield increased by approximately 4.76 t ha -1 or 14.8 % in
comparison with the control variants.
263

Having no possibilities to fertilize table potatoes additionally with the blend of
complex fertilizer Atgaiva-2 and growth regulators Humicop, good results are also
achieved by additional foliar fertilization with liquid complex fertilizer Atgaiva-2
solely. Under the influence of this additional fertilization, bulky potatoes made up
60 % of the yield structure, whereas the rest of the yield contained 40 % of small and
medium sized potatoes. Under the influence of complex fertilizer, a greater number
of tubers per plant develop compared to the case when the blend of Atgaiva-2 and
Humicop is applied. After additional foliar fertilization with fertilizer Atgaiva-2
solely, the yield significantly increased by 8.54 t ha -1 or 26.5 % in comparison with
the control variants.
Discussion. The investigations of the phytohormones at molecule, cell and plant
organism level makes it possible to solve the problem of growth regulators in plant
growing. Long-term data on various potato varieties (‘Vokė’, ‘Nida’, ‘Vilma’, etc.)
with respect to ripening time revealed that treatments with optimum growth regulator
TA – 12, TA – 14 concentrations and optimum treatment time (4 th organogenesis stage)
resulted in activation of the organogenesis process. It is well established that these
combinations modify both the formation and growth of the stolons not only quantatively
but also stimulate earlier root formation through shortening the period of the stolon
growth what has positive effect on potato productivity (Dargevičienė et al., 2002).
Leaf spray fertilization with complex fertilizers also stimulated more intensive
potato growth and formation of the structural yield elements. Additional fertilization
with complex mineral fertilizers with microelements (Rainys et al., 2005) contributed to
higher potato productivity. Even better results were obtained using growth regulators at
the time of additional leaf spray fertilization. Growth regulators stimulated metabolism
processes, the plants assimilated nutrients better and in turn formed higher and better
quality yield (Jakienė et al., 2008).
Treatments with growth regulators have been more and more widely used in
plant growing. The treatments of the winter wheat with growth regulators resulted
in longer wheatears and increase in 1 000 grain mass. Chlorophyll biosynthesis was
more intensive and higher chlorophyll concentration was determined in the plant
leaves (Auрkalnienė, 2005).
Growth regulators had significant effect on cabbage biometric parameters,
improved their productivity and disease resistance. Treatments of the cabbage leaves
with the considered growth regulator (epin, lizar) solutions 1.5 × 10 – 4 % during
vegetation period resulted in 10 cm plant height increase, 8 mm flower diameter
increase, 62 % pulse increase. The yield was by 230 kg ha -1 or by 32.9 % higher, ripe
seeds were of better quality (Danilevič, 2005).
More and more often liquid complex and microelement fertilizers have been used
for additional fertilization in plant growing technologies. Leaf spray fertilization of
the beetroot spouts with calcium saltpeter, nitrabor or nutrifol + microelements during
the first part of the vegetation resulted in root and diameter increase (Bundinienė
et al., 2007). It is well established that leaf spray fertilization with various nutritive
and biologically active substances affect summer rape biomass, seed quality and
profitability. The most profitable was found to be rape fertilization with the fertilizer
‘Aton AZ’ containing microelements and amino acid, and “Boramin Ca” – the profit
from one hectare in the considered years made at the average 62 Lt (Staugaitis et al.,
2007).
264

The experiments on additional winter wheat spray fertilization and growth
regulator use carried out on the experimental station at Lithuanian University of
Agriculture revealed that two time fertilization with 30 kg ha -1 nitrogen resulted in
4.27–4.84 t ha -1 yield increase on the plots of mean intensity technologies, and up to
7.33–9.25 t ha -1 on the plots of the intensive technologies. On the plots of the intensive
technologies the most effective was found to be fertilization with microelement complex
nutrifol and growth regulator cycocel. The yield in this variant made 9.25 t ha -1 . The
grain contained 15.8 % of protein and 30.1 % gluten (Šiuliauskas et al., 2002).
Additional leaf spray fertilization using liquid complex fertilizers and growth
regulators had positive effect on potatoes. Growth regulators Penergetic-P and Stilite-
123 activated the cells participating in metabolism process and the plants assimilated
nutrients better. Potato productivity under the influence of these regulators was by
9.12 t ha -1 or by 28 % (Penergetic-P) and by 6.28 t ha -1 or 19.5 % (Stilite-123) higher than
in the control. Additional potato fertilization with the mixture of the liquid fertilizers
Atgaiva-2 and growth regulator Humicop modified the growth of the tubers effectively.
Big potatoes on the potato bushes on these plots made 78 %. The productivity was by
4.76 t ha -1 or by 14.8 % higher than in not fertilized variants.
Conclusions. 1. In the process of potato growing, it is advisable to apply for
potatoes additional foliar fertilization with the blend of complex fertilizers and growth
regulators within the bloom formation stage.
2. The greatest addition to the yield (8.54–9.12 t ha -1 ) and the bulkiest potatoes
(approximately 60 % of potatoes weighed over 80 g each) has grown after additional
fertilization with liquid complex fertilizers Atgaiva-2 and Atgaiva-P.
3. After spraying the potatoes during the bud stage with the solutions of growth
regulators Stilitas-123 or Penergetic-P, bulky potatoes made up 55.9–58.4 % of the
yield structure, whereas the rest of the tubers contained approximately the equal
number of medium sized and small potatoes. The productivity under the influence of
these growth regulators significantly increased by 5.34–6.28 t ha -1 or 16.6–19.5 % in
comparison with the control variants.
4. It is advisable to fertilize table potatoes additionally through leaves with the
blend of solutions of complex fertilizer Atgaiva-2 and growth regulator Humicop.
After additional fertilization of potatoes with this blend, bulky potatoes made up even
78.8 % of the yield structure, whereas medium sized potatoes amounted just to 19.7 %
and small potatoes were almost not found (1.5 %) at all. Under the influence of this
additional fertilization, the potato yield significantly increased by 4.76 t ha -1 or 14.8 %
in comparison with the control variants.
5. After additional spraying of potatoes with the solution of growth regulator
Humicop solely, bulky potatoes made up app. 52.7 % of the yield structure, whereas
the rest of the tubers contained the equal shares of medium sized and small potatoes.
Application of this regulator had no any significant effect on productivity.
Gauta 2008 03 05
Parengta spausdinti 2008 04 14
265

References
1. Auškalnienė O. 2005. Augalų augimo reguliatoriaus modus mišinių įtaka
žieminių kviečių derliui ir jo struktūros elementams. Mokslo darbai. Žemdirbystė.
2(90): 48–60.
2. Bulvininkystė Lietuvos ūkiuose. Statistikos departamentas prie Lietuvos
Respublikos vyriausybės. 1991. Vilnius, 12–35.
3. Bundinienė O., Viškelis P., Zalatorius V. 2007. Papildomo tręšimo per lapus įtaka
raudonųjų burokėlių derliui ir šakniavaisių kokybei. Mokslo darbai. Sodininkystė
ir daržininkystė, 26(1): 108–118.
4. Danilevich J. 2005. Augimo reguliatorių poveikis kopūstų sėkloms. Mokslo darbai.
Sodininkystė ir daržininkystė, 24(3): 55–62.
5. Darginavičienė J., Novickienė L. 2002. Augimo problemos šiuolaikinėje augalų
fiziologijoje. Lietuvos mokslų akademijos leidykla, Vilnius, 58–62.
6. Jakienė E. 2006. Papildomo tręšimo mikroelementinėmis trąšomis ir augimo
reguliatoriais įtaka bulvių produktyvumui. Lauko augalų tręšimo aktualijos.
Šiuolaikinės augalininkystės technologijos. Akademija, 1–5.
7. Jakienė E., Venskutonis V. 2008. Augimo reguliatoriai augalininkystėje.
Akademija.
8. Makaravičiūtė A. 2003. Tręšimo įtaka bulvių derliui, krakmolo ir sausųjų medžiagų
kiekiui gumbuose. Žemės ūkio mokslai. 2: 35–42.
9. Rainys K., Rudokas V. 2005. Bulvių augimo sąlygų ir veislės įtaka derliui ir jo
kokybei. Mokslo darbai. Žemdirbystė. 1(89).
10. Ražukas A. 2002. Bulvės: biologija, selekcija, sėklininkystė. LŽI, Dotnuva,
Akademija.
11. Simanavičienė O. 1999. Bulvių auginimas. Elmininkai.
12. Staugaitis G., Laurė R. 2007. Lapų trąšų įtaka vasarinių rapsų sėklų derliui,
kokybei ir pelningumui. Mokslo darbai. Žemdirbystė. 3(94).
13. Šiuliauskas A., Vagusevičienė I., Liakas V. 2002. Žieminių kviečių tręšimo per
lapus agroekonominis įvertinimas. Žemės ūkio mokslai. 2.
14. Tarakanovas P., Raudonius S. 2003. Agronominių tyrimų duomenų statistinė
analizė taikant kompiuterines programas. Akademija.
15. Венскутонене Э., Якене Э., Даугела Г. 2004. Влияние регуляторов роста на
динамику прорастания семенных клубней картофеля. Vagos. LŽŪU Mokslo
darbai. 64(17).
16. Меркис А., Новицкене Л., Милювене Л. 1994. Регуляция роста картофеля,
сахарной свеклы, ячменя и пшеницы. Žemės ūkio mokslai. 3.
266

SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Papildomo tręšimo skystosiomis kompleksinėmis trąšomis ir
augimo reguliatoriais įtaka bulvių produktyvumui
E. Jakienė, V. Venskutonis, V. Mickevičius
Santrauka
Tyrimai atlikti 2004–2006 metais Lietuvos žemės ūkio universiteto bandymų stotyje.
Tirta skystųjų kompleksinių trąšų ir augimo reguliatorių įtaka bulvių augimui, stiebagumbių
formavimuisi ir derlingumui.
Nustatyta, kad didžiausias derliaus priedas (8,54–9,12 t ha -1 ) gautas ir apie
60,0–59,2 proc. stambių bulvių kere užaugo žiedų formavimosi tarpsnio pabaigoje bulves
papildomai per lapus patręšus „ARVI fertis“ skystosiomis kompleksinėmis trąšomis Atgaiva-2
arba Atgaiva-P.
Bulves apipurškus augimo reguliatorių Penergetic-P lapams
arba Stilito-123 tirpalais, stambios
bulvės derliaus struktūroje sudarė 55,9–58,4 proc. Derlius šių augimo reguliatorių įtakoje
patikimai padidėjo 5,34–6,28 t ha -1 arba 16,6–19,5 proc., lyginant su kontrole.
Maistui auginamas bulves tikslinga papildomai patręšti kompleksinių trąšų
Atgaiva-2 ir reguliatoriaus Humicop mišiniu. Papildomai per lapus patręрus šiuo mišiniu,
stambios bulvės derliaus struktūroje sudarė net 78,8 proc., o smulkių beveik nerasta
(tik 1,5 proc.). Bulvių derlius šio papildomo tręšimo įtakoje patikimai padidėjo 4,76 t ha -1 arba
14,8 proc., lyginant su kontrole.
Reikšminiai žodžiai: augimo reguliatoriai, bulvės, produktyvumas, skystos kompleksinės
trąšos.
267

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Correlation between chlorophyll fluorescence of
primrose (Primula malacoides Franch.) and DNA
polymorphic bands
Vytautas Šlapakauskas 1 , Vidmantas Stanys 2 ,
Judita Varkulevičienė 3
1
Department of Botany University of Agriculture, Studentų 11, Akademija,
LT-53067, Kauno distr., Lithuania , e-mail: Vytautas.Slapakauskas@lzuu.lt
2
Biotechnological Laboratory, Lithuanian Institute of Horticulture, Kauno 30, LT-
54333 Babtai, Kaunas distr., Lithuania, e-mail: V.Stanys@ lsdi.lt
3
Kaunas Botanical Garden of Vytautas Magnus University, Ž. E. Žilibero 6,
LT-46324 Kaunas, Lithuania, e-mail: j.varkuleviciene@bs.vdu.lt
Plant material of primrose (Primula malacoides Franch.) varieties as well as hybrids
where grown in the greenhouse.
The total DNA was isolated from leaf material according Doyle and Doyle (1990). Eleven
primers were used for DNA amplification. Chlorophyll fluorescence was recorded on portable
fluorometer (PAM-210, Walz, Germany). Correlation coefficient and regression equations
were used to demonstrate reciprocity among distribution of polymorphic DNA fragments and
fluorescence parameters.
A number of common polymorphic markers obtained for varieties and hybrids of primrose
using 11 DNA primers were demonstrated to have low negative correlation to electron transport
rate (ETR) of photo system (PS II) of the plants. Polymorphic bands generated using DNA
primers 1A and 3C had a negative correlation to ETR for varieties of primrose, while hybrids
displayed a positive correlation. DNA markers characteristic to every variety or hybrid were
obtained after choosing two or more primers that enabled not only to identify them but also to
differentiate the state of photosynthetic machinery.
Key words: DNA polymorphism, electron transport rate (ETR), fluorescence, primrose.
Introduction. While solving problems of decorative plant introduction,
acclimatization and biodiversity enrichment, it is crucial not only to cultivate newly
introduced decorative plants, but also to develop new hybrids suitable for growing
conditions in Lithuania (Varkulevičienė, 2002). Primrose (Primula malacoides Franch.)
has been cultivated in the Kaunas Botanical Garden since the start of a breeding
program in 1946. Five varieties and a large number of hybrids have been developed
using a method of sexual hybridization (Varkulevičienė, 1999).
Detection of random amplified polymorphic DNA markers in varieties and hybrids
of primrose display genetic similarity of varieties and hybrids of Lithuanian origin
(Stanys et al., 2005). The ornamental varieties and hybrids of primrose demonstrate
correlation between genetic characteristics of plants and photosynthesis machinery,
269

as well. Photosynthesis may be considered the most fundamental biological process.
Chlorophyll fluorescence measurements show the quantitative relationship between
the fluorescence and efficiency of photosynthetic energy conversion. It opens new
ways to characterize plants in process of breeding of new varieties and hybrids
(Šlapakauskas, Ruzgas, 2005). The DNA amplification and localization identified
unique signs characteristic to individual varieties and hybrids that were related to
photosynthesis genes. These regulatory elements can be positive or negative. Gene
regulatory elements of rbcs and cab families located 35 to – 2 bp from the transcription
start site of light inducible genes and were shown to regulate expression of the gene
and the positive quantitative element. A negative regulatory element was located
between 107 to 56 bp.
In this study, we investigated correlation between distribution of random amplified
polymorphic DNA markers and electron transport rate (ETR) of photosystem II of the
P. malacoides varieties and hybrids.
Object, methods and conditions. Plant material of primrose (Primula malacoides
Franch.) varieties ‘Jadvyga’, ‘Linkėjimai Latvijai’, ‘Lietuvaitė’ and ‘Jaunystė’ as well as
hybrids ‘Žydrė’, ‘Vakarė’, ‘Rubinas’ and ‘Margutė’ where grown in the greenhouse.
The total DNA was isolated from leaf material according Doyle and Doyle
(Doyle and Doyle, 1990; Schreiber and Bilger, 1993; Rohaček, 2002). The PCR
mixture contained: 1 unit of Tag DNA polymerase (MBI Fermentas), 1.5 mM MgSO 4
,
0.2 mM dNTP and 1 µM of each oligonucleotide primer. Eleven primers were used
for DNA amplification: OPA – 15; 3472 – 02 (Operon Technologies, Inc.), 1A; 2B;
3C (MBI Fermentas), 2064; 2066; 2067; 2068; 2069; 2070 (Carl-Roth GmbH). The
amplification was carried out according to the following scheme: 1 cycle of 94° for
5 min, for 45 sec 94 °, 45 sec for 32 °C, 72 °C V for 50 sec. The amplified products
were separated on 1% agarose gel in TAE buffer (pH 8.0).
Chlorophyll fluorescence was recorded on portable fluorometer (PAM-210, Walz,
Germany). Chlorophyll fluorescence measurements were performed with the light
adapted leaves. An actual chlorophyll fluorescence yield at any time of induction by
an actinic light fluorescence (F t
) and a maximal fluorescence (F m’
) was measured for
every saturation pulse. The saturation pulse of 3 500 мmol quanta per square meter
per second (мmol m -2 s -1 ) was used. The quantum yield of electron transport (Y) was
estimated according to the relationship: Y = (F m
’
- F t
): F m
’
= ∆F: F m
’
and electron transport
rate (ETR): ETR = c . 0.5 . PAR . Y. Using this equation, it is assumed that 84 % of
the incident quanta were absorbed and 50 % of the absorbed quanta were distributed
to PSII (6.7). Statistical significance was estimated using one way ANOVA (Tukey
test) method and STATISTICA v. 6 software. Correlation coefficient and regression
equations were used to demonstrate reciprocity among distribution of polymorphic
DNA fragments and fluorescence parameters.
Results. Numerous experiments show that light is one of the most important
environmental factors influencing gene expression in the process of photosynthesis.
A sensitivity of photosynthetic apparatus to environmental changes has been reflected
measuring chlorophyll fluorescence. Thus it has been established that genes of the
270

photosynthetic machinery components demonstrate light dependent expression. It has
become clear that photosynthesis itself is an important contributor to the light controlled
gene expression regulation (Pfannschmidt et al., 2001). Hence, the fluorimetry might
be useful tool assessming physiological plant response to different conditions, as well
as characteristics of genetic polymorphism. For instance, chlorophyll fluorescence
parameter as indicator of actual efficiency of PSII photochemistry can give a measure
of the linear electron transport rate and thus the indication of overall efficiency of
photosynthesis (Maxwell and Johnson, 2000). In addition, this parameter correlates
with the quantum yield of carbon fixation.
The chlorophyll fluorescence technique has many applications in analysis of plant
productivity, ornamental features and polymorphic parameters, as well (Baker and
Rosenqvist, 2004). Nevertheless, the ETR determined for all varieties and hybrids of
primrose had a low correlation to total number of polymorphic bands obtained using
all of the 11 primers in this study (Fig. 1).
Fig. 1. Correlation between ETR and genetic polymorphism data obtained using
polymorphic primers for varieties and hybrids of primrose
1 pav. Koreliacija tarp ETR ir genetinio polimorfizmo duomenų, naudojant raktažolės veislių
ir hibridų polimorfinius žymenis
Only polymorphic bands obtained using primers 1A + 3C (table) demonstrated
correlation to electron transport rate of PSII (Fig. 2). The responding markers had
different correlation between the ETR and genetic polymorphism for varieties and
hybrids of primrose. Correlation was found to be negative for varieties and positive
for hybrids.
271

Table. Polymorphic markers of P. malacoides varieties and hybrids obtained
using 1A ir 3C primers
Lentelė. P. malacoides veislių ir hibridų polimorfiniai žymenys gauti su 1A ir 3C
pradmenimis
Fig. 2. Correlation between ETR and genetic polymorphism data obtained using
primers 1A and 3C for varieties and hybrids of primrose
2 pav. Koreliacija tarp ETR ir genetinio polimorfizmo duomenų, naudojant 1A ir 3C
polimorfinių pradmenų skaičiaus
Discussion. With the development of genetic and molecular research tools,
new ways to study chlorophyll fluorescence and cultivar genotype differences, DNA
polymorphism (including random amplified polymorphism) and correlation between
272

chlorophyll fluorescence have been opened (Rascher et al., 2000).
DNA polymorphism was evaluated of light subspecies of Triticum turgidum L.
(Kokindova, Kraic, 2005). The average values of diversity index and polymorphic
content indicate that microsatellites located in the A genome generated higher
polymorphism than microsatellites from B genome. Microsatellite profiles confirmed
full identity of all the four analyzed genotypes for subspecies-specific alleles were
identified.
Marker genes have been shown to be very useful for establishing variation in
fluorescence levels among different tissues and organs, evaluation and improvement
of transformation procedures for plants (Hraрka et al., 2006). Random amplified
polymorphic markers were used for determination of the genetic stability and no
genetic instability was determined during 4-year period using 4 selected primers with
the high polymorphisms of Norway spruce (Hanaček et al., 2002).
Earlier investigation of genetic characteristics of newly developed varieties and
hybrids of primrose showed a close relation between varieties ‘Margutė’, ‘Jadvyga’,
‘Linkėjimai Latvijai’, ‘Lietuvaitė’ and hybrids ‘Rubinas’, ‘Žydrė’, ‘Vakarė’, ‘Jaunystė’.
A number of common polymorphic bands for these plants was estimated to be 18.5–
51.9 %. In this study, we demonstrated that correlation between main fluorescence
parameter of the PSII electron transport rate and genetic polymorphism was more
specific for varieties and hybrids. Our results suggested that examination of fluorescence
parameters of different polymorphic plants might represent a more informative
(and faster) method for practical characterization of newly developed varieties
and hybrids. A further details of examination of the relation between fluorescence
measurements corresponding electron transport rate of PSII and genetic background
of the P. malacoides plants would be prospective to advance this method.
Conclusions. 1. Genes associated with photosynthetic apparatus demonstrated
light dependent expression and fluorimetry might be useful food assessing physiological
response to genetic characterization of plants.
2. Electron transport ratio (ETR) determined low correlation to total number
of polymorphic bands using all of 11 primers; using 1A + 3C primers demonstrated
correlation to ETR. Markers of primrose varieties were found negative and positive
of hybrid correlation between ETR and genetic polymorphism.
3. Correlation between main fluorescence parameter of the electron transport rate
and genetic polymorphism was more specific for varieties and hybrids.
References
Gauta 2008 04 02
Parengta spausdinti 2008 04 28
1. Baker N. R., Rosenqvist E. J. 2004. Applications of chlorophyll fluorescence
can improve crop production strategies: an examination of future possibilities.
J. ExP. Bot. 44: 1 607–1 621.
2. Chlorophyll a fluorescence. A signature of photosynthesis. 2004. G. C. Papageorgiou
and Govindjee (eds.). Springer, Illinois USA.
273

3. Doyle J. J., Doyle J. L. 1990. Isolation of plant DNR from fresh tissue. Focus,
12(1): 13–15.
4. Hanaček P., Havel L., Truksa M. 2002. Characterization and determination of
genetic stability of early somatic embryo culture clones of Norway spruce using
RAPD markers. Biologia, Bratislava, 57: 517–522.
5. Hraрka M., Rakousky S., Čurn V. 2006. Green fluorescent protein as a vital
marker for non-destructive detection of transformation events in transgenic plants.
Biologia Plantarum, 86(3): 303–318.
6. Kokindova M., Kraic J. 2005. Genetic variation of Triticum turgidum accessions
characterized by DNA markers. Biologia, Bratislava, 60: 451–456.
7. Maxwell K., Johnson G. N. 2000. Chlorophyll fluorescence – a practical guide.
J. ExP. Bot. 51: 659–668.
8. Nedbal L., Soukupova J., Kaftan D., Whitmarsh J., Trtilek M. 2000. Kinetic
imaging of chlorophyll fluorescence using modulated light. Photosynthesis
Research, 66: 3–12.
9. Pfannschmidt T., Allen J. F., Oelmüller R., 2001. Principles of redox control in
photosynthesis gene expression. Physiologia Plantarum. 112: 1–9.
10. Rascher U., Liebig M., Lüttge U. 2000. Evaluation of instant light-response curves
of chlorophyll fluorescence parameters obtained with a portable chlorophyll
fluorometer on site in the field. J. Plant, cell and environment, 23: 1 397–1 405.
11. Rohaček K. 2002. Chlorophyll fluorescence parameters: the definitions,
photosynthetic meaning and mutual relationships. Photosynthetica, 40(1): 13–29.
12. Schreiber U., Bilger W.1993. Progress in chlorophyll fluorescence research:
major developments during the past years in retrospect. Progress in Botany,
54: 151–173.
13. Šlapakauskas V., Ruzgas V. 2005. Chlorophyll fluorescence characteristics of
different winter wheat varieties (Triticum aestivum L.). Agronomy Research,
3(2): 203–209.
14. Stanys V.,Gelvonauskis B., Zalatorienė G., Stanienė G.,Varkulevičienė J. 2005.
Švelniosios raktažolės (Primula malacoides Franch.) lietuviškų veislių DNR
polimorfizmas. Sodininkystė ir daržininkystė, 24(2): 105–112.
15. Varkulevičienė J. 1999. Švelniosios raktažolės (Primula malacoides Franch.)
selekcija.VDU Kauno botanikos sodo raštai. Kaunas, IX: 53–56.
16. Varkulevičienė J. 2002. Švelniosios raktažolės (Primula malacoides Franch.)
biologinių savybių tyrimų metodika. Vagos, X: 47–55.
17. Zhang H. Y., Liu X. Z., He C. S. 2005. Random amplified DNA polymorphism
of Nicotiana tabacum L. cultivars. Biologia Plantarum, 49(4): 605–607.
274

SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Koreliacija tarp raktažolės (Primula malacoides Franch.) chlorofilo
fluorescencijos ir polimorfinių DNR žymenų
V. Šlapakauskas, V. Stanys, J. Varkulevičienė
Santrauka
Koreliacija tarp raktažolės veislių ir hibridų bendro polimorfinių žymenų, gautų su 11 DNR
pradmenų skaičiaus ir fotosintetinių sistemų elektronų transporto greičio (ETR) buvo silpnai
negatyvi. Polimorfinių raktažolės DNR žymenų, gautų su pradmenimis 1A ir 3C skaičius
veislems su ETR turėjo negatyvų, o hibridams – pozityvų ryšį. Polimorfinių žymenų gretinimas
su ETR gali paspartinti naujų augalų hibridų ir veislių įvertinimą ir atrinkimą.
Iš panaudotų 11 DNR pradmenų gautų bendrų polimorfinių žymenų skaičius turi nežymią
neigiamą koreliaciją su raktažolių lapų antrosios fotosintetinės sistemos elektronų transporto
greičiu (ETR). Polimorfiniai žymenys, gauti panaudojant 1A ir 3C pradmenis, turėjo neigiamą
koreliacinį ryšį su raktažolių veislių fotosintetinės sistemos ETR ir teigiamą ryšį su šių augalų
hibridų fotosintetinių sistemų ETR. Raktažolių veislių ir hibridų DNR žymenų charakteristika
gauta panaudojant du arba daugiau pradmenų ir įgalina nustatyti ne vien augalų savitumą, bet
ir fotosintetinių sistemų skirtumus.
Reikšminiai žodžiai: DNR polimorfizmas, elektronų transporto greitis (ETR),
fluorescencija, raktažolė.
275

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF HOR-
TICULTURE AND LITHUANIAN UNIVERSITY OF AGRICULTURE.
SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Changes of some biochemical parameters during the
development of sweet pepper fruits
Maria Leja, Gabriela Wyżgolik, Iwona Kamińska
Departament of Plant Physiology, Faculty of Horticulture, Agricultural University,
29 Listopada 54, 31-425 Krakуw, Poland
E-mail: mleja@bratek.ogr.ar.krakow.pl
Sweet pepper (Capsicum annuum) of cultivar ‘Spartacus’ was grown in the foil tunnel of
two different PAR intensities. Nitrate nitrogen as well as its reduced form (ammonium and urea)
were applied by the fertigation technique. Fruits were harvested in three maturity stages: green,
turning and red. The contents of total phenols, total carotenoids and evolution of endogenous
ethylene were determined.
The content of soluble phenol was detected by the photometric method with Folin’s reagent,
carotenoid level in hexan extracts was measured photometrically, and ethylene evolution was
determined by GC with flame-ionisation detector.
The significant accumulation of both phenols and carotenoids was accompanied by the
increase in ethylene production. The most distinct synthesis of carotenoids was observed when
fruits were converted to the full maturity stage (red colour), while soluble phenol constituents
were accumulated gradually during the whole ripening period. Neither light nor nutrient nitrogen
form affected the above mentioned parameters.
Key words: carotenoids, ethylene, maturation, phenols, sweet pepper.
Introduction. Sweet pepper fruits are an excellent source of health promoting
substances, particularly antioxidants of both hydrophilic and hydrophobic phase.
Ascorbic acid, flavonoids and phenolic acids as well as carotenoids present in pepper
fruits have a strong ability to neutralize active oxygen species (Howard et al., 2000).
In different pepper types these authors found the wide spectrum of carotenoids such
as β-cryptoxanthin, α- and β-carotene, capsanthin, lutein and zeaxanthin. Quercetin
and luteolin are dominating among flavonoids, a significant number of cinamic acid
derivatives and hydroxy-substituted benzoic acids were also observed in the fruit
tissues (Howard et al., 2000). High activity of enzymes associated with antioxidative
system (superoxide dismutase, catalase, xanthine oxidase and glutathione reductase)
in peroxisomes of pepper fruit was reported by Mateos et al. (2003).
Pepper fruits are harvested and consumed at different maturity stages (green, red
and not-fully ripe). During ripening some substances of important nutritional quality,
particularly carbohydrates and vitamin C are accumulated; changes in phenols and
carotenoids are also noted (Zhang, Hamauzu, 2003; Navarro et al., 2006).
In many species fruit ripening is accompanied by endogenous ethylene production,
peppers, in general, are classified as non-climacteric fruits (Saltveit, 1977), however,
277

according to findings of Biles et al. (1993) in peppers of New Mexican type
concentration of internal ethylene was high enough to initiate ripening, and in some
cultivars of hot pepper respiratory climacteric was found (Gross et al., 1986).
The aim of the present studies was to determine the content of total soluble phenolic
compounds and carotenoids in sweet pepper fruits, harvested as green, turning and red
ones, with additional measurements of endogenous ethylene evolution. The effect of
some growth conditions (light and form of nitrogen nutrient) was also investigated.
Object, methods and conditions. Sweet pepper (Capsicum annuum)
of cultivar ‘Spartacus’ was grown in 2007, in the foil tunnel of two different
light intensities, obtained by application of Gemme 4S and Ginegar foils (light
transparency 90 % and 86 %, light diffusion 15 % and 58 %, respectively). The PAR
permeability measured by infrared analyser of CO 2
was 300–320 µmol · m -2 s -1 and
950–1 000 µmol · m -2 s -1 for the cloudy and sunny days, respectively (Gemme
4S), in the case of Ginegar foil these parameters were 200–220 µmol · m -2 s -1 and
650–750 µmol · m -2 s -1 . Ginegar foil tunnel was described as A, Gemme 4S foil tunnel
as B.
Nutrient nitrogen was used either as N-NO 3
(100 %) or as the reduced form
(N-NO 3
: N-NH 4
: N-NH 2
in ratio 50:13:37 %). In both treatments the average nitrogen
level was 210 mg of N · dm -3 of nutrient solution. The other mineral constituents were
introduced in concentrations recommended for pepper cultivation. The rockwool
was used as the substrate; nutrient solution was supplied by the automatic fertigation
system. Nutrient parameters were systematically controlled by measurements of pH
and EC values.
Fruits were harvested at three maturity stages: green, turning (brownish) and
red (fully ripe) on 2 nd , 5 th and 6 th of July, respectively. The mean of 30 fruits sample
was used for analytical procedure. Total phenols were determined by the photometric
method with Folin’s reagent (Swain, Hills, 1959). Total carotenoids were detected in
acetone extracts prepared from previously frozen tissue by the photometric method
recommended by Polish Norm (PN-90). For ethylene measurement the 5 cm 3 air
samples were withdrawn from the cavity of attached fruit and injected into a gas
chromatograph (Chrom 4) with a flame ionization detector and 1.2 m long column
filled with aluminium oxide at 60 °C.
All analyses were made in three replications and results were statistically verified
by the Duncan’s test.
Results. During fruit ripening the considerable increase in carotenoids was
observed. Accumulation of these compounds was particularly distinct in red fruits
(Fig. 1). The differences between A and B tunnels as well as between NO 3
and NO 3
-
NH 4
-NH 2
treatments were non-significant, except fruits of turning (brownish) phase
where slightly higher level of carotenoids in fruits treated with the reduced nitrogen
form grown in B tunnel was observed.
278

Fig. 1. Carotenoid content in sweet pepper fruits
1 pav. Karotinoidų kiekis saldžiosios paprikos vaisiuose
Soluble phenols accumulated gradually in developing fruits reaching the highest
content in the full maturity stage. In green fruits the differences both between A and B
tunnels and nitrogen form treatments were non-significant, in the case of turning and
red ones slightly higher content of total phenols was noticed in fruits of pepper plants
grown in B tunnel and fertilized with the reduced form of nitrogen (Fig. 2).
Fig. 2. Total phenol content in sweet pepper fruits
2 pav. Bendras fenolių kiekis saldžiosios paprikos vaisiuose
According to the results presented in Figure 3 at the early stage of ripening the
endogenous ethylene production was very low. In pepper fruits of the turning phase
ethylene evolution was considerably higher and reached the maximum in the fully
maturity stage.
279

Fig. 3. Ethylene production in sweet pepper fruits
3 pav. Etileno gamyba saldžiosios paprikos vaisiuose
The effect of radiation (tunnels A and B) as well as that of applied nitrogen
form in most cases was non-significant, only in fruits collected in the turning phase
slightly higher content of carotenoids was found in tunnel B under treatment with
the reduced nitrogen form in comparison with tunnel A. Significantly higher level of
total phenolics was observed in fruits of turning stage treated with ammonium-urea
nitrogen as compared to NO 3
application in both tunnels, similar effect was noticed
in red fruits grown in tunnel B.
Discussion. Increase in carotenoids in development of sweet pepper fruits, was
reported previously by many authors. In general, ripening of pepper fruits is strongly
associated with carotenoid accumulation (Markus et al., 1999). Navarro et al. (2006)
observed the significant increase of β-carotene and lycopen contents in developing fruits
of cultivar ‘Orlando’. Howard et al. (2000) who determined the individual carotenoids
in the ripening pepper fruits of various types by HPLC method noted extensive increase
of β-cryptoxanthin, α- and β-carotene, capsanthin and zeaxanthin in all pepper types,
while content of lutein decreased.
Significant accumulation of total soluble phenols during pepper fruit ripening was
confirmed by findings of Howard et al. (2000), however, flavonoid concentration was
either unaffected by maturity or decreased in respect to investigated cultivars. Zhang
and Hamauzu (2003) observed the highest level of these constituents in green pepper
fruits, Navarro et al. (2006) did not find any significant differences in total phenols
determined in three maturity phases (green, turning and red).
As described earlier pepper is not considered as the climacteric plant, however,
the presented results show evolution of internal ethylene corresponding with fruit
maturation. According to studies of Villavicencio et al. (2001) on ethylene production
by the attached fruits of three pepper cultivars during their ripening, all cultivars
seemed to be intermediate between climacteric and non-climacteric fruits. The
authors observed the undetectable level of C 2
H 4
in the mature green stage followed
by increasing at the breaker stage and remaining high in red fruits and decreasing in
mature light red fruits.
Synthesis of internal ethylene in developing fruits was accompanied by
accumulation of total phenols and also by carotenoids. The role of ethylene as the
plant hormone inducing ripening of fruits is, among others, associated with its effect on
280

carotenoid synthesis. Exogenous ethylene treatment of pepper fruits caused enhanced
carotenoid accumulation (Fox et al., 2005).
The effect of light (foils of various permeability, as described earlier) on
accumulation of carotenoids and phenolic compounds was negligible; ethylene
production determined in the attached fruits was irrespective to the applied foils.
Cox et al. (2003) observed higher lycopene concentration in tomato fruits, harvested
green-mature and exposed to 24 h light than in those radiated for 8 h. In our previous
investigations with broccoli heads of the spring growing cycle (extensive PAR intensity)
both radical scavenging activity and content of phenols were higher in comparison
with plants grown in autumn at poor radiation (Leja et al., 2002). No effect of radiation
intensity on numerous antioxidant parameters (flavonoids, carotenoids, ascorbic acid,
antioxidant activity) was found in ripening tomato fruits (Raffo, 2003).
As described earlier the slight effect of the reduced nitrogen form on higher
accumulation of phenolic compounds in pepper fruits of turning and red phase was
noticed, while no influence of this factor on ethylene evolution was found. Some
influence of the nutritive nitrogen form on the antioxidant system (antioxidants and
enzymes) of various vegetable species such as lettuce, broccoli, carrot, red cabbage,
particularly during their short-term storage was reported previously (Leja et al., 1994;
1997; 1998; 2005).
In general, further investigations might explain the effect of both light intensity
and nutritive nitrogen form on changes of phenols, carotenoids and ethylene evolution.
The present experiment is the preliminary study of long-term project and it will be
continued.
Conclusions. 1. Significant accumulation of phenolic constituents and carotenoids
was observed in ripening sweet pepper fruits.
2. Sweet pepper of cultivar ‘Spartacus’ can be considered as the climacteric plant:
low ethylene production detected in green fruits increased with their maturity.
3. In most cases reaction to light conditions as well as to applied form of nutritive
nitrogen was negligible, only in fruits of turning (brownish) maturity stage the slight
effect was found.
Acknowledgment. The study was financed by the State Committee for Scientific
Research, Poland, under project No. 2 P06R 021 30
References
Gauta 2008 04 03
Parengta spausdinti 2008 04 28
1. Biles C. L., Wall M. M., Blackstone K. 1993. Morphological and physiological
changes during maturation of New Mexican type peppers. Journal of the American
Society for Horticultural Science, 118(4): 476–480.
2. Cox S. E., Stushnoff C., Sampson D. A. 2003. Relationship of fruit colour and
light exposure to lycopene content and antioxidant properties of tomato. Canadian
Journal of Plant Science, 83(4): 913–919.
281

3. Fox A. J., Pozo-Insfran D., Lee J. H., Sergent S. A., Talcott S. T. 2005. Ripeninginduced
chemical and antioxidant changes in bell peppers as affected by harvest
maturity and postharvest ethylene exposure. Hortscience, 40: 732–736.
4. Gross K., Watada A. E., Kang M. S., Kim S. D., Kim K. S., Lee S. W. 1986.
Biochemical changes associated with the ripening of hot pepper fruit. Physiologia
Plantarum, 66: 31–36.
5. Howard L. R., Talacott S. T., Brenes C. H., Villalon B. 2000. Changes in
phytochemical and antioxidant activity of selected pepper cultivars (Capsicum
Species) as influenced by maturity. Journal of Agricultural and Food Chemistry,
48: 1 713–1 720.
6. Leja M., Wojciechowska R., Mareczek A., Kunicki E. 1997. The effect of
fertilization with different forms of nitrogen on certain senescence indices in
short-term stored broccoli heads. Folia Horticulturae, 9(1): 85–96.
7. Leja M., Mareczek A., Starzynska A. 2002. Some antioxidant and senescence
parameters of broccoli as related to its maturity stages. Acta Physiologiae
Plantarum, 24(3): 237–241.
8. Leja M., Rozek S., Mareczek A. 1998. Effect of fertilization with reduced
nitrogen forms on phenolic metabolism in carrot roots. Umbelliferae Improvement
Newsletter, 8: 17–19.
9. Leja M., Rozek S., Myczkowski J. 1994. The effect of fertilization with different
forms of nitrogen on greenhouse lettuce quality and its changes during storage.
III. Phenolic metabolism. Folia Horticulturae, IV/1: 63–72.
10. Leja M., Wyzgolik G., Mareczek A. 2005. Phenolic compounds of red cabbage
as related to different forms of nutritive nitrogen. Horticulture and Vegetable
Growing, 24(3): 421–428.
11. Marcus F., Daood H. G., Kapitany J., Biacs P. A. 1999. Change in the carotenoid
and antioxidant content of spice red pepper (paprika) as a function of ripening
and some technological factors. Journal of Agricultural and Food Chemistry,
47: 100–107.
12. Mateos R. M., Leon A. M., Sandalio L. M., Gomez M., del Rio L. A.,
Palma J. M. 2003. Peroxisomes from pepper fruits (Capsicum annuum L.):
purification, characterisation and antioxidant activity. Journal of Plant Physiology,
160: 1 570–1 516.
13. Navarro J. M., Flores P., Garrido C., Martinez V. 2006. Changes in the contents
of antioxidant compounds in pepper fruits at different ripening stages, as affected
by salinity. Food Chemistry, 96: 66–73.
14. Polish Norm (PN-90).
15. Raffo A., Salucci M., Azzini E., Bertone A., Quaglia G. B., Fogliano V.,
Graziani G., Malfa G. 2003. Nutritional characteristics of greenhouse cherry
tomatoes. Acta Horticulturae, 614(2): 681–686.
16. Saltveit Jr. M. E. 1977. Carbon dioxide, ethylene and colour development ripening
mature green bell peppers. Journal of the American Society for Horticultural
Science, 102(5): 523–525.
17. Swain T., Hills W. E. 1959. Phenolic constituents of Prunus domestica.
I. Quantitative analysis of phenolic constituents. Journal of the Science of Food
and Agriculture, 10: 63–68.
282

18. Villavicencio L. E., Blankenship S. M., Sanders D. C., Swallow W. H. 2001.
Ethylene and carbon dioxide concentrations in attached fruits of pepper cultivars
during ripening. Scientia Horticulturae, 91: 17–24.
19. Zhang DongLin, Hamauzu Y. 2003. Phenolic compounds, ascorbic acid,
carotenoids and antioxidant properties of green, red and yellow bell peppers.
Journal of Food, Agriculture & Environment, 1(2): 22–27.
SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Kai kurių biocheminių parametrų kitimai saldžiosios paprikos
vaisių vystimosi metu
M. Leja, G. Wyżgolik, I. Kamińska
Santrauka
Saldžiosios paprikos veislė ‘Spartacus’ auginta veikiant dviem skirtingais PAR
intensyvumo lygiais. Tręšiama nitrato azotu ir jo redukuota forma (amoniu ir šlapalu). Vaisių
derlius nurenkamas trijose nokimo stadijose: žali vaisiai, nokstantys ir raudoni. Nustatytas
bendras fenolių ir karotinoidų kiekis bei etileno kitimas.
Tirpių fenolių kiekis nustatytas fotometriniu metodu su Folino reagentu, karotinoidai –
heksano ekstrakte matuoti fotometriрkai, etileno kitimas nustatytas GC metodu.
Reikšmingа fenolių ir karotinoidų kaupimа lydėjo etileno didėjimas. Skirtingiausia
karotinoidų sintezė stebėta, vaisiams esant pilnos brandos tarpsnyje (raudona spalva), o tirpūs
fenoliai laipsniрkai kaupėsi viso nokimo metu. Nei šviesa, nei maitinimas azotu aptartiems
parametrams įtakos nedarė.
Reikšminiai žodžiai: brendimas, etilenas, fenoliai, karotinoidai, saldžioji paprika.
283

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
The influence of various substratum on the quality of
cucumber seedlings and photosynthesis parameters
Julė Jankauskienė, Aušra Brazaitytė
Lithuanian Institute of Horticulture, Kauno 30, LT-54333 Babtai, Kaunas distr.,
Lithuania, e-mail: j.jankauskiene@lsdi.lt
Cucumber hybrids ‘Mandy’ were grown in the greenhouse covered with double polymeric
film at the Lithuanian Institute of Horticulture in 2004–2006. Cucumber seedlings were grown
in different substratum: peat, peat + perlite (1 : 1), peat + perlite (2 : 1), peat + zeolite (1 : 1),
peat + zeolite (2 : 1). During the experiment seedling biometrical measurements were carried
out, the amount of dry matter and pigments in seedling leaves, photosynthesis productivity was
established, cucumber yield calculations were fulfilled. Seedlings grown in peat are higher,
have bigger leaf area than the seedlings grown in peat-perlite, peat-zeolite substratum, but in
leaves and roots they accumulate less dry matter and plant fresh weight also is smaller. When the
amount of zeolite and perlite in peat is smaller, cucumber seedlings accumulate in leaves more
photosynthesis pigments. When zeolite is mixed into peat substratum, seedling photosynthesis
productivity becomes bigger than this of the cucumbers grown in peat. Nevertheless, the mixing
of zeolite and perlite into seedling substratum do not have positive influence on cucumber
yield.
Key words: cucumber, dry matter, yield, peat, perlite, photosynthesis pigments,
photosynthesis productivity, seedlings, zeolite.
Introduction. Substratum selection is important factor, influencing seedling
quality. For the seedling growing, peat of high type most often is used. When
growing plants in peat substratum it isn’t easy to keep the optimal air-water regime.
In order to create the more favourable air-water regime for plants, peat is mixed
with perlite, vermiculite et al (Sawan, Eissa, 1996). Zeolites are crystalline, hydrated
aluminosilicates of alkali and earth metals that possess infinite, three-dimensional
crystal structures. This is ecologically clean, inert and non-toxic substance. It has the
ability to absorb and hold plant nutrients due to its crystal lattice structure (Mumpton,
1999). Perlite is derived from siliceous volcanic rock that is crushed and heated to
in a furnace 982 °C until it expands to form the white particles that make up perlite.
These expanded particles provide for air-filled pore space in a substratum, provide
little water-holding capacity, have a negligible cation-exchange capacity and have a
pH of approximately 7.5. Perlite is considered chemically inert and has little effect on
substratum pH (Thomas, Thomas, 1988).
In the opinion of Russian scientists, one of the most perspective trends of plantgrowing
is the use of natural zeolite as substratum for seedling and vegetable growing
(Перфильева, 1988; 1991). In Russia there are created technologies of cucumber,
285

tomato and greens growing in zeolite substratum (Постников et al, 1991). Studies with
zeolite substratum were carried out in other countries – Bulgaria, Greece, Yugoslavia,
UK, etc. (Harland et al, 1999; Mumpton, 1999; Stamatakis et al, 2001). Markovic et al
compared different substratum for pepper seedling production – compost, peat and
enriched zeolites (Markovic et al., 1994). Manolov and other scientist investigated
the possibilities for growing of vegetable seedlings in zeolite substratum based on
Jordanian zeolitic tuff and compared it with zeolite substratum based on Bulgarian
zeolite (Manolov et al, 2005). Cattivello investigated the possibilities of zeolite use for
the growing of vegetable seedlings and pot plants. Zeolite didn’t influence positively
the quality of lettuce, tomato, and melon seedlings and the earliness of yielding.
Cyclamen and primroses grew better when 7 % of zeolite was mixed into substratum
(Cattivello, 1995).
Perlite is widely used for the rooting of decorative plants, flower growing, also in
the mixture with peat when growing flower seedlings and in small-volume vegetable
growing technology (Arenas et al., 2002; Grillas et al., 2001). The possibilities of the
use of zeolite and perlite as the ingrediens of substratum for seedling growing, are
still not investigated in Lithuania.
The aim of the study is to establish the influence of zeolite and perlite mixed into
peat substratum on the development of cucumber seedlings, photosynthesis productivity
and total yield.
Object, methods and conditions. Investigations were carried out in the
greenhouse covered with polymeric film at the Lithuanian Institute of Horticulture
in 2004–2006. Cucumber seedlings were grown in polymeric pots with prepared
peat substratum. Sowing time – the beginning of February. Cucumber seedlings
were grown in seed-plot on boards, the duration of growing – 30 days. The object of
investigation – hybrid ‘Mandy’. Seedlings were grown in different substratum: a 0
– peat,
a 1
– peat + perlite (1 : 1), a 2
– peat + perlite (2 : 1), a 3 –
peat + zeolite (1 : 1), a 4
– peat +
zeolite (2 : 1). Seedlings were planted in the greenhouse in the middle of March. In
the greenhouse plants were grown in 25 l capacity peat bags (1 bag – 2 plants). Plant
density – 2.5 plant. m -2 . In the greenhouse cucumbers were fertilized with “Nutrifol”
(green and brown), magnesium sulphate, calcium and ammonium nitrate taking into
account the stage of growth. For water souring there was used nitrogen acid. Salt
concentration in the nutritional solution – EC 2.5–2.8, acidity – pH 5.5–5.8. The end
of cucumber vegetation – the middle of June. Area of record plot – 4.8 m 2 . The trial
was established in randomized block design with three replications.
During investigation seedling biometrical observations were carried out, amount
of pigments and dry matter in seedling leaves and photosynthesis productivity were
established. The amount of photosynthesis pigments in fresh weight was established
preparing 100 % acetone extracts and analyzing them by spectrophotometrical
Wettstein’s method (Wettstein, 1957). There was used spectrophotometer Genesys 6
(Thermospectronic, JAV). Assimilation area was measured with leaf area measurer
CI-202 (CID Inc., USA). Plant dry weight was established drying at the temperature
of 105 °C.
Pure photosynthesis productivity (F pr
) was calculated according to the formula:
286

F pr
= 2(M 2
- M 1
) / (L 1
+ L 2
)T (1)
Here (M 2
- M 1
) – increase of dry weight during certain period; L 1
and L 2
– leaf area
at the beginning and at the end of the period; T – duration of period 24 h (Bluzmanas
et al, 1991). There was carried out cucumber yield calculation. Cucumbers were picked
for three times per week and sorted into standard and not standard. Yield data was
processed by statistical methods (Tarakanovas, Raudonius, 2003).
Results. Cucumber seedlings grown in peat were 36.5 % higher than the seedlings
grown in peat-zeolite (2 : 1) substratum (essential difference) and 30.1 % higher than
the seedlings grown in peat-zeolite (1 : 1) substratum (Table 1). Cucumber seedlings
grown in peat also were higher than the seedlings grown in peat-perlite substratum.
They were 35.7 % higher then seedlings grown in peat-perlite (2 : 1) substratum
(essential difference) and 17.0 % higher than the seedlings grown in peat-perlite (1 : 1)
substratum. The lowest seedlings were of plants grown in peat-zeolite (2 : 1) substratum.
The biggest number of leaves formed the seedlings, which were grown in peat-perlite
(1 : 1) substratum. Seedlings grown only in peat formed the biggest leaf area. When it
was mixed perlite and zeolite into peat substratum, leaf area was smaller thant this of
seedlings grown in peat. But when perlite was inserted into peat substratum, leaf area
was bigger than this of seedlings grown in peat-zeolite substratum. Seedlings grown
in peat enriched with zeolite and perlite (with the exception of peat-perlite (2 : 1)
substratum) produced the bigger plant and root fresh weight.
Table 1. Biometric data of cucumber seedlings grown in the different
substratum
1 lentelė. Agurkų daigų, augintų skirtinguose substratuose, biometrija
Cucumber seedlings grown in peat-zeolite (1 : 1) substratum the biggest amount of
dry matter accumulated in leaves and roots (Fig. 1). Their amount was correspondingly
3.7 % bigger than this in the leaves of seedlings grown in peat and 1.6 times bigger
than in the roots of seedlings grown in peat. Cucumber seedlings grown in peat-zeolite
and peat-perlite substratum accumulated in roots more dry matter than these grown in
peat. Seedlings grown in peat-perlite (2 : 1) substratum accumulated the least amount
287

of dry matter. Seedlings grown in peat-perlite substratum accumulated in roots more
dry matter than these grown in peat-zeolite substratum.
Fig. 1. The amount of dry matter in leaves and roots of cucumber seedlings grown
in the different substratum: 1 – peat; 2 – peat + perlite (1 : 1);
3 – peat + perlite (2 : 1); 4 – peat + zeolite (1 : 1); 5 – peat + zeolite (2 : 1)
1 pav. Sausųjų medžiagų kiekis agurkų daigų, augintų skirtinguose substratuose, šaknyse bei
antžeminėje dalyje: 1 – durpė; 2 – durpė + perlitas (1 : 1);
3 – durpė + perlitas (2 : 1); 4 – durpė + ceolitas (1 : 1); 5 – durpė + ceolitas (2 : 1)
Seedlings grown in peat-zeolite (2 : 1) substratum accumulated in leaves the
biggest amount of chlorophylls (Table 2).
Table 2. Chlorophyll contents in the leaves of cucumber seedlings grown in
different substratum
2 lentelė. Chlorofilų kiekis agurkų daigų, augintų skirtinguose substratuose, lapuose
There was more 13.7 % of them than in the leaves of seedlings grown in peat.
Nevertheless, the ratio of chlorophylls a and b was the same as in the leaves of
cucumbers grown in peat. Cucumber seedlings grown in peat-perlite and peat-zeolite
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(1 : 1) substratum accumulated in leaves less chlorophylls than seedlings grown in
peat. Nevertheless, the ratio of chlorophylls a and b was the bigger in the leaves of
cucumber grown in various substratum than this in the leaves of seedlings grown
only in peat.
The amount of carotenoids in the leaves of cucumber seedlings grown in peatzeolite
substratum (2 : 1) was 11.4 % bigger than this in the leaves of seedlings grown
only in peat (Fig. 2). In the leaves of cucumber grown in peat-perlite (2 : 1) substratum
there was as much carotenoids as in the leaves of seedlings grown in peat, but after
insertion of the bigger amount of perlite (1 : 1), the amount of carotenoids in leaves
decreased.
Fig. 2. Carotenoid content in the leaves of cucumber seedlings grown in the
different substratum: 1 – peat; 2 – peat + perlite (1 : 1); 3 – peat + perlite (2 : 1);
4 – peat + zeolite (1 : 1); 5 – peat + zeolite (2 : 1)
2 pav. Karotinoidų kiekis agurkų daigų, augintų skirtinguose substratuose, lapuose:
1 – durpė; 2 – durpė + perlitas (1 : 1); 3 – durpė + perlitas (2 : 1);
4 – durpė + ceolitas (1 : 1); 5 – durpė + ceolitas (2 : 1)
The bigger amount of zeolite and perlite (1 : 1) determines the fact that cucumber
seedlings accumulate in leaves smaller amount of pigments, i. e. chlorophylls and
carotenoids, but in roots and leaves accumulate more dry matter. When the amount of
zeolite or perlite in peat is less (2 : 1), cucumber seedlings accumulate in leaves more
pigments and less dry matter.
Photosynthesis productivity was the biggest one of the seedlings grown in peatzeolite
(1 : 1) substratum (Fig. 3). It was 1.5 times bigger than this of seedlings grown
in peat. Photosynthesis productivity of seedlings grown in peat-zeolite (2 : 1) and
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peat-perlite (1 : 1) substratum also was bigger than this of seedlings grown in peat.
Photosynthesis productivity of seedlings grown in peat-perlite (2 : 1) substratum was
almost the same as this of seedlings grown in peat. When there was mixed bigger
amount of perlite and zeolite into peat substratum (1 : 1), seedling photosynthesis
productivity was bigger than in the case of smaller amount of zeolite and perlite in
peat, i. e. 2 : 1.
Fig. 3. Photosynthesis productivity of cucumber seedlings grown in the different
substratum: 1 – peat; 2 – peat + perlite (1 : 1); 3 – peat + perlite (2 : 1);
4 – peat + zeolite (1 : 1); 5 – peat + zeolite (2 : 1)
3 pav. Agurkų daigų, augintų skirtinguose substratuose, fotosintezės produktyvumas:
1 – durpė; 2 – durpė + perlitas (1 : 1); 3 – durpė + perlitas (2 : 1);
4 – durpė + ceolitas (1 : 1); 5 – durpė + ceolitas (2 : 1)
The mixing of perlite and zeolite into peat substratum didn’t influence positively
cucumber yield (Fig. 4).
Fig. 4. Total yield of cucumbers which seedlings were grown in different
substratum: 1 – peat; 2 – peat + perlite (1 : 1); 3 – peat + perlite (2 : 1);
4 – peat + zeolite (1 : 1); 5 – peat + zeolite (2 : 1)
4 pav. Agurkų, kurių daigai auginti skirtinguose substratuose, suminis derlius:
1 – durpė; 2 – durpė + perlitas (1 : 1); 3 – durpė + perlitas (2 : 1);
4 – durpė + ceolitas (1 : 1); 5 – durpė + ceolitas (2 : 1)
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The yield of cucumber, which seedlings were grown in peat-zeolite (2 : 1)
substratum, was the same as this of the plants, which seedlings were grown in peat.
The yield of cucumber, which seedlings were grown in peat-zeolite (1 : 1) and peatperlite
(2 : 1) substratum, was smaller than this of cucumber, which seedlings were
grown only in peat.
Discussion. The selection of substratum influences not only seedlings quality, but
also plant yield, the earlines of their yielding and fruit quality (Lopez et al., 2004).
Zeolite and perlite is used as the addition of other peat substratum. According to the
data of Arenas and other scientists, the mixing of perlite, vermiculite, coco fibers into
peat substratum influenced tomato development. Seedlings grown in peat-vermiculite
and peat-perlite substratum had bigger root weight, stem diameter, leaf area (Arenas
et al., 2002). The data of other scientists showed that the highest seedling quality
was achieved using the mixture of substratum, peat (2/3) and enriched zeolite (1/3)
(Markovic et al., 1995). Pepper seedlings grown in peat substratum, enriched with
1/3 zeolite, were higher, had more leaves and dry matter (Markovic et al., 2000). The
amount of zeolite also influences seedling growth and development. Stem diameter,
leaf area and dry weight of seedlings increased with the increased amount of zeolite
(Song et al., 2004). According to the data of Güler et al, tomato seedlings grown in
peat-perlite and other substratum and grown only in peat were the same (Güler, Büyük,
2007). According to the data of Eltez and other scientists, the seedlings of aubergine
and pepper grown in peat-perlite substratum didn’t differe from seedlings grown in
peat (Eltez et al., 1994). According to the data of Demirer and Kuzucu investigations,
perlite positively influenced the growth and development of lettuce, cucumber, tomato
seedlings (Demirer, Kuzucu, 2000). According to the data of our investigation, zeolite
and perlite, mixed into peat substratum influenced biometrical parameters of seedlings.
Cucumber seedlings grown in peat-perlite and peat-zeolite substratum were lower and
had smaller leaf area thant the seedlings grown in peat. Seedlings grown in peat-perlite
and peat-zeolite substratum had bigger fresh weight.
In order to evaluate the influence of the used substratum on seedlings it was
established the amount of photosynthesis pigments in green cucumber leaves. The
amount of chlorophylls in plant leaves is one of the parameters of potential productivity.
It is often used in order to established as some method of growing and environmental
conditions influence plant photosynthesis system. If growth conditions aren’t suitable
chlorophylls concentration and the ration of chlorophylls a and b decreases. Chlorophyll
a is more important to photosynthesis process. It more quickly reacts to the changing
environmental conditions (Gabryś et al., 1998; Hay, Andrew, 1989). Addition of
zeolite to the substratum also had some effects on the photosynthetic pigment contents,
photosynthetic parameters (Song et al., 2004). According to the data of Güler and other
scientists, the amount of chlorophylls in the leaves of cucumber seedlings grown in
peat-perlite (1 : 1) substratum was smaller than this of the leaves of seedlings grown
in peat (Güler, Büyük, 2007). In our investigations the addition of perlite in peat
also slightly inhibited the synthesis of photosynthesis pigments. Small amount of
zeolite (peat:zeolite 2 : 1) stimulated the synthesis of these pigments in the leaves of
cucumber seedlings. The amount of perlite and zeolite in peat influenced the amount of
photosynthesis pigments in leaves. When the bigger amount of zeolite was mixed into
291

peat substratum (1 : 1), cucumber seedlings accumulated in leaves more carotenoids.
When zeolite and perlite was mixed into peat (ratio 1 : 1), the amount of chlorophylls in
cucumber leaves was smaller. When the ratio of peat and zeolite was 2 : 1, the amount
of chlorophylls in leaves was bigger than this in the leaves of seedlings grown in peat.
The mixing of zeolite into peat substratum increased net photosynthesis productivity.
The amount of photosynthesis pigments and photosynthesis productivity are one of
plant productivity parameters, which influence their final productivity. According
to the data of some scientists, when zeolite is mixed into seedling substratum, there
was obtaines bigger pepper yield (Markovic et al., 2000). According to the data
of our investigations, seedling growing in peat-zeolite substratum didn’t increase
cucumber yield. Even though cucumber seedlings grown in peat-perlite substratum
didn’t distinguished themselves with bigger amount of photosynthesis pigments or
photosynthesis productivity, the yield of these seedlings was similar to this one of
plants, which seedlings were grown in peat.
Cucumber seedlings grown in peat-perlite and peat-zeolite substratum are compact,
have smaller leaf area, but their above-ground and root weight is bigger than this
of seedlings grown in peat. The bigger amount of zeolite and perlite (1 : 1) in peat
determines the fact that cucumber seedlings accumulate in leaves smaller amount of
pigments, but in roots and leaves accumulate more dry matter. When the amount of
zeolite or perlite in peat is less (2:1), cucumber seedlings accumulate in leaves more
pigments and less dry matter.
Conclusions. 1. Cucumber seedlings grown in peat substratum were higher, had
bigger leaf area than the seedlings grown in peat-perlite and peat-zeolite substratum, but
the seedlings grown in peat-zeolite (1 : 1 and 2 : 1) and peat-perlite (1 : 1) substratum
had bigger fresh weight and root weight.
2. Cucumber seedlings grown in peat-zeolite and peat-perlite substratum
accumulated in leaves and roots more dry matter than the seedlings grown only in
peat.
3.The addition of perlite in peat decreased the synthesis of photosynthesis
pigments, and small amount of zeolite (peat:zeolite 2 : 1) stimulated the synthesis in
the leaves of cucumber seedlings.
4. The biggest photosynthesis productivity was in the substratum of seedlings
grown in peat-zeolite (1 : 1).
5. The mixing of perlite and zeolite into substratum of seedlings didn’t influence
positively cucumber yield.
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SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Įvairių substratų įtaka agurkų daigų kokybei bei fotosintetiniams
rodikliams
J. Jankauskienė, A. Brazaitytė
Santrauka
Darbo tikslas – nustatyti ceolito bei perlito, įmaišyto į durpių substratа, įtaką agurkų
daigų vystymuisi bei fotosintezės produktyvumui ir suminiam derliui. 2004–2006 m. Lietuvos
sodininkystės ir daržininkystės institute dviguba polimerine plėvele dengtame šiltnamyje auginti
‘Mandy’ hibridiniai agurkai. Agurkų daigai auginti skirtinguose substratuose: durpė, durpė +
perlitas (1 : 1), durpė + perlitas (2 : 1), durpė + ceolitas (1 : 1), durpė + ceolitas (2 : 1). Bandymo
metu atlikti daigų biometriniai matavimai, nustatytas sausųjų medžiagų, pigmentų kiekis daigų
lapuose, nustatytas fotosintezės produktyvumas, atlikta agurkų derliaus apskaita. Daigai, auginti
durpėje, yra aukštesni, turi didesnį lapų asimiliacinį plotą negu daigai, auginti durpių-perlito,
durpių-ceolito substratuose, tačiau jie lapuose ir šaknyse kaupia mažiau sausųjų medžiagų, žalia
augalo masė taip pat yra mažesnė. Kai ceolito ir perlito kiekis durpėje yra mažesnis, agurkų
daigai lapuose kaupia daugiau fotosintezės pigmentų. Į durpių substratą įmaišius ceolitą, daigų
fotosintezės produktyvumas didesnis negu augintų durpėje. Tačiau ceolito bei perlito įmaišymas
į daigų substratą neturi teigiamos įtakos agurkų derliui.
Reikšminiai žodžiai: agurkai, ceolitas, daigai, derlius, durpės, fotosintezės pigmentai,
fotosintezės produktyvumas, perlitas, sausosios medžiagos.
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SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Evaluation of the methods of soil cultivation growing
dessert strawberries in beds
Nobertas Uselis, Juozas Lanauskas, Vytautas Zalatorius,
Pavelas Duchovskis, Aušra Brazaitytė, Akvilė Urbonavičiūtė
Lithuanian Institute of Horticulture, Kauno 30, LT-54333 Babtai, Kaunas distr.,
Lithuania, e-mail: n.uselis@lsdi.lt
The aim of the study was to investigate the influence of the methods of soil cultivation
on strawberry plant development, plant generative development, their physiological processes
and berry yield. The investigations of the methods of strawberry plantation soil cultivation
were carried out at the Lithuanian Institute of Horticulture in 2005–2007 growing strawberries
according to the scheme: 1) not mulched with film, not irrigated, 2) not mulched with film,
irrigated, 3) mulched with film, not irrigated, 4) mulched with film, irrigated. Strawberries were
grown for the usual (not covered) and earlier (covered with agrofilm) yield. It was established
that in the case of low temperature (~ -30 °C) and little amount of snow, strawberries mulched
with white film wintered best of all. In the beds mulched with film all the plants wintered,
and in not mulched beds 64–97 % of plants survived. The biggest amounts, up to 30 %, of
strawberry flowers were frost damaged in the mulched beds. In not mulched beds there were
frost-damaged only 10 % of strawberry flowers. The positive influence of mulching on the
number of leaves per plant, fresh weight and assimilation area was significant only in the first
year of growth. The methods of soil supervision and irrigation do not influence significantly the
amount of chlorophylls in strawberry leaves. In the first year of yielding, strawberries mulched
with film produced the biggest amount of berries. In the second year of yielding, berry number
per plant essentially didn’t depend on soil mulching or irrigation. In the first year of yielding,
strawberries grown in the beds mulched with white film yielded better. In the second year of
yielding, the productivity of strawberries grown without cover didn’t differ. Irrigation didn’t
influence strawberry productivity.
Key words: growth, strawberry, soil cultivation methods, yield.
Introduction. After Lithuania entered the European Union, purchasing power
of population increased and high quality fruits and vegetables became more popular.
Strawberries are among the most popular berries in our country. Strawberry growing
requires much manual labour. In recent years, a lot of working power emigrated or
went to work into the biggest cities of the country. Under such social and demographic
situation, it is necessary to apply in strawberry growing maximally mechanized growing
technologies, which lessen the expenditures of manual labour and allow growing
competitive production.
For the time being in the other European Union countries strawberries most
often are grown for one year in high single row beds mulched with film and irrigated
by capillary method. Berries grown in annual strawberry plantations are big and of
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attractive marketable appearance. Such growing of dessert strawberries is rather
expensive, even though maximally mechanized (manually are picked only berries).
According to the data, the mulching of strawberries grown in beds with various
plastic films helps to preserve water in the soil, controls weed growth, decreases the need
of herbicides and improves berry quality (Kasperbauer, 2000; Fernandez, 2001). Other
authors state that the use of such mulch accelerate berry ripening (Pritts et al., 1992),
extend growing season (Pollard, Cundari, 1988), protects plants against frosts during
flowering (Hochmuth et al., 1993), increases strawberry productivity (Gast, Pollard,
1991; Pritts et al., 1992). Strawberry mulching in autumn improves inflorescence
development and increases yield (Gast, Pollard, 1991). Mulching positively influences
pigment synthesis in strawberry leaves (Wang et al., 1998; Kirnak et al., 2002).
Mulching coordinated with irrigation increases strawberry productivity (Renquist et al.,
1982). Most often strawberries are mulched with black plastic films (Lamont, 1993).
It is established that mulch of black polyethylene improves the quality of strawberry
berries and the yield (Wittwer, Castilla, 1995). It is stated in the literature that even
though such strawberry growing method is expensive, but the bigger productivity and
smaller work expenditures allow obtaining higher income (Butler et al., 2002).
Lately Lithuanian farmers quickly change extensive strawberry growing
technologies on the flat soil surface to more intensive and progressive ones on the
profiled surface. Unfortunately, there is changeable snow cover in Lithuania and big
winter frosts occur, therefore when growing strawberries in not mulched beds, plants
may be more frost bitten. Earlier investigations showed that weed destroying in
strawberry plantation both mechanically and chemically is rather expensive (Uselis,
Kulikauskas, 2004). Under the expansion of trade strawberry growing it is necessary
to look for more effective methods of soil supervision in strawberry plantations.
The aim of the study was to investigate the influence of the methods of soil
cultivation on strawberry plants development, plant generative development, their
physiological processes and berry yield.
Object, methods and conditions. The investigations of the strawberry plantation
soil cultivation methods were carried out at the Lithuanian Institute of Horticulture in
2005–2007 growing strawberries according to the scheme:
1. Strawberries not mulched with film, not irrigated.
2. Strawberries not mulched with film, irrigated.
3. Strawberries mulched with film, not irrigated.
4. Strawberries mulched with film, irrigated.
Strawberries were grown for the usual (not covered) and earlier (covered with
agrofilm) yield.
Strawberry cultivar ‘Elkat’ was grown in low three row beds, planting scheme –
(1.0 + 0.35 + 0.35 × 0.2 m) (87 719 unt./ha). Strawberries, which weren’t mulched
with film, before picking were mulched with straw. Experiment variants were carried
out in 3 replications. Experimental strawberry plantation was cultivated on August
15–20, 2005.
During investigation such parameters were established: strawberry plants
destruction by frost (%); berry amount (unt. plant -1 ); berry yield (t ha -1 ); amount of
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photosynthesis pigments in fresh strawberry leaves (established in 100 % acetone
extraction with spectrophotometer (Genesis 6) according Wettstein (Wettstein, 1957).
Leaf area was measured with automatic measurer (WinDias). Plant weight was
calculated by gravimetrical method. The measurements of strawberries of cultivar
‘Elkat’ were carried out in the beginning of their ripening. Data significance was
evaluated by the method of one-factor dispersion analysis, applying the program
ANOVA (Tarakanovas, Raudonius, 2003).
Results. Wintering of strawberry shrubs. During strawberry
investigation in 2005–2006, in the beginning of winter there was period when there
almost wasn’t snow cover and air temperature all the week was -30 °C. Under such
conditions, strawberries, especially these, which grew in beds, might be frost damage.
Investigations showed that in the case of cold winter and small amount of snow,
strawberry mulched with white film winter best of all. After mulching with film all the
plants wintered, and in not mulched beds only 64–97 % of plants survived (Table 1).
Before frosts, the inter-rows were mulched with straw.
Table 1. The amount of wintered strawberries.
1 lentelė. Peržiemojusių braškių kiekis
Babtai, 2006
In 2007 winter during frosts there was snow cover and strawberry weren’t frost
damaged. In spring, on May 1–6, when temperature fell down to -4–-6°C, blooms of
fully covered strawberries, which then were at full bloom, were very frost damaged.
This was determined by strawberry plantation soil cultivation method. The biggest
amounts, up to 30 % of strawberry flowers, were frost damaged in beds mulched with
film. In not mulched beds essentially less strawberry flowers were frost damaged
(10 %).
G r o w t h v i g o u r a n d d e v e l o p m e n t o f s t r a w b e r r y p l a n t s.
When strawberries ‘Elkat’ were grown without cover, they had essentially more
leaves, then they were mulched with film than not mulching them and irrigating. In
the second year of yielding the number of leaves per plant completely didn’t depend
on strawberry cultivation method. Under cover there weren’t essential differences of
soil cultivation methods (Table 2).
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Table 2. The number of strawberry leaves (unt. per plant)
2 lentelė. Braškių lapų skaičius, vnt. aug. -1 Babtai, 2006–2007
In both years of investigation the fresh weight of overground part of strawberry
cultivar ‘Elkat’ under cover was smaller than this of uncovered strawberries.
Strawberries grown without cover produced the least amount of fresh weight when
they weren’t mulched and irrigated. Not mulched but irrigated strawberries produced
slightly bigger fresh weight. Irrigation didn’t influence the fresh weight of mulched
strawberries. The analogical fresh weight change tendencies of the strawberries
grown under cover were observed only in the first year. On the contrary, in the second
year mulched strawberries (in comparison with the not mulched ones) and irrigated
strawberries (in comparison with the not irrigated ones) produced less fresh weight
(Table 3).
Table 3. Fresh weight of strawberry shrubs (g)
3 lentelė. Braškių kerelių žalioji masė, g
Babtai, 2006–2007
The cultivation methods didn’t influence essentially the leaf area of not covered
strawberries, but it was the biggest of strawberries mulched with film and not irrigated.
Mulched strawberries under cover in the first year of growth produced much bigger leaf
area. In the second year there weren’t essential differences, but the biggest leaf area
was formed by mulched and not irrigated strawberries. It became clear that irrigation
didn’t increase plant leaf area (Table 4).
Not mulched and irrigated strawberries ‘Elkat’ in the first year of yielding had
essentially less inflorescences than these mulched and irrigated (Table 5).
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Table 4. Leaf area of strawberries (cm 2 )
4 lentelė. Braškių asimiliacinis plotas, cm 2 Babtai, 2006–2007
Table 5. Number of strawberry inflorescences (unt. per plant)
5 lentelė. Braškių žiedynų skaičius, vnt. aug. -1 Babtai, 2006–2007
Strawberry yield depends on strawberry development. In the first year of yielding
essentially the biggest amount of berries produced strawberries mulched with film.
Essentially the least amount of berries produced strawberries in not mulched, not
irrigated and not covered with agrofilm beds (Table 6). In the second year of strawberry
yielding the number of berries essentially didn’t depended on soil mulching or irrigation
(Table 6).
Table 6. Number of strawberry berries (unt. per plant)
6 lentelė. Braškių uogų skaičius, vnt. aug. -1 Babtai, 2006–2007
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Total chlorophylls content in leaves. Soil cultivation methods didn’t influence
essentially the amount of chlorophylls in strawberry leaves. There were established
slightly more chlorophylls in the leaves of ‘Elkat’ strawberries grown under cover.
In the second year of growing there was established more chlorophylls in the leaves
of strawberries mulched with film than in the leaves of not mulched strawberries
(Table 7).
Table 7. Total chlorophylls content in strawberry leaves (mg g -1 )
7 lentelė. Bendras chlorofilų kiekis braškių lapuose, mg g -1 Babtai, 2006–2007
Strawberry yield. In the first year of yielding strawberry yield very depended on
strawberry plantation soil cultivation method. In all the cases strawberries grown in
bed mulched with white film yielded essentially better. In the second year of yielding
the productivity of not covered strawberries essentially didn’t differ, and strawberries
under agrofilm, mulched with white film yielded worse because their flowers were
frost damaged. Strawberry irrigation didn’t influence essentially their productivity
(Table 8).
Table 8. Berry yield (t ha -1 )
8 lentelė. Uogų derlius, t ha -1 Babtai, 2006–2007
Discussion. The conditions for strawberry growing in Lithuania, in comparison
with the most EU countries, are more complicate. In most West and South Europe
countries winters are warm with variable and often thin snow cover. In Lithuania,
in different winter periods snow cover also is thin or there is no snow at all. Air
300

temperature may fall down to -30 °C and lower. Therefore, in strawberry growing
technology it is necessary to use the means, which allow decreasing negative cold
influence in winter periods without snow. Our investigations showed that in the case of
cold winter with very thin snow cover, strawberries mulched with white film wintered
best of all (Table 1). Other authors also indicate that mulching helps strawberries to
winter in North countries (Gast, Pollard, 1989; Pollard et al., 1989; Dalman, Matala,
1997). Nevertheless, we find in the literature contradictional data about the influence
of mulching when protecting plants against spring frosts. Some authors maintain that
mulching protects against spring frosts (Hochmuth et al., 1993), but more authors state
that mulched strawberries suffer from spring frosts (Pollard et al., 1989; Fernandez,
2001; Plekhanova, Petrova, 2002). Earlier strawberries are especially susceptible to
frosts (Plekhanova, Petrova, 2002).
According to the data presented in literature, mulching positively influences
vegetative strawberry productivity components, such as diameter of crowns, number
of leaves per crown and plant, leaf area, etc., which indirectly influence yield (Renquist
et al., 1982; Gast, Pollard, 1989; Fernandez, 2001; Kirnak et al., 2001; Sharma,
Sharma, 2003; Singh et al., 2005). Under the conditions of our investigation, the
positive influence of mulching on the number of leaves per plant, fresh weight and
assimilation area was evident only in the first year of growth. In the second year of
yielding these indices almost didn’t depend on soil supervision method (Tables 2–4).
Literature presents contradictional data about the influence of mulching in the first and
second year of growth. Renquist with co-authors (1982) state that positive influence
of mulching on vegetative growth in the second year became even more evident,
and according to the data of Fernandez (2001), such influence became clear only in
the second year. Some authors present the data that irrigation improves the positive
influence of mulching on strawberry growth (Renquist et al., 1982), but the other didn’t
establish such influence in their investigations (Pires et al., 2006). Such influence in our
investigations also wasn’t established. The lack of such influence probably is connected
with the sufficient amount of natural precipitation during experiments.
According to the data of many authors, mulching increases the number of
inflorescences and berries, stimulates berry ripening ahead of time, increases yield
(Fernandez, 2001; Plekhanova, Petrova, 2002; Singh et al., 2005), but does not
influence berry size (Fernandez, 2001; Mohamed, 2002; Kivijarvi, Prokkola, 2003).
According to the data of our investigations, mulching didn’t influence inflorescence
number, but increased the amount of berries, the weight of one berry and the yield in
the first year of growing. In the second year of growing there wasn’t established the
influence of different soil supervision methods on strawberry yield. Mulching with
irrigation insignificantly increased the mentioned indices. Irrigation in the first year
insignificantly increased the yield of not mulched strawberries also (Tables 5, 6, 8,
9). Other scientists obtained the similar results (Renquist et al., 1982; Krüger et al.,
2002; Kirnak et al., 2003).
The amount of photosynthesis pigments in plant leaves is one of the potential
indices of productivity. Basing on this indice it is ofter established how environmental
conditions or plant development affect their photosynthesis system (Hay and Andrew,
1989; Kirnak et al., 2002; Keutgen et al., 2005). The creation of the suitable conditions
301

for the optimal photosynthesis allows increasing plant yield (Sharma-Natu, Ghildiyal,
2005). In our investigations soil cultivation methods and irrigation didn’t influence
essentially the amount of chlorophylls in strawberry leaves and the conditions for
strawberry growing were suitable (Table 7).
Conclusions. 1. Under low temperature (~ -30 °C) and thin snow cover strawberries
mulched with white film wintered the best of all. All the plants wintered in beds mulched
with film, and in not mulched beds remained only 64–97 % of shrubs.
2. During spring frosts the biggest amount, up to 30 %, of strawberry flowers
were frost damaged in mulched beds. In the beds not mulched with film there were
frost damaged only 10 % of strawberry blooms. Blooms of strawberries grown in the
open field without agrofilm cover weren’t frost damaged at all, because during frosts
they didn’t have inflorescences yet.
3. The positive influence of mulching on the number of leaves per plant, fresh
weight and leaf area was evident only in the first year of growth. In the second year
of yielding these indices almost didn’t depend on soil cultivation method.
4. Soil cultivation methods and irrigation didn’t influence the total chlorophylls
content in strawberry leaves.
5. In the first year of yielding strawberries mulched with agrofilm produced the
biggest amount of berries. Strawberries not covered with agrofilm and grown in not
mulched and not irrigated bed produced the least amount of berries. In the second
year of strawberry yielding berry number per plant do not depend on soil mulching
or irrigation.
6. In the first year of yielding strawberries mulched with white film yielded better.
In the second year of yielding the productivity of strawberries grown without cover
didn’t differ, and strawberries under agrofilm cover mulched with white film yielded
worse because their flowers were frost damaged. Strawberry irrigation didn’t influence
essentially their productivity.
Acknowledgement. Authors are grateful to the State Science and Studies
Foundation for financial support.
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Gauta 2008 04 23
Parengta spausdinti 2008 04 30
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SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Dirvos priežiūros būdų įvertinimas auginant desertines braškes
lysvėse
N. Uselis, J. Lanauskas, V. Zalatorius, P. Duchovskis, A. Brazaitytė,
A. Urbonavičiūtė
Santrauka
Darbo tikslas – ištirti dirvos priežiūros būdų įtaką braškių kerelių išsivystymui, augalų
generatyvinei raidai, jų fiziologiniams procesams bei uogų derliui. Braškyno dirvos priežiūros
būdų tyrimai atlikti Lietuvos sodininkystės ir daržininkystės institute 2005–2007 m., auginant
braškes pagal schemą: 1) nemulčiuotos plėvele, nelietintos, 2) nemulčiuotos plėvele, lietintos,
3) mulčiuotos plėvele, nelietintos, 4) mulčiuotos plėvele, lietintos. Braškės augintos įprastiniam
(nedengtos) ir paankstintam (dengtos agroplėvele) derliui gauti. Nustatyta, kad esant žemai
temperatūrai (~ -30 °C) ir mažai sniego geriausia peržiemojo balta plėvele mulčiuotos braškės.
Plėvele mulčiuotose lysvėse peržiemojo visi augalai, o nemulčiuotose lysvėse išliko tik 64–97 %
kerelių. Pavasarinių šalnų metu daugiausiai, iki 30 %, žiedų pašalo mulčiuotose lysvėse augusių
braškių. Plėvele nemulčiuotose lysvėse pašalo tik 10 % braškių žiedų. Teigiamas mulčiavimo
poveikis lapų skaičiui augale, žaliajai masei ir asimiliaciniam plotui buvo žymus tik pirmaisiais
augimo metais. Chlorofilų kiekiui braškių lapuose dirvos priežiūros būdai ir lietinimas esminės
304

įtakos neturi. Pirmaisiais derėjimo metais daugiausia uogų išaugina plėvele mulčiuotos braškės.
Antraisiais braškių derėjimo metais uogų skaičius iš augalo iš esmės nepriklauso nei nuo dirvos
mulčiavimo nei nuo lietinimo. Pirmaisiais derėjimo metais balta plėvele mulčiuotose lysvėse
auginamos braškės derėjo gausiau. Antraisiais derėjimo metais be priedangų auginamų braškių
derlingumas nesiskyrė. Braškių lietinimas jų derlingumui įtakos neturėjo.
Reikšminiai žodžiai: augimas, braškės, dirvos kultivavimo būdai, derlius.
305

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Influence of climatic conditions of northeastern Poland
on growth of bower actinidia
Zdzisław Kawecki, Anna Bieniek
Department of Horticulture, University of Warmia and Mazury in Olsztyn, ul.
Prawocheсskiego 21, 10 – 719 Olsztyn, Poland
E-mail: anna.bieniek@uwm.edu.pl
Studies of the growth of vegetative and generative shoots of bower actinidia (Actinidia
arguta Sieb. et Planch.): ‘Purpurowaja Sadowaja’, ‘Kijewskaja Gibrydnaja’, ‘Kijewskaja
Krupnopщodnaja’, ‘Figurnaja’ and ‘Sientiabrskaja’ were carried out in the Experimental Garden
of University of Warmia and Mazury in Olsztyn in 2005–2007. The shoot length and diameter
as well as the number of leaves on one-year-old generative and vegetative shoots were recorded
every two weeks from the beginning of vegetation.
As a result of three years of observation, varieties ‘Sientiabrskaja’ and ‘Kijewskaja
Gibrydnaja’ gave the longest one-year-old vegetative shoots (103.62 cm and 77.79 cm). The most
intensive growth of shoots was observed in 2006 and 2007. The shortest vegetative shoots were
in variety ‘Figurnaja’ (31.3 cm). The longest generative shoots were in variety ‘Sientiabrskaja’
(mean for 3 years: 34.23 cm), the shortest shoots were in variety ‘Kijewskaja Gibrydnaja’
(14.44 cm). The highest diameter of generative shoots was in variety ‘Purpurowaja Sadowaja’
(0.51 cm). The lowest increasement was recorded in variety ‘Kijewskaja Gibrydnaja’ (0.42 cm).
The largest number of leaves on the vegetative shoots was in variety ‘Sientiabrskaja’, but the
smallest number of leaves was on generative shoots of variety ‘Purpurowaja Sadowaja’.
The most resistant to spring ground frost (freezing of flowers) was ‘Purpurowaja Sadowaja’
and ‘Kijewskaja Gibrydnaja’. Typical to 2007 were series of days with severe ground frosts in
the first and last decade of May, which might have affected a lack of fruit setting on shoots of
most of the actinidia varieties.
Key words: climate, bower actinidia, north-eastern Poland, plant morphology,
varieties.
Introduction. Bower actinidia Actinidia arguta (Siebold et Zucc.) Planch. Ex
Miq belongs to the genus Actinidia and the family of Actinidiaceae. This genus
includes 30–40 species of climbing plants. Actinidia arguta belongs to the section
Leiocarpae that encompasses species adapted for low temperatures in the winter season
(Latocha, 2006a). Depending on the variety, it survives frosts ranging from -23 °C to
-35 °C (Kawecki et al., 2004). On commencing the vegetative seasons, it is capable
of surviving slight frosts to -3 °C. In order to delay its vegetation, its cultivation on a
western or south-western exposure is recommended (Kawecki et al., 2007). To bear
fruits, bower actinidia plants need 150 days without ground frosts (Latocha, 2006 a).
Under the moderate climate conditions of Poland, especially of its more chill regions
including the province of Warmia and Mazury, it poses a great challenge to gardeners.
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Currently, these fruit-bearing climbers are relatively uncommon, yet their outstanding
taste and health-promoting values are arousing the interest of consumers on the fruit
market (Werner, 2002). Fruits of bower actinidia ripen at the end of September and
berries are 2–5 cm in length depending on variety, with hairless and smooth skin.
They occur of the following colors: green, green with blush and red (Bieniek et al.,
2006). As compared to kiwi fruits, they are a richer source of vitamin C; they are also
claimed to be tastier. In most cases, plants of bower actinidia are free of diseases and
pests and may be cultivated with ecological methods (Latocha, 2006 b). Nevertheless,
bower actinidia should not be treated only as a fruit plants as they also serve peculiar
decorative functions. The plants are readily planted in allotments and home gardens
along walls or other constructions.
The objective of the study was to characterize the growth of vegetative and
generative shoots as well as to determine the number of leaves, flowers and fruits on those
shoots in 5 hybrid varieties of bower actinidia: ‘Figurnaja’, ‘Kijewskaja Gibridnaja’,
‘Kijewskaja Krupnopщodnaja’, ‘Purpurowaja Sadowaja’ and ‘Sientiabrskaja’, under
climatic conditions of the city of Olsztyn (north-eastern Poland) in 2005–2007.
Object, methods and conditions. Five varieties of bower actinidia (Actinidia
arguta): ‘Purpurowaja Sadowaja’, ‘Kijewskaja Krupnopщodnaja’, ‘Kijewskaja
Gibrydnaja’, ‘Figurnaja’, ‘Sientiabrskaja’ were planted at a spacing of 1.5 × 2 m in
the Educational and Experimental Station of the University of Warmia and Mazury in
Olsztyn in autumn of 1996. For each variety, five bushes were planted. Male bushes
of varieties form were used as pollinators; they were used in the ratio of 1 : 5. The
first yield of fruit was obtained in the fourth year after planting. The plants grew in
corn-fodder strong complex IV class soil. It is strong clay sand of pH 6.2–6.8. Weeds
were removed manually three times per year during the vegetation period. The plants
did not have any diseases or pests; therefore no chemical treatment was applied.
Morphology of one-year-old vegetative and generative shoots of the examined
varieties of actinidia was determined in 200–2007. Results are mean values of three
samples measured simultaneously. Each year, from May 16th till September, 19th the
following morphological traits were measured every two weeks: length of vegetative
shoots, diameter of vegetative shoots, the number of leaves on a vegetative shoot,
length of generative shoots, diameter of generative shoots, the number of leaves
as well as flowers and next fruits on a generative shoot. The manuscript provides
final experimental results of 19th of September 2005, 2006 and 2007. Atmospheric
conditions occurring in the area of Olsztyn in 2005–2007 were described with data of
the Meteorological Station in Tomaszkowo (Table 1 and 2).
Results of measurements of morphological traits were analyzed statistically by
calculating the significance of differences between mean values with Duncan’s test at
a significance level of p = 0.05 for a two-factor experiment. The statistical software
package “Statistica 6.0” was used for calculations.
Results. Analyses of climatic conditions in particular years of morphological
measurements of the vegetative and generative shoots of the investigated actinidia
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varieties demonstrated that the highest mean temperature of the vegetative season
(from April till September) occurred in 2007 and reached 16.0 °C (Table 1). The lowest
mean temperature of the vegetative season, i. e. 13.1 °C, was recorded in 2006. As
compared to the multiannual mean of 1961–2000, it was lower by 0.5 °C. In the year
2006, especially low temperatures were reported in winter months: -8.5 °C in January,
-3.4 °C in February and -2.5 °C in March. In turn, temperatures of the summer months,
July and August of 2006 in particular, appeared to be the highest and reached 20.9 °C
and 18.2 °C, respectively. When compared to the multiannual mean, they were higher
by 3.7 °C in July and by 2.4 °C in August.
Table 1. The mean monthly values of weather factors in 2005–2007 and
multiannual mean (data of Meteorological Station in Tomaszkowo)
1 lentelė. Klimato veiksnių vidutinės mėnesio reikšmės 2005–2007 m. ir daugiamečiai
vidurkiai (Meteorologinės stoties Tomaškove duomenimis)
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In all experimental years, mean temperatures recorded in April were at a similar
level, i. e. 7.4 °C, 7.3 °C and 7.5 °C, and were higher than the multiannual mean, which
accounted for 7.0 °C. The highest mean temperature of May (13.8 °C) was recorded in
2007. In that year, the highest mean minimum ground temperature was also recorded.
In turn, the growth and development of actinidia and especially its blooming were
significantly affected by low ground temperatures, which between the 1 st and 5 th of May
reached -6.0 °C, -7.1 °C, -5.8 °C, -3.0 °C and -1.0 °C, respectively (Table 2). Ground
frosts were also recorded in the second decade of May, in which there were 7 days
with temperatures below zero. In the last two days of May 2007, ground temperatures
accounted for -1.4 °C and -3.5 °C, respectively.
Table 2. Spring ground frosts in 2005–2007 (data of Meteorological Station in
Tomaszkowo)
2 lentelė. Pavasarinės priežemio šalnos 2005–2007 m. (Meteorologinės stoties Tomaškove
duomenimis)
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In the first months of the vegetative season (April, May) the lowest total
precipitation accounting for 10.9 mm and 24.7 mm, was recorded in 2005. Total
levels of precipitation in May of 2006 and 2007 were similar and reached 25.6 and
24.7 mm, i. e. less by ca. 10 mm than the multiannual mean (35.4 mm). In the discussed
experimental years, May was characterized by a higher total level of precipitation:
89.2 mm and 93.5 mm, in respect of the multiannual mean that accounted for 57.6 mm.
In the summer months, the driest turned out to be July of 2006 with a total precipitation
of 29.3 mm. The subsequent month, August, was more abundant in precipitation (as
compared to levels recorded in 2005 and 2006 and to the multiannual mean), which
total level reached 165 mm. The record of total precipitation was broken in July of 2007,
when the total precipitation level reached 173.7 mm. The year 2005 was characterized
by the lowest total precipitation in August (33.1 mm), whereas September of 2005
appeared to be the most humid with a total precipitation of 78.4 mm. In the two
subsequent years, the total precipitation of September approximated the multiannual
mean (50 mm). The lowest level of precipitation in vegetative season was recorded
in 2005, i. e. 297.3 mm. In 2006 and 2007 levels of total precipitation in vegetative
season were similar and reached 556.9 mm and 564.5 mm, respectively. They were
higher than the multiannual mean, which accounted for 378.3 mm.
April of 2005 and 2006 was characterized by a similar number of days with slight
ground frosts, i. e. 14 and 15, respectively (Table 2). In April of 2007, ground frosts
were reported until the 14th of April, whereas the subsequent days of vegetative season
were warmer and no minus temperatures were noted. In the first days of May there
was a series of 5 days with severe ground frost and negative ground temperatures were
still recorded between the 15th and 31st of May. In the two previous years, May frosts
were small and occurred in two and three subsequent days.
The statistical analysis demonstrated significant differences in all the examined
morphological traits both between varieties and between experimental years (Table 3,
4, 5, 6, 7, 8, 9).
Table 3. Mean length (cm) of vegetative shoots of bower actinidia in
2005–2007
3 lentelė. Smailialapės aktinidijos vegetatyvinių ūglių vidutinis ilgis (cm) 2005–
2007 m.
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Table 4. Mean length (cm) of generative shoots of bower actinidia in
2005–2007
4 lentelė. Smailialapės aktinidijos generatyvinių ūglių vidutinis ilgis (cm) 2005–
2007 m.
Table 5. Mean diameter (cm) of vegetative shoots of bower actinidia in
2005–2007
5 lentelė. Smailialapės aktinidijos vegetatyvinių ūglių vidutinis skersmuo (cm)
2005–2007 m.
Table 6. Mean diameter (cm) of generative shoots of bower actinidia in
2005–2007
6 lentelė. Smailialapės aktinidijos generatyvinių ūglių vidutinis skersmuo (cm)
2005–2007 m.
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Table 7. Mean number of leaves on vegetative shoot of bower actinidia in
2005–2007
7 lentelė. Smailialapės aktinidijos vegetatyvinių ūglių vidutinis lapų skaičius (cm)
2005–2007 m.
The longest and the thickest vegetative shoots and generative ones with the
highest number of leaves, and the lowest numbers of flowers and fruits on generative
shoots were recorded in 2007. Observations carried out in three years of the study
demonstrate that the longest vegetative shoots were reported for variety ‘Sientiabrskaja’
(103.62 cm), followed by ‘Kijewskaja Gibrydnaja’ (77.79 cm). In turn, the shortest
vegetative shoots were noted in variety ‘Figurnaja’, i. e. 31.3 cm. Values of the mean
length of one-year-old vegetative and generative shoots appeared to be the lowest
in 2005 and reached 31.13 cm (Table 3) and 103.2 cm (Table 4), respectively. In
contrast, the highest value of the mean length of the shoots was noted in 2007, i.e.
109. 86 cm (Table 3) and 29.36 cm (Table 4), respectively. In the experimental years,
the variety with the highest values of the mean length of generative shoots appeared
to be ‘Sientiabrskaja’. The only exception was 2007 when longer shoots (37.7 cm)
were observed in variety ‘Figurnaja’. The statistical analysis of results obtained in
that year did not demonstrate any significant differences between varieties, which
together with variety ‘Kijewskaja Gibrydnaja’ of 2006, constituted a homogenous
group with the lowest values. In contrast to the subsequent years of the study, in 2005
variety ‘Sientiabrskaja’ was characterized by the shortest generative shoots: 8.6 cm
(Table 4). In plants of this cultivar the longest generative shoots were recorded in
2006: 61.1 cm. Mean values computed for the 3 experimental years for all varieties
indicate that the variety with the shortest generative shoots (14.44 cm) was ‘Kijewskaja
Gibrydnaja’. The greatest diameters of vegetative and generative shoots were noted in
2007: 0.77 cm (Table 5) and 0.53 cm (Table 6), respectively, whereas the smallest one
in 2006: 0.41 cm (Table 5) and 0.38 cm (Table 6), respectively. The greatest diameter
of vegetative shoots (0.69 cm, Table 5) in the 3 experimental years was observed in
variety ‘Sientiabrskaja’. The varieties ‘Kijewskaja Gibrydnaja’ and ‘Purpurowaja
Sadowaja’ generated vegetative shoots with the same mean diameter, i.e. 0.61 cm. The
smallest diameter was recorded for shoots of variety ‘Figurnaja’: 0.44 cm. The greatest
diameter of generative shoots during 3 years of the study, i. e. 0.51 cm, was noted in
variety ‘Purpurowaja Sadowaja’. Shoot diameters of varieties ‘Figurnaja’, ‘Kijewskaja
Krupnopщodnaja’ and ‘Sientiabrskaja’ belonged to the same homogenous group and
ranged from 0.45 cm to 0.46 cm. Shoots with the smallest diameter (0.42 cm) were
formed by variety ‘Kijewskaja Gibrydnaja’.
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In 2005–2006 the number of leaves on both the vegetative and generative shoots of
varieties under study turned out to be the lowest (Table 7 and 8). In 2007, the number
of leaves on both types of shoots was significantly higher and reached 30.1 (Table 7)
and 15 (Table 8), respectively.
Table 8. Mean number of leaves on generative shoots of bower actinidia in
2005–2007
8 lentelė. Smailialapės aktinidijos generatyvinių ūglių vidutinis lapų skaičius (cm)
2005–2007 m.
Table 9. Mean number of fruits on generative shoots of bower actinidia in
2005–2007
9 lentelė. Smailialapės aktinidijos generatyvinių ūglių vidutinis vaisių skaičius (cm)
2005–2007 m.
Determination of the number of leaves on vegetative and generative shoots
consisted in calculating their density per 10 cm of the shoots (Table 10). The highest
number of leaves, on both vegetative and generative shoots, was reported for variety
‘Purpurowaja Sadowaja’, i. e. 4.8 leaves on vegetative shoots and 6.8 leaves on
generative shoots. The smallest leaf density determined on vegetative shoots (2.2)
was noted for variety ‘Kijewskaja Gibrydnaja’, whereas that calculated on generative
shoots (2.4) was for variety ‘Sientiabrskaja’.
In 2007, due to high ground frost, most of analyzed varieties did not bloom.
Nevertheless, 3 fruits were set on shoots of varieties ‘Kijewskaja Gibrydnaja’ and
‘Purpurowaja Sadowaja’ (Table 9). In respect of all plants examined, they were the
only shoots with fruits.
The highest number of flowers and then fruits was set on generative shoots in
2005, i.e. on average 18.07. As compared to the other cultivars, highly significantly
314

different appeared to be the variety ‘Purpurowaja Sadowaja’, on the shoots of which
there were set 36 fruits. Twenty fruits on the generative shoot were reported for variety
‘Sientiabrskaja’. Other varieties did not differ significantly in terms of the number
of fruits, which ranged from 10 in variety ‘Kijewskaja Gibrydnaja’ to 13 in variety
‘Kijewskaja Krupnopщodnaja’. The mean results of the 3 experimental years indicate
that generative shoots of variety ‘Purpurowaja Sadowaja’ were characterized by a
significantly higher number of fruits, i.e. 14.8, which when expressed per 10 cm of the
generative shoots accounted for 8.2 fruits. Varieties ‘Figurnaja’ and ‘Sientiabrskaja’
constituted another homogenous group in terms of the number of fruits per shoot, i.e.
9.7 and 7.7 fruits respectively, which when expressed per 10 cm of the shoot gave 4.5
and 3.9 fruits (Table 10).
Table 10. Mean number of leaves and fruits expressed per 10 cm of vegetative
and generative shoots of actinidia varieties examined in 2005–2007
10 lentelė. Aktinidijos veislių vidutinis lapų ir vaisių skaičius 10 cm vegetatyvinių ir
generatyvinių ūglių ilgyje 2005–2007 m.
Varieties Kijewskaja ‘Gibrydnaja’ and ‘Kijewskaja Krupnopщodnaja’ constituted a
homogenous group with the lowest number of fruits on generative shoots: 4.7 and 5
fruits (Table 9), which when expressed per 10 cm of the shoot accounted for 3.3 and
2.3 fruits.
Discussion. Investigations into the morphology of vegetative and generative
shoots of five hybrid varieties of bower actinidia were conducted under climatic
conditions of north-eastern Poland, namely in Olsztyn – the capital of the province of
Warmia and Mazury. In contrast to other Polish regions, the area of Olsztyn is poorer
in horticultural crops, which is due to frequent spring frosts – recorded on average
until the 15th of May, in the extreme until 30th of May and locally in June (Grabowski,
Grabowska 1983).
One of the most detrimental meteorological phenomena faced, among others, by
horticulture are claimed to be frosts. Data of 1971–2000 demonstrated that the length
of the frost-free period determined at the altitude of 200 cm in the region of the northeastern
Poland ranged from 143 days in Gołdap to 173 days in Mikołajki. In turn, the
mean length of the frost-free period determined 5 cm above the ground ranged from 112
days in Suwałki to 145 days in Mikołajki (Dragańska et al., 2004). The most common
series of frosty days in the Warmia and Mazury province are a one-day series followed
by a two-day series. The number of days with frost is observed to diminish along with
their length (Dragańska et al., 2004). On May of 2007, there were series of days with
315

frost that were noted both in the first and last days of the month. It contributed to a
lack of fruit setting on shoots of the examined varieties of actinidia.
The climate of the north-eastern Poland, determined mainly by air masses inflowing
from the eastern border of the country, has been observed to change in recent years. One
of the reasons of this change is global warming, which has led to an increase in the mean
annual temperature and, consequently, to a decrease in the total annual precipitation
and soil humidity. The noticeable increase of temperature resulted in the elongation of
the meteorological vegetative season by 8 days (from 192 to 200 days) in the region
of the north-eastern Poland. Undeniably, it is a positive aspect of the contemporary
climatic changes, yet the resultant potential deficiency of soil moisture content at low
annual precipitation observed so far seems worrying (Ziernicka, 2004). Analyzing
the effect of climatic factors on the growth and development of actinidia, the utmost
attention should be paid to data referring to temperatures recorded in the winter and
summer months. Severe freezing in the winter or very low temperatures in the summer
impair the farming of fruit plants (Pieniążek, 2000) by contributing to their poor fruit
bearing. One of the negative traits typical of the course of agroclimatic conditions of
north-eastern Poland are late spring frosts. The extreme dates of their occurrence have
been recorded even in the first half of June (Atlas klimatyczny, 1990).
In Actinidii arguta (Siebold et Zucc.) Planch. Ex Miq, there are two types of
shoots, namely: those with a relatively short period of length gain – including shortshoots
and some long-shoots, and those with a ceaseless growing period which develop
far more strongly and when uncut may reach several meters per season – including
long-shoots (Latocha, 2006a). In the present study, measurements were performed
on shoots terminating their growth and developing to a weaker extent. According to
Chojnowska (1998), annual gains are from 1 m to 3 m in length.
As claimed by Plechanowa (1982), different varieties of bower actinidia are
characterized by varied growth strength and season of particular phenophases. Processes
of growth and development are determined by the whole array of extrinsic factors,
including light, temperature, water, gravitational force, touch, edaphic factors and
chemical stimuli. Out of those factors, a significant role is attributed to light, which,
by acting on photosynthetic and other pigments, affects all processes occurring in plant
tissues (Nowakowska, Tretyn, 1996).
Conclusions. 1. In Olsztyn (north-eastern Poland) during vegetative seasons of
the experimental years 2005–2007 the highest temperatures were recorded in the last
year of the study. Typical to that year were series of days with severe ground frosts in
the first and last decade of May, which might have affected a lack of fruit setting on
shoots of most actinidia varieties.
2. The weakest growth of vegetative and generative shoots was observed in 2005,
which was characterized by the lowest precipitation in vegetative season.
3. In 2005–2007, the longest vegetative and generative shoots were observed in
variety ‘Sientiabrskaja’, followed by variety ‘Kijewskaja Gibrydnaja’.
4. The highest number of flowers and then fruits was observed on shoots of
the examined actinidia varieties in 2005. In terms of the number of fruits, variety
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‘Purpurowaja Sadowaja’ with an average of 8.2 fruits on shoots differed significantly
from the other studied varieties.
5. In 2007 a few flowers were set on shoots of varieties ‘Purpurowaja Sadowaja’
and ‘Kijewskaja Gibrydnaja’, hence it may be assumed that they are the most resistant
to spring frosts.
Acknowledgement. This work was supported by grant 2 PO6R 100 28.
References:
Gauta 2008 04 10
Parengta spausdinti 2008 04 28
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2. Bieniek A., Kawecki Z., Łojko R., Stanys V. 2005. Owocodajne drzewa i krzewy
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3. Chojnowska E.1998. Mrozoodporne pnаcza. Działkowiec, 12: 7.
4. Dragańska E., Rynkiewicz I., Panfil M. 2004. Częstotliwość Częstotliwość
intensywność występowania przymrozków w Polsce północno – wschodniej w
latach 1971–2000. Acta Agrophysica, 3(1): 35–41.
5. Grabowski J., Grabowska K. 1983. Zagrożenie kwitnаcych jabłoni przez
przymrozki w rejonie Olsztyna. Mat. XIX Ogólnopolsk. Konf. Agrometr.,
Szczecin, Poland, 68–74.
6. Kawecki Z., Bieniek A., Stanys V. 2004. Plonowanie i skład chemiczny owoców
aktinidii ostrolistnej w warunkach klimatycznych Olsztyna. Folia Univ. Arric.
Stein. Agricultura, 240(96): 91–96.
7. Kawecki Z., Щojko R., Pilarek B. 2007. Mało znane rośliny sadownicze. Wyd.
UWM, Olsztyn.
8. Latocha P. 2006a. Aktinidia – roślina ozdobna i owocowa. Hortpress Sp. zo. o.,
Warszawa.
9. Latocha P. 2006b. Aktinidia – jako ciekawa i wartościowa roślina owocowa
nadająca się do uprawy w Polsce. Materiały z II Międzynarodowych targów
Agrotechniki sadowniczej. Warszawa, Poland, 69–78.
10. Nowakowska A., Tretyn A. 1996. Mutanty fotomorfogenetyczne. Wiad. Bot.
40(1): 37–51.
11. Pieniążek S. A. 2000. Sadownictwo. PWRiL, Warszawa.
12. Plechanowa M. N. 1982. Aktinidia, limonit, żimołost. Leningrad, 64–92.
13. Werner T. 2002. Aktinidia na plantacje towarowe. Hasło Ogrodnicze, 11: 34.
14. Ziernicka A. 2004. Globalne ocieplenie a efektywność opadów atmosferycznych.
Acta Agrophysica, 3(2): 393–397.
317

SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Šiaurės rytų Lenkijos klimato sąlygų įtaka smailialapės aktinidijos
augimui
Z. Kawecki, A. Bieniek
Santrauka
Smailialapės aktinidijos (Actinidia arguta Sieb. et Planch.) veislių ‘Purpurowaja Sadowaja’,
‘Kijewskaja Gibrydnaja’, ‘Kijewskaja Krupnopщodnaja’, ‘Figurnaja’ and ‘Sientiabrskaja’
vegetatyvinių ir generatyvinių ūglių augimo tyrimai buvo atlikti 2005–2007 m. Varmijos ir
Mazurijos universiteto Olštine eksperimentiniame sode. Ūglių ilgis, skersmuo ir lapų skaičius
ant vienų metų generatyvinių ir vegetatyvinių ūglių matuotas kas dvi savaitės nuo vegetacijos
pradžios. Olštine (šiaurės rytų Lenkija) aukščiausia temperatūra vegetacijos sezono metu
užregistruota paskutiniais metais. Tais metais kelios dienos pirmoje ir paskutinėje gegužės
dekadoje buvo su stipriomis šalnomis, kurios paveikė daugelio aktinijos veislių vaisių
užmezgimа. Labiausia atsparios pavasario šalnoms buvo veislės ‘Purpurowaja Sadowaja’ ir
‘Kijewskaja Gibrydnaja’. Trejų metų tyrimų duomenimis veislių ‘Sientiabrskaja’ ir ‘Kijewskaja
Gibrydnaja’ augalai užaugino ilgiausius vienų metų vegetatyvinius ūglius. Trumpiausi
vegetatyviniai ūgliai buvo veislės ‘Figurnaja’ augalų. Ilgiausi generatyviniai ūgliai buvo
‘Sientiabrskaja’ veislės augalų, o trumpiausi – ‘Kijewskaja Gibrydnaja’ veislės augalų.
Reikšminiai žodžiai: augalų morfologija, smailialapė aktidija, klimatas, šiaurės rytų
Lenkija, veislės.
318

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Expression of yeast Saccharomyces cerevisiae K2
preprotoxin gene in transgenic plants
Brigita Čapukoitienė 1 , Vidmantas Karalius 2 , Elena Servienė 1 ,
Juozas Proscevičius 2, 3 , Vytautas Melvydas 1
1
Institute of Botany, Laboratory of Genetic
2
Laboratory of Cell Engineering, Žaliųjų ežerų 49, Vilnius 2021, Lithuania
E-mail: vytautas.melvydas@botanika.lt
3
Vilnius Pedagogical University, Department of Natural Sciences. Studentų 39,
Vilnius 08105, Lithuania, e-mail: juozas.proscevicius@gmail.com
The aim of this work was to investigate expression possibilities of yeast K2 preprotoxin
gene in plant Nicotiana tabacum.
All experiments were performed using general methods of gene engineering, microbiology
and molecular biology.
Plant transformation vector pGA/ADH1-Kil2 carrying S. cerevisiae K2 preprotoxin gene
under control of yeast ADH1 promoters as well as yeast plasmids pAD/CGT-Kil2D and pAD/
CGT-Kil2R (K2 under control of Cauliflower mosaic virus CaMV promoter) were constructed.
Analysis of expression of K2 gene controlled by CaMV promoter in yeast showed 72–75%
stability of plasmid and weak killer activity with suicidal phenotype. Study of peculiarities of
functional activity of K2 killer gene in yeast demonstrated that expression of this gene is not
strictly dependent on the context of regulatory sequence.
The plant transformation vector bearing K2 type killer preprotoxin gene under
transcriptional control of ADH1 promoter pGA/ADH1-Kil2 was introduced into plant Nicotiana
tabacum via Agrobacterium mediated transformation. The transgenic plants possessed active
K2 type toxin. Such results allow conclude that promoter of yeast gene ADH1 is transcriptional
active in plant as well as preprotoxin can be proceeded in plant cell. Since K2 type toxin can
prevent developing of some pathogen fungus it may be adopted in future to construct diseaseresistant
plants.
Key words: Saccharomyces cerevisiae, K2 preprotoxin gene, plant Nicotiana tabacum,
transgenic plants.
Introduction. Plants are constantly exposed to a great variety of potentially
pathogenic organisms, such as viruses, fungi, bacteria, protozoa, mycoplasma and
nematodes, and can be affected by adverse environment conditions (Castro, Fontes,
2005). Although they do not have immune system, plants have evolved a variety of
potent defense mechanisms including synthesis of low-molecular-weight compounds
(phytoalexins), proteins (chitinases, ureases) and peptides (thionins, defensins, heveinlike
proteins, knottin-like peptides) that have antibacterial or antifungal activity (Claude
et al., 2001; Becker-Ritt et al., 2007).
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Similarly, some bacteria, fungi or mammals synthesize a number of proteins
and peptides with antiphytopathogenic properties (Selitrennikoff, 2001; Mandryk
et al., 2007). There are discovered a number of yeast (Saccharomyces cerevisiae,
Ustilago maydis, Kluyveromyces lactis) secreted proteins that are lethal to fungal cells
(Magliani et al., 1997; Melvydas et al., 2006) or microbial-originated substances having
antibiotic features (Melvydas et al., 2007; Mandryk et al., 2007). These proteins appear
to be involved in either constitutive or induced resistance to bacteria or fungi. The
mechanism of the action are as varied as their sources and include cell wall degradation
of pathogens, membrane channel and pore formation, damage to cellular ribosomes,
inhibition of DNA synthesis and inhibition of the cell cycle.
Many different genetic strategies have been proposed to engineer plant resistance
to diseases, including producing antibacterial or antifungal proteins of non-plant
origin, inhibiting microbial pathogenicity or virulence factors, enhancing natural
plant defense and artificially inducing programmed cell death at the site of infection
(Mourgues et al., 1998). Unlike classical breedings, genetic engineering allows the
modification or introduction of one or more resistant traits into susceptible varieties
(Mourgues et al., 1998). These days many genes encoding antibacterial proteins (as
lytic peptides from insects, lyzocimes, lactoferrin, Pichia anomala killer toxin) have
been cloned and expressed in plants (Donini et al., 2005). However, there are some
problems associated with the efficiency of expression of foreign peptides and their
toxicity. Considerable effort has been made for optimizing production of recombinant
proteins. This research includes molecular technologies to increase replication,
boost transcription, direct transcription in tissues for protein accumulation, stabilize
transcript, optimize translation, target proteins to subcellular locations optimal for their
accumulation and engineer proteins to stabilize them (Streatfield, 2007).
Previously antiphytopathogenic effect of different yeast species isolated from
natural apples and grapes habitats was reported. Specifically, ability of S. cerevisiae
K2 toxin to kill Aspergillius culture was demonstrated (Melvydas et al., 2006). Taking
in account widespread appearance of killer yeasts and their attractive features, the aim
of this work was to investigate expression possibilities of yeast K2 preprotoxin gene
in plant Nicotiana tabacum and peculiarities of its functional manifestation depending
on different transcriptional regulation.
Object, methods and conditions. The yeast expression plasmids pAD4 and
pYEX12 (Gulbinienė et al., 2004) and plant vectors pCGT (Jefferson et al., 1987)
and pGA482 (Proscevičius, Žukas, 1999) were used for construction of recombinant
plasmids pCGT-Kil2 and pGA/ADH1-Kil2 (carrying S. cerevisiae K2 preprotoxin
gene under control of Cauliflower mosaic virus CaMV and yeast ADH1 promoters)
as well as yeast plasmids pAD/CGT-Kil2D and pAD/CGT-Kil2R (K2 under control
of CaMVpr.). General procedures for the construction and analysis of recombinant
DNAs were performed as described by Sambrook et al., (2001). All restriction
enzymes (SalI, SmaI, XbaI, Eam1105I, EheI, Ecl136II), T4 DNA ligase, bacterial
alkaline phosphatase, Klenow fragment and DNA size marker (GeneRuler TM DNA
Ladder mix) were obtained from UAB “Fermentas” (Vilnius) and used according to
the manufacturer’s recommendations.
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The S. cerevisiae strain ά 1 (MATį leu2-2 (KIL-0)), sensitive to all killers
(Čitavičius et al., 1972), was transformed by plasmids of interest according to (Gietz
et al., 2002). Transformants were selected by complementation of LEU2 auxotrophy.
Killer phenotype-selective indicative medium (MB) (Sherman et al., 1986) was used
to test killer toxin production and the immunity of transformants. Transformants were
checked for toxin production in a killing zone plate assay following replica-plating
of transformants onto a lawn of the sensitive strain ά 1. Immunity was tested by the
streaking the standard K1, K2 and K28 killer strains on the lawn of transformed cells.
Stability of the Leu + and K2 + phenotype of transformants was analysed growing cell
colonies on non-selective YEPD medium (Sherman et al., 1986) 3 days at 30 °C and
plating onto selective media: minimal – in case of LEU2; indicatory MB with layer
of ά 1 – in case of K2 preprotoxin gene.
Three parental crosses were performed to introduce the plant transformation vector
pGA/ADH1–Kil2, possessing yeast killer toxin gene under control of yeast promoter
ADH1, into Agrobacterium tumefaciens GV 3101 containing Ti plasmid PMP90RK
(Koncz et al., 1986). Fresh overnight cultures of Agrobacterium tumefaciens GV 3101,
E. coli DH5α containing vector pGA/ADH1-Kil2 and E. coli DH5α possessing helper
plasmid Lelpm7623 were mixed for conjugation on solid YE medium (Draper et al.,
1991). Agrobacterium possessing vector pGA/ADH1-Kil2 was selected on medium
containing 50 mg/L kanamycin, 10mg/L tetracycline and 50 mg/L rifampicin and was
used for plant transformation.
Leaf explants of Nicotiana tabacum SR1 grown in vitro on MS medium (Murasige
et al., 1962) were inoculated with overnight suspension culture of Agrobacterium and
co-cultivated on MSD-4 medium (MS supplemented with 0.1 mg/L naphtaleneacetic
acid (NAA) and 1 mg/L benzylaminopurine (BAP)). After 3–5 days leaf explants were
washed and replaced on MSD-4 medium supplemented with 400 mg/L cefatoxim to
kill Agrobacterium. Latter, callus-forming explants were replaced on the same medium
supplemented with 50 mg/L kanamycin to select transgenes. Shoots regenerated on
survived explants were rooted in MS medium with kanamycin. Plants possessing
resistance to kanamycin were rooted and selected as transgenes.
Results. In the previous study it was determined that cDNA of the S. cerevisiae
K2 preprotoxin gene expressed under control of constitutive ADH1 promoter confer
both killer and immunity phenotypes. Also, functional activity of K2 killer gene in
yeast was demonstrated, gene expression was not strictly dependent on the context of
regulatory sequence (Gulbiniene et al., 2004). Therefore, we decided to investigate
expression of K2 gene controlled by ADH1 promoter in plant Nicotiana tabacum. For
this purpose recombinant plasmid pGA/ADH1-Kil2 (K2 expression controlled by yeast
ADH1 promoter) based on plant vector pGA482 was constructed (Fig. 1).
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Fig. 1. Principal scheme of plant expression plasmid pGA/ADH1-Kil2
1 pav. Principinė pGA/ADH1-Kil2 plazmidės schema
K2 preprotoxin gene surrounded by ADH1 promoter and terminator was isolated
from pYEX12 plasmid by the using XbaI restriction enzyme and inserted into pGA482
linearized by identical endonuclease. It is interesting to point out that obtained plasmids
are suitable for plant transformation but not transformable into yeast, therefore can’t
be replicated in S. cerevisiae.
To detect possibility of yeast killer toxin gene to be expressed in plant it was
introduced into tobacco N. tabacum SR1 via Agrobacterium-mediated transformation.
Callus formation on leaf explants started two weeks after co-cultivation with
Agrobacterium GV 3101 containing plasmid pGA/ADH1-Kil2. Since plasmid
pGA/ADH1–Kil2 possessed gene nptl, it was used for transgenes selection.
Replacement of callus forming explants on kanamycin containing MSD-4 medium
allowed growing and regeneration of transgenes. Co-cultivation with Agrobacterium
during transformation slightly reduced viability of leaf explants. Control (without
Agrobacterium treatment) callus formed 85.7 % of tested explants, after transformation
procedures callus formed 77.2 % explants. Almost half of callus obtained after
transformation was able to grow on medium with kanamycin. However, later only 9
out of 56 canamycin- resistant calluses survived and regenerated shoots. All of them
were rooted on medium with canamycin and selected as transgenes (Fig. 2).
Explants were analysed for production of active K2 toxin either by the placing of
small leafs directly onto yeast λ′1 layer or after the grinding of tested leafs in liquid
nitrogen and spotting of biomass on medium inoculated with sensitive to killer toxins
yeast (Fig. 3). Appearance of small clear lysis zones around leafs (Fig. 3 b) allowed
conclude that yeast ADH1 promoter is transcriptional active in plant as well as
preprotoxin can be preceded in plant cell. However, production of K2 toxin regulated
by specific to yeast ADH1 promoter is barely detectable in plants and required stronger
regulation by using more specific promoter as CaMV. Also, different processing of
protein in plant comparing to yeast can decrease killer production and activity.
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Fig. 2. Root systems of kanamycin-resistant transformants
and regenerated control plants
2 pav. Atsparių kanamicinui transformantų ir kontrolinių
regenerantų šaknų sistemos
Fig. 3. Analysis of explants for production of active K2 toxin
3 pav. Aktyvaus K2 toksino produkavimo eksplantuose tyrimas
In order to analyze expression of K2 preprotoxin gene controlled by CaMV
promoter in plants recombinant plasmids pAD/CGT-Kil2D and pAD/CGT-Kil2R
possessing possibility to analyze this gene expression in yeast was created (Fig. 4).
For construction of abovementioned plasmids at first pCGT-Kil2D and pCGT-
Kil2R constructs was obtained, by introducing of 1196 bp K2 preprotoxin gene
(digested with SalI endonuclease and blunted with T4 DNA polymerase) into vector
pCGT linearized by SmaI and Ecl136II restriction enzymes. Next, Eam1105I – EheI
fragment bearing CaMV promoter, K2 preprotoxin gene and poliA nos terminator (from
pCGT-Kil2D and pCGT-Kil2R respectively) inserted into pAD4 plasmid, resulting in
pAD/CGT-Kil2D and pAD/CGT-Kil2R (Fig. 4). New constructs were transformed into
323

yeast ά 1. It was determined that stability of these plasmids (monitored by maintaining
the Leu + K2 + phenotype) under both non-selective and leucine-selecting conditions
reached 72–75%.
Fig. 4. Map of the yeast plasmids pCGT-Kil2D and pAD/CGT-Kil2D
4 pav. Mielių plazmidžių pCGT-Kil2D ir pAD/CGT-Kil2D genolapiai
It is important to point out that both ά 1 [pAD/CGT-Kil2D] and ά 1 [pAD/CGT-
Kil2R] transformants (K2 gene placed in direct or reverse orientation to promoter
sequence) shows weak killer activity and were sensitive not only to wt K2 toxins as
well as to their own product (partial suicide phenotype) (Fig. 5).
Fig. 5. Testing of killer and immunity functions of ά 1 [pAD/CGT-Kil2D] yeast
5 pav. ά 1 [pAD/CGT-Kil2D] mielių kileriškumo ir imuniškumo tyrimas
Discussion. Since plants are exposed to many different pathogenic organisms it
is important to construct transgenic cultures possessing defence abilities. Resistance
324

mechanism can be realized by self-production of antibacterial or antifungal
proteins.
Yeast Saccharomyces cerevisiae K2 toxin, bearing antipathogenic properties, was
selected for introducing into Nicotiana tabacum cells. Our investigation showed that
transgenic plants possessed active K2 type toxin. Such results allow conclude that
promoter of yeast gene ADH1 is transcriptional active in plant as well as preprotoxin
can be proceeded in plant cells. However, toxin activity was weak and it is not clear
reason of that: possible incorrect transcriptional regulation (non-specific to plants
ADH1 promoter), plant reaction to the toxin formation inside the cell, different protein
secretion mechanisms of yeast and plants. Also it was detected, that the functioning of
foreign toxin protein may influence the plant vitality as the root system of transformants
was faintly developed and formation of the callus was delayed. One of the possibilities
to increase K2 killer production in plant is to use desirable promoter (exp. Cauliflower
mosaic virus CaMV promoter) for transcriptional regulation. Given the capability of
K2 type toxin to prevent developing of some pathogen fungus this feature may be
adopted in future to construct disease resistant plants. It is reliable, that this research
will provide important insights into more general aspects of foreign gene functioning
in transgenic organisms.
Conclusions. 1. New plant transformation vector pGA/ADH1-K2 with K2 type
killer preprotoxin gene under transcriptional control of yeast-specific ADH1 promoter
was successfully introduced into plant Nicotiana tabacum via Agrobacterium mediated
transformation. K2 toxin activity in transgenic plants was demonstrated.
2. Expression of K2 gene controlled by plant-specific CaMV promoter
(pCGT/ADH1-Kil2) in yeast shows production of defective killer protein with reduced
killer activity and immunity functions.
Acknowledgements. This study is supported by Lithuanian State Sciences and
Studies Foundation.
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Gauta 2008 03 27
Parengta spausdinti 2008 05 09
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326

SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Mielių Saccharomyces cerevisiae K2 preprotoksino geno raiška
transgeniniuose augaluose
B. Čapukoitienė, V. Karalius, E. Servienė, J. Proscevičius, V. Melvydas
Santrauka
Šio darbo tikslas buvo patikrinti mielių Saccharomyces cerevisiae kilerinio K2 tipo
preprotoksino geno raiškos galimybes augaluose.
Eksperimentai atlikti pasinaudojant pagrindiniais genų inžinerijos, mikrobiologijos ir
molekulinės biologijos metodais.
Sukonstruotos rekombinantinės plazmidės: augalų transformacijos vektorius
pGA/ADH1-Kil2, turintis mielių alkoholdehidrogenazės ADH1 promotoriumi reguliuojamą
S. cerevisiae K2 preprotoksino geną, bei mielių plazmidės pAD/CGT-Kil2D ir
pAD/CGT-Kil2R (K2 raiška kontroliuojama žiedinių kopūstų mozaikos viruso CaMV
promotoriumi). Atlikus išsamią CaMV promotoriumi reguliuojamo kilerinio geno raiškos
analizę mielėse, parodytas 72–75 % siekiantis plazmidinis stabilumas bei nustatyta, kad
produkuojamas baltymas pasižymi silpnomis kilerinėmis savybėmis ir nesugeba išlaikyti
visaverčio imuniteto nei K2 tipo kileriams, nei savo paties produkuojamam toksinui. K2 geno
funkcionavimo mielėse tyrimai atskleidė, kad šio geno raiška nėra griežtai priklausoma nuo
reguliacinės sekos konteksto.
Sėkmingai atlikta modelinio augalo Nicotianum tabacum transformacija pGA/ADH1-Kil2
vektoriumi (K2 preprotoksino geno raiška reguliuojama ADH1 promotoriumi) panaudojant
Agrobacterium palaikomą sistemą. Gauti aktyvų K2 toksinа produkuojantys transgeniniai
augalai. Tyrimų rezultatai leidžia teigti, kad mielių ADH1 promotorius yra veiklus augaluose
bei preprotoksinas gali būti ekspresuojamas augalų lаstelėse. K2 toksino sugebėjimas inhibuoti
kai kurių patogeninių grybų vystymаsi gali būti sėkmingai panaudotas ateityje, kuriant atsparius
ligoms augalus.
Reikšminiai žodžiai: Saccharomyces cerevisiae, K2 preprotoksino genas, augalas
Nicotiana tabacum, transgeniniai augalai.
327

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Transformations of chemical compounds during apple
storage
Bogumił Markuszewski, Jan Kopytowski
Department of Horticulture, University of Warmia and Mazury, Olsztyn, Ul.
Prawocheсskiego 21, 10-719 Olsztyn, Polska
E-mail: bogumil.markuszewski@uwm.edu.pl
An experiment was conducted in Rakowice (Province of Warmia and Mazury) in 2003–
2005 involving apple tree cv. ‘Szampion’ grafted on M.26 and MM.106 rootstocks and ‘Gloster’
grafted on M.26 rootstock and a seedling of ‘Antonowka’ with a B.9 insert. Six manners of
soil cultivation were applied under the trees in rows. Analyses of fruit chemical composition
were conducted every year on fresh fruit and after four months of storage in an ordinary cold
storage warehouse. The fruit was analysed for dry matter, ascorbic acid, total sugars and simple
sugars as well as for organic acids.
The chemical compounds’ contents in fruit depended on the time of analyses, year of the
study, cultivar, rootstock and the manner of soil cultivation. After the storage period, the fruit
contained less dry matter, ascorbic acid and organic acids and more total sugars and simple
sugars. Small amounts of rainfall during vegetation period at higher temperatures favoured
the accumulation of dry matter, ascorbic acid and organic acids in fruit. The highest contents
of the components under study were detected in cultivar ‘Szampion’ on MM.106. The fruit
quality was also affected by the manner of soil cultivation, especially in the combinations with
polypropylene fabric and manure.
Key words: apple tree, cultivated cultivar, rootstock, mulching, chemical compounds,
storage.
Introduction. Organic compounds’ content in fruit depends on fruit cultivar,
ripeness, physiological condition of a tree as well as soil and weather conditions
(Lange, Ostrowski, 1992; Rutkowski et al., 2006). The chemical composition of an
apple may vary, depending on fruit size, rootstock, fertilization, tree age, yield and
pruning (Rejman, 1994; Stutte et al., 1994). Following their picking from the tree,
fruit are still live organisms, what is indicated by the processes that go on in them,
especially breathing and transpiration. The processes that take place in fruit depend on
the conditions after the storage period (Lange, Ostrowski, 1992). According to these
authors, the quality of apples and their storability depends mainly on weather conditions
in a given period of vegetation, but they also emphasize the great importance of tree
properties, fertilization, cultivation system, spraying with pesticides and the time of
apple picking. Tomala and Piestrzeniewicz (2001) report that apple storability depends
primarily on the genetic characteristics of the cultivar. According to Kruczyсska, (1998)
and Rejman (1994), cultivars ‘Szampion’ and ‘Gloster’ may be stored in an ordinary
cold storage warehouse for up to 4 months. After a longer period, the fruits of these
cultivars considerably lose their taste value.
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The aim of this study was to perform an analysis of chemical composition of fresh
and stored apples from trees grafted on various rootstocks with different systems of
soil cultivation.
Object, methods and conditions. The experiment was conducted in an orchard
near Lubawa, in the province of Warmia and Mazury in 2003–2005. The fruit was
grown by the integrated method. The experiment involved 12-year-old apple trees
of cv. ‘Szampion’ grafted on M.26 and MM.106 rootstock, cv. ‘Gloster’ on M.26
rootstock and seedlings of ‘Antonowka’ with the B.9 insert. Trees of both cultivars
grew in belts with a two-row spacing 4 + 1.25 × 2.5 (1 538 tree ha -1 ). Tree crowns were
maintained in the shape of a spindle. The soil on which the trees grew originated from
slightly loamy sand with the silt and clay content of 8.8 % and IVb quality class. The
following cultivation combinations were applied in the rows of growing trees: control,
bark, sawdust, black polypropylene fabric, manure and herbicide fallow. The soil in
the control group was maintained by manual weeding. The mulches were laid in the
tree rows in a 2.25 m wide belt. Organic mulches were laid in a 15 cm thick layer.
The bark and sawdust were obtained from coniferous trees. Following the application
of mulch, an additional dose of nitrogen fertilizer (urea) was applied (50.0 % bigger
than the applied dose). Weeds on the herbicide fallow were controlled with a mixture
of Roundup 360 SL at 5 l/ha and Chwastox 450 SL at 1.5 l/ha in the second decade of
May and July. Grass was grown between the rows of trees.
The chemical composition of fruit was analysed every year; fresh fruit and fruit
after 4 months of storage in a cold storage warehouse (temp. 1.5–2.5 °C, humidity
85.0–90.0 %). Medium-sized fruit was taken for analysis, 50 items from each
combination as a mixed sample. The following were determined: dry matter at 105 °C,
total and simple sugars – by the Luff-Schoorl method, organic acids – according to
Petersburski, converted to malic acid, and ascorbic acid, by Tillmans method, modified
by Pijanowski.
Results. The dry matter content was higher in fresh fruit (Table 1). On both dates,
the highest level of the component was recorded in 2003 and the lowest one – in 2005.
The higher content of dry matter in the first and third year of the study was caused by
the weather conditions.
These were the periods of vegetation with the highest daily temperatures and the
lowest rainfall levels. During the harvest period, the fruit contained more dry matter
in cultivar ‘Gloster’ on both rootstocks (10.81–10.90 %) and ‘Szampion’ on MM.106
rootstock (10.90 %). After the storage period, the component level was the highest
only in fruit of cultivar ‘Szampion’ on MM.106 rootstock (9.30 %). It can be claimed
that this cultivar in combination with MM.106 rootstock restricted the dry matter loss
in fruit during the storage period. In particular years, the dry matter content in fruit
significantly differed from one cultivar-rootstock combination to another, both during
the harvest period and after storage.
The dry matter content in fruit was significantly different dependently on the
manner of soil cultivation. Its highest level in fresh fruit was measured on the soil
mulched with manure (11.15 %), and the lowest – when the soil was mulched with
pine tree bark (10.51 %). After the storage period, the highest value of the component
was measured in the fruit from trees on soil mulched with fabric (9.40 %). The fruit
in this combination contained a higher amount of dry matter, which resulted in the
lowest loss of this component in storage.
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Table 1. Dry matter content in freshly picked fruit and in fruit after storage
dependently on the type of rootstock and the manner of soil cultivation (%)
1 lentelė. Sausųjų medžiagų kiekis šviežiuose vaisiuose ir po jų laikymo, priklausomai
nuo poskiepio ir dirvos priežiūros būdų, %
The chemical analysis of fruit revealed a higher ascorbic acid content in freshly
picked fruit (3.7–4.6 mg 100 g -1 ) than after storage (Table 2). The highest amounts
of the component in both times of the analysis were determined in the fruit picked
in the first year of the study, and the lowest was in those picked in the second year.
As with the dry matter, higher ascorbic acid content was favoured by better weather
conditions. No differences were found between the ascorbic acid content in fresh fruit
for different experimental variants.
The effect of the examined factors on the value of the feature proved to be
significant after the fruit storage. The highest level of ascorbic acid was recorded in
fruit of cultivar ‘Szampion’ on MM.106 rootstock (3.5 mg 100 g -1 ), what indicates the
lowest loss of the component in this combination. A similar relationship in the ascorbic
acid content was found to be a result of the applied methods of soil cultivation. The
lowest ascorbic acid loss after storage was found in the samples taken from the trees
in the soil mulched with fibre.
331

Table 2. Ascorbic acid content in freshly picked fruit and in fruit after
storage dependently on the type of rootstock and the manner of soil cultivation
(mg 100 g -1 )
2 lentelė. Askorbino rūgšties kiekis šviežiuose vaisiuose ir po jų laikymo, priklausomai
nuo poskiepio ir dirvos priežiūros būdų, mg 100 g -1
The total sugar content in fruit was much higher after storage and amounted on
average to 23.1–29.4 % (Table 3). More sugar was found in the first and third year of
the study. A higher total sugar content during the harvest period was measured only
in 2003. The fruit of cultivar ‘Szampion’ on MM.106 rootstock contained the highest
amounts of the component, both during the harvest period (11.5 %) and after storage
(30.1 %). A high level of sugar after storage was also found in the fruit of cultivar
‘Gloster’ on M.26 rootstock. In particular years, in analyses performed in both times,
the total sugar content varied. The effect of the manner of soil cultivation on the total
sugar content became noticeable only during the harvest period.
332

Table 3. Total sugar content in freshly picked fruit and in fruit after storage
dependently on the type of rootstock and the manner of soil cultivation (%)
3 lentelė. Bendras cukrų kiekis šviežiuose vaisiuose ir po jų laikymo, priklausomai nuo
poskiepio ir dirvos priežiūros būdų, %
The highest sugar concentrations were determined in the fruit picked from the
trees under which fibre had been laid, and the lowest concentrations – from the tress
grown on the herbicide fallow. The strongest effect of the manner of soil cultivation
on the total sugar content was apparent during the first year of the study.
The chemical analysis of the fruit after storage revealed a higher simple sugar
content –12.6–16.9 % (Table 4). During that period, their highest amounts were found
in the fruit obtained in 2003, and the lowest was in 2004. During the harvest period,
the relationship was reversed – the highest level of simple sugars was determined in
the fruit obtained in 2004 (9.5 %). A high content of the sugars under study during
the period was favoured by a cool and rainy vegetation period. The highest amounts
of simple sugars were found after storage of fruit of cultivar ‘Szampion’ grafted on
M.26 rootstock (17.2 %), whereas during the harvest period the fruit of ‘Szampion’ on
MM.106 rootstock contained more sugar (9.1 %) than other cultivars. In particular years
of the study, the level of simple sugars was mainly cultivar-dependent. The effect of the
method of soil cultivation on the level of simple sugars proved to be significant after
333

storage. The highest level of the sugars was determined in fruit from the combination
with manure and the lowest was in those from the control trees. A similar relationship
at the highest values of the feature was recorded in 2003.
Table 4. Simple sugar content in freshly picked fruit and in fruit after storage
dependently on the type of rootstock and the manner of soil cultivation (%)
4 lentelė. Paprastų cukrų kiekis šviežiuose vaisiuose ir po jų laikymo, priklausomai nuo
poskiepio ir dirvos priežiūros būdų, %
The organic acid content in fruit decreased after their storage (Table 5). In the
chemical analyses performed at both times, the highest amounts of this component
were recorded in 2005 and the lowest – in 2004. The level of organic acids in the
second year of the study was negatively affected by adverse weather conditions. The
highest acid content was determined in cultivar ‘Gloster’, regardless of the type of
rootstock and time of performing the chemical analysis. A similar relationship was
recorded in all the years of the study. The highest organic acid content in fruit of the
studied cultivars was determined in the control trees and in combination with pine
bark (0.56 %), and the lowest was in fruit from the trees under which the soil was
mulched with pine sawdust, fibre and manure. The fruit from the control combination
contained the highest amounts of acid after storage.
334

Table 5. Organic acid content in freshly picked fruit and in fruit after storage
dependently on the type of rootstock and the manner of soil cultivation (%)
5 lentelė. Organinių rūgščių kiekis šviežiuose vaisiuose ir po jų laikymo, priklausomai
nuo poskiepio ir dirvos priežiūros būdų, %
Discussion. The quality of fruit, determined by the chemical composition of the
examined cultivars after harvesting was affected by the weather conditions in a given
year. In warmer years, when the amount of rainfall was smaller, the fruit contained
more dry matter, ascorbic acid and organic acids, but less simple sugars. The amount
of sugars was the highest only in the first year. The weather conditions significantly
affected the amount of nutrients in the years of the study, which has been recorded by
Kawecki et al. (1999) and Ważbińska and Brych (2003). The greatest amounts of dry
matter were found in fruit of cultivar ‘Gloster’ on the two rootstock combinations and
in those of cultivar ‘Szampion’ on MM.106. The differences in accumulation of dry
matter in the fruit of the examined cultivar on different rootstocks have been found
by Ostapenko (2006). Kawecki et al. (1997) examined the effect of the method of soil
cultivation and found smaller amounts of dry matter in fruit from the trees where soil
was mulched with bark, which was corroborated by this study. A higher amount of
the component when the soil was mulched with manure was confirmed in a study of
cultivation of chokeberry (Tomaszewska, Kopytowski, 2003). The highest amounts
of ascorbic acid were found in the fruit of cultivar ‘Szampion’ on MM.106. A similar
335

elationship was shown to exist by Ostapenko (2006), who obtained more ascorbic
acid in fruit from the trees on MM.106 than on M.26. Waźbińska and Brych (2003)
showed the content of the component to be significantly cultivar-dependent. During
the period of the study, sweeter apples were obtained from cultivar ‘Szampion’ on
MM.106. Maćkowiak (1989) found the cultivar to strongly differentiate sugar content
in the fruit. Ostapenko (2006) examined the effect of rootstocks on the total content
of sugars and did not find significant differences, what was contrary to the results
shown in this experiment for cultivar ‘Szampion’. The effect of the method of soil
cultivation on sugar content in fruit was the strongest when fibre was applied; a similar
relationship was shown to exist by Kawecki et al. (1997). Cultivar ‘Gloster’ contained
more organic acids than ‘Szampion’, regardless of the used rootstock. Significant
cultivar-dependence of the acid content was shown by Maćkowiak (1989). A study
conducted by Ben and Błaszczyk (1994) found MM.106 rootstock to increase the acid
content more than M.26, what was not corroborated in the current study where trees
of cultivar ‘Szampion’ grew on those rootstocks. The organic acid content was also
related to a method of soil cultivation. The trees for which pine tree bark was applied,
as well as the control, tended to contain more organic acids; similar to the findings of
a study by Kawecki et al. (1999).
After being stored in a cold storage warehouse, the fruit usually contained less
dry matter, ascorbic acid and organic acids and more total and simple sugars. Similar
tendencies in the contents of these components were observed by Ben and Błaszczyk
(1994) and Kviklienė et al. (2006). The extent of the content change for most nutrients
was cultivar and rootstock-dependent; it was also affected by the applied method
of soil cultivation. Fruit of cultivar ‘Szampion’ on MM.106 contained more dry
matter as compared to the other cultivar-rootstock combinations. Its different extent,
depending on the cultivar, was also shown by Chojnacka et al. (1999). In an experiment
conducted by the authors with trees on M.26 rootstock, a higher sugar concentration
was determined, what is contrary to the findings of Ben and Błaszczyk (1994), who
did not observe any differences between the sugar content in fruit from trees on M.26
and MM.106 rootstocks. Fruit of cultivar ‘Szampion’ accumulated less organic acids
than ‘Gloster’, regardless of the used rootstock. According to Lange and Ostrowski
(1992), organic acid content is largely cultivar-dependent.
Conclusions. 1. Nutrient content in the fruit of the studied apple trees varied
dependently on weather conditions, rootstock and the method of soil cultivation. A
period of vegetation with higher temperatures and lower level of rainfall favoured the
accumulation of dry matter, ascorbic acid and organic acids in the fruit, while at the
same time reducing the amount of simple sugars in them.
2. The highest amount of dry matter in fresh fruit was found in cultivar ‘Szampion’
on MM.106 and in ‘Gloster’ on both of the applied rootstocks. The total and simple
sugar content was the highest in fruit of cultivar ‘Szampion’ on MM.106. The highest
amount of organic acids was found in fruit of cultivar ‘Gloster’.
3. Soil mulching with pine tree bark reduced the dry matter content in fruit and
increased their acidity. Mulching with fibre favoured the accumulation of sugars and
manure increased the dry matter content. The fruit from the control combination
contained large amounts of organic acids, while the trees on the herbicide fallow bore
336

fruit with the lowest concentration of sugars.
4. After storage, fruit of cultivar ‘Szampion’ on M.26 rootstock contained the
lowest amounts of ascorbic acid and organic acids, and the highest amounts of simple
sugars, whereas those grafted on the MM.106 rootstock contained the highest amounts
of dry matter, ascorbic acid and total sugars. Cultivar ‘Gloster’, regardless of the
rootstock used, accumulated the lowest amounts of dry matter and simple sugars, with
the highest acid concentration in the fruit.
5. Soil mulching with pine sawdust increased the dry matter content and fibre
mulching did the same with ascorbic acid in the stored fruit. The fruit in the control
combination contained the highest concentration of acids and the lowest concentration
of ascorbic acid. The fruit in the bark combination contained the lowest amounts of
ascorbic acid.
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Lublin, Polska, 14–20.
5. Kruczyńska D. 1998. Nowe odmiany jabłoni. Hortpress. Warszawa.
6. Kviklienė N., Kviklys D., Viškelis P. 2006. Chenges in fruit quality during
ripening and storage in the apple cultivar ‘Auksis’. J. Fruit Ornam. Plant Res.,
14(2): 195–202.
7. Lange E., Ostrowski W. 1992. Przechowalnictwo owoców. PWRiL Warszawa.
8. Maćkowiak M. 1989. Wpływ podkładek oraz systemu uprawy gleby na wzrost
i plonowanie dwóch odmian jabłoni. Rocz. AR Poznaс. Rozp. Nauk. Zesz.,
186: 52–54.
9. Ostapenko V. 2006. The influence of rootstock on productivity and fruit quality of
apple-tree cultivar ‘Florina’ under conditions of South Russia. SODININKYSTĖ
ir daržininkystė, 25(3): 207–211.
10. Rejman A. 1994. Pomologia. Odmianoznawstwo roślin sadowniczych. PWRiL
Warszawa.
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11. Rutkowski K. P., Kruczyńska D. E., Krzesak P. 2006. Jakość i zdolność
przechowalnicza jabłek z grupy ‘Fuji’. XLIV Ogólnopolska Naukowa Konferencja
Sadownicza. Skierniewice, Polska, 153–154.
12. Stutte G. W., Baugher T. A., Walter S. P., Leach D. W., Glenn D. M.,
Tworkowski T. J. 1994. Rootstock and training system affect dry-matter and
carbohydrate distribution in ‘Golden Delicious’ apple trees. J. Amer. Soc. Hort.
Sci., 119: 492–497.
13. Tomala K., Piestrzeniewicz C. 2001. Skład chemiczny owoców z ich
zdolność przechowalnicza. VI Ogólnopolskie Spotkanie w Grójcu. Grójec,
Polska, 66–74.
14. Tomaszewska Z., Kopytowski J. 2003. Wpływ różnych sposobów utrzymania
gleby na plonowanie i zawartość związków organicznych w owocach aronii.
Biul. Nauk., 22: 265–268.
15. Waкbiсska J., Brych A. 2003. Skщad chemiczny owocуw niektуrych odmian
jabщoni. Biul. Nauk., 22: 269–272.
SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Cheminių junginių pokyčiai laikant obuolius
B. Markuszewski, J. Kopytowski
Santrauka
Tyrimuose naudotos tokios obelys: veislių ‘Szampion’, įskiepytos į M.26 ir MM.106
poskiepius, ‘Gloster’, įskiepytos į M.26 poskiepį, bei ‘Antonуwka’ sėjinukai su B.9 intarpu.
Šeši dirvos priežiūros būdai buvo naudoti po medžių eilėmis. Vaisių cheminių junginių analizės
atliktos kiekvienais metais šviežiuose vaisiuose ir po keturių mėnesių laikymo saugykloje.
Buvo nustatyta sausosios medžiagos, askorbino rūgštis, bendri ir paprasti cukrūs bei organinės
rūgštys. Po laikymo periodo vaisiai turėjo mažiau sausųjų medžiagų, askorbino rūgšties ir
organinių rūgščių bei daugiau bendrų ir paprastų cukrų. Mažas kritulių kiekis vegetacijos metu
ir aukštos temperatūros palankiai veikė sausųjų medžiagų, askorbino rūgšties ir organinių
rūgščių kaupimаsi vaisiuose. Didesnis šių komponentų kiekis nustatytas veislės ‘Szampion’ su
MM.106 poskiepiu vaisiuose. Vaisių kokybę lėmė ir dirvos priežiūros būdai, ypač polypropyleno
medžiagos ir trąšų derinys.
Reikšminiai žodžiai: obelys, veislės, poskiepiai, mulčias, cheminiai junginiai,
laikymas.
338

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Effect of harvest maturity on quality and storage
ability of apple cv. ‘Ligol’
Nomeda Kviklienė, Alma Valiuškaitė, Pranas Viškelis
Lithuanian Institute of Horticulture, Kauno 30, LT-54333 Babtai, Kaunas distr.,
Lithuania, e-mail: n.kvikliene@lsdi.lt
The effect of fruit maturity on apple cv. ‘Ligol’ storage ability and rot development was
investigated at the Lithuanian Institute of Horticulture in 2003–2004. Fruits for storage were
harvested 5 times at weekly intervals before, during and after predictable optimum harvest
date. Fruit internal and external quality changes were measured during harvest period, and the
presence of storage disorders and mass losses at the end of storage. During investigation period
fruits quality parameters changed according to harvest date and were specific for each trial year.
Later harvested fruits were softer. Content of soluble solids did not depend on harvest time.
Fruit storage ability was closely connected to fruit maturity. After 180 days of storage apples
picked one week before climacteric peak were of the best quality and with the smallest mass
losses caused by decay and water loss.
Key words: Malus Ч domestica, fruit firmness, rots, soluble solids content, storage,
weight loss.
Introduction. A different picking date of apple fruits during the harvest season
may have a significant impact on fruit quality (Vielma et al., 2008; Rizzolo et al., 2006;
Kviklienė, 2001; Franelli, Casera, 1996; Meresz et al., 1996; Streif, 1996). To ensure
the highest fruit quality at the end of long storage, apples must be harvested when
mature but not when fully ripe. If harvested too early fruits are smaller, have reduced
flavour and colour, and are more susceptible to scald, bitter rot and internal breakdown.
Mass reduction by water loss is greater in earlier picked apples because waxy surface
is not completely formed at this moment (Zerbini et al., 1999; Juan et al., 1999). Early
picked fruits are smaller and their surface in a storage unit is larger. Because water
transpiration depends on fruit surface area too, small fruits loss their weight faster.
Another reason of more intensive evaporation is structure of fruit cuticle, which is not
fully developed when fruits are harvested too early. At the same time the cuticle is the
first barrier that pathogens have to challenge (Ihabi et al., 1998). Later picked apples
often are over mature and all physiological processes are underway what complicate
storage, even under optimal conditions (Ingle et al., 2000; Braun et al., 1995). Apples
harvested too late are vulnerable to mechanical injures, sensitive to low temperature
breakdown, watercore and more rot (Hribar et al., 1996). At optimal harvest time
picked apples have the organoleptic qualities (Casals et al., 2006), which enable them
to survive more than six months of storage.
The objective of this study was to investigate the effect of harvest time on fruit
quality and storage ability of cv. ‘Ligol’ apples.
339

Object, methods and conditions. Investigations were carried out with apple
cv. ‘Ligol’ on M.26 rootstock in 2003 and 2004. The measurements of fruit quality
changes were performed 2–3 weeks before and after the predictable optimum harvest
date. Apples for long storage were harvested 5 times at weekly intervals. The experiment
was carried out with 4 replications and 5 trees per plot.
On each picking date 10 fruits from each replication were taken for laboratory
measurements: respiration intensity (mg CO 2
/kg h, measured with gas analyzer
‘Anagas 95’), fruit firmness (kg/cm 2 , measured with penetrometer FT-327 with 11 mm
diameter probe), soluble solids content (%, with refractometer).
On each picking date 100 fruits from each replication were taken in order to
measure storability (firmness, soluble solids concentration, weight loss, storage
disorders and rots). Fruits were stored for 180 days.
Variance analysis of main quality characters was done using ‘ANOVA’ statistical
program.
Results. It was found that respiration pattern during the maturation period was
typical for climacteric fruits and was similar during both years of investigation (Fig. 1).
Early picked apples had reduced respiration. Respiration intensity increased until 4th
harvest and reached climacteric maximum, thereafter strong decrease in respiration
was recorded.
Fig. 1. Respiration intensity at different harvest time
1 pav. Skynimo laiko įtaka vaisių kvėpavimo intensyvumui
During ripening period fruit firmness decreased by 13 % in 2003 and by 22 %
in 2004 (Table 1). In 2003 significant differences were recorded starting from the
3 rd harvest. In 2004 fruits were firmer and did not differ significantly from 1 st to 3 rd
harvest times. In both year of investigation the highest softening rate was observed
at the last week.
At the end of the storage differences between fruit firmness were not so big.
Significant differences were established comparing last two harvest dates with earlier
340

ones only. The difference in fruit firmness between first and last harvest date was 9 %
in 2003, and only 5 % in 2004.
On average fruit firmness at harvest time gradually decreased according harvest
time and in most cases differences between dates were significant. After the storage
there were no differences between 1st and 3rd, and between 4th and 5th harvest dates.
During storage, fruit firmness decreased on average from 21 to 37 % of its original
value. The highest reduction of fruit firmness was recorded at 4th harvest.
Table 1. Effect of harvest time on fruit firmness (kg cm -2 )
1 lentelė. Skynimo laiko įtaka obuolių minkštumui, kg cm -2
The dynamic of soluble solid content (SSC) every year was different (Table 2). In
2003 SSC increased linearly to harvest time. The highest amount of SSC was observed
in the last week of measurements, though significant difference was established only
with 1st harvest date. In 2004 SSC reached maximum at 3rd harvest after which it
leveled off.
In 2003 there were no significant differences in fruit SSC after the 180 days of
cold storage. During the storage period decrement of SSC was recorded for most of
harvest dates, while in 2004 opposite tendencies were defined. Almost at all harvest
dates SSC showed a tendency to increase.
Table 2. Effect of harvest time on SSC (%)
2 lentelė. Skynimo laiko įtaka tirpių sausųjų medžiagų kiekiui, %
341

Weight losses during storage were dissimilar every year (Fig. 2) and depended
on harvest time. In 2003 the largest weight losses were estimated for earlier picked
apples. Apples picked at 4 th harvest lost by 27 % less their mass in comparison with
apples picked at earliest time. In 2004 the largest weight loss was estimated for earlier
and later picked apples. Mass reduction was lowest of apples picked at 3 rd harvest.
Fig. 2. Effect of harvest time on fruit weight losses during storage
2 pav. Skynimo laiko įtaka natūraliems masės nuostoliams laikymo metu
Apple mass loss by rots and decay were not large in 2003 (Fig. 3). Up to 9.6 %
of apples rotted during 180 days of storage period. Too late picked fruits rotted more.
Significantly less amount of rotten fruits was recorded when apples were picked at 2 nd
and 3 rd harvest. Up to 33.9 % of apples rotted in 2004 year. The extent of loss linearly
depended on harvest time. At each picking the significant increase of rotten apples was
recorded and the maximum of damaged fruits was estimated of latest picked apples. At
this stage picked apples rotted by 4.8 times more, in comparison with apples picked
at earliest harvest.
Cv. ‘Ligol’ apples were infected by Monilinia sp., Gloeosporium spp. and
Penicillium spp. In both trial years apples were mostly infected by fungus of
Gloeosporium genus.
342

Fig. 3. Effect of harvest time on fruit rots incidence during storage
3 pav. Skynimo laiko įtaka obuolių puvimui laikymo metu
Discussion. During investigation period fruit quality parameters changed
according to harvest date and were specific for each trial year. Later harvested fruits
were softer and had higher content of soluble solids. Fruit storage ability was closely
connected to fruit maturity too. After 180 days of storage apples picked one week
before climacteric peak were of the best quality and with the smallest mass losses
caused by decay and water loss.
The softening rate of apple fruit vary from cultivar to cultivar, depending on the
presence and expression of genes, which regulate the activity of hydrolytic enzymes
(Ingle et al., 2000; Konopacka, Plocharski, 2002; Johnston et al., 2002). In our trials
measurements of firmness showed that cv. ‘Ligol’ belongs to the group of naturally
firm fruits. Softening rate during ripening and storage was low. In comparison with
other investigated cultivars as ‘Auksis’, ‘Lobo’ and ‘Lodel’ fruits of cv. ‘Ligol’ tended
to lose their firmness slower (Kviklienė, 2001; Kviklienė et al., 2006). In our trials the
highest softening of fruits was observed when apples picked at climacteric peak, and
the lowest – one week before. Apples harvested at optimal harvest time usually tend to
loose their firmness during the storage much slower what agree with results of Castro
et al. (2007), Kviklienė (2004), Hribar et al. (1996), and Meresh et al. (1993).
Usually, later picked apples show higher SSC value not only at harvest time, but at
the end of storage too (YongSoo et al., 1998). In our study SSC varied during the years
so it is difficult to draw a conclusion on the change pattern. Similar tendencies were
obtained in different trials (Kviklienė et al., 2006; Wargo, Watkins, 2004; Echeverria
et al., 2002; Braun et al., 1995).
The results of our investigations showed that mass reduction by water loss and
decay was closely connected with fruit maturity. On average the lowest loss were
observed of apples picked one week before climacteric peak. The bigger weight loss
at early stage of maturation can be explained by not fully developed waxy surface
343

and cuticle (Ihabi et al., 1998; Sass and Lakner, 1998). The higher incidence of rots
in later picked apples can be explained by more intensive all physiological processes
in overmature fruits. Similar results were recorded with other apple cultivars (Dris &
Niskanen, 1999; Elgar et al., 1999; Ingle et al., 2000; Kviklienė, 2001).
Conclusions. 1. Harvest time has a significant effect on fruit internal quality and
fruit physiological processes at harvest time and during the storage.
2. Fruit SSC depended more on growing season conditions neither on fruit
maturity.
3. Incidence of decay and rots depended linearly on fruit maturity – more late
harvest more fruit loss.
4. Cv. ‘Ligol’ apples harvested one week before climacteric maximum have the
best storage ability.
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Gauta 2008 04 77
Parengta spausdinti 2008 04 29
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SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Skynimo laiko įtaka ‘Ligol’ obuolių kokybei vaisiams nokstant ir
juos laikant
N. Kviklienė, A. Valiuškaitė, P. Viškelis
Santrauka
2003 ir 2004 m. Lietuvos sodininkystės ir daržininkystės institute tirta veislės ‘Ligol’
obuolių kokybė vaisiams nokstant ir juos laikant. Vaisiai buvo skinti kas savaitę penkis kartus.
Vaisiams nokstant buvo matuota: kvėpavimo intensyvumas, minkštimo kietumas, ir tirpių sausųjų
medžiagų kiekis. Obuolius laikant matuotas jų minkštimo kietumas, tirpių sausųjų medžiagų
kiekis ir apskaičiuoti masės nuostoliai. Laikymo pabaigoje nustatytas optimalus skynimo laikas
atsižvelgiant į tai, kurie vaisiai geriausiai išsilaikė. Nustatyta, kad vaisių kokybė skynimo ir
laikymo metu priklausė nuo sunokimo laipsnio. Vėliau nuskinti vaisiai buvo minkštesni ir
skynimo metu, ir laikymo pabaigoje. Tirpių sausųjų medžiagų kiekio dinamika tyrimų metais
buvo skirtinga. Natūralius masės nuostolius ir nuostolius dėl puvinių taip pat lėmė skynimo
laikas. Geriausiai laikėsi vaisiai, nuskinti vieną savaitę prieš klimakteriumo maksimumą.
Reikšminiai žodžiai: Malus × domestica, krakmolo susiskaidymas, laikymas, minkštimo
kietumas, tiršios sausosios medžiagos, puviniai.
346

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Identification of scab resistance genes in apple trees by
molecular markers
Oksana Urbanovich, Zoya Kazlovskaya 1
Institute of Genetic and Cytology, NASB, 27, Akademichiskaya str., Minsk, 220072,
Belarus, e-mail: O.Urbanovich@igc.bas-net.by
1
Institute of Fruit Growing, 2, Samokhvalovitchi, Kovaleva str., Minsk region,
223013, Belarus, e-mail: zoya-kozlovskaya@tut.by
Apple scab is a widespread and one of the most harmful fungal diseases of apple trees in
Belarus. Molecular markers were used for detection of resistance genes in 130 apple accessions
including old, modern and introduced cultivars. The presence of genes Vm, Vr1 and Vh2 were
detected using the molecular markers OPB12STS, AD13 and OPL19 respectively. Gene Vf was
identified with the markers VfC, AL07 and AM19.
The gene Vr1 was detected in modern cultivars as well as in old ones grown in Belarus
from 19 th century, such as ‘Papirovka’, ‘Bely naliv’, ‘Korobovka krupnoplodnaya’, etc. The
marker OPL19 presents in genome of 81 accessions. Gene Vm was revealed in 7 accessions
related to the parental cultivars McIntosh and SR0523. Gene Vf, which was transferred from
M. Xfloribunda 821 and introduced into cultivated cultivars, was identified in 41 cultivars of
different breeding (France, USA, Poland, Russia, Belarus, etc.). The presence of this gene
provides a high scab resistance in apple cultivars. This was verified by field trials carried out
during 2004–2007. The cultivars containing gene Vf and some cultivars with polygenic resistance
shown a high scab resistance both in leaves and fruits.
Key words: apple scab, Malus accession, resistance genes.
Introduction. Apple scab is a widespread and one of the most harmful fungal
disease of apple trees in Belarus. This disease is caused by an ascomycetes fungus
Venturia inaequalis. In the years of intensive expansion that happens once per 3 years
or even more often, scab causes premature leaf loss of trees and 100 % fruit lesion
in susceptible cultivars if chemical protection is not used. Its control in co mMercial
orchards can require up to 15 fungicide treatments per year. Such a large amount of
chemical treatments raise numerous ecological problems and consumer health concerns,
in addition to the economic cost (Lespinasse et al., 2002). An alternative approach is
the use of resistant cultivars, which can be grown with much less pesticide treatment.
Apple breeding is aimed for developing cultivars with durable scab resistance genes.
There are many progra mMes for creating new cultivars with durable resistance of
apple to scab (Crosby et al., 1992; Janick, 2002; Lespinasse et al., 2002).
Scab resistance is a complicated biological character determined genetically and
depending on environmental conditions during morphogenesis. Various sources of apple
scab resistance have been found (Williams, Kuc, 1969). Several major scab resistance
347

genes originated from small fruited asiatic Malus spp. The genes Vbj from Malus
baccata jackii, Vb from M. baccata, Vm from M. Xmicromalus and M. Xatrosanguinea
804, Vr from M. pumila (Russian Seedling) R12740-7A, Vf from M. Xfloribunda 812
have been introgressed into breeding lines and selections to make them available for
breeding purposes (Liebhard et al., 2003). Until now, mostly the Vf resistance has been
incorporated into co mMercially available cultivars. The sources of scab resistance are
‘Golden Delicious’ (Vg), GMAL 2473 (Vr2), ‘Durello di Forli’ (Vd), differential host
2 (Vh2) and host 4 (Vh4) (derived from M. pumila R12740-7A), M. sylvestris W193b
(Vh8) too (Durel et al., 2000; Patocchi et al., 2004; Tartarini et al., 2004; Bus et al.,
2004; Bus et al., 2005a, 2005b).
Most known major resistance genes are “recognition genes” (Bergelson et al.,
2001; Jones, 2001). The resistance genes differ in their level of resistant. Durable
scab resistant cultivars were created combining major resistance genes and polygenic
resistance. It is important to identify genes resistance to scab in apple cultivars and
determine its significance for breeding.
Cultivars with multiple resistance genes can be easily selected with molecular
markers associated with the resistance genes. The molecular markers have been
developed for identification of gene resistance to scab in apple genome (Gessler et al.,
2006). They are an excellent instrument for identification of durable genes and creation
resistance cultivars using marker assisted selection.
The aim of this study was to identify durable genes leading to high level resistance
to scab in the collection of apple accessions grown in Belarus and to determine valuable
cultivars for breeding process. The molecular markers were used for determining scab
resistance genes.
Object, methods and conditions. Plant material and estimation.
The collection of apple accessions from the orchard of the Institute of Fruit Growing
of Belarus, which include 130 accessions including old, modern and introduced
cultivars, was studied in this investigation. The assessment of apple scab lesion was
made according to the Program and methods of fruit, small fruit and nut cultivar
assessment (Sedov, Ogoltsova, 1999).
DNA isolation. DNA was extracted from frozen leaves in all evaluated genotypes.
DNA was isolated by Genomic DNA Purification Kit (Fermentas) according to
manufacturer’s instructions.
PCR markers. The molecular markers OPB12STS, AD13 and OPL19 linked to
genes Vm, Vh2 and Vr1 (Cheng et al., 1998; Bus et al., 2005a; Boudichevskaia et al.,
2006) were used respectively. The gene Vf was identified with the markers VfC, AL07
and AM19 (Afunian et al., 2004; Tartarini et al., 1999) (Table 1).
348

Table 1. Markers for detection of scab resistance genes
1 lentelė. Žymenys rauplėms atsparių genų nustatymui
Amplification. Amplification was performed in 20 мl final volume
containing: 40 ng of genomic DNA as template, 67 mM Tris-НCl рН 8.8, 16 mM
(NH 4
) 2
SO 4
, 1.5 mM MgCl 2
, 0.2 mM of each dNTP, 0.25 мM of each primers, and
1U Taq DNA Polymerase. The program of amplification was: 3 min at 94 °С; followed
by 40 cycles of 94 °С for 40 s, Tєan for 1 min, 72 °С for 1 min; and a final extension
of 72 °С for 10 min.
Amplified DNA fragments were analyzed by horizontal electrophoresis on a
standard 1.0 % agarose gel in Tris-acetate-EDTA buffer (Sambrook et al., 1989). DNA
was visualized by ethidium bromide. Gels were photographed with BioRad camera
under UV light. GeneRuler tm 100 bp DNA Ladder Plus (Fermentas) was used as a
molecular weight marker.
Results. The gene Vr1/Vh4/Vx was identified in the Malus scab resistance
source R1740-7A (Russian seedling) (Bus et al., 2005b). This gene was mapped on
linkage group 2. The codominant, multiallelic molecular marker AD13-SCAR have
been developed by Boudichevskaia et al. (2006). Marker AD13-SCAR was used for
identification of gene Vr1 in 130 accessions of apple trees and presence of gene Vr1
was detected in 34 accessions. AD13-SCAR was detected in both modern cultivars
and old cultivars grown in Belarus from 19 th century, such as ‘Papirovka’, ‘Bely naliv’,
‘Korobovka krupnoplodnaya’, etc. (Table 2). Presence of the locus from ‘Papirovka’
was detected in modern cultivars ‘Mechta’ and ‘Narodnoe’. This marker also was found
in old cultivar ‘McIntosh’ and in its progenies ‘Wijcik’, ‘Auksis’, ‘Melba’.
The SCAR marker OPL19 developed by Bus et al. (2005b), was applied
for testing locus Vr2/Vr-A. This locus of linkage group 2 contains linked genes
Vr2/Vr-A and Vh8 (Bus et al., 2005a). The marker OPL19 dispose at a distance of 1.3 cM
from Vh8. SCAR maker OPL19 was identified in 81 accessions. Possibly this marker
do not let to test the gene in collection, but it is very important for testing breeding
forms. Information about this locus can be used in breeding process for testing new
accessions, if their parents contained this locus.
The sources of the gene Vm in breeding are M. Xatrosanguinea 804 and
M. Xmicromalus 245-38 (Dayton, Williams, 1970). The marker OPB12 STS have been
developed for Vm by Cheng et al. (1998). The OPB12 STS being placed at about 6 cM
349

from Vm is an excellent indicator for the presence of Vm in germplasm collection.
This marker is mapped on apple linkage group 17 (Patocchi et al., 2005). The gene
Vm was revealed by OPB12 STS marker in 7 accession including ‘McIntosh’ and its
progeny ‘Wijcik’. This marker also detected presence of the gene Vm in ‘SR0523’ and
its progenies ‘Orlovim’, ‘Pervinka’, ‘84–39/58’, ‘84–50/9’.
Table 2. The list of observed cultivars
2 lentelė. Tirtų veislių sąrašas
350

Table 2 continued
2 lentelės tęsinys
351

Table 2 continued
2 lentelės tęsinys
Gene Vf originates from the wild apple accession M. Xfloribunda 821. Vinatzer
et al. (1998) have cloned a cluster of resistance genes HcrVf from the Vf locus of the
chromosome 1. The cluster consisted of four homologous genes encoding receptor-
352

like protein. It is possible that one of them is related to resistant (Belfanti et al., 2004).
Marker VfC have been designed based on conserved regions in the Vf candidate genes
from HcrVf members (Afunian et al., 2004). Three fragments of 646, 484 and 286 bp
in length were revealed in the result of PCR with VfC marker. The presence of the
gene is determined by the presence of the fragment of 286 bp in length.
Tartarini et al. (1999) suggested using two primers pair AL07 and AM19
for identification of the gene Vf. This marker allows Vf homozygous resistant, Vf
heterozygous resistant and susceptible genotypes to be reveled. Amplification fragments
of 466 and 526 bp-long indicate gene Vf. Amplification fragment of 724 bp-long
indicate gene vf.
Markers VfC, AL07 and AM19 were detected in genomes of 41 accessions of
diverse breeding – France, USA, Poland, Russia, Belarus, etc. (Table 2). Cultivars
‘Relinda’, ‘Freedom’ and ‘Jonafree’ contain the genotype VfVf. The other resistant
cultivars contain genotype Vfvf.
Comparison of the obtained results with apple pedigree made it possible to
determine that the line BM41497 was the source of the gene Vf in cultivars of Belarus
breeding: ‘Belorusskoe sladkoe’, ‘Darunak’, ‘Nadzeiny’, ‘Pamyat Kovalenko’,
‘Pospeh’. The line BM41497 inherited this gene from M. Xfloribunda 821. Cultivar
‘Imant’ inherited the gene from ‘Liberty’. Line 814 was a source of gene Vf in cultivars
of Russian breeding ‘Afrodita’, ‘Jubilyar’, ‘Orlovskoe polesye’, ‘Solnyshko’, ‘Start’,
‘Stroevskoe’, ‘Venyaminovskoe’.
Levels of resistance detected for tested accessions in Belarus are presented
in Table 2. 41 cultivars demonstrated resistant to apple scab, 18 – field resistant,
43 – low susceptive, 25 – moderate susceptive, 3 – high susceptive of the 130 tested
cultivars.
Discussion. Great scab lesion was found in the cultivars ‘McIntosh’, ‘SR0523’,
‘Melba’. Presence of gene Vm in genome of ‘McIntosh’ and ‘SR0523’ did not protect
them from scab lesion. Gene Vm has been overcome by V. inaequalis race 5, an event
first discovered in England (Williams and Brown, 1968). Scab affection for these
cultivars was the same as for the cultivar ‘Melba’, which has been originated from
the cultivar ‘McIntosh’ but do not contained gene Vm.
Old cultivars ‘Antonovka obyknovennaya’, ‘Babushkino’, ‘Bely naliv’,
‘Borovinka’, ‘Gravenstein’, ‘Korobovka krupnoplodnaya’, ‘Koshtelya’, ‘Papirovka’,
‘Pepin litovsky’ had low or moderate susceptibility to scab. Some of them do not contain
the markers tested; the markers OPL19 and (or) AD13-SCAR were detected in others
(Table 2). Inversely the source of resistance of different genotypes of ‘Antonovka’
reported to be monogenic and polygenic (Williams and Kuc, 1969; MacHardy, 1996;
Quamme et al., 2003).
The accessions, in which the markers OPL19 and AD13-SCAR were detected,
shown different levels of scab resistance: from low level in ‘SR0523’ up to high
level in ‘78-14/245’. It is possible that impact of these loci on scab resistance is not
determinative.
The field resistance to scab demonstrated 17 cultivars, such as ‘Hislop’, ‘Jay
Darling’, ‘Minkar’, ‘Nora’, ‘Pamyat Vavilova’, ‘Kovalenkovskoe’, ‘Verbnoe’ and
others, in which the markers studied were not detected. Sasnauskas et al. (2006) have
353

characterized cultivars of Belarus breeding ‘Verbnoe’, ‘Kovalenkovskoe’ and ‘Pamyat
Syubarovoi’ (carrying the markers OPL19) as having a stable resistance to scab in
Lithuania in 2003–2005. Therefore, scab resistance in these cultivars is determined
by other genes or can by polygenic.
All cultivars with gene Vf were detected to be resistance to scab during the period
of study in Belarus except cultivar ‘Amulet’, which had low scab susceptibility. It has
been proposed that the variation in resistance reactions of Vf-carrying plants may be
due to the presence and action of modifier genes (Belfanti et al., 2004). Resistance
to scab was found in the cultivars of Russian breeding ‘Bolotovskoye’, ‘Yubilyar’,
‘Svezhest’ in Lithuania (Sasnauskas et al., 2006). Cultivar ‘Freedom’ demonstrated
resistance in other region of growing (Sestras, 2003). An unspecified ‘Antonovka’
clone is included in the pedigree of ‘Freedom’. The large proportion of resistant
seedlings observed in progenies derived from ‘Freedom’, suggested that this cultivar
is containing two resistance genes (Lamb et al., 1985). Later Zini (2005) mapped the
scab-resistance gene from ‘Antonovka’ at 20–25 cM from Vf .
Vf gene has been used most extensively in apple breeding programs around the
world. Vf locus confers resistance to five races V. inaequalis. However, it have been
investigated that Vf resistance has been overcome by V. inaequalis races 6 and 7 (Parisi
et al., 1993; Benaouf and Parisi, 2000). All major resistances in apple are ephemeral.
It is only a question of time until the pathogen with the matching virulence appears
and overcomes such a resistance (MacHardy et al., 2001). The durability of resistance
depends on the pathogen’s evolutionary potential (McDonald and Linde, 2002).
Actually, it is necessary to create new cultivars with two or more resistance sources.
New sources of resistance should be discovered and tools to exploit these resistances in
breeding programs (Liebhard et al., 2003). The combination of different resistances in
the same genotype was proposed as a possible way to obtain a durable scab resistance
for a long time (Lespinasse et al., 1999). This strategy was named ‘pyramiding’ and
the role of molecular markers in its implementation is very important as they allow to
detected necessary genes in different stages of ontogenesis.
Conclusion. Molecular methods of resistance gene detection can be successfully
used in breeding programs. The use of them can significantly reduce time for genotype
estimation and improve reliability of necessary gene detection.
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Gauta 2008 04 17
Parengta spausdinti 2008 04 29
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Va a ticchiolatura. PhD thesis, DCA-BO, Italy, 126.
SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Rauplėms atsparių genų nustatymas obelyse naudojant
molekulinius žymenis
O. Urbanovich, Z. Kazlovskaya
Santrauka
Obelų rauplės yra viena labiausiai paplitusių ir viena žalingiausių grybinių ligų
Baltarusijoje. Molekuliniai žymenys buvo naudojami atsparumo genų nustatymui 130-tyje
obelų pavyzdžių, įskaitant senas, naujas ir introdukuotas veisles. Genų Vf, Vm, Vr1 ir Vh2 buvo
nustatytas naudojant molekulinius žymenis OPB12STS, AD13 ir OPL19, kurie atitinkamai susiję
su genais Vm, Vr1 ir Vh2. Genas Vf buvo identifikuotas žymenimis VfC, AL07 ir AM19.
Genas Vr1 buvo nustatytas ir naujose, ir senose veislėse, kurios auginamos Baltarusijoje
nuo 19 a., tokios kaip ‘Papirovka’, ‘Bely naliv’, ‘Korobovka krupnoplodnaya’ ir kt. Žymuo
OPL19 buvo 81 pavyzdžio genome. Genas Vm buvo nustatytas 7 pavyzdžiuose susijusiuose su
tėvinėmis veislėmis ‘Mcintosh’ ir SR0523. Genas Vf, kuris buvo perkeltas iš M. Xfloribunda
821 ir įvestas į auginamas veisles, buvo identifikuotas 41 skirtingos selekcijos (Prancūzijos,
JAV, Lenkijos, Rusijos, Baltarusijos ir kt.) veislėje. Šio geno buvimas duoda didelį obelų veislių
atsparumą rauplėms. Tai buvo patikrinta lauko bandymuose 2004–2007 m. Veislės, turinčios
geną Vf, ir kai kurios veislės su poligenišku atsparumu parodė didelį atsparumą rauplėms ir
ant lapų, ir ant vaisių.
Reikšminiai žodžiai: atsparumo genai, Malus pavyzdžiai, obelų rauplės.
357

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Prospects for using of isozyme markers in identification
of apple cultivars
Alena Biruk, Zoya Kazlovskaya
The National Academy of Sciences of Belarus, The Institute for Fruit Growing,
Kovalev str. 2, Samokhvalovichy, Minsk reg., Belarus, 223013
E-mail: biohimbel@rambler.ru
The question of plant cultivar identification is important both for researcher’s work and
for decision of questions of fundamental science. The analysis of cultivar isozymes provides
fast way for identifying plant cultivars.
A preliminary analysis of the discriminating power of isozyme electrophoresis for
identification of apple cultivars is presented. The research objects were 57 apple cultivars. The
material was leaves and buds. Leaves were collected in June; buds were collected in November.
The polymorphism in five enzyme systems, peroxidase (PRX), malat dehydrogenase (MDH),
alcohol dehydrogenase (ADH) and glucose phosphate isomerase (GPI) was analysed using
polyacrylamide gel electrophoresis. The highest number well detectable bands were obtained
for PRX. Only peroxidase showed considerable variation among investigated apple cultivars.
The polymorphism of PRX was sufficient for identification of most samples.
Key words: apple, isozyme, peroxidase, malat dehydrogenase, glucose phosphate
isomerase, alcohol dehydrogenase, electrophoresis.
Introduction. The breeding process of horticultural plants is closely linked not
only with traditional methods but new methods as well enabling to change purposefully
heritability, to speed up the breeding process and obtain reliable information on
genotype properties. Plant physiology methods occupy an important place in solving
theoretical and practical problems of breeding and seed production (Duchovskis, 2001;
Gelvonauskis et al., 2004). For solving many problems of plant breeding and seed
production, the most convenient, in both technical and methodological aspects, are
seed storage proteins (Kонарев, 2001; Монархович, Яковлева, 2005). Nevertheless,
seed storage proteins can be used only when the plant enters a fruiting stage, while
actual problems remain an estimation of an initial and selection material. In this case
the most available are isozyme systems.
Biochemical markers such as isozymes have been extensively studied in plants
and many important agricultural characters have been correlated with isozyme
polymorphism as a result of the high heterozygosity and high level of polymorphism
that exist in apple, isozyme techniques have provided a reliable method for cultivar
identification in scion and rootstock. Evidence for the allopolyploid nature of the apple
genome was demonstrated using apple pollen enzyme system (Antonius-Klemola,
1999; Barnes, 1993; Protopapadakis, Paranikolaou, 1999; Vladova et al., 2000;
Weeden, 1989).
Isozyme polymorphism was examined in many fruit tree species: Malus, Prunus,
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Pyrus and others. Many studies have focused on cultivar identification, genetic linkage
and isozyme inheritance. Molecular markers have very good potential for plant breeders
(Ramos-Cabrer, Pereira-Lorenzo, 2005). A high level of isozymes polymorphism has
been detected in apple and more than 20 polymorphic isozyme loci were identified
(Gelvonauskis, Šikšnianienė, 2001).
The objective of our work was to evaluate polymorphism of peroxidase, malat
dehydrogenase, alcohol dehydrogenase and glucose phosphate isomerase in apple
cultivars.
Object, methods and conditions. Buds of 57 apple cultivars were collected in
November; leaves were collected in June. Samples of leaves (0.5 g) and buds (0.2 g)
were homogenized at 4 °C in Tris-HCL buffer (pH 6.8) containing 10 % sucrose,
0.2 % EDTA Na, 0.05 % 2-mercapto-ethanol, 0.1 % ascorbic acid. The homogenate
was centrifuged at 5 000 g for 30 min at 4 °C. The clear supernatants were used for
electrophoresis analysis. Protein concentrations were determined according to the
method of Bradford (1976) using bovine serum albumin as standard. Electrophoresis
was performed in vertical polyacrylamide gels. After electrophoresis gels were stained
for peroxidase (PRX) in solution containing 45 mg EDTA Na, 45 mg nitroprusside
sodium salt, 5 ml C 2
H 5
OH, 75 mg benzidine and 150 ml 0.2 M acetic buffer; for malat
dehydrogenase (MDH) in 40 ml 0.05 M Tris HCl buffer (pH 8.0) containing 200 mg
malic acid, 20 mg NAD, 10 mg nitrotetrazolium blue chloride, 0.6 mg PMS; for glucose
phosphate isomerase (GPI) in 10 ml 0.05 M Tris HCl buffer (pH 8.0) containing 10 mg
fructose-6-phosphate, 9 µl glucose-6-phosphate dehydrogenase, 15 mg NADP, 10 mg
nitrotetrazolium blue chloride, 0.3 mg PMS, 100 mg mgCl 2
10 mg agarose; for alcohol
dehydrogenase (ADH) in 30 ml 0.1 M Tris HCl buffer (pH 8.0) containing 10 ml
C 2
H 5
OH, 20 mg NAD, 20 mg nitrotetrazolium blue chloride, 0.6 mg PMS, 10 ml
distilled water (Гончаренко et al., 1989).
Results. Peroxidase provides sufficient diversity for the differentiation of most
of the cultivars examined in this study (Fig. 1. a, b). PRX isozymes occurred in three
regions of gels. The middle region is shown in migration isozyme bands of considerable
variability. The differences between cultivars of apple were also in the zone of the
slowly moving components. The number of PRX components varied from 6 to 12. The
components with Rf = 0.24, 0.26, 0.38, 0.60 and 0.65 occurs in almost all cultivars.
360

a: 1 – ‘Mechta’, 2 – ‘Byelorusskii synap’, 3 – ‘Freedom’, 4 – ‘Vesyalina’, 5 –
‘Alyesya’, 6 – ‘Byelorusskoye malinovoye’, 7 – ‘Slava pobeditelyam’, 8 – ‘Papirovka’,
9 – ‘Luchezarnoy’, 10 – ‘Pamyat Sikory’, 11 – ‘Verbnoye’, 12 – ‘Zaslavskoye’,
13 – ‘Bananovoye’, 14 – ‘Nadzeiny’, 15 – ‘Tellissaare’, 16 – ‘Syeruel’, 17 – ‘Imrus’,
18 – BM41497, 19 – ‘Melba’, 20 – ‘Antonovka’, 21 – ‘Kovalenkovskoye’, 22 –
‘Chulanovka’, 23 – ‘Pepinka zolotistaya’, 24 – ‘Wealthy’, 25 – ‘Koshtelya’, 26 – ‘Osenneye
polosatoye’, 27 – ‘Minskoye’, 28 – ‘Antei’, 29 – ‘Zarya Alatau’, 30 – ‘Sinap orlovskii’,
31 – ‘Byelorusskoye sladkoye’, 32 – ‘Imant’, 33 – ‘Auksis’, 34 – ‘Pamyat Syubarovoi’, 35 –
‘Charavnitsa’, 36 – ‘Pamyat Kovalenko’.
b: 1 – ‘Pospeh’, 2 – ‘Darunak’, 3 – ‘McIntosh’, 4 – SR0523, 5 – ‘Florina’, 6 – ‘Lawfam’,
7 – ‘Witos’, 8 – ‘Elstar’, 9 – ‘Gravenstein’, 10 – ‘Syabryna’, 11 – ‘Empire’, 12 – ‘Sawa’,
13 – ‘Kent’, 14 – ‘Red Boskop’, 15 – ‘Borovinka’, 16 – ‘Jonafree’, 17 – ‘Peppin litowskii’,
18 – ‘Idared’, 19 – ‘Redfree’, 20 – ‘Pinova’, 21 – ‘Elena’.
Fig. 1. Fingerprints of apple cultivars using PRX
1 pav. Obelų veislių PRX izoformų elektroforegrama
A total of 57 bands were recognized, and 49 phenotypes were observed among
all cultivars. ‘Byelorusskoye sladkoye’, ‘Nadzeiny’ and ‘Wealthy’ had the same PRX
banding pattern. ‘Antei’ and ‘Posreh’ showed the uniform banding pattern, respectively.
Similar bands had also ‘Kovalenkovskoye’ and ‘Pinova’, ‘Zaslavskoye’ and ‘Syabryna’,
‘Tellissaare’ and ‘Pamyat Syubarovoi’, ‘Imrus’ and ‘Sinap orlovskii’, ‘Freedom’ and
‘Chulanovka’, respectively.
The resolution of glucose phosphate isomerase was good, two areas of GPI were
identified, slower migrating region and faster migrating region. It was found that all
cultivars contain only one or two faster moving components in electrophoretic spectra.
The slowly migrating region shows variability between cultivars. However, it was found
only 1–4 components for slowly migrating region. 9 phenotypes were observed among
all the cultivars. The cultivars, which had similar PRX banding pattern (‘Byelorusskoye
sladkoye’, ‘Nadzeiny’ and ‘Wealthy’), had the different GPI banding pattern (Fig. 2).
‘Kovalenkovskoye’ and ‘Pinova’, ‘Zaslavskoye’ and ‘Syabryna’, ‘Tellissaare’ and
‘Pamyat Syubarovoi’ had the different GPI banding pattern too.
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1 – ‘Antonovka’, 2 – ‘Kovalenkovskoye’, 3 – ‘Chulanovka’, 4 – ‘Pepinka zolotistaya’,
5 – ‘Wealthy’; 6 – ‘Osenneye polosatoye’, 7 – ‘Minskoye’, 8 – ‘Auksis’, 9 – ‘Nadzeiny’, 10 –
‘Sinap orlovskii’, 11 – ‘Byelorusskoye sladkoye’, 12 – ‘Imant’.
Fig. 2. Fingerprints of apple cultivars using GPI
2 pav. Obelų veislių GPI izoformų elektroforegrama
Electrophoretic spectra of malat dehydrogenase of investigated cultivars were
similar. It was found two areas of MDH, but only some bunds were variable between
cultivars (Fig. 3). ADH did not yield any detectable variation among the cultivars
tested.
1 – ‘Bananovoye’, 2 – ‘Nadzeiny’, 3 – ‘Tellissaare’, 4 – ‘Syeruel’, 5 – ‘Imrus’,
6 – ‘BM41497’, 7 – ‘Melba’, 8 – ‘Antonovka’, 9 – ‘Kovalenkovskoye’, 10 – ‘Chulanovka’,
11 – ‘Pepinka zolotistaya’, 12 – ‘Wealthy’, 13 – ‘Kent’, 14 – ‘Red Boskop’, 15 – ‘Borovinka’,
16 – ‘Jonafree’, 17 – ‘Peppin litowskii’, 18 – ‘Idared’, 19 – ‘Redfree’, 20 – ‘Pinova’,
21 – ‘Elena’
Fig. 3. Fingerprints of apple cultivars using MDH
3 pav. Obelų veislių MDH izoformų elektroforegrama
Discussion. The search of protein markers of species, varieties and genotypes is
an urgent problem in modern breeding. Isoenzyme electrophoresis has been shown
to be applicable to apple cultivar identification. Several isozymes have been found to
detect useful amounts of variation in species. Isozymes analysis has also revealed some
cases of obvious mislabelling of plant material. The differences between electrophoretic
362

patterns could be observed with simple visual analysis, but the smaller differences
were revealed only by densitometer analysis.
The method of biochemical markers allow to solve whole number of problems for
selection such as identification of cultivars, analysis of hybrids, selection of valuable
genotype. The most investigations of enzyme polymorphism for fruits were curry out
for evaluation of cultivars. Isozyme used for evaluation of cultivars of peach, cherry,
almonds, plum, pear (Романова, Высоцкий, 2007).
The objective of our study was to design a practical procedure for examining
isozyme variation apple cultivars. Four isozyme systems were tested for their
resolvability and usefulness for discriminating apple cultivars. We carry out research
on peroxidase, malat dehydrogenase, alcohol dehydrogenase and glucose phosphate
isomerase. The results of this study showed considerable variability of PRX isozyme
bands, 49 cultivars of apple have unique of PRX bands. ADH did not yield any
detectable variation among the cultivars tested. GPI have two areas slower and faster
migrating regions. Although were define only several components for slowly migrating
region, we found 9 phenotypes GPI among all the cultivars.
Conclusions. Isozyme analysis offers a possible method for cultivars identification.
The results of this investigation show sufficient of peroxidase polymorphism, which
allow unique identification of the most tested cultivars. Although four cultivar pairs
(‘Kovalenkovskoye’ and ‘Pinova’, ‘Zaslavskoye’ and ‘Syabryna’, ‘Tellissaare’ and
‘Pamyat Syubarovoi’, ‘Byelorusskoye sladkoye’, ‘Nadzeiny’ and ‘Wealthy’) had
similar PRX banding patterns, they had the different GPI banding pattern. Using two
enzyme systems (peroxidase and glucose phosphate isomerase) allowed identification
of 51 cultivars from 57.
References
Gauta 2008 04 01
Parengta spausdinti 2008 05 09
1. Antonius-Klemola K. 1999. Molecular markers in Rubus (Rosaceae) research and
breeding. Journal of Horticultural Science & Biotechnology. 74(2): 149–160.
2. Barnes M. F. 1993. Leaf peroxidase and catechol oxidase polymorphism and
identification of commercial apple varieties. New Zealand Journal of Crop and
Horticultural Science. 21: 207–210.
3. Bradford M. M. 1976. A rapid sensitive method for the action of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem.
72: 248–254.
4. Duchovskis P. 2001. Physiological principles for breeding efficiency of
horticultural plants. Sodininkystė ir daržininkystė. 20(3): 3–14.
5. Gelvonauskis B., Šikšnianienė J. B. 2001. Peroxidase and polyphenoloxidase
polymorphism in apple cultivars of different scab resistance. Sodininkystė ir
daržininkystė. 20(3)–1: 37–43.
363

6. Gelvonauskis B., Šikšnianienė J. B., Duchovskis P. 2004. Genetic control of
resistance to fungal diseases, winterhardiness and productivity in orchard plants
and the use of DNA and isoenzyme markers for donor identification. Biologia.
2: 15–18.
7. Protopapadakis E., Paranikolaou X. 1999. Use of four isozymatic systems in
lemon and lemon-like citrus cultivars to detect their genetic diversity. Horticulture
Science and Biotechnology. 74(1): 26–29.
8. Ramos-Cabrer A. M., Pereira-Lorenzo S. 2005. Genetic relationship between
Castanea sativa Mill. Trees from north-western to south Spain based on
morphological traits and isoenzymes. Genetic Resources Crop Evolution.
52: 879–890.
9. Vladova R., Pandeva R., Petcolicheva K. 2000. Seed storage proteins in Capsicum
annuum cultivars. Biologia Plantarum. 43(2): 291–295.
10. Weeden N. F. 1989. Application of isozymes in plant breeding. Plant Breeding
Rev. 6: 1 154.
11. Гончаренко Г. Г., Падутов В. Е., Потенко В. В. 1989. Руководство по
исследованию хвойных видов методом электрофоретического анализа
изоферментов. Гомель. 158 c.
12. Kонарев В. Г. 2001. Морфогенез и молекулярно-биологический анализ
растений. Ст.-Петербург. 417 c.
13. Монархович С. В., Яковлева Г. А. 2005. Влияние различных факторов на
электрофоретические профили белков клубней картофеля. Вести НАН
Беларуси. Серия аграрных наук. 4: 60–63.
14. Романова О. В., Высоцкий В. А. 2007. Методика молекулярно-генетической
идентификации косточковых культур. Москва. 71 с.
SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Izozimų žymenų naudojimas obelų veislių identifikavimui
A. Biruk, Z. Kazlouskaya
Santrauka
Augalų veislių identifikavimas yra svarbus ir tyrėjų darbe, ir sprendžiant fundamentalaus
mokslo klausimus. Veislių izozimų analizė lemia greitą jų identifikavimą. Šiame darbe pristatyta
preliminari obelų veislių izozimų elektroforezės analizė. Buvo tirtos 57 obelų veislės, kurių buvo
analizuoti lapai ir pumpurai. Lapai buvo renkami birželio mėn., o pumpurai – lapkričio mėn.
Buvo tirta penkių fermentų sistemos polimorfizmas – peroksidazės (PRX), malat dehidrogenazės
(MDH), alkoholio dehidrogenazės (ADH) ir glukozės fosfato izomerazės (GPI) – naudojant
poliakrilamido gelio elektroforezę. Didžiausias skaičius gerai matomų juostų buvo nustatyta
PRX. Tik peroksidazė parodė žymią variaciją tarp tirtų obelų veislių. PRX polimorfizmas buvo
pakankamas identifikuojant daugelį pavyzdžių.
Reikšminiai žodžiai: alkoholio dehidrogenazė, elektroforezė, gliukozės fosfato izomerazė,
izozimai, malat dehidrogenazė, obelys, peroksidazės.
364

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
The use of black currant buds for the production of
essential oils
Edita Dambrauskienė, Pranas Viškelis, Audrius Sasnauskas
Lithuanian Institute of Horticulture, Kauno 30, LT-54333 Babtai, Kaunas distr.,
Lithuania, e-mail: e.dambrauskiene@lsdi.lt
Investigations of black currant (Ribus nigum L.) buds were carried out at the Lithuanian
Institute of Horticulture in order to establish their productivity and suitability for the production
of essential oils. There were investigated six cultivars more often grown in Lithuania: early
‘Joniniai’; mid-season ‘Almiai’ and ‘Gagatai’; late ‘Ben Alder’, ‘Ben Lomond’ and ‘Ben Nevis’.
It was established that at various months the cutting of currant branches, cultivars ‘Almiai’
and ‘Ben Nevis’ produced the biggest amount of essential oils (approximately 1.5 %); a little
less – ‘Gagatai’ and ‘Ben Lomond’ (approximately 1.2 %). Cultivar ‘Almiai’ had the biggest
buds. Cultivar ‘Joniniai’ forms the biggest number of buds on the branch (averagely 28.6).
Since the significant changes of essential oils’ amount in various months of rest period weren’t
established, the period from March up till December is being kept as suitable for currant shoot
cutting. As perspective cultivars for the extraction of essential oils in Lithuania, it may be grown
black currant cultivars ‘Almiai’ and ‘Ben Nevis’.
Key words: black currants, buds, cultivars, essential oils.
Introduction. Black currants are popular berry bushes, which berries because of
good biochemical and culinary properties are very valued in food industry. But black
currants, which distinguish themselves with strong aroma, also may be used in the
production of essential oils. For that purpose buds are gathered from young currants
and they became aroma raw material. Specific currants aroma is valued in perfumery,
medicine and culinary industry (Piry, Pribela, 1991). Such studies in the world are not
new, but there weren’t such investigations so long about black currant bud application
in the extraction of essential oils in Lithuania.
In foreign studies there are often accented valued chemical composition of black
currant berry, abundant of ascorbic acid and of anthocyanins, the change of these
indices depends on cultivar or berry ripening (Heiberg, Mage, 1992; Rubinskienė
et al., 2006). There were carried out the investigations with bud extracts (Kerslake,
Menary, 1985). Lately there was paid attention to such product as black currant bud
essential oil. The most valued black currant parts in respect of essential oils are their
buds (Nishimura, 1987).
For the production of essential oils not only the bud quality of black currant, but
also bud number per shoots, productivity of one plant or bigger massif is important.
Therefore, the amount of essential oils often is directly influenced by the conditions
of growing the berry bush and the methods of the increasing of their productivity
365

(Sasnauskas, Buskienė, 2006; Šikšnianas, Sasnauskas, 2002). The earlier studies
established that one of the essential indices is genetic origin of the plants. Therefore
both foreign (Koltowski et al., 1999) and Lithuanian (Misevičiūtė, 1997; Sasnauskas
et al., 2004; Šikšnianas, Sasnauskas, 1998) cultivars of black currants distinguish
themselves different not only in berry yield, but also qualitative and quantitative bud
parameters. The investigations of essential oils’ compositions are carried out already
for two decades in the world, thus their importance is unquestionable (Latrasse,
Lantin, 1976; Le, Latrasse, 1986; 1990; Rigaud et al., 1986). In Lithuania such studies
are started not long ago (Dvaranauskaitė et al., 2007). The aim of this study was to
establish the suitability of the black currant cultivars more often grown in Lithuania
for the production of essential oils.
Object, methods and conditions. For the investigations there were selected six
black currant cultivars of various earliness: ‘Joniniai’ (Lithuania), ‘Almiai’ (Lithuania),
‘Gagatai’ (Lithuania), ‘Ben Alder’ (Great Britain), ‘Ben Lomond’ (Great Britain) and
‘Ben Nevis’ (Great Britain). ‘Joniniai’ is an early cultivar. The fruits are of large size
(1.2 g). The average harvest – 5.0 t ha -1 . ‘Almiai’ is a mid-season cultivar. The average
fruits are of medium size (0.9 g). The average harvest was 8.2 t ha -1 (Misevičiūtė,
1997). ‘Gagatai’ is a mi d-season cultivar. The fruits are of large size (1.3 g). The
average harvest was 3.8 t ha -1 (Šikšnianas, Sasnauskas et al., 1998). ‘Ben Alder’, ‘Ben
Lomond’, ‘Ben Nevis’ are late season cultivars. Fruits are small (0.9 g), the average
harvest – 5.0 t ha -1 (Sasnauskas, Buskienė, 2006).
For the samples the shoots of black currant were taken from the currant plantations
of the Lithuanian Institute of Horticulture. They were cut for five times, each month,
from December 2005 up till the middle of April 2006. The average bud number per
shoot (unt.) and the average weight of one bud (g) is established. The essential oils
(%) of currant bud were obtained by hydrodistillation using apparatus of Clevenger
type (AOAC, 1990). The obtained results were processed by statistical methods, using
the program ANOVA.
Results. It was planed to carry out the experiment from the December up till the
middle of April, but the practice showed that black currant buds already became green
in the middle of April. After the analysis of such burst buds it was obtained that the
amount of essential oils, which was for five or six times smaller (0.18–0.28 %) than
that of the other investigation months (Table 1). Therefore analyzing the amount of
essential oils we do not base on the results of fifth (V) shoot cutting.
In various currant shoot cutting months constantly abundant amount of essential
oils (approximately 1.5 %) was established in buds of cultivars ‘Almiai’ and ‘Ben
Nevis’, a little smaller (approximately 1.2 %) in cultivars ‘Gagatai’ and ‘Ben Lomond’.
The amount of essential oils in the buds of cultivar ‘Joniniai’ in different months was
very different – from 0.78 % in March, up to 1.75 % in February. The smallest output
of essential oils among the investigated black currant cultivars was established in the
buds of ‘Ben Alder’ – from 0.86 up to 1.05 % (Table 1).
366

Table 1. The amount (%) of essential oils in black currant buds
1 lentelė. Eterinių aliejų kiekis (%) juodųjų serbentų pumpuruose
Babtai, 2005–2006
After calculations it was established that currant cultivar ‘Joniniai’ forms the
biggest number of buds per one shoot (on the average 28.6 unt.). Approximately 24–25
buds were found on the shoots of cultivars ‘Gagatai’, ‘Ben Alder’, ‘Ben Lomond’,
22–23 buds were found on shoots of cultivars ‘Almiai’ and ‘Ben Nevis’ (Table 2).
Table 2. The average number of buds per shoot (unt.)
2 lentelė. Vidutinis pumpurų skaičius ant ūglio, vnt.
Babtai, 2005–2006
Table 3. The average weight of one bud (g)
3 lentelė. Vidutinis vieno pumpuro svoris, g
Babtai, 2005–2006
367

When the weight of one bud was estimated, the most massive black currant buds
were acknowledged these of ‘Almiai’ – 0.043 g. The weight of ‘Ben Alder’ buds weight
on the average 0.034 g, ‘Joniniai’ – 0.028 g, ‘Gagatai’ and ‘Ben Nevis’ – 0.026 g. The
buds of ‘Ben Lomond’ weighted the least of all – 0.021 g (Table 3).
Discussion. According to the data of the investigation in 2005–2006, it is possible
to state that the amount of essential oils in the fresh buds of black currant depends on
their cultivar. Out of more popular six black currant cultivars grown in Lithuania the
biggest amount of essential oils in buds accumulate averagely early ‘Almiai’ and late
‘Ben Nevis’. Late cultivar ‘Ben Alder’ is the least productive from this point of view.
We didn’t observe the constant change of essential oils’ amount dependently on the
earliness of black currant cultivars. Both Lithuanian and foreign cultivars differ from
each other according to the amount of essential oils. Therefore selecting the suitable
cultivar for the production of essential oils is important to pay attention not to the
origin of the cultivar, but to its genetic properties.
The number of black currant buds on the shoots is an important parameter, basing
on which, it is possible to decide about the total productivity of certain cultivar. The
average data of the investigated cultivars fluctuated between 22.84 and 25.12 units
per shoot. These similar parameters on our opinion do not have big influence on the
amount of essential oils per unit of area. Only early ‘Joniniai’ distinguished themselves
with more abundant (28.62 unt.) number of buds. The weight of one bud among the
investigated black currant cultivars fluctuated from 0.021 up to 0.043 g. The heaviest
were mid-season ‘Almiai’, the lightest – the late ‘Ben Lomond’.
When choosing the time of cutting of currant shoots all the period of plant
rest is suitable – from December up till March. During investigations there weren’t
established the regular changes of essential oils’ amount in different winter months.
When plant vegetations starts, especially in April, currant buds burst and appear the
first green leaves, the amount of essential oils according to our data decreases for
four-six times.
The use of black currant for the extraction of essential oils is the new and
perspective type of business, but for that purpose it would be necessary to cultivate
rather big and productive black currant massifs. Early ‘Joniniai’ of Lithuanian origin
(Misevičiūtė, 1997) and mid-season ‘Almiai’ and ‘Gagatai’ (Šikšnianas, Sasnauskas,
1998) according to their parameters would be suitable for such application. Even
though berry productivity of the mentioned cultivars is rather high and may compete
with the foreign cultivars (Heiberg et al., 1992), under suitable conditions other black
currant application also may be perspective.
Conclusions. 1. It was established that in various months of black currant cutting
cultivars ‘Almiai’ and ‘Ben Nevis’ synthesize the biggest amount of essential oils
(approximately 1.5 %); slightly less – ‘Gagatai’ and ‘Ben Lomond’ (approximately
1.2 %).
2. Black currant cultivar ‘Almiai’ has the biggest buds (on the average of 0.043 g).
The buds of other investigated cultivars – ‘Joniniai’, ‘Gagatai’, ‘Ben Nevis’ – the
weight 0.026–0.028 g. The weight of buds of cultivar ‘Ben Lomond’ was the least of
all – 0.021 g.
368

3. Currant cultivar ‘Joniniai’ forms the biggest amount of buds per one shoot
(on the average 28.6 unt.). On the shoots of other cultivars it was found from 22 up
to 25 buds.
4. Since the significant changes of essential oils’ amount in various months of
rest period weren’t established, the period from March up till December is being kept
as suitable for currant shoot cutting.
5. As perspective cultivars for the extraction of essential oils in Lithuania, it may
be grown black currant cultivars ‘Almiai’ and ‘Ben Nevis’.
References
Gauta 2008 04 14
Parengta spausdinti 2008 04 30
1. AOAC, 1990. Official Methods of Anglysis. Arlington,. 15 th ed. 962. 17: 1 001.
2. Dvaranauskaitė A., Venskutonis P. R., Raynaud C., Talou T., Viškelis P.,
Dambrauskienė E. 2007. Chemical composition and antioxidant activity of six
blackcurrant buds cultivars. Footbalt – 2007: 2 nd Baltic conference on food science
and technology. Kaunas, 20.
3. Heiberg N., Mage F. 1992. Chemical composition of ten blackcurrant (Ribes
nigrum L.) cultivars. Acta Agriculturae Scandinavica Section B Soil and Plant
Science 42(4): 251–254. {a} Ullensvang Res. Stn., Substation Njos, N-5840
Hermansverk, Norway.
4. Kerslake M. F., Menary R. C. 1985. Varietal differences of extracts from
blackcurrant buds (Ribes nigrum L.). Journal of the Science of Food and
Agriculture. 36(5): 343–351.
5. Koltowski Z., Pluta S., Jabłoński B., Szklanowska K. 1999. Pollination
requirements of eight cultivars of black currant (Ribes nigrum L.). Journal of
Horticultural Science and Biotechnology. July 74(4): 472–474.
6. Latrasse A., Lantin B. 1976. Composition of essential oil of black currant buds –
its variability and inheritance. Acta Horticulturae. 60: 183–196.
7. Le Q. J. L., Latrasse A. 1990. Composition of the essential oils of blackcurrant buds
(Ribes nigrum L.). Journal of Agricultural And Food Chemistry. 38(1): 3–10.
8. Le Q. J. L., Latrasse A. 1986. Identification of dextro-spathulenol in the essential
oil of blackcurrant buds (Ribes nigrum). Sciences Des Aliments. 6(1): 47–59.
9. Misevičiūtė A. 1997. Naujos juodųjų serbentų veislės – Almiai, Laimiai, Vyčiai,
Pilėnai, Joniniai. Sodininkystė ir daržininkystė, 16: 3–15.
10. Nishimura O., Masuda H., Mihara S. 1987. Hydroxy nitriles in blackcurrant
buds absolute (Ribes nigrum L.). Journal of Agricultural and Food Chemistry.
35(3): 338–340.
11. Piry J., Pribela A. 1991. Distribution of aromatic substances in black currants.
Biologia. 46(3): 185–192.
12. Rigaud J., Etievant P., Henry R., Latrasse A. (1986). 4-Methoxy-2-methyl-2-
mercaptobutane, a major constituent of the aroma of the blackcurrant bud (Ribes
nigrum). Sciences Des Aliments. 6(2): 213–220.
369

13. Rubinskienė M., Viškelis P., Jasutienė I., Duchovskis P., Bobinas Č. 2006. Changes
in biologically active constituents during ripening in black currants. Journal of
fruit and ornamental plant research, 14(2): 237–246.
14. Sasnauskas, A., Šikšnianas, T., Rugienius R. 2004. New black currant cultivars
from Lithuania. Acta Horticulturae, 649: 323–326.
15. Sasnauskas A., Buskienė L. 2006. Genėjimo būdų įtaka juodojo serbento augumui
ir derėjimui. Sodininkystė ir daržininkystė, 25(1): 29–38.
16. Šikšnianas T., Sasnauskas A. 1998. Juodųjų serbentų veislės Gagatai, Kriviai ir
Kupoliniai. Sodininkystė ir daržininkystė, 17(4): 23–33.
17. Šikšnianas T., Sasnauskas A. 2002. Heritability of productivity and resistance to
fungal diseases in black currant. Acta Horticulturae, 585(1): 399–404.
SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Juodųjų serbentų pumpurų panaudojimas eterinių aliejų gamybai
E. Dambrauskienė, P. Viškelis, A. Sasnauskas
Santrauka
Lietuvos sodininkystės ir daržininkystės institute atlikti juodųjų serbentų (Ribus nigum L.)
tyrimai, siekiant nustatyti pumpurų produktyvumа ir tinkamumа eterinių aliejų gavybai.
Tirti šešių Lietuvoje dažniau auginamų veislių serbentai: labai ankstyvi ‘Joniniai’; vidutinio
ankstyvumo ‘Almiai’ ir ‘Gagatai’; vėlyvi ‘Ben Alder’, ‘Ben Lomond’ ir ‘Ben Nevis’. Nustatyta,
kad įvairiais serbentų ūglių pjovimo mėnesiais gausiausiai eterinių aliejų – apie 1,5 % sintetina –
‘Almiai’ ir ‘Ben Nevis’ veislių serbentai, kiek mažiau – apie 1,2 % ‘Gagatai’ ir ‘Ben Lomond’.
Masyviausi – juodųjų serbentų ‘Almiai’ pumpurai. Daugiausiai pumpurų ant ūglio – vidutiniškai
28,6 vnt. suformuoja ‘Joniniai’ serbentai. Nenustačius ženklių eterinių aliejų kiekio pokyčių
įvairiais ramybės periodo mėnesiais, tinkamu serbentų ūglių pjovimo tarpsniu laikome laikotarpį
nuo gruodžio iki kovo mėnesių. Kaip perspektyvios veislės eterinių aliejų gamybai, Lietuvoje
gali būti auginami ‘Almiai’ ir ‘Ben Nevis’ veislių juodieji serbentai.
Reikšminiai žodžiai: eteriniai aliejai, juodieji serbentai, pumpurai, veislės.
370

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Genetic resources of European small berries according
to GENBERRY project
Beatrice Denoyes-Rothan 1 , Audrius Sasnauskas 2 ,
Rytis Rugienius 2 , Philippe Chartier 3 , Aurelie Petit 3 ,
Stuart Gordon 4 , Julie Graham 4 , Alison Dolan 4 , Monika Hцfer 5 ,
Walther Faedi 6 , Maria Luigia Maltoni 6 , Gianluca Baruzzi 6 ,
Bruno Mezzetti 7 , Jose F. Sanchez Sevilla 8 , Edward Zurawicz 9 ,
Margaret Korbin 9 , Mihail Coman 10 , Paulina Mladin 10
1
UREF – INRA, 71 Avenue Edouard Bouleau, BP81, 33883,
Villenave d’Ornon, France
2
LIH, Kauno 30, LT-54333 Babtai, Kaunas distr., Lithuania
3
CIREF, Maison Jeannette, 24140, Douville, France
4
SCRI, Dundee DD2 5DA, Scotland, Great Britain
5
JKI, Pillnitzer Platz 3a, D-01326 Dresden, Germany
6
CRA-FRF, Via La Canapona 1bis, 47100, Forlм, Italy
7
SAPROV – UNIVPM, Plazza Roma 22, 60121, Ancona, Italy
8
IFAPA, Tabladilla s/n, 41071, Sevilla, Spain
9
INSAD, Pomologiczna 18, PO Box 105, 96-100, Skierniewice, Poland
10
FRIP, Marului street 402, PO Box 73, 1, Pitesti, Romania
E-mail: A.Sasnauskas@lsdi.lt
Small berries are vital fruit crops for maintaining activities in European rural areas. During
the last few years it has become apparent that there is a need for new varieties specifically adapted
to local environmental conditions. Therefore it is important to record and evaluate currently
available genetic resources. GENBERRY project, partly funded by the European Community,
has been designed to ensure that agricultural biodiversity of small berries will be preserved,
characterized and used to improve varieties adapted to local European regions. Strawberry
(Fragaria × ananassa) and raspberry (Rubus ideaus) represent the two main cultivated small
berries. The project is divided in different objectives, which represent different work-packages.
This project will help to improve conservation of small berry genetic resources by construction
of core collections, development of a passport data list, selection and definition of appropriate
primary and secondary descriptors, characterization of genotypes using molecular markers,
identification of health nutritional compounds and diseases evaluation for a large subset of
collections and establishment of European small berry database sustained by continuous long
term network.
Key words: strawberry, raspberry, database, descriptors, molecular markers, diseases,
genetic resources.
371

Introduction. For the most important cultivated small berry species, strawberry
and red raspberry, domestication has resulted in a reduction of both morphological
and genetic diversity with modern cultivars being genetically similar (Graham and
McNicol, 1995). Therefore, it is important to keep all the variability available today
in order to limit the decrease of the genetic basis of small berry genetic resources.
This restricted genetic diversity is of serious concern for future small berry
breeding, especially when seeking durable host resistance to intractable pests and
diseases for which the repeated use of pesticides in some regions is ineffective or
unacceptable. Therefore, it is now essential to enhance our appreciation of all small
berry genetic resources in order to improve these varieties to the new challenge of
agriculture: i. e. respect of the environment by using natural control factors, taking in
account of the global change, health nutrition.
The main objective of this project is to ensure that agricultural biodiversity of
small berries would be preserved, characterized and used to improve productivity.
The long-term objective of this project is to promote these genetic resources for future
utilizations, e. g. improving actual varieties for a sustainable agriculture. By creating
synergy with the COST 863 “Euroberry” results of this project will be available to all
the European community of breeders.
Object, methods and conditions. For small berries, the only work coordinated in
the past concerned strawberry genetic resources. First, a European Fragaria germplasm
Repository was developed in 1992/1995 in the AIR concerted action (Roudeillac,
Boxus, 1997). Then, with the COST 836 “Integrated Research in Berries”, the database
included 2819 entries of 1056 varieties (Geibel et al., 2004) (http://193.205.128.6/
agraria/ricerca/prog_ric/cost836.htm, EuroGerm reported 822 accessions, and EvalTrai
reported passport-authentication list). However, due to large change or less persons
responsible of European genetic resources, to lack of money and to phytosanitary
problems, works of genetic resources are today endangered.
The project is funded half-and-half by the participating institutes using their national
financing and by ‘the Community programme on the conservation, characterization,
collection and utilization of genetic resources in agriculture’ established by the
European Commission (EC).
Several levels of coordination will be worked out (Fig. 1). The participating
institutes are French National Institute for Agricultural Research (France, as
coordinating partner), Marche Polytechnic University (Italy), Unitą di ricerca per la
frutticoltura (Forlì) (Italy), Research Institute of Pomology and Floriculture (Poland),
Lithuanian Institute of Horticulture (Lithuania), Création Variétale Fraises – Fruits
Rouges (France), Federal Research Center for Cultivated Plants – Julius Kuehn
Institute (Germany), Scottish Crop Research Institute (Great Britain), Instituto Andaluz
deInvestigaciуn y Formación Agraria, Pesquera, Alimentaria y de la Producción
Ecológica (Spain) and Research Institute for Fruit Growing Pitesti – Maracineni
(Romania).
372

Fig. 1. The structure of the project
1 pav. Projekto schema
Results. Acquisition of the collection composition toward
a rationalization and conservation of ex situ collections and towards
selection of European core collection. The first step of this work-package will consist of
bringing together the list of accessions, allowing for the further steps the identification
of misnaming, synonyms and homonyms.
The species considered is the cultivated strawberry (F. × ananassa) and the
red raspberry (R. idaeus). The conservation techniques of the genetic resources are
373

shared by partners: integrated procedure for limiting pests and diseases in the ex situ
collections, use of in vitro culture, use of virus free runners, etc.
The outcome of this task will be lists of the small berries genetic resources from
which 96 genotypes will be chosen as representative of the diversity (based on different
priorities) of the collections. These lists named as “core collections-like” will be used
for molecular characterization and evaluation of both health compounds and disease
resistances. In addition, cultural techniques in order to improve the conservation of
the genetic resources will be shared.
P a s s p o r t d a t a l i s t a n d s e l e c t i o n a n d d e f i n i t i o n o f
a p p r o p r i a t e p r i m a r y a n d s e c o n d a r y d e s c r i p t o r s. The idea of this
work is to pursue the work already done for the AIR concerted action on 92 in which
CIREF was the coordinator and of the COST 836 were CIREF was involved, for
strawberry, and to take advantage of this experience for the other species.
The first step of this work-package will consist of choosing the more pertinent
passport data and the primary (notated by each partner, and therefore quite independent
of environmental factor) and secondary (facultative) descriptors. These lists will be used
the first year for the project on the collections of each partner before to be definitively
adopted according to remarks of the different partners. A definitive list will be set up.
The outcome of this task will feed the establishment of the database.
Characterization of the genetic diversity of representative part of the collections
with molecular markers, using microsatellites already developed. The first step of this
work-package consists of analyzing a large set of microsatellites in order to choice the
more relevant for studying genetic resources (Davis et al., 2006).
The further steps will be the analyses of the sub-set of microsatellites on the “core
collections-like” determined in WP1, and some other genotypes (controls of diversity,
misnaming, etc.). The outcome of this task will be a standardization of SSR-marker
analysis results for variety distinction and identification.
Characterization of biochemical components linked to health nutritional values
(total antioxidants and vitamin C) and evaluation of resistances. Small berries
represent an important source of bioactive compounds with antioxidant capacity
such as phenolics, with pharmacological and clinical activities (Manach et al., 2004,
Santos-Buelga, Scalbert, 2000). The objective of this WP is to evaluate and screen
large collections, populations, and core collections for identifying optimal source fruit
quality and of bioactive compounds for conferring health benefits.
Soil pathogens of strawberry such as Verticillium dahliae and Phytophthora
cactorum (Ertus et al., 2001) were controlled by methyl of bromide, which is today
forbidden. For red raspberry, the most important diseases or pests are caused by
Phytopthora fragaria var. rubi, Botrytis cinerea and Didymella applanata.
The objective is to evaluate collections of strawberry and raspberry in order to
identify very resistant accessions that could be further used in breeding programmes.
Breeding for resistance appears to be the most efficient and practical control strategy
for limiting or suppressing disease epidemics.
The outcome of this task will be a better characterization of the European genetic
resources for determining genotypes interesting to breeding.
Dissemination of the results to all publics, which include scientists, professional
horticulturists, and breeders via meetings and a WEB site. Dissemination will go beyond
common publications and workshops along the project. By organising workshops as
374

joint meeting with the COST working group 1, these workshops will allow to share
objectives and results with all the European and associated countries involved in small
berries breeding.
A web site publishes periodically overview documents that highlight the
achievements of the project.
Elaboration of living European small berry database sustained by continuous
long-term network cooperation. The first step consists of identifying carefully passport
data and descriptors according to the WP2. This list is sent to all experts of COST 863
working group 1 for their suggestions.
The further steps will be the establishment of database documenting the small
berry accessions of European collections according to the one developed on Prunus:
“The European Prunus database (EPDB)” developed and actually managed by INRA-
Bordeaux (http://cbi.labri.fr/outils/EPDB/index.html).
The outcome of this task will be an internet-accessible database, with differential
access of contributors and general public. Then, the long-term conservation of the
database and the up-date of the acquisition will be then performed by other partners.
Management and establishment of partners’ network. The objective of this work
package is to coordinate efforts of individual European countries to conserve genetic
resources.
A material transfer agreement is defined to exchange material between partners
during the project. For resources in the public domain, FAO recommendations and
its provisional MTA model are adopted. Second MTA will be used for resources not
in the public domain. Partners will define the access (open, restricted, etc.) to the
different level of information of the database for the different categories of users,
and will study measures to protect new information with an interest for industrial or
commercial applications.
During the course of the project and at the end of it, the partners commit themselves
to publish the results of the project in scientific and vulgarization journals, and make
a final summary meeting and report for the benefit of the end-users.
Discussion. Small berries are vital for maintaining activities in European rural
areas, despite they can be considered as minor species compared to other fruit species.
These activities generate sufficient incomes even in small farms and generate labour
specially for harvesting. In addition, they have an important high-value horticultural
industry in many European countries, providing employment in agriculture, but also
in food processing and confectionary. Many European and associated countries are
working on genetic improvement of small berries. Major objectives of breeding
programmes are, in addition to a fruit production in adequation with profits, disease
resistances and fruit quality such as health quality (Soria et al., 2008; Simpson,
Hammond, 2008; Faedi et al., 2008).
In this project, the evaluation on plant disease resistance and on fruit nutritional
quality will bring to the identification of new genotypes, which can be further used
as parents in European breeding programmes for improving disease resistances, and
fruit quality, ones of which high content of bioactive compounds useful for human
health.
The interest of a shared conservation network also lies in the possibility to build
safety duplicates. Europe will be stronger and more stable if the resources are shared
375

and protected Europe-wide rather than at small local levels.
By using molecular markers on small berries, this project will allow to reduce
the gap between the classical breeders and the molecularists and to reduce the gap
between countries that developed largely the use of molecular markers with countries
that just start or do not yet start to use them. One outcome of this project will be
the standardization of microsatellites for studying genetic diversity and varietal
identification, and the choice of core collections based on molecular and agronomical
characters.
For small berries, domestication has resulted in a reduction of both morphological
and genetic diversity with modern cultivars being genetically similar. GENBERRY
will lead to the monitoring of long term conservation of large small berries genetic
resources ex-situ and it will be of benefit for, the scientific progress, and the future
small berries generations.
Acknowledgements. Financial support provided by the European Commission
(Agricultural Commission DG AGRI) and the national governmental supports to
participating institutes are acknowledged.
References
Gauta 2008 03 26
Parengta spausdinti 2008 04 11
1. Davis T., Denoyes-Rothan B., Lerceteau-Kцhler E. 2006. Strawberry. In: Kole
C (ed), The Genomes: A Series on Genome Mapping, Molecular Breeding and
Genomic of Economic Species. Vol. IV. Science Publishers. New Hampshire,
Plymouth. (in Press).
2. Ertus C., Markocic M., Roudeillac P., Denoyes-Rothan B., Molinera V.,
Navatel J. C., et Lavialle O. 2001. Amйlioration du fraisier pour la lutte contre
Phytophthora cactorum. Phytoma, 537: 42–45.
3. Faedi V., Ballini L., Baroni G., Baruzi G., Baudino M., Giordano R., Lucchi P.,
Maltoni M. L., Migani M., Placchi L. 2008. Advances in strawberry breeding
for the north of Italy. Book of abstracts VI international strawberry symposium.
ISHS. Huelva, Spain. 3–7 March, 56.
4. Geibel M., Roudeillac P., Masny A., Trajkovski K., Coman M., and Simpson D.
2004. The European Strawberry Database and Building up a European Core
Collection. Acta Horticulturae, 649: 41–44.
5. Graham J., McNicol R. J. 1995. An examination of the ability of RAPD markers
to determine the relationships within and between Rubus species. Theoretical
Applied Genetics, 90: 1 128–1 132.
6. Manach C., Scalbert A., Morand C., Remesy C., Jimenez L. 2004.
Polyphenols: food sources and bioavailability. American Journal Clinical
Nutrition, 79: 727–747.
376

7. Roudeillac P., Boxus P. 1997. Preservation of F. × ananassa genetic resources
in Europe: organization of a European strawberry genebank. Acta Horticulturae,
439: 43–46.
8. Santos-Buelga C., Scalbert A. 2000. Proanthocyanidins and tannin-like
compounds – nature, occurrence, dietary intake and effects on nutrition and health.
Journal Science Food Agriculture, 80: 1 094–1 117.
9. Simpson D., Hammond K. 2008. A breeding strategy for improving resistance to
blackspot (Colletotrichum acutatum) in the United Kingdom. Book of abstracts
VI international strawberry symposium. ISHS. Huelva, Spain. 3–7 March, 53.
10. Soria C., Medina J. J., Sanchez-Sevilla J. F., Galvez J., Miranda L., Villalba R.,
Lopez-Aranda J. M. 2008. Current situation of the Spanish public strawberry
breeding program. Book of abstracts VI international strawberry symposium.
ISHS. Huelva, Spain. 3-7 March, 50.
SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Europos uoginių augalų genetiniai resursai pagal GENBERRY
projektа
B. Denoyes-Rothan, A. Sasnauskas, R. Rugienius, P. Chartier,
A. Petit, S. Gordon, J. Graham, A. Dolan, M. Hцfer, W. Faedi,
M. Luigia Maltoni, G. Baruzzi, B. Mezzetti, J. F. Sanchez Sevilla,
E. Zurawicz, M. Korbin, M. Coman, P. Mladin
Santrauka
Uoginiai augalai yra labai svarbūs Europos kaimo regionų plėtros atžvilgiu. Pastaraisiais
metais ypatingas dėmesys skiriamas naujoms, tinkamoms augti vietinėse agroklimato sąlygomis,
veislėms. Dėl tos priežasties genetinių resursų aprašymas ir įvertinimas yra fundamentalūs.
Projektas GENBERRY, iš dalies finansuojamas Europos Bendrijos, yra skirtas išsaugoti,
charakterizuoti ir pagerinti uoginių augalų genofondа vietiniuose Europos regionuose. Braškės
(Fragaria × ananassa) ir avietės (Rubus ideaus) pristatomos kaip dvi pagrindinės uoginių
augalų rūšys. Projektas darbo grupėse turi skirtingus tikslus. Uoginių augalų genetiniai resursai
saugomi ir gerinami sukuriant bendras kolekcijas, augalo pasų duomenų bazes, pradinius ir
pagrindinius deskriptorius, naudojant molekulinius žymenis, nustatant biocheminę sudėtį
bei įvertinant ligas. Kuriama Europos uoginių augalų duomenų bazė kaip Europinio tinklo
panaudojimas tarp mokslo institucijų.
Reikšminiai žodžiai: braškės, avietės, duomenų bazė, aprašai, molekuliniai žymenys,
ligos, genetiniai resursai.
377

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Effect of different mineral nitrogen and compost
nutrition on some compounds of corn salad
(Valerianella locusta (L.) Latter.)
Anna Kołton 1 , Agnieszka Baran 2
1
Department of Plant Physiology, Faculty of Horticulture, Agricultural University,
29 Listopada 54, 31-425 Krakуw, Poland
E-mail: koltona@ogr.ar.krakow.pl
2
Department of Agricultural Chemistry, Faculty of Agriculture and Economics,
Agricultural University, Mickiewicza 21, 31-120 Krakуw, Poland
During spring and autumn in 2007 corn salad ‘Noordhollandse’ was grown in containers
under shading cloth. The containers were filled with the clay loam soil. Before the both sowing
dates mineral nutrition was supplemented to the level of 250 kg NPK · ha -1 in ratio 2 : 1 : 2.
Mineral fertilizers and compost of the known composition were used as a source of nitrogen.
The following treatments were applied in the experiment: 1 – control (without fertilization),
2 – Ca(NO 3
) 2
, 3 – NH 4
NO 3
, 4 – compost. In both dates of growth climatic conditions were
different.
Contents of phenols, soluble sugars, chlorophylls a and b and carotenoids were analysed
in fresh material. Corn salad leaves harvested in autumn contained significantly more sugars
and phenols than the spring ones. Mineral and compost fertilization decreased soluble sugar
concentration as compared with control sample in both growing cycles, but only mineral
fertilization decreased content of phenols. Compost treatment significantly increased content
of phenols in corn salad in comparison with mineral fertilization and increased soluble sugar
concentration as related to Ca(NO 3
) 2
application. Corn salad leaves of spring experiment had
more chlorophyll and carotenoids than those of autumn one. During both growing periods,
mineral fertilization increased carotenoid and chlorophyll concentrations as compared to
control. In spring the level of pigments was higher in the case of compost treatment than in the
control sample, however, in autumn no significant differences were observed. Cultivation of
corn salad fertilized with either mineral or compost may bring high quality yield both in spring
and autumn growing cycles.
Key words: corn salad, nitrogen fertilizers, soluble sugars, chlorophyll, carotenoids.
Introduction. Leafy vegetables are an important element of human diet. They are
an excellent source of vitamins, minerals, sugars, folic acid and they have not much
calories. The everyday consumption of leafy vegetables lowers risk of cancer and
heart diseases, prevents tiredness, helps keeping well condition, prevents senescence.
Unfortunately, the consumption of leafy vegetables is still too little (Orłowski, 2000;
Wierzbicka, 2002).
Corn salad belongs to the family of Valerianaceae. It is an annual plant, which
leaves form a rosette (Martyniak-Przybyszewska, 2005).
379

Corn salad is not very popular in Poland. It is eaten like other salads. Leaves of corn
salad are delicate, without bitter flavor, with large amount of ascorbic acid, carotenoids,
folic acid, carbohydrates and mineral salts (Fajkowska, Wolfowa, 1985). Corn salad
leaves may accumulate large amounts of nitrates but there are known methods of
cultivation to decrease level of nitrates (Rożek, 2000). For every leafy vegeTable an
important factor of quality is the concentration of chlorophyll (Kaukounaras et al.,
2007; Michałek, Rukasz, 1998). Corn salad could be grown either under covers in
many cycles or in the field conditions – being frost resistant (Gapiński, 1993; Gonnella
et al., 2004)
Green leafy vegetables are healthy in human diet. Some researchers have found
that vegeTable extract with chlorophyll is antimutagenic and anticarcinogenic and
also has some antioxidant properties (Ma, Dolphin, 1999). Thus, chlorophyll and
carotenoids have specific dietary activities and these pigments are sensitive to growing
conditions (Caldwell, Britz, 2006).
Vegetables are source of naturally occurring antioxidants, which plays an important
role as a health protecting factors such as phenols, considered as more powerful
antioxidants than vitamin C or carotenoids to decrease disease risk (Larson et al.,
1988; Vinson et al., 1998).
There are a lot of data concerning method of growth, nitrate accumulation in
leaves and shelf life of corn salad (Fontana et al., 2004). However, study about effect
of different nitrogen fertilizers on the crop quality is incomplete. Growing of corn salad
is an important production in Italy, France, Netherlads, Germany, Belgium (Nicola
et al., 2004; Martyniak-Przybyszewska, 2005) and most of available corn salad in
Poland is from these countries.
The aim of the study was to investigate the effect of different nitrogen fertilizers
(mineral in two form of nitrogen and compost) on content of soluble sugars, chlorophylls
a and b, carotenoids and total phenols in corn salad ‘Noordhollandse’.
Object, methods and conditions. The study was carried out at the Agricultural
University in Krakуw in 2007. The experiment included growing of corn salad
(Valerianella locusta (L.) Latter.) ‘Noordhollandse’ in openwork containers. The
dimension of containers was: 60 Ч 40 Ч 20 cm and one container contained 45 dm 3
of soil. The containers were kept under clothing shadow. There were two growing
periods in 2007: the first one started on April 10 th (sowing date) and finished on
June 19 th (harvest date). The second started on July 20 th and finished on October 3 rd .
Containers were filled with clay loam soil (3 % of sand, 28 % of silt, 37 % of clay).
Before both sowing dates, the soil was analyzed. According to corn salad requirements
the soil was rich enough in P, K, Ca, mg, and the pH in KCl was about 6.5 in both
growing periods. Soil nitrogen was supplemented to the level of 2.25 g N · 45 dm -3
before sowing. Mineral fertilizers (Ca(NO 3
) 2
, NH 4
NO 3
) and compost (made by
Ekokonsorcjum Efekt Sp. z o. o. in Krakуw) were used as a source of nitrogen for
plants. Compost was made from green waste and contained macro and micronutrients
necessary for plants. The concentrations of minerals in compost were known
(in g · kg -1 of compost dry matter: N = 25, P 2
O 5
= 7, K 2
O = 35, Ca = 32, mg = 5). The
380

experiment included four treatments with regard to form of nitrogen fertilization:
1 – control – without any fertilization
2 – fertilized with Ca(NO 3
) 2
3 – fertilized with NH 4
NO 3
4 – fertilized with compost.
Experiment was randomly arranged in four replications of each treatment. The
spacing of plants and nursing were done in accordance with recommendation (Orłowski,
2000; Rekowska, 2001). Depending on weather conditions plants were irrigated.
Some climate factors were collected and analyzed (Table). Climate factors in both
growing periods were similar but not the same. During first growing period ground
frost was recorded. However, first growth period started with low and finished with
high temperatures, while during the second growing period the temperature conditions
were opposite. Mean temperature was similar for both growing cycles; however, in the
second period the higher relative humidity was noticed. Total rainfalls in first growing
period (from April to June) were nearly twice lower than in the second growing period
(from July to September) – 143.7 and 261.2 mm, respectively. Only in September
rainfalls were more intensive than during the whole first growing cycle. Insolation
was slightly higher during first growing period (674.3 and 609.3 hours, respectively).
The lowest insolation was observed in September.
Table. Climate factors during corn salad growth in 2007 (from IMGW and own
sensors)
Lentelė. Meteorologinės sąlygos salotinės sultenės auginimo 2007 m. metu (iš IMGW
ir savų jutiklių)
Immediately after harvest leaves of corn salad were randomly collected for
chemical analysis. Soluble sugars were determined by colorimetric method with
anthron reagent (Yemm, Wills, 1954). Total phenols were estimated according to the
photometric method with Folin’s reagent (Swain, Hillis, 1959). Chlorophylls a and
b and total carotenoids were determined by spectophotometric methods in acetone
solvent (Wellburn, 1994). Results of analysis (all in three replications) were statistically
evaluated using ANOVA and Fisher (LSD) test for the significance α = 0.05.
381

Results. A . Soluble sugars. Corn salad leaves harvested in autumn (second
growing period) accumulated significantly more sugars than the spring ones
(first growing period). Mineral and compost fertilization significantly decreased
accumulation of sugars in comparison with control treatment. Compost significantly
increased concentration of soluble sugars as compared to Ca(NO 3
) 2
fertilization in
both periods but only during first growing cycle as related to NH 4
NO 3
fertilization.
Mean soluble sugar concentration for spring cycle was 580.31 mg · 100 g -1 f. w. and
for autumn harvest – 906.25 mg · 100 g -1 f. w. (Fig. 1).
Fig. 1. Content of soluble sugars and total phenols in corn salad leaves
depending on nitrogen fertilization in 2007
(letters represented homogenous groups, LSD for α = 0.05)
1 pav. Tirpių cukrų ir bendras fenolių kiekis sultingosios saulenės
lapuose priklausomai nuo tręšimo azotu, 2007 m.
(raidės rodo homogenines grupes, R α = 0.05)
B . T o t a l p h e n o l s. Similarly like in the case of sugars corn salad leaves
accumulated significantly more phenols in autumn than in spring and mean concentration
was 342.96 and 185.66 mg · 100 g -1 f. w., respectively. During first growing cycle the
highest content of phenols contained leaves from compost treatment but in the second
one – thus of control. Mineral fertilization significantly decreased accumulation of
phenols in comparison with compost treatment in both growth periods (Fig. 1).
C. Chlorophylls a and b and carotenoids. Spring corn salad leaves contained
significantly more pigments than those harvested in autumn. Mean concentration for
first and second growth period was respectively: chlorophyll a: 0.415 and 0.272 mg · g -1
f. w., chlorophyll b: 0.249 and 0.185 mg · g -1 f. w., total chlorophyll (a + b): 0.664 and
0.457 mg · g -1 f. w. and carotenoids 0.157 and 0.121 mg · g -1 f. w. Mineral fertilization
382

significantly increased concentration of chlorophyll in comparison with control in
both periods. In most cases corn salad leaves from compost treatment had similar
chlorophyll concentration as control, except of chlorophyll b and total chlorophyll
from spring harvest (Fig. 2).
Concentration of carotenoids in corn salad leaves from spring harvest was
significantly higher in the case of mineral and compost fertilization. However, in
leaves from autumn harvest only mineral fertilization affected increase of carotenoids.
Plants from compost and control treatments in second growing period had the same
concentration of carotenoids (Fig. 2).
Fig. 2. Content of chlorophylls a and b, total chlorophyll and carotenoids
in corn salad leaves depending on nitrogen fertilization in 2007
(letters represented homogenous groups, LSD for α = 0.05)
1 pav. Chlorofilo a bei b, bendras chlorofilo ir karotinoidų kiekis sultingosios
saulenės lapuose priklausomai nuo tręšimo azotu, 2007 m.
(raidės rodo homogenines grupes, R, kai a = 0,05)
Discussion. Form of nitrogen fertilizer could have on important influence on
the accumulation of chlorophyll in plants. Lettuce plants fertilized with two form of
nitrogen (NO 3
+ NH 4
) had significantly more chlorophylls a and b than that fertilized
with nitrate nitrogen; and plants fertilized with ammonium nitrogen had the highest
concentration of chlorophylls (Michałek, Rukasz, 1998). In the present experiment
results did not confirm that dependence. Higher radiation increased concentration of
chlorophylls (Caldwell, Britz, 2006). Corn salad plants from spring period where the
insolation was higher had higher chlorophylls content in presented study. Nitrogen
fertilization stimulates accumulation of chlorophyll. Mihalovic et al. (1997) reported
383

that ammonium ions in nutrient solution increased concentration of chlorophylls a and
b in wheat leaves in comparison with nitrate ions. Presented results did not confirm
that conclusion, however, in the present experiment mixed nitrogen fertilizer was used
(not only NH 4
form) and drought stress was not observed. The higher total chlorophyll
concentration of marigold leaves was observed when compost or vermicompost were
added to the commercial horticultural plant growth medium than in pure medium
(Atiyeh et al., 2000). According to these authors, compost and vermicompost had a
potential for improving plant growth. However, they observed differences between
specific vermicomposts and composts. In the present experiment compost improved
soluble sugars and phenols content as compared with mineral fertilization, but this
effect was not observed in the case of pigment accumulations. Michałek and Rukosz
(1998) found that ammonium nitrogen increased accumulation of phenols. Influence
of ammonium and nitrate nitrogen applied together on phenol concentration depended
on cultivar. In some cases using the pure nitrate form or both forms of nitrogen (NO 3
+ NH 4
) as fertilizers may bring the same results, which were observed in the present
study. Total phenol content in lettuce was in good correlation with antioxidant activity
but might increase browning of lettuce (Altunkaya, Gökmen, 2008). Low temperature
caused higher carbohydrate accumulation in timothy leaves (Thorsteinsson et al., 2002)
and also in leaves of pelargonium cuttings (Druege, Kadner, 2008). The same effect
was observed in corn salad leaves. Rożek et al. (1994) reported that fertilization with
reduced form of nitrogen increased content of sugars in leaves of lettuce in comparison
to plants fertilized only with NO 3
. The same effect was observed in corn salad leaves
in the case of second growing period. Quality of lettuce crop was better when treated
with vermicompost and compost than with minral fertilization (higher vitamin C
concentration and lower nitrate accumulation) (Premuzic et al., 2002). In presented
experiment other quality factors increased in the case of compost treatments.
Conclusions. 1. Nitrogen fertilization and weather conditions together influenced
quality of corn salad.
2. Mineral fertilization increased carotenoid and chlorophyll concentrations as
compared to control.
3. Compost treatment increased content of phenols in corn salad in comparison
with mineral fertilization.
4. Corn salad leaves harvested in autumn contained more sugars and phenols
than the spring ones.
5. Cultivation of corn salad fertilized with either mineral nutritives or with compost
may bring high quality yield both in spring and autumn growing cycles.
Gauta 2008 04 17
Parengta spausdinti 2008 04 30
384

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SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Skirtingo mineralinio azoto ir kompostinių trąšų poveikis salotinės
sultenės (Valerianella locusta (L.) Latter.) biocheminei sudėčiai
A. Kołton, A. Baran
Santrauka
2007 m. pavasarį ir rudenį Valerianella locusta (L.) Latter. veislė ‘Noordhollandse’ buvo
auginama uždengtuose konteineriuose. Jie buvo pripildyti priemoliu. Ir prieš vienа, ir prieš kitа
sėjа buvo tręšta 250 kg NPK · ha -1 santykiu 2 : 1 : 2. Mineralinės trąšos ir žinomos sudėties
kompostas buvo naudojamas kaip azoto šaltinis. Bandymo schema buvo tokia: 1 – kontrolė
(be trąšų), 2 – Ca(NO 3
) 2
, 3 – NH 4
NO 3
, 4 – kompostas. Klimato sąlygos abejais auginimo
laikotarpiais skyrėsi.
Žaliuose augaluose nustatytas fenolių, tirpių cukrų, chlorofilo a ir b bei karotinoidų kiekis.
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Salotinės sultenės, augintos rudenį, turėjo žymiai daugiau cukrų ir fenolių negu pavasarinės.
Mineralinis ir kompostinis tręšimas sumažino tirpių cukrų koncentracijа jose palyginus su
kontrolės pavyzdžiais abejų auginimo ciklų metu, tačiau tik mineralinis tręšimas sumažino
fenolių kiekį. Salotinės sultenės lapai pavasarinio eksperimento metu turėjo daugiau chlorofilų
ir karotinoidų negu augintos rudenį. Abejais auginimo periodais mineralinis tręšimas padidino
karotinoidų ir chlorofilų koncentracijа palyginus su netręštais augalais. Tręšiant kompostu, tik
pavasarį šių augalų lapuose buvo daugiau pigmentų palyginus su kontrole. Salotinės sultenės
auginimas, naudojant ir mineralinį trąšą, ir kompostа, leidžia gauti aukštos kokybės derlių per
abu auginimo ciklus.
Reikšminiai žodžiai: azoto trąšos, chlorofilai, karotinoidai, salotinė sultenė, tirpūs
cukrūs.
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SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Small berry research according to COST 863 Action
Audrius Sasnauskas, Rytis Rugienius, Tadeušas Šikšnianas,
Nobertas Uselis, Laimutis Raudonis, Alma Valiuškatė,
Aušra Brazaitytė, Pranas Viškelis, Marina Rubinskienė
Lithuanian Institute of Horticulture, Kauno 30, LT-54333 Babtai, Kaunas distr.,
Lithuania, e-mail: A.Sasnauskas@lsdi.lt
Strawberry and blackcurrant cultivars with promising hybrids according to COST 863
Action were investigated at the Lithuanian Institute of Horticulture.
Strawberry cultivars ‘Dangė’, ‘Saulenė’ and ‘Elsanta’ had the biggest number of crowns,
leaves and runners, while ‘Elsanta’ and ‘Kent’ had the biggest amount of flower clusters and
berries. The most productive were strawberries of ‘Elsanta’ and ‘Kent’. ‘Venta’ and ‘Rosie’
had highest average berry size in strawberry collection. The best appearance was of ‘Rosie’
and J973854, berry firmness – 005004 and ‘Rosie’, best taste of 64 and ‘Venta’. ‘Dangė’ and
‘Honeoye’ strawberries distinguished themselves with intensive photosynthesis at period of
full blooming.
The highest bushes had blackcurrant cultivars ‘Titania’ and ‘Ben Lomond’, the lowest –
‘Gagatai’ and ‘Almiai’. The biggest bush width had ‘Öjebyn’ and ‘Ben Nevis’, narrow – ‘Ben
Tron’, ‘Ben Alder’ and ‘Ben Tirran’. Highest average yield was received from ‘Ben Tirran’,
‘Titania’ and ‘Öjebyn’. ‘Joniniai’, ‘Vyčiai’ and ‘Laimiai’ had the biggest berries. Biological
efficiency of fungicide Signum WG against blackcurrant powdery mildew (Sphaerotheca morsuvae
(Schw.) Berk. et Curt), leaf spot (Mycosphaerella ribis Lind.) and insecticide-acaricide
Envidor 240 SC against two-spotted spider mite (Tetranychus urticae Koch.) was investigated.
Applying the rates of 0.5–1.0 kg/ha four times per vegetation Signum WG effectively prevented
powdery mildew and leaf spot. Applying the rates of 0.3–0.6 l/ha three times per vegetation
Envidor 240 SC was effective against two-spider mite.
Sequence specific marker (SCAR) associated with strawberry resistance to red stele
(Phytophthora fragariae) Rpf1 gene was developed after cloning and sequencing of RAPD
marker. Using SCAR marker occurrence of Rpf1 gene in different strawberry cultivars and
seedlings were estimated. According to these data, cultivars ‘Redgauntlet’, ‘Anapolis’, ‘Tristar’,
‘Dangė’ and promising hybrids have this gene. Expression of COR47 gene homologues in
strawberry was evaluated during cold acclimation in vitro. It was established that maximal
accumulation of this gene transcript occurs on 30th day of cold acclimation.
Key words: strawberry, blackcurrant, trial evaluation, physiology, pest and diseases,
markers.
Introduction. The COST 863 Action has a new and advanced approach to promote
the integration of research, production systems, quality control, added nutritional value
and consumer acceptance. The action organized with a new integrated approach: from
laboratory via farm to consumer table.
The importance of this COST Action on berry research is supported by the large
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group of countries involved in the cultivation of berries (Faedi, 2004; Karhu, Hytönen,
2006; Spak et al., 2006; Özuygur et al., 2006; Rugienius, Sasnauskas, 2006). This will
improve co-operation between research programmes, including high level science and
technology, to develop European berry production system characterized by a reduction
of chemical inputs that are harmful to the environment along with lower production
costs (reduced labour costs) and increased benefits for the consumer (healthy fruits).
The aim of this study was to investigate small berry cultivars, efficiency of
fungicide and insecticide, photosynthesis period of full blooming, to estimate SCAR
marker occurrence of Rpf1 gene in different strawberry cultivars and seedlings
and expression of COR47 gene homologues in strawberry during cold acclimation
in vitro.
Object, methods and conditions. S t r a w b e r r y t r i a l s. The following
strawberry cultivars were compared in unheated plastic greenhouse: ‘Elsanta’, ‘Kent’,
‘Elkat’, ‘Honeoye’, ‘Saulenė’ and ‘Dangė’. Strawberries were established with frigo
plants in early spring. Peat-filled plastic sacks were used as substrate. Sacks were
located on shelving of 1.3 m height. Plant growth (units/plant), development (units/
plant) and yield (kg/m 2 ) were evaluated. The trial was established in four replications.
Each plot contained 21 plants.
The following strawberry cultivars were investigated in the collection:
‘Anapolis’, ‘Krymsakaja Remontantnaja’, ‘Wega’, ‘Sara’‚ ‘Venta’‚ ‘Rosie’, F. virg
glauca, hybrid clones 005004 (‘Selen’ × (F. chiloensis D. N. × ‘Tribute’)), J973854
(K88-4 × ‘Mohawk’), 64 (‘Guardian’ × ‘Pegasus’). The trial was established in four
replications. Each plot contained 5 plants. Beginning of yield (month, day), berry size
(scores), appearance (scores), firmness (scores) and taste (scores) were evaluated.
Scores 1–9 were used (1 – lowest, 9 – highest).
Assimilation area was measured with leaf area measurer CI-202 (CID Inc., USA).
Plant dry weight was established drying at the temperature of 105 °C. Photosynthesis
intensity was measured using PorTable Photosynthesis System (CI-310). Measurements
were made during flowering.
Polymorfic primer OPO16 suggested by Van de Weg, (1997) was used for PCR
in aim to develop SCAR marker of strawberry red stele (Phytophthora fragariae)
resistance gene Rpf1.
Total DNA was isolate from strawberry cultivars and hybrid clones 005001-2
(‘Selen’ × ‘Tristar’) ‘Anapolis’, ‘Redgauntlet’, 940101 (‘Guardian’ × ‘Pegasus’)‚
‘Elsanta’‚ ‘Selen’‚ ‘Tristar’ using CTAB protocol.
For COR47 gene expression investigations total RNA was isolated from strawberry
plants incubated at + 2 °C temperature for 1, 2, 4, 8 and 14 days. Copy DNA of COR47
gene was synthesized using RevertAid H Minus First Strand cDNA Synthesis Kit
(Fermentas).
Blackcurrant trials. The following blackcurrant cultivars were compared:
‘Joniniai’, ‘Kupoliniai’, ‘Zagadka’, ‘Gagatai’, ‘Almiai’, ‘Vyčiai’, ‘Laimiai’, ‘Öjebyn’,
‘Kriviai’, ‘Titania’, ‘Pilėnai’, ‘Ben Tron’, ‘Ben Lomond’, ‘Ben Alder’, ‘Ben More’,
‘Ben Nevis’ and ‘Ben Tirran’.
The bushes were planted at the distance of 3 × 0.6 m. The trial was established
in tree replications. Each plot contained 3 bushes. In the trial there was established
bush height and width (cm), berry yield (kg/bush) and berry size (g). Fungicides were
sprayed four times: until blooming, two times after blooming and after harvesting.
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Insecticides were sprayed three times: until blooming, after blooming and after
harvesting. Sprayer STIHL SR 400 was used for spraying, water volume – 250 l ha -1 .
Growing, fertilizing, weed control, soil cultivation, pruning and care of blackcurrant
cultivars were maintained as recommended for commercial orchards (Intensyvios
uoginių augalų auginimo technologijos, 2002).
Data were elaborated by analysis of variance, followed by Fisher’s Protected LSD
and Duncan’s Multiple-Range t-test at P = 0.05.
Results. Strawberry growth vigour. In both years of investigation cv.
‘Dangė’ (3.8 units) was distinguished for the highest crown per plant. Within the range
of tested cultivars ‘Elsanta’ (22.8 unts) had more leaves the same as cv. ‘Dangė’ (6.3
units) and ‘Saulenė’ (3.7 units) runners per plant (Table 1).
Table 1. Strawberry growth vigour (units/plant)
1 lentelė. Braškių kerelių augumas, vnt./aug.
Babtai, 2003–2004
Strawberry development. ‘Elsanta’ (2.7 units) had more inflorescences
in comparison with other investigated cultivars (Table 2). ‘Kent’ (2 units) and ‘Elkat’
(1.9 units) had medium, while ‘Dangė’ (0.6 units) had the smallest inflorescences per
plant over two years.
During the years of investigations the highest berries produced cv. ‘Elsanta’ (19.1
units). The lowest berries were of cvs. ‘Dangė’ (4 units) (Table 2).
Table 2. Strawberry development
2 lentelė. Braškių kerelių išsivystymas
Babtai, 2003–2004
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Y i e l d o f s t r a w b e r r y c u l t i v a r s. The highest yield was from ‘Elsanta’
(2.41 kg/m 2 ) and ‘Kent’ (2.25 kg/m 2 ). ‘Elkat’ (1.56 kg/m 2 ) and ‘Honeoye’ (1.18 kg/m 2 )
were more productive than ‘Dangė’ (0.40 kg/m 2 ) and ‘Saulenė’ (0.80 kg/m 2 ).
Table 3. Strawberry yield
3 lentelė. Braškių derlius
Babtai, 2003–2004
S t r a w b e r r y c u l t i v a r e v a l u a t i o n i n c o l l e c t i o n. F. virg glauca,
‘Krymskaja remontantnaja’, J973854, ‘Wega’ and ‘Venta’ ripened early, while 005004 –
latest (Table 4). The biggest berries produced cvs. ‘Venta’ and ‘Rosie’ (11 g), smallest –
F. virg glauca (3.5 g). The berries of cvs. ‘Rosie’ (10 scores) and J973854 (9.5 scorers)
produced extremely good appearance berries; 005004 and ‘Rosie’ (9.5 scorers) – berries
of good firmness; 64 and ‘Venta’ (9.5 scorers) – berries of very good taste.
Table 4. Strawberry cultivar evaluation in collection
4 lentelė. Braškių veislių tyrimas kolekcijoje
Babtai, 2007
B u s h h e i g h t a n d w i d t h c h a r a c t e r i s t i c s a n d b e r r y q u a l i t y
p a r a m e t e r s o f b l a c k c u r r a n t s. The highest bushes had blackcurrant cultivars
‘Titania’ (130 cm) and ‘Ben Lomond’ (125 cm), the lowest – ‘Gagatai’ (90 cm) and
‘Almiai’ (91.7 cm) (Table 5).
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Table 5. Bush height and width characteristics and berry quality parameters
5 lentelė. Juodųjų serbentų uogakrūmių augumas ir uogų kokybės parametrai
Babtai, 2007
The biggest bush width had ‘Цjebyn’ (165 cm) and ‘Ben Nevis’ (168.3 cm); ‘Ben
Tron’ (96.7 cm), ‘Ben Alder’ (103.3 cm) and ‘Ben Tirran’ (105 cm) were the narrowest.
‘Joniniai’ (140.7 g), ‘Vyčiai’ (129.5 g) and ‘Laimiai’ (124.5 g) had the biggest berries,
while ‘Öjebyn’ (68.7 g) and ‘Ben Alder’ (70.4 g) – smallest. ‘Joniniai’ (1.97 g) and
‘Vyčiai’ (1.88 g) distinguished themselves with the biggest berry size; ‘Titania’ (0.23 g)
and ‘Kupoliniai’ (0.29 g) berries were the smallest.
Y i e l d o f b l a c k c u r r a n t c u l t i v a r s. During the year of investigation
the late spring frost at the beginning of bloom injured blossoms and average yield was
0.73 kg/bush (Fig.1). The yield of blackcurrant cultivars ranged from 0.2 to 1.86 kg/
bush. ‘Ben Tirran’ (1.86 kg/bush), ‘Titania’ (1.59 kg/bush) and ‘Öjebyn’ (1.21 kg/
bush) produced a higher yield. Cvs. ‘Ben Alder’ (0.88 kg/bush), ‘Kriviai’ (0.86 kg/
bush), ‘Zagadka’ (0.81 kg/bush) and ‘Almiai’ (0.76 kg/bush) produced higher yield
in comparison with the average of investigated cultivars. Cvs. ‘Vyčiai’ (0.2 kg/bush),
‘Laimiai’ (0.25 kg/bush), ‘Pilėnai’ (0.26 kg/bush) and ‘Gagatai’ (0.27 kg/bush) had
the lowest yield.
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Fig. 1. Yield of blackcurrant cultivars, kg/bush
1 pav. Juodųjų serbentų veislių derlius, kg/krūmo
Babtai, 2007
P h o t o s y n t h e s i s i n t e n s i t y. ‘Dangė’ and ‘Honeoye’ strawberries
distinguished themselves with intensive photosynthesis at period of full blooming;
while in leaves of ‘Elsanta’ and ‘Saulenė’ dominate breathing (Fig. 2).
Fig. 2. Net photosynthesis intensity at period of full blooming
(PAR average – 698 µmol m -2 s -1 ; air temperature average – 26 °C)
2 pav. Neto fotosintezės intensyvumas braškių gausiausio žydėjimo metu (vidutinė FAR –
698 µmol m -2 s -1 ; vidutinė oro temperatūra – 26 °C)
Babtai, 2004
B l a c k c u r r a n t p r o t e c t i o n f r o m d i s e a s e s a n d p e s t.
Signum WG 1.0, 0.5 kg/ha applied 4 times during growing season (until blooming,
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after blooming and after harvesting) was effective against diseases. Rate of fungicide
Signum WG 1.0 kg/ha efficiency of preparation was as follows: against powdery
mildew – 90.5 %, against leaf spot – 91.6 %. Lower rate of fungicide Signum WG 0.5 kg/
ha reduced powdery mildew efficiency to 88.4 %, against leaf spot – 88.7 %. Efficiency
of standard fungicide Candit 0.2 kg/ha was also high: against powdery mildew – 89.5 %,
against leaf spot – 90.9 %. There were found 95 % powdery mildew symptoms and
70 % leaf spot symptoms on leaves in unsprayed plots (Tables 6–7).
Table 6. Impact of fungicide Signum on powdery mildew
6 lentelė. Fungicido Signum efektyvumas nuo miltligės
Babtai, 2004–2005
Table 7. Impact of fungicide Signum on leaf spot
7 lentelė. Fungicido Signum efektyvumas nuo šviesmargės
Babtai, 2004–2005
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Rate of insecticide-acaricide Envidor 240 SC 0.3 l/ha efficiency of preparation
against two-spotted spider mite ranged between 63.8–100 % (Table 8). Envidor 240 SC
0.6 l/ha and 0.3 l/ha applied 2 months after spraying efficiency was 87.7–83.4 %, while
standard insecticide Karate 0.5 l/ha efficiency against two-spotted spider mite was
only 63.9 %. In both treatments with Envidor 240 SC 0.6 l/ha and 0.3 l/ha number of
two-spotted spider mite on 1 leaf not statistically differences were found.
Table 8. Efficiency of insekticide-acaricide Envidor 240 SC against two-spotted
spider mite
8 lentelė. Insekticido akaricido Envidoro 240 g/l efektyvumas nuo paprastųjų voratinklinių
erkių
Babtai, 2004–2005
Molecular investigations of strawberry. Using OPO16 polymorfic
primer, DNA fragment specific for red stele susceptible strawberry was isolated. This
DNA fragment was cloned to pTZ57 plasmid and sequenced. DNA sequence specific
(SCAR) primers were selected. Newly developed SCAR marker allows excluding
susceptible genotypes lacking of red stele resistance gene Rpf1. According PCR data,
genotypes ‘Redgauntlet’, ‘Anapolis’; ‘Tristar’, ‘Dangė, 005001; 005002 have Rpf1
gene, other genotypes: ’Honeoye’; ‘Elsanta’, Venta’, ‘Kama’; 940101; ‘Selen’; ‘Elkat’
and ‘Senga Sengana’ – have not (Fig. 3).
Homologues of Arabidopsis thaliana (L.) Heynh cold response (COR47) gene
were isolated from different fruit and berry plants. Expression of those genes during
acclimation was investigated. It was established that maximal accumulation of COR47
transcript occurs 30 after start of cold acclimation (Fig 4).
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Fig. 3. Strawberry electrophoregram after PCR using newly developed Rpf1 gene
SCAR marker (size 400 bp)
3 pav. Braškių DNR elektroforegrama po PGR, taikant naujai išskirtа geno Rpf1 žymenį
(dydis 400 bp) M – GeneRuler TM 1kb DNA Ladder, 1 – ‘Honeoye’; 2 – ‘Redgauntlet’,
3 – ‘Elsanta’, 4 – ‘Venta’, 5 – ‘Kama’; 6 – ‘Anapolis’; 7 – ‘Tristar’; 8 – 940101;
9 – ‘Selen’; 10 – 005001; 11 – 005002; 12 – ‘Elkat’, 13 – ‘Senga Sengana’; 14 – ‘Dangė’.
Fig. 4. Dynamics of cold responsive COR47 gene expression in strawberry during
cold acclimation in vitro
4 pav. Šalčio indukuojamo COR47 geno homologų transkripcijos kitimas užsigrūdinimo
metu braškėse in vitro. 1, 3, 5, 7, 9, 11 – ‘Melody’. 2, 4, 6, 8, 10, 12 – ‘Holiday’.
1–2 – before acclimation/ augalai prieš grūdinimа; 3–4 – cold acclimated for
6 days/ 6 dienas grūdinti augalai; 5–6 – 12 days/ 12 dienų;
7–8 – for days 18/ 18 dienų, 8–9 – 24 days/ 24 dienas; 11–12 – 30 days/ 30 dienų;
13 – O‘GeneRulerTM 1 kb DNA Ladder.
Discussion. Small berry yield and quality parameter has recently become more
and more important for the scientists, consumers and producers (Faedi et al., 2002;
Roudeillac, Trajkovski, 2004; Sousa et al., 2008; Voca et al., 2008). Our data show that
different genotypes might have different strategy for these characters. During the years
of investigation, strawberry cvs. ‘Elsanta’, ‘Kent’ and blackcurrant cvs. ‘Ben Tirran’,
‘Titania’, ‘Öjebyn’ produced the highest yield. The investigation shows that ‘Elsanta’
and ‘Kent’ had the biggest amount of flower clusters and berries in peat-filled plastic
sacks like ‘Venta’ and ‘Rosie’ for the same investigated character in the collection.
On the other hand, Lithuanian blackcurrant cultivars ‘Joniniai’, ‘Vyčiai’ and ‘Laimiai’
had the biggest berries in comparison with introduced cultivars. Appearance, taste and
firmness are quality attribute. Results of organoleptic evaluation show that berries of
397

cvs. ‘Rosie’ and J973854 were of extremely good appearance; 005004 and ‘Rosie’ – of
good firmness; 64 and ‘Venta’ – of very good taste.
New chemicals help in blackcurrant pests and diseases control (Rašinskienė,
Šikšnianas, 1995; Locke, Koomen, 1999; Raudonis, 2002; Locke et al., 2003; Worley,
2003). The investigation shows that applying the rates of 0.5–1.0 kg/ha four times per
vegetation Signum WG effectively prevented powdery mildew and leaf spot. On the
other hand, applying the rates of 0.3–0.6 l/ha three times per vegetation Envidor 240
SC was effective against two-spotted spider mite.
Biotechnological methods (in vitro, marker assisted selection) helps to speed
up breeding process, screen gene pool and evaluate of fruit and berry plants holding
valuable traits and also investigate biological mechanisms of disease and cold
resistance.
Conclusions. 1. Strawberry cultivars ‘Dangė’, ‘Saulenė’ and ‘Elsanta’ had the
biggest number of crowns, leaves and runners.
2. ‘Elsanta’ and ‘Kent’ had the biggest amount of flower clusters and berries.
3. Most productive were strawberries of ‘Elsanta’ and ‘Kent’.
4. The biggest fruits produced cvs. ‘Venta’ and ‘Rosie’. The berries of cvs. ‘Rosie’
and J973854 were of extremely good appearance; 005004 and ‘Rosie’ – of good
firmness; 64 and ‘Venta’ – of very good taste.
5. The highest bushes had blackcurrant cultivars ‘Titania’ and ‘Ben Lomond’,
the lowest – ‘Gagatai’ and ‘Almiai’. The biggest bush width had ‘Öjebyn’ and ‘Ben
Nevis’; ‘Ben Tron’, ‘Ben Alder’ and ‘Ben Tirran’ were the narrowest. Highest average
yield was received from ‘Ben Tirran’, ‘Titania’ and ‘Öjebyn’. ‘Joniniai’, ‘Vyčiai’ and
‘Laimiai’ had the biggest berries.
6. ‘Dangė’ and ‘Honeoye’ strawberries distinguished themselves with intensive
photosynthesis at period of full blooming.
7. Applying the rates of 0.5–1.0 kg/ha four times per vegetation
Signum WG effectively prevented powdery mildew and leaf spot. Applying the rates
of 0.3–0.6 l/ha three times per vegetation Envidor 240 SC was effective against twospotted
spider mite.
8. Strawberry cultivars ‘Redgauntlet’, ‘Anapolis’, ‘Tristar’, ‘Dangė’ and promising
hybrids have Rpf1 gene.
9. Maximal accumulation of COR47 gene transcript occurs on 30th day of cold
acclimation.
Acknowledgement. We’re grateful to Agency for International Science and
Technology Development Programmes in Lithuania for help and support realizing
COST 863 Action.
Gauta 2008 04 14
Parengta spausdinti 2008 04 30
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2. Faedi F. 2004. COST: Past, Present, Future. Acta Horticulturae, 649: 21–24.
3. Intensyvios uoginių augalų auginimo technologijos. 2002. N. Uselis. (sudaryt.).
Lietuvos Sodininkystės ir daržininkystės institutas, Babtai.
4. Karhu S. T., Hytönen T. P. 2006. Nursery plant production controlled by
prohexadione-calcium and mechanical treatments in strawberry cv. ‘Honeoye’.
The Journal of horticultural science and biotechnology, 81(6): 937–942.
5. Locke T., Koomen I. 1999. Can spore trapping be of help in black currant mildew
control Acta Horticulturae, 505: 333–336.
6. Locke T., Bobbin P., Atwood J., Owen J. 2003. Effect of strobilurin fungicides on
disease control and yield in blackcurrants. Acta Horticulturae, 585: 394–396.
7. Özuygur M., Paydaŗ K. S., Kafkas E. 2006. Investigation on yield, fruit
quality and plant characteristics of some local, European and American
strawberry varieties and their hybrids. Agriculturae conspectus scientificus,
71(4): 175–180.
8. Raudonis L. 2002. Monitoring of harmful insects and mites of strawberries.
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11. Rugienius R., Sasnauskas A. 2006. Braškių veislių tyrimas Lietuvoje pagal
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12. Sousa M. B., Curado T., Trigo M. J., Vasconcelos F. N., Nunes T. 2008. Strawberry
quality: effect of cultivars, harvest date and storage. Book of abstracts VI
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13. Spak J., Navrátil M., Karelová R., Pribylová J., Válová P., Kucerová J.,
Kubelková D., Fialová R., Spaková V. 2006. Occurrence, symptom variation and
yield loss caused by full blossom disease in red and white currants in the Czech
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14. Voca S., Duralija B., Družic J., Dobričevic N., Dragovic-Uzelac V. 2008. Quality
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SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Uogininkystės tyrimai pagal COST 863 programą
A. Sasnauskas, R. Rugienius, T. Šikšnianas, N. Uselis, L. Raudonis,
A. Valiuškatė, A. Brazaitytė, P. Viškelis, M. Rubinskienė
Santrauka
Lietuvos sodininkystės ir daržininkystės institute pagal tarptautinę COST 863 programą
tirtos braškių bei juodųjų serbentų veislių ir hibridinių klonų biologinės ir ūkinės savybės.
‘Dangės’, ‘Saulenės’ ir ‘Elsantos’ veislių braškės pagal ragelių, lapų ir ūsų skaičių augo
vešliausiai. Pagal žiedynų ir uogų skaičių geriausiai išsivysčiusios buvo braškės ‘Elsanta’
ir ‘Kent’. Derlingiausios braškės ‘Elsanta’ ir ‘Kent’. Braškių kolekcijoje pagal didžiausiа
vidutinę uogų masę išsiskyrė ‘Venta’ ir ‘Rosie’. Gera uogų išvaizda pasižymėjo ‘Rosie’ ir
J973854; uogų kietumu – 005004 ir ‘Rosie’; geru uogų skoniu – selekcinis klonas 64 ir ‘Venta’.
Gausiausio braškių žydėjimo metu intensyviausia fotosintezė vyko ‘Dangės’ ir ‘Honeoye’
braškių lapuose.
Palyginus skirtingas juodųjų serbentų veisles nustatyta, kad aukščiausi užauga ‘Titania’
ir ‘Ben Lomond’, o žemiausi – ‘Gagatai’ bei ‘Almiai’ veislių serbentų krūmai. Plačiausiais
krūmais išsiskyrė ‘Öjebyn’ ir ‘Ben Nevis’ veislės, siauriausiais – ‘Ben Tron’, ‘Ben Alder’
bei ‘Ben Tirran’ juodųjų serbentų krūmai. Gausiausiai derėjo juodųjų serbentų veislių ‘Ben
Tirran’, ‘Titania’ ir ‘Öjebyn’ uogakrūmiai. Stambiausias uogas išaugino ‘Joniniai’, ‘Vyčiai’
ir ‘Laimiai’. Juodųjų serbentų veislės ‘Joniniai’ bei ‘Vyčiai’ išsiskyrė didžiausiomis uogomis.
Fungicidas Signum 334 g/kg v. d. g. 0,5–1,0 kg/ha yra efektyvus juodųjų serbentų apsaugai
nuo miltligės (Sphaerotheca mors-uvae (Schw.) Berk. et Curt) ir šviesmargės (Mycosphaerella
ribis Lind.) purškiant keturis kartus per vegetacijа: prieš žydėjimа, po žydėjimo ir nuėmus
derlių. Insekticidas akaricidas Envidoras 240 g/l k. s. 0,3 – 0,6 l/ha juodųjų serbentų apsaugai
nuo paprastųjų voratinklinių erkių (Tetranychus urticae Koch.) yra efektyvus purškiant 2 –3
kartus per vegetacijа: prieš ir po žydėjimo bei nuėmus derlių, taip pat apsaugai nuo serbentinių
amarų.
Pagal RAPD žymens DNR sekа sukurtas jai specifinis SCAR žymuo, susijęs su braškių
atsparumo fitoftorozei (Phytophthora fragariae) Rpf1genu. Naudojant šį žymenį įvertintas
geno Rpf1 buvimas skirtingose braškių veislėse ir sėjinukuose. Pagal šiuos duomenis veislės
ir hibridiniai klonai ‘Redgauntlet’, ‘Anapolis’; ‘Tristar’, ‘Dangė’, 005001; 005002 turi Rpf1
genа.
Ištirta COR47 geno raiška braškėse užsigrūdinimo metu in vitro. Didžiausias COR 47
geno transkripto kiekis braškėse susidaro po 30 dienų grūdinimo žemose teigiamose
temperatūrose.
Reikšminiai žodžiai: braškės, juodieji serbentai, veislių tyrimas, fiziologija, ligos ir
kenkėjai, molekuliniai žymenys.
400

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Growing, yielding and quality of different ecologically
grown pumpkin cultivars
Rasa Karklelienė, Pranas Viškelis, Marina Rubinskienė
Lithuanian Institute of Horticulture, Kauno 30, LT-54333 Babtai, Kaunas distr.,
Lithuania, e-mail: R.Karkleliene@lsdi.lt
Investigations were carried out at the Lithuanian Institute of Horticulture in the greenhouse
of ecological seed-growing covered with double polymeric film, in the natural soil – in loam on
loam, more deeply epihypogleyic luvisol (IDg 8-k, /Calc(ar)i – Epihypogleyc Luvisols – LVgp-w-cc),
enriched with peat-compost substrate. There was grown Cucurbita pepo L. cultivar
‘Beloruskaja’ and two cultivars of Cucurbita maxima Duch. – ‘Gele Reuzen’ and ‘Bambino’.
Morphological indices of pumpkin fruits were fixed in three stages. It was established that
the main pumpkin fruit growth took place in August. Then they already had fruits characteristic
to the cultivar. In September ripening processes occurred in fruits, therefore fruit weight didn’t
differ strongly from the second weighting of pumpkin fruits. Investigations showed that pumpkin
cultivar ‘Gele Reuzen’ produced fruits, which weighted on the average from 8.0 to 8.7 kg.
Pumpkins accumulate on the average 4.73 % of dry soluble solids. Cultivar ‘Beloruskaja’
distinguished itself with bigger their amount. Sugar concentration in pumpkins changed from
3.1 % to 4.29 %. The least amount of sugars was established in pumpkins of cultivar ‘Bambino’,
the biggest one – in pumpkins of cultivar ‘Beloruskaja’. These vegetables accumulate little amount
of ascorbic acid – on the average 3.33 mg 100 g -1 . Dependently on the cultivar, the amount of
the accumulated nitrates in pumpkins change in wide limits – from 105 (‘Gele Reuzen’) up
to 636 (‘Bambino’) mg kg -1 . The amount of carotenoids was investigated in the edible part of
pumpkins. More of them was established in cultivar ‘Beloruskaja’ (5.94 mg 100 g -1 ).
Key words: chemical composition, colour parameters, cultivars, growth, yield,
morphological indices, pumpkin.
Introduction. Lithuanian climate is excellent for pumpkin growing. It is suitable
for them rich, soft soil, which quickly gets warm. Pumpkins grow faster, when stems,
which start fruits, are shortened. When growing large-fruit pumpkins, it is left on
plant 3–4 started fruits (Елацкова, 2005). Fruit quality is significantly influenced by
the conditions of growing, fertilization and other factors (Paulauskienė et al., 2005).
When growing pumpkins, it is very important to select the suitable cultivars, which
genetype influences taste properties. Pumpkins suitable for using should be completely
ripen, with hard skin and, with the exception of several striped species, of uniform
external colour. The flesh of the pumpkins, which are of good quality, is brightly yellow
or orange with excellent juicy structure; there are much dry soluble solids, sugars
(1.15–14 %), starch (1.5–20 %), pectin (4.8–12.8 %) and cellular tissue (0.7–0.95 %)
in them. In the pumpkins of some cultivars there is more carotene than in carrots (up
to 16 mg 100 g -1 ) (Cantwell and Suslow, 1998; Prohens, Nuez, 2007). Because of the
401

ig amount of pigments, these vegetables are valuable and widely spread (Hazara
et al., 2007; Kidmose et al., 2006). There are these vitamins in pumpkin fruits: vitamin
C (8 mg 100 g -1 ), B1 (0.03 mg 100 g -1 ), PP (0.5 mg 100 g -1 ). It is found in pumpkin
vitamin T (0.07–0.08 mg %), which improves assimilation of nutrients. Pumpkins have
one of the biggest amounts of iron among vegetables. They help to assimilate food,
which is hard to digest, and they have little calories (Лебедева, 1989).
The flesh of overripen fruits is dryer and have more fibres. The time of pumpkin
harvesting coincides with fruit physiological maturity, the features of which are
observed visually. When the surface of skin loses its brilliance and becomes hard and
resistant to mechanical effect, the fruit is easily to pick from the stem. When the yield
is gathered too late, pumpkin fruits during storage are more injured by root diseases
(Hawthorne, 1990).
The aim of investigation is to select ecologically grown pumpkin cultivar, which
are distinguished for good nutritional properties and are suitable to process.
Object, methods and conditions. Investigations were carried out at the Lithuanian
Institute of Horticulture in the greenhouse of ecological seed-growing covered with
double polymeric film, in the natural soil – in loam on loam, more deeply epihypogleyic
luvisol (IDg 8-k, /Calc(ar)i – Epihypogleyc Luvisols – LVg-p-w-cc), enriched with
peat-compost substrate. There was grown Cucurbita pepo L. cultivar ‘Beloruskaja’
and two cultivars of Cucurbita maxima Duch. – ‘Gele Reuzen’ and ‘Bambino’.
Pumpkins were sown in the heated nursery on May 4, 2007. Into the constant
growing place in the greenhouse shoots were planted at the distances of 140 Ч 90 cm,
three plants per each replication, on May 29. The area of experimental field was
3.78 m 2 . Experiment was carried out in three replications. Pumpkins of technical
maturity were gathered on September 20. During pumpkin growing, morphological
parameters of fruits were evaluated: length, diameter, weight and the average yield.
Morphological indices of pumpkin fruits were evaluated in three stages. The first
pumpkin yield evaluation was on July 20 (fruit diameter 4.0–6.0 cm), other – on
August 20 (fruit diameter 15.0–25.0 cm) and the third one – on September 20 (fruit
diameter 20.0–30.0 cm).
In the beginning of June, 2007 weather was a little bit cooler, and this might
influence pumpkin flower formation, therefore they started flowering only in the
middle of June and started fruits in the first half of July. Ecologically grown pumpkins
were fertilized with natural fertilizer Biokal 01 and Biojodis (for three times). When
pumpkins started forming flowers, they were fertilized with Ekoplant and after a week
they were sprinkled with the solution of potassium sulfate and magnesium sulfate. At
the end of July plants were for two times sprayed with biological preparation (Nimazal
0.5 % concentration solution) from pests. Pumpkins during their growth each week
were sprinkled with water. It was weeded and hoed with manual implement for nine
times.
The quality of pumpkin cultivars ‘Beloruskaja’ ‘Gele Reuzen’ and ‘Bambino’ of
technical maturity was evaluated at the laboratory of biochemistry and technology
applying chemical and physical methods of investigations. Ascorbic acid was measured
402

y titration with 2.6- dichlorphenolindophenol sodium chloride solution (Ермаков
et al., 1987); dry soluble solids - by digital refractometer (ATAGO PR-32, Atago Co.,
Ltd., Tokyo, Japan); sugar content (inverted sugar and sucrose) – by Bertrand method
(AOAC, 1990); total amount of dry matter was established gravimetrically – by
drying fruits at the temperature of 105 °C up to unchangeable mass (Manuals, 1986);
the amounts of nitrates – potentiometrically, with ionselective electrode (Metodiniai
nurodymai, 1990). Carotenoids were extracted from fresh pumpkins by hexane, and
their amount was expressed by β-carotene equivalent. The amount of carotenoids was
established spectrophotometrically according to Scott (Scott, 2001).
Pumpkin fruit surface colour was measured with a spectrophotometer
MiniScan XE Plus (Hunter Associates Laboratory, Inc., Reston, Virginia, USA).
CIEL*a*b* colour parameters were recorded as L* (lightness), a* (+ redness),
and b* (+ yellowness). The chroma (C* = (a* 2 + b* 2 ) 1/2 ) and hue angle (h° = arctan(b*/a*))
were also calculated (McGuiere, 1992). Data were presented as the averages of the
three measurements. Colour parameters were processed with program Universal
Software V. 4–10.
Investigations were carried out in three replications. Averages of the data of
experiments and standard deviations were calculated using MS Excel program packet.
For the evaluation of data significance there was used statistical program ANOVA
(Tarakanovas, Raudonius, 2003).
Results. The investigated pumpkins are the varieties of common and large
pumpkin. Fruits of pumpkin cultivars ‘Beloruskaja’ and ‘Bambino’ are oval or roundoval,
and these of ‘Gele Reuzen’ – flat. During all the stages of harvest gathering
cultivar ‘Gele Reuzen’ produced the biggest fruits (Table 1). The evaluation of pumpkin
cultivars’ fruit morphological parameters showed that in the beginning of fruit growth
these parameters differ from each other only slightly. The differences of cultivar
morphological parameters became clear, when the intensive fruit growth took place
(from July 20 up to August 20). During this period the biggest average fruit weight
produced pumpkins of cultivar ‘Gele Reuzen’ (6.8 kg). Fruits of the ripen pumpkins
distinguished themselves with the features characteristic to the cultivar.
After evaluation of the productivity of the investigated pumpkin cultivars, it
was established that pumpkin cultivar ‘Gele Reuzen’ produced the biggest total yield
(27.6 kg m -2 ) (Table 2). Out of the investigated pumpkin cultivars, ‘Bambino’ pumpkins
distinguished themselves with the smallest total yield.
403

Table 1. Evaluation of pumpkin morphological parameters
1 lentelė. Moliūgų morfologinių požymių rodikliai
Babtai, 2007
Table 2. Estimation of ripen pumpkin yield
2 lentelė. Subrendusių moliūgų derliaus įvertinimas
Babtai, 2007
The amount of total sugar in pumpkins was small – from 3.1 up to 4.29 %.
Monosaccharides dominate among sugars. The amount of sugars in pumpkins of
cultivars ‘Bambino’ and ‘Gele Reuzen’ essentially didn’t differ. Fruits of cultivar
‘Beloruskaja’ had more sugars (Fig. 1).
The amount of dry soluble solids in pumpkin fluctuates from 3.4 up to 5.9 %. Fruits
of cultivar ‘Beloruskaja’ accumulated the biggest amount of them (5.9 %) (Fig. 2).
It was found only little amount of ascorbic acid in the investigated pumpkins – on
the average 3.33 mg 100 g -1 . More of it accumulated fruits of cultivars ‘Gele Ruzen’
and ‘Beloruskaja’ (Fig. 2). Pumpkins of these cultivars also contain more dry matter.
Essentially less amount of dry matter was established in ‘Bambino’ fruits (Fig. 2).
404

Fig. 1. Sugars content of pumpkin fruits
1 pav. Cukrų kiekis moliūguose
Fig. 2. Change of biochemical composition in pumpkin fruits
2 pav. Moliūgų vaisių biocheminė sudėtis
There were established essential differences among cultivars according to the
accumulated amount of nitrates. During the years of investigations fruits of cultivar
‘Gele Reuzen’ had the least amount of nitrates (105 mg kg -1 ), more of them it was
established in ‘Bambino’ pumpkins (636 mg kg -1 ) (Fig. 3).
Pumpkins of the analysed cultivars essentially differ from each other by the amount
of accumulated carotenoids (Fig. 4). ‘Gele Reuzen’ fruits have very little amount of
pigments – 1.7 mg 100 g -1 . But ‘Beloruskaja’ pumpkins accumulate 3.5 times more
carotenoids and also exceed cultivar ‘Bambino’ by the amount of pigments.
405

Fig. 3. Nitrates content in pumpkin fruits
3 pav. Nitratų kiekis moliūguose
Fig. 4. Carotenoids content in pumpkin fruits
4 pav. Karotinoidų kiekis moliūguose
Carotenoid concentration in pumpkin flesh essentially determined the parameter
of their lightness index L*, which changed from 63.43 (‘Beloruskaja’) up to 71.0
(‘Bambino’) (Table 3).
Table 3. Colour parameters in pumpkins fruits
3 lentelė. Moliūgų vaisių spalvos rodikliai
The parameters of ‘Gele Ruzen’ and ‘Beloruskaja’ fruit flesh redness coordinate a*
essentially do not differ. The flesh of ‘Bambino’ has more expressed red colour and
the meaning of its parameter of yellowness coordinate b* is essentially the least
one (54.92). Though there are essential differences between the meanings of ‘Gele
Ruzen’ and ‘Beloruskaja’ pumpkins’ coordinate b*, the meanings of yellowness of
406

the mentioned cultivars in the space of colours are similar.
‘Gele Ruzen’ fruits essentially distinguished themselves with chroma (C*) (69.1).
There were established essential differences among cultivars’ hue (h°), which is shown
by the ratio between colour coordinates a* and b*. ‘Bambino’ fruits have the smaller
ratio of redness and yellowness and ‘Gele Ruzen’ fruits – the bigger one.
Discussion. Pumpkin fruit external quality, size, colour depends on cultivar’s
genetic nature, soil, fertilization, etc. Pumpkin cultivar ‘Beloruskaja’ distinguishes
itself with oval or round-oval fruits, which ripe weight about 6–8 kg and the total
yield per ha reaches about 50 t ha -1 (Хлебородов et al., 2005). Our investigations
showed that when growing pumpkin cultivars ecologically it is possible to obtain
qualitative yield. After the evaluation of morphological parameters of pumpkin fruits
in different pumpkin growth stages, it was established that in the beginning of fruit
growing pumpkin cultivars do not differ. During intensive fruit growing they obtain
the features, characteristic to that cultivar. Ripen pumpkins distinguished themselves
with the features characteristic to that cultivar and qualitative parameters of the fruit.
This shows our and other investigators’ data (Hazara et al., 2007). Cultivar’s genetype
very influences pumpkin taste properties. Pumpkin flesh colour intensity depends on
the amount of accumulated carotenoids, and the amounts of sugars and dry soluble
solids are among fruit maturity parameters, which strongly correlate among themselves
and influence pumpkin taste properties (Daniel et al., 1995; Harvey et al., 1997).
Pumpkin fruits, grown as usually, ripen accumulate from 6.48 up to 20.63 mg 100 g -1
of ascorbic acid (Danilčenko et al., 2003). Pumpkins of cultivar ‘Beloruskaja’ grown
in 2007 distinguished themselves with the parameters of chemical composition. In the
flesh of its fruits it was established the biggest amount of sugars (4.29 %), dry soluble
solids (5.9 %), and carotenoids (5.94 mg 100 g -1 ). The bigger pigments’ concentration
determined the exceptionality of the flesh colour parameters of this cultivar. Fruit flesh
of cultivar ‘Beloruskaja’ was of brightest orange colour.
Dependently on the climate conditions and growing technologies, fruits of
cultivar ‘Bambino’ grown in Lithuania accumulate up to 20.63 mg 100 g -1 of ascorbic
acid and 7.98–9.94 % of dry matter (Danilčenko et al., 2003). During the years of
investigation the fruits of this cultivar weren’t vitaminous and accumulated less dry
matter. Comparing with the cultivars grown in 2007, the parameters of ‘Bambino’
pumpkin chemical composition, with the exception of the accumulated amount of
pigments, essentially were worse. Our data confirms the results of other investigators
that there is found only a small amount of carotenoids (3.45 mg 100 g -1 ) in the flesh
of the mentioned cultivar. It is necessary to mention that during the cultivars’ flesh
colour analysis it was established the exceptionality of ‘Bambino’ pumpkin colour.
The parameters of coordinate a* of ‘Bambino’ flesh redness in the space of colours
are more in the red zone. Though there was found less carotenoids in the flesh of this
cultivar in comparison with ‘Beloruskaja’ fruits, we think that there may be more red
b-carotene in the composition of ‘Bambino’ pumpkin carotenoids. In human organism
this carotene directly participate in the process of the formation of vitamin A molecules
(Kidmose et al., 2006).
Analysing the parameters of pumpkin cultivar colour quality it was established
that the parameters of flesh colourness coordinate a*, which influenced the changes
407

of hue (h°), fluctuated more.
Conclusions. 1. Pumpkins of cultivar ‘Gele Reuzen’ distinguished themselves
with oval form, produced on the average 8.4 kg of marketable fruits, were productive
(27.6 kg m -2 ) and accumulated little amount of nitrates (105 mg kg -1 ).
2. Ripe pumpkins of cultivar ‘Beloruskaja’ essentially distinguished
themselves with the parameters of chemical composition. There was found in their
flesh more dry soluble solids (5.9 %), sugars (4.29 %), ascorbic acid (4 mg 100 g -1 ), and
carotenoids (5.94 mg 100 g -1 ). The amount of the accumulated nitrates (341 mg kg -1 )
was less than the average one (361 mg kg -1 ).
3. Out of the colour parameters, the parameters of flesh colourness coordinate a*,
which influenced the changes of hue (h°), fluctuated more.
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Gauta 2008 04 17
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SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Skirtingų ekologiškai auginamų moliūgų veislių augimas, derėjimas
ir kokybė
R. Karklelienė, P. Viškelis, M. Rubinskienė
Santrauka
Tyrimai vykdyti Lietuvos sodininkystės ir daržininkystės instituto ekologinės sėklininkystės
dviguba polimerine plėvele dengtame šiltnamyje, natūraliame dirvožemyje – limnoglacialiniame
priemolyje ant moreninio priemolio, giliau glėjiškame išplautžemyje (IDg 8-k, /Calc(ar)
i – Epihypogleyc Luvisols – LVg-p-w-cc), papildytame durpių-komposto substratu. Auginta
paprastojo moliūgo (Cucurbita pepo L.) veislė ‘Beloruskaja’ ir didžiojo moliūgo (Cucurbita
maxima Duch.) dvi veislės ‘Gele Reuzen’ ir ‘Bambino’.
Moliūgų vaisių morfologiniai rodikliai tirti trimis etapais. Pagrindinis moliūgų vaisių
augimas buvo rugpjūčio mėnesį, kai jie jau buvo suformavę veislei būdingus vaisius. Rugsėjo
mėnesį vaisiuose vyko brendimo procesai, todėl vaisių svoris nedaug skyrėsi nuo antrojo moliūgų
vaisių svėrimo. Tyrimai parodė, kad ‘Gele Reuzen’ veislės moliūgai suformuoja vidutiniškai
nuo 8,0 iki 8,7 kg vaisius.
Moliūgai sukaupia vidutiniškai 4,73 % tirpių sausųjų medžiagų. Didesniu jų kiekiu išsiskyrė
‘Beloruskaja’ veislė. Cukrų koncentracija moliūguose kito nuo 3,1 % iki 4,29 %. Mažiausiai
409

cukrų nustatyta ‘Bambino’, daugiausiai – ‘Beloruskaja’ veislės moliūguose. Šios daržovės
sukaupia mažа askorbo rūgšties kiekį – vidutiniškai 3,33 mg 100 g -1 . priklausomai nuo veislės,
sukauptų nitratų kiekiai moliūguose kinta plačiose ribose – nuo 105 (‘Gele Reuzen’) iki 636
(‘Bambino’) mg kg -1 . Karotinoidų kiekis buvo tiriamas valgomoje moliūgų dalyje. Daugiau jų
nustatyta ‘Beloruskaja’ (5,94 mg 100 g -1 ) veislėje.
Reikšminiai žodžiai: augimas, cheminė sudėtis, derlius, moliūgai, morfologiniai rodikliai,
spalvos parametrai, veislės.
410

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Effect of abiotic factors on risk of Venturia inaequalis
infection depending on apple tree growth stages
Laimutis Raudonis, Alma Valiuškaitė, Elena Survilienė
Lithuanian Institute of Horticulture, Kauno 30, LT-54333 Babtai, Kaunas distr.,
Lithuania, e-mail: l.raudonis@lsdi.lt
The influence of abiotic factors on risk of apple scab infection depending on fruit tree
growth stages was studied in 2006–2007. Scab warning equipment Metos D in 2006 and Internet
based system iMETOS in 2007 were used for prediction of infection risks of apple scab. Long
duration of leaf wetness, depending on air temperature induced light, medium and strong scab
conidia infection. Conidia infections start to occur at 69 (end of flowering) growth stage, when
leaves, young shoots and other parts of apple tree are developed. Peak of conidia infections
occurs from 71 (fruit size up to 10 mm) and it lasts till beginning of 74 (fruit diameter up to
40 mm) or 75 (fruit about half final size) growth stages. The next peak of infections starts from
the end of 75 or the beginning of 77 (fruit about 70 % of final size) growth stages and it lasts till
the end of the season. There are no conidia infections from 69 till 71 growth stages and from
the beginning of 74 or 75 till the end of 75 or the beginning of 77 growth stages.
Key words: air temperature, conidia infection, growth stage, leaf wetness, relative
humidity, scab.
Introduction. Venturia inaequalis causal agent of apple scab is the most important
disease of apple and its control depends almost exclusively on frequent use of
fungicides. 75 % of the pesticide use in apple production is related to control of fungal
diseases, in which apple scab has a share of 70 % (Creemers, Laer, 2006). European
agriculture faced the strict demands to decrease the use of pesticides in order to reduce
any human or environmental hazards; therefore, the need for evaluation and reduction
of the use of pesticides is expressed. To obtain the reduction of the use of fungicides
without any significant damage to the crop, the control of apple scab should be based
on registration of climatic data, scouting of biotic parameters, infection risks and
simulation disease models. The bioecology of V. inaequalis has been widely studied in
many countries. The development of infection risks of apple scab, highly depends on
apple cultivar susceptibility, inoculum of the pathogen in orchards, growth stage of the
tree, ascospore release and the influence of parameters as air temperature, duration of
leaf wetness, relative humidity or light (Gadoury et al., 1998; Stensvand et al., 1998;
Li, Xu, 2002; Rossi et al., 2003; Holb et al., 2004; Holb et al., 2005; Carisse et al.,
2007). Based on air temperature duration of leaf wetness and other parameters apple
scab models have been developed, evaluated and recently successfully used for the
disease control in apple orchards. Met stations equipped with sensors for registration
and transmission of data about temperature, relative humidity, rainfall, leaf wetness
411

and others for prediction of apple scab infection risks (Berrie, 1994; Butt, Xu, 1994;
Bьhler, Gesle, 1994; Rossi et al., 1999; Raudonis, 2002; Raudonis et al., 2003; Holb
et al., 2003; Xu, Robinson, 2005; Atlamaz et al., 2007; Rossi, Bugiani, 2007). However,
little is known about effect of abiotic factors on risk of apple scab infection depending
on fruit tree growth stages.
Therefore, the objective of this study was to evaluate the influence of abiotic
factors on risk of apple scab infection depending on fruit tree growth stages.
Object, methods and conditions. Scab warning equipment Metos D in 2006 and
Internet based system iMETOS in 2007 (G. Pessl, Austria) were used for the record
of meteorological data and apple scab infection periods.
Apple scab infection periods were measured at different growth stages according
to BBCH scale (Meier, 1997). Abbreviations for tree growth stages given above
are 69 = end of flowering: all petals fallen; 71 = fruit size up to 10 mm, fruit fall
after flowering; 73 = second fruit fall; 74 = fruit diameter up to 40 mm, fruit erect
(T-stage, underside of fruit and stalk forming – T); 75 = fruit about half final size;
77 = fruit about 70 % of final size; 81 = beginning of ripening, lightening of cultivar
specific fruit color; 85 = advanced ripening, increase in intensity of cultivar specific
fruit color; 86 = fruit ripe for picking and 89 = fruit ripe for consumption.
The field trials were carried out in the apple tree orchard at the Lithuanian Institute
of Horticulture in 2006–2007. The apple trees of variety ‘Conel Red’ were planted in
1995; 1250 seedlings per hectare. The soil was fertilized with N – 70 kg ha -1 before
flowering.
Assessments of scabbed leaves were made after primary and secondary infection
periods as follows: on VI.21 and VIII.18 in 2006 and on VII.09 and VIII.06 in 2007.
There were 200 leaves to assess the incidence and intensity of apple scab on leaves in
each plot. 100 fruits in each plot were assessed at harvest on X.09 and IX.27 in 2006
and 2007 and percentage of fruits injured by apple scab was recorded.
Incidence of apple scab on leaves and fruits was calculated: P = a × 100/N;
P – disease incidence; a – number of scabbed leaves; N – total assessed leaves. Intensity
of apple scab on leaves and fruits was calculated: R = Sa × b × 100/NK; R – disease
intensity, a – the number of leaves or fruits scabbed the same level, Sa × b – the sum
of scabbed leaves or fruits assessed by the same score, N – total assessed leaves or
fruits, K – the highest score of the scale (5).
Scab incidence and intensity on leaves and fruits was compared among treatments
in this study with a single factor analysis of variance (ANOVA). Specific differences
were identified with Duncan’s multiple range test.
Results. Mean data of air temperature, relative humidity and duration of leaf
wetness per day in 2006 and 2007 are presented in Fig. 1 and 2. In 2006 the mean
air temperature on average ranged from 15 to 20 °C at the moment of 69–71 growth
stages (end of flowering – fruit size up to 10 mm) (Fig. 1). Relative humidity was high
(75–100 %). Duration of leaf wetness was high just after flowering. Later duration of
leaf wetness was short or leaves were dry. Though air temperature was comparatively
lower, but high relative humidity and long duration of leaf wetness resulted light,
medium and even strong conidia infection of scab at 69 growth stage of apple tree
(Fig. 3). Later, till 71 growth stage no conidia infections were observed. Mean air
412

temperature increased and relative humidity dropped between 65–75 % after 71 till
the beginning of 74 (fruit diameter up to 40 mm) growth stages. Duration of leaf
wetness increased and this resulted light or medium conidia infection during 71–74
growth stages. No leaf wetness occurred from 74 till the end of 75 (fruit about half
final size) growth stages and no conidia infection was recorded during this period for
18 days. After 75 growth stage till the 86 (fruit ripe for picking) mean air temperature
started gradually decrease, meanwhile relative humidity grew up. The duration of leaf
wetness was highest and often lasted more than 20 hours per day during this period.
Long duration of leaf wetness, though air temperature started slightly decrease, often
resulted light, medium and strong scab conidia infections from the end of 75 growth
stage till the end of the season in 2006 (Fig. 3).
Similar patterns were recorded in 2007. Strong conidia infection was induced by
long duration of leaf wetness at 69 growth stage (Fig. 2, 4). Later, approximately for
two weeks, till 71 growth stage no high duration of leaf wetness and conidia infection
was observed (Fig. 2). After 71 till the beginning of 75 growth stages the mean air
temperature varied between 15–20 °C and relative humidity started increase. The
duration of leaf wetness started to grow up and often reached 20 hours per day. Apple
scab conidia infections were often and strong during these grow stages (Fig. 4). No
leaf wetness occurred from the beginning of 74 till the beginning of 77 (fruit about
70 % of final size) growth stages and no conidia infection was recorded during this
period for near 2 weeks. After the beginning of 77 growth stage till 86 and later the
duration of leaf wetness per day was long and it often resulted light, medium and
strong scab conidia infections. Except, there was no conidia infection before and
during 85 (advanced ripening, increase in intensity of cultivar specific fruit color)
growth stage in 2007.
Scab conidia infection periods resulted in direct damage of leaves and fruits of
apple tree (Table). More often and strong conidia infections were recorded, particularly
between 71 and the beginning of 75 growth stages in 2007 comparing with 2006.
The scab incidence and intensity on leaves was 64–85 % and 34–49 % in 2007,
respectively. The incidence and intensity of scab on fruits was 84.7 and 45.7 %. There
were statistically less damaged leaves and fruits by apple scab in 2006.
Table. An incidence and intensity of apple scab
Lentelė. Obelų rauplių paplitimas ir intensyvumas
Babtai, 2006–2007
413

Fig. 1. Dynamics of meteorological parameters influencing
scab development, 2006
1 pav. Meteorologinių parametrų įtakojančių rauplių
plitimą dinamika. 2006 m.
Fig. 2. Dynamics of meteorological parameters influencing
scab development, 2007
2 pav. Meteorologinių parametrų įtakojančių rauplių
plitimą dinamika. 2007 m.
414

Fig. 3. Scab infection periods and intensity at different apple growth
stages in 2006
3 pav. Obelų rauplių infekcija ir intensyvumas skirtinguose obelų
augimo tarpsniuose. 2006 m.
Fig. 4. Scab infection periods and intensity at different apple growth
stages in 2007
4 pav. Obelų rauplių infekcija ir intensyvumas skirtinguose obelų
augimo tarpsniuose. 2007 m.
415

Discussion. Air temperature, relative humidity and leaf wetness are one of factor
the most influencing development of apple scab infections (Gadoury et al., 1998;
Hartman et al., 1999; Aylor, Qiu, 1996; Rossi, Bugiani, 2007; Laer, Creemers, 2004;
Hamada, 2005; FRöhling et al., 2005; Xu, Robinson, 2005). Refereeing these abiotic
factors scab warning systems for prediction of ascospores or conidia infection and scab
control are developed (Berrie, 1994; Bьhler, Gesle, 1994; Butt, Xu, 1994; Raudonis,
2002; Raudonis et al., 2003; Atlamaz et al., 2007). Seasonal patterns of scab conidia
infections in our investigations are in accordance with other studies on V. inaequalis.
Depending on air temperature, relative humidity and leaf wetness the conidia infections
start to occur at 69 (end of flowering) growth stage, when leaves, young shoots and
other parts of apple tree are developed. No fruits are developed during this stage, but
young leaves can be easy infected and later it could be as infection source for small
fruits. Depending on growth stage the susceptibility of apple tree to scab infection is
very different. Some authors had stated that only newly emerged leaves of apple tree
are susceptible to scab infection by ascospores or conidia (Sanogo, Aylor, 1997; Li,
Xu, 2002; Holb et al., 2004; Holb et al., 2005). The same patterns of scab infection
severity depending maturity of fruits were observed (Xu, Robinson, 2005). Therefore,
apple tree have to be controlled against scab at 69 growth stage. There were no conidia
infections from 69 till 71 growth stages and fungicide sprays could be omitted. Peak
of conidia infections occur from 71 and it lasts till the beginning of 74 or 75 growth
stages. An intensive scab control system using protective and curative fungicides
should be used, especially when small fruits start to develop during this period. No
infections were observed from the beginning of 74 or 75 till the end of 75 or the
beginning of 77 growth stages. The next peak of infections starts from the end of 75
or the beginning of 77 growth stages and it lasts till the end of the season. Control
system applying curative or protective fungicides depending on severity and duration
of conidia infection should be applied.
Conclusions. 1. Depending on air temperature, relative humidity and leaf wetness
the conidia infections start to occur at 69 (end of flowering) growth stage, when leaves,
young shoots and other parts of apple tree are developed. Peak of conidia infections
occur from 71 and it lasts till beginning of 74 or 75 growth stages. The next peak of
infections starts from the end of 75 or the beginning of 77 growth stages and it lasts
till the end of the season.
2. There are no conidia infections from 69 till 71 growth stages and from the
beginning of 74 or 75 till the end of 75 or the beginning of 77 growth stages.
References
Gauta 2008 04 23
Parengta spausdinti 2008 04 30
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416

3. Berrie A. 1994. Practical experiences with the Ventem TM system for managed
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17. Meier U. 1997. Growth stages of Mono- and Dicotyledonous plants. Berlin.
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19. Raudonis L., Valiuškaitė A., Rašinskienė A., Survilienė E. 2003. Pests and disease
management model of apple-trees according to warning equipment. Sodininkystė
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SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Abiotinių faktorių įtaka Venturia inaequalis infekcijos rizikai
priklausomai nuo obelų augimo tarpsnių
L. Raudonis, A. Valiuрkaitė, E. Survilienė
Santrauka
Abiotinių faktorių įtaka obelų rauplių infekcijos rizikai priklausomai nuo obelų augimo
tarpsnių buvo tirti 2006–2007 metais. Rauplių prognozavimo prietaisas Metos D 2006 m. ir
internetinė sistema iMETOS 2007 m. buvo panaudoti prognozuojant obelų rauplių infekcijos
riziką. Ilgai trunkanti lapų drėgmė, priklausomai nuo oro temperatūros sukelia silpną, vidutinę
ir stiprią rauplių konidijų infekciją. Konidijų infekcija prasideda 69 augimo tarpsnyje (žydėjimo
pabaiga), kai lapai, jauni ūgliai ir kitos dalys vystosi. Konidijų infekcijos maksimumas nustatytas
nuo 71 augimo tarpsnio (vaisius padidėja iki 10 mm) ir tęsias iki 74 pradžios (vaisiaus diametras
padidėja iki 40 mm) arba 75 augimo tarpsnio (vaisius pasiekia pusę būdingo dydžio). Kitas
infekcijos pikas prasideda nuo 75 augimo tarpsnio pabaigos arba 77 pradžios (vaisius pasiekia
apie 70 % būdingo dydžio) ir tęsias iki sezono pabaigos. Nenustatyta konidijų infekcija nuo
69 augimo tarpsnio iki 71 ir nuo 74 augimo tarpsnio pradžios iki 75 pabaigos arba 77 augimo
tarpsnio.
Reikšminiai žodžiai: augimo tarpsnis, konidijų infekcija, lapų drėgnumas, oro temperatūra,
rauplės, santykinė drėgmė.
418

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Biological control of potato against
Rhizoctonia solani (Kühn)
Halina Kurzawińska, Stanisław Mazur
University of Agriculture, Department of Plant Protection, 31-425 Krakow,
Al. 29 Listopada 54, Poland, e-mail: hkurzaw@ogr.ar.krakow.pl,
smazur@ogr.ar.krakow.pl
The aim of three years field investigations was to evaluate the effect of bio-preparations
Polyversum (B.A.S. Pythium oligandrum) and Biochikol 020 PC (B. A. S. chitosan) applied
on infected tubers by Rhizoctonia solani sclerots during vegetation period. As a standard
fungicide Vitavax 2000 FS (B.A.S. karboxin and thiuram) was used. The effect of the mentioned
preparations applied at three different concentrations on R. solani mycelium linear growth
was investigated under in vitro conditions. The examinations were made using Kowalik and
Krechniak method (1961). During potato vegetation period all applied preparations affected
both the lower tubers’ infestation degree by R. solani sclerots and the lower tuber infestation
percent by these pathogens.
According to the results obtained from conducted in vitro examinations, it was found
that Polyversum preparation, chemical preparation (Vitavax 2000 FS) and Biochikol 020 PC
but applied only at the highest concentration (2.0 %) significantly reduced mycelium linear
growth of R. solani.
Key words: Rhizoctonia solani, Pythium oligandrum, chitosan, karboxin and thiuram
bioprotection, potato.
Introduction. In Poland potato belongs to economically important cultures. This
plant is infected by many agrophags. Fungi Rhizoctonia solani, which causes black
scurf of potato tubers, belong to commonly appearing potato pathogens. Sclerots of
the mentioned fungi occurring on sets can be the source of infection for plants and
descendant tubers. Moreover, it make their quality worsen (Ahrenniemi et al., 2005).
Potato yield losses caused by this disease amounted even to 50 % (Hдni et al., 1998).
Besides, rhizoctoniosis constitutes distinct aesthetic defect, which decreases market
value of potatoes intendent for consumer purpose or for food industry (Lutomirska,
2007).
R. solani is soil born pathogens, which affects sets and contributes not only to yield
losses but to fungi accumulation in soil (Bogucka, 1993). According to this author,
interference with chemical preparations by sets dressing is still in use because of lack of
discussed disease-resistant varieties. However, increasing environmental contamination
through use pesticides among others things incline us for using alternative methods to
fight with plant pathogens. In modern plant protection an interest of biological methods,
which consist of replacing pesticides with bio-preparations based on plant’s extracts,
organic compounds and antagonistic microorganisms to pathogens, is increasing. To
419

the mentioned antagonistic organisms belongs myco-parasite Pythium oligandrum,
biologically active substance of bio-preparation Polyversum. Moreover, there are
high possibilities of Biochikol 020 PC (B. A. S. chitosan) application in protection of
many plants against diseases, especially as plant immunity inducer (Bell et al., 1998;
Orlikowski et al., 2002, Pięta et al., 2006).
Therefore, the objective of the presented study conducted as field experiment
was the test of reduction of R. solani occurrence by application of bio-preparation
Polyversum (contained Pythium oligandrum oospors) and Biochikol 020 PC (B. A. S.
chitosan). As standard fungicide Vitavax 2000 FS (B. A. S. karboxin and thiuram) was
used. Besides, the effect of mentioned bio-preparations on the R. solani mycelium
linear growth was investigated under in vitro conditions.
Object, methods and conditions. Field experiment was conducted at the
Experimental Station at Mydlniki near Kraków owned by the Department of Plant
Protection Academy of Agriculture, on brown soil, in 2005–2007. The winter wheat
was the forecrop. During the investigations potatoes of mid-early cv. ‘Ibis’ cultivated
according to recommendation of proper agro-technique was used. The experiment
was established in third decade of April with the method of random squares in four
replications (100 tubers on each plot) and in following combinations: 1 – control –
plants derived from tubers without any protection treatment; 2 – plants derived from
tubers dressed with Biochikol 020 PC (B. A. S. chitosan) at concentration of 2.5 %;
3 – dressed tubers + 4 times plants spraying with Biochikol 020 PC at concentration
of 2.5 %; 4 – plants derived from tubers dressed with Polyversym (B. A. S. Pythium
oligandrum) in dose 10 g/kg tubers; 5 – dressed tubers + 4 times plants spraying with
Polyversum at concentration of 0.05 %; 6 – plants derived from tubers dressed with
Vitavax 2000 FS (B. A. S. karboxin and thiuram) in dose 5 ml/kg tubers.
After harvesting 100 tubers from each plot were randomly chosen, collected and
put in storage (temperature 6–7 °C, relative air humidity 80–85 %). The analysis of
degree and percent of tubers infested by R. solani sclerots were directly made after
harvesting (in September) and later every 3 months (in December and March) during
the storage period. The degree of infestation by above mentioned pathogens was defined
according to following scale (Kapsa et al., 1998): 0 – lack of symptoms, 1 – disease
symptoms founded on 5–25 % of tubers’ surface, 2 – disease symptoms on 26–50 %
of tubers’ surface, 3 – disease symptoms appearing on 51–75 % of tubers’ surface, 4 –
disease symptoms including over 75 % of surface. Received results were subjected to
statistical analysis using analysis of variance. The multiple t-Duncan test was used to
estimate the differences between mean values at significance level α = 0.05.
The Kowalik and Krechniak (1961) method was used to the in vitro studies.
R. solani was isolated from potato tubers. Each of examined preparations were applied
at three different concentrations: Biochikol 020 PC at concentrations: 0.5 %, 1.0 %,
2.0 %; Polyversum at concentrations: 0.05 %, 0.1 %, 0.2 %; Vitavax 2000 FS in doses:
0.025 %, 0.05 %, 0.1 %.
After pouring PDA agar to Petri dishes and after its silidified, the agar disk
with mycelium of 14-days old culture of examined fungi was placed centrally into
each dish. The control constituted Petrie dishes with medium PDA and agar disk
with R. solani mycelium but without amendments. The test was carried out in five
repetitions for each combination (10 Petri dishes for 1 repetition). Measurements
420

were started when first increase of mycelium in control dishes was observed and were
conducted until the complete overgrowth of mentioned dishes was noted down. The
crosswise measurement of colony diameter was made, and then the arithmetic mean
for repetitions was calculated. The percent of inhibition of mycelium linear growth
on medium with amendments of appropriate preparation compared to the growth of
fungi on control Petri dish was used to measure the preparations activity (Kowalik
and Krechniak, 1961). Received results were treated statistically using the analysis of
variance followed by Duncan’s test (α = 0.05).
Results. According to the received results during three years of investigations the
favourable effect of both sets dressing and potato plant spraying with tested preparations
on the reduction of black scurf on tubers was observed (Table).
Table. The effect of bio-preparations on potato tuber Rhizoctonia solani, 2005–
2007
Lentelė. Biopreparatų poveikis bulvių stiebagumbių Rhizoctonia solani, 2005–2007 m.
Application of chemical standard preparation Vitavax 2000 FS to sets dressing had the
best influence on both inhibition of tubers’ infestation degree and tubers’ infestation
percent by R. solani sclerots during the storage period. In all terms of analysis of tubers
421

from this combination, the percent of tubers’ infestation by R. solani sclerots was the
lowest and amounted from 1.0 (September) to 1.2 (March) – Table. Also the percent
of tubers infestation by discussed pathogen was the lowest and reached from 11.1 %
(September) to 16.8 in March (Table).
In comparison to the control Polyversum and Biochikol 020 PC preparations
significantly reduced R. solani growth on stored tubers of tested potato variety in all
terms of analysis (Table). Statistical calculations showed the important influence of
all preparations under consideration on the inhibition of both degree and percent of
infested tubers by R. solani sclerots (Table).
Figure represents the percent of R. solani mycelium linear growth inhibition.
Among examined preparations Polyversum applied at all concentrations (0.05 %,
0.1 %, 0.2 %) the most limited R. solani mycelium linear growth. The percent of
inhibition appropriately came to 89.5 %, 91.1 %, 92.0 % (Fig.). Also in combination
where Vitavax 2000 FS was used in all tested doses the important inhibition of abovementioned
pathogen mycelium linear growth was found but the best inhibition was
noted down at the highest concentration of this preparation, which is 0.1 % (87.5 %) –
Fig. The weakest in vitro effect of reduction R. solani mycellium linear growth was
found in combinations where Biochikol 020 PC was used. Mentioned preparation
limited R. solani mycelium linear growth applied only at the highest concentration
(2.0 %), whereas in doses 0.5 % and 1.0 % there was not any statistically important
effect of Biochikol 020 PC application on the discussed pathogen mycelium linear
growth (Fig.).
Fig. Percent of inhibition of Rhizoctonia solani growth
Pav. Rhizoctonia solani augimo inhibicijos procentas
According to statistical calculations, the significant influence (in comparison to the
control) of preparations under examination (with the exception of Biochikol 020 PC
422

at concentration 0.5 % and 1.0 %) on the percent of R. solani mycelium linear growth
inhibition was noted down.
Discussion. Applied preparations limited the black scurf of tubers of examined
potato variety. Investigations conducted by Gajda and Kurzawińska (2004) confirmed
favourable effect of sets dressing as well as Polyversum bio-preparation and chemical
preparation on the inhibition of R. solani sclerots occurrence on potato tubers. Similar
results were obtained by Bernart and Osowski (2006). Findings from conducted
experiments showed the possibility of the use of both preparations Polyversum and
Biochikol 020 PC in protection of potatoes against R. solani. Previous test data
demonstrated that application of Polyversum bio-preparation to sets dressing limited
contamination of descended tubers by R. solani sclerots. Under in vitro conditions
above-mentioned preparation showed strong antifungal activity in relation to R. solani
(Gajda, Kurzawińska, 2004). It is believed that the application of micro-biological
material on the seeds (tubers) surface is the most effective method of prevention through
infestation. According to Martin and Hancock (1987), Pythium oligandrum colonizes
the ecological niche in the soil and successfully competes with plant pathogens. The
authors showed that Pythium oligandrum have an influence on pathogenic factors.
It resulted that the great number of pathogenic factors in the soil, on seeds, tubers,
bulbs and rootstocks may decrease to the degree when the significant increase of
plant health is noted down. According to Deacon (1991), Pythium oligandrum
demonstrates destructive and parasitic character to many pathogenic fungi. Bell
et al. (1998), Kurzawińska (2007), Nawrocki and Mazur (2007), Pięta et al. (2006)
confirmed efficiency of chitosan in vegetable protection. The results of investigations
conducted by above-mentioned authors showed that efficiency of protective chitosan
activity applied in plant prevention is much better than applied in plant intervention.
Effective influence of chitosan on phyto-pathogens depends on its concentration and
the virulent of infectious factor. Followed to Poъpieszny (1997), chitosan induces
immunity of many plants through cell wall lignification, phytoalexins production and
synthesis of proteinase inhibitors. The treatment of the cells with chitosan stimulates
their intensification by production of additional structures and accumulation of phenol
substances, which are harmful for fungi. Chitosan applied together with fungicides
may complete their activity reducing the doses and fungi immunization against them
(Pośpieszny, 1997). Among the tested preparations under in vitro conditions biopreparation
Polyversum the most limited R. solani mycelium linear growth. Chemical
preparation Vitavax 2000 FS showed slightly weak effect. The least inhibition of
R. solani mycelium linear growth was found in combinations where Biochikol 020 PC
was used. Followed to Orlikowski et al. (2002) most of studies on mechanism of
chitosan activity to pathogenic fungi showed that mentioned substance do not inhibit
the mycelium growth and spores’ germination under in vitro conditions. However,
according to Pośpieszny (1997), under in vitro conditions chitosan inhibits growth of
several fungi and microbes but not all of them. Exploitation of preparations based on
natural substances, which can limit plant pathogens development comes into higher and
higher prominence, especially restricting traditional chemical preparation application.
It results from comparable efficiency of bio-preparations to pesticides.
423

Conclusions. 1. Polyversum and Biochikol 020 PC bio-preparations significantly
(in comparison with control) reduced degree and percent of tuber infestation by
R. solani.
2. Application of chemical standard preparation karboxin and thiuram mixture to
sets dressing had the best influence on inhibition of tubers infestation by R. solani.
3. Among all the tested preparations under in vitro conditions the most effective
in reduction R. solani mycelium linear growth turned out to be Polyversum biopreparation.
In vitro response of the tested pathogen depended on the type of preparation
and its concentration.
Acknowledgements. The studies were financed by the Ministry of Science and
Information within grant No. PO6R 00129.
References
Gauta 2008 03 31
Parengta spausdinti 200805 05
1. Ahrenniemi P. M., Lehtonen M. J., Wilson P. S., Valkonen J. P. T. 2005. Influence of
flarming system and black scurf infestation level of seed tubers on stem canker and
bleach scurf (Rhizoctonia solani) of potato. Abstracts of 16 th Triennial Conference
EAPR Bilbao, 17–22.07: 335–338.
2. Bell A. A., Hubbard C. J., Liu L., Davis M. R., Subbarao V. K. 1998. Effects of
chitin and chitosan on the incidence and severity of Fusarium yellows of celery.
Plant Disease. 82: 322–328.
3. Bernat E., Osowski J. 2006. Wpływ różnych terminów zaprawiania na
tempo wschodów i zdrowotność ziemniaka. Progress in Plant Protection.
46(1): 401–408.
4. Bogucka H. 1993. Rizoktonioza ziemniaka. Ziemniak Polski. 2: 18–20.
5. Deacon J. W. 1991. Significance of ecology in the development of biocontrol
agents against soil-borne plant pathogens. Biocontrol Science and Technology.
1: 5–20.
6. Gajda I., Kurzawińska H. 2004. Potential use of Pythium oligandrum and
imazalil in the protection of potato against Rhizoctonia solani. Phytopathol. Pol.
33: 47–52.
7. Häni F., Popow G., Reinhard A. 1998. Ochrona roślin rolniczych w uprawie
integrowanej. PWRiL Warszawa.
8. Kapsa J., Lewosz W., Osowski J., Gawińska H. 1998. Parch srebrzysty
(Helminthosporium solani) i jego występowanie w Polsce. [W]: Ochrona
ziemniaka, Konferencja, Kołobrzeg 21–22 kwietnia, IHAR – Oddział w Boninie,
21–24.
9. Kowalik R., Krechniak E. 1961. Szczegółowa metodyka biologicznych
laboratoryjnych badań środków grzybobójczych. Biul. Inst. Ochr. Roślin Poznań,
63–66.
424

10. Kurzawińska H. 2007. Potential use of chitosan in the control of lettuce pathogens.
Progress on Chemistry and Application of Chitin and its Derivatives Monograph
XII: 173–178.
11. Lutomirska B. 2007. Wpływ odmiany i czynników meteorologicznych okresu
wegetacji na ospowatość bulw ziemniaka. Progress in Plant Protection. 47(2): 173–
177.
12. Martin F. N., Hancock J. G. 1987. The use of Pythium oligandrum for biological
control of preemergence damping-off caused by P. ultimum. Phytopathology.
77: 1 013–1 020.
13. Nawrocki J., Mazur S. 2007. Effectiveness of Biochikol 020 PC in the control of
carrot and parsley pathogens. Progress on Chemistry and Application of Chitin
and its Derivatives Monograph XII: 211–215.
14. Orlikowski L. B., Skrzypczak Cz., Wojdyła A., Jaworska-Marosz A. 2002. Wyciągi
roślinne i mikroorganizmy w ochronie roślin przed chorobami. Zesz. Nauk. AR
Kraków. 387(82): 19–32.
15. Pięta D., Patkowska E., Pastucha A. 2006. Influence of Biochikol 020 PC used as
seed dressing of bean on healthiness and yield of plants. Progress on Chemistry
and Application of Chitin and its Derivatives Monograph XI: 159–170.
16. Pośpieszny H. 1997. Niektóre aspekty stosowania chitozanu w ochronie roślin.
Progress in Plant Protection. 37/1: 306–309.
SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Bulvių biologinė kontrolė prieš Rhizoctonia solani (Kühn)
H. Kurzawińska, S. Mazur
Santrauka
Lauko tyrimai atlikti 2005–2007 m. Žemės ūkio akademijos Augalų apsaugos skyriaus
Midlnikų tyrimų stotyje netoli Krokuvos. Tyrimų tikslas buvo ištirti biopreparatų Polyversum
(B. A. S. Pythium oligandrum) ir Biochikol 020 PC (B. A. S. chitosanas), naudojamų bulvių
vegetacijos metu, poveikį stiebagumbių infestacijai Rhizoctonia solani skleročiais. Kaip kontrolė
buvo naudojamas fungicidas Vitavax 2000 FS (B. A. S. karboksinas ir tiuramas). Buvo tirtas
in vitro minėtų preparatų poveikis Rhizoctonia solani micelio linijiniam augimui. Gauti rezultatai
parodė, kad testuoti preparatai per bulvių vegetacijа nulėmė mažesnę infestaciją Rhizoctonia
solani skleročiais. Stiebagumbių infestacijos laipsnis šiais patogenais, panaudojus testuotus
preparatus buvo žymiai mažesnis palyginus su kontrole. Pagal gautus rezultatus iš in vitro
testų, Rhizoctonia solani micelio linijinio auginimo slopinimas pasireiškė panaudojus visus
preparatus, tačiau naudojant didрtesnę (2 %) koncentraciją.
Reikšminiai žodžiai: bioapsauga, bulvės, chitosanas, karboksinas, Pythium oligandrum,
Rhizoctonia solani, tiuramas.
425

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
In vitro efficiency of bio-preparations against
Stewartia pseudocamellia (Max.) pathogens
Halina Kurzawińska, Joanna Duda-Surman
University of Agriculture, Department of Plant Protection, 41-425 Krakуw, Al. 29
Listopada 54, Poland, e-mail: hkurzaw@ogr.ar.krakow.pl, joanad@go2.pl
Conducted investigation aims at determination the typical composition of fungi occurred
on diseased Stewartia pseudocamellia seedlings and the in vitro effect of biological preparations
based on natural substances on the mycelium linear growth of fungi potentailly pathogenic to
Stewartia plants.
There were examined preparations Bioczos BR (B.A.S. garlic extract), Biochikol 020 PC
(B. A. S. chitosan), Biosept 33 SL (33 % extract from grapefruit seeds and pulp), each at 3
concentrations. As standard fungicide Bravo 500 SC (B. A. S. chlorotalonil) was applied.
Estimation of the preparation effect on the mycelium linear growth of dominant species isolated
from the Stewartia seedlings was carried out using Kowalik and Krechniak method (1961).
In the laboratory experiment from root and stem bases with observed disease symptoms
14 fungi species and 15 bacteria were isolated. The dominant fungi were Alternaria alternata,
Cylindrocarpon radicicola, Fusarium oxysporum, Fusarium avenaceum, Phomopsis thea.
Received results showed that all the preparations taken under consideration significantly
reduced mycelium linear growth of tested fungi. The effect of preparations was diversed and
depended on concentration and the type of preparation.
Key words: chitosan, chlorotalonil, fungi, grapefruit extract, garlic extract, seedlings,
Stewartia pseudocamellia.
Introduction. In the last few years dynamic develompent of decorative nursery
was observed in Poland. Many little know or completely new species of ornamental
shrubs have appeared. Stewartia pseudocamellia Max. belongs to them. In case of
reproduction of Stewartia pseudocamellia from seed, sprouts, and seedlings can be
destroyed by the fungi occurred in the soil. The soil-born pathogens can also attack
slightly elder plants, quicksets during taking root and perennial shrubs as well. The
following fungi were most frequent isolated from the sore seedlings: Fusarium spp.,
Cylindrocarpon spp., Alternaria spp., Phytophthora spp., Rhizoctonia spp., and
Verticillium spp. (Gajda et al., 2002).
The most common method of plant protection against phyto-pathogens is chemical
method, which is based on fungicide application to seeds and shoot dressing or shoot
spraying. However, work for limitation of pestidides application and replacement them
with biological preparations based on atagonistic microorganisms, organic compounds,
plant’s extracts is observed (Orlikowski, Skrzypczak, 2003; Pięta et al., 2006; Rose
et al., 2003; Wojdyła, 2004). Among biological preparations, the effective influence
showed: Bioczos BR, Biochikol 020 PC and Biosept 33 SL.
427

Biosept 33 SL containig 33 % of grapefruit seeds and pulp extract has a direct
influnce on pathogenic factor and as well induces plants’ immunity to some pathogens
(Orlikowski et al., 2002). Mentioned activity shows 7-geranoksycumarin enclosed
in grapefruit extract (Orlikowski, Skrzypczak, 2003). According to Caccioni et al.
(1998), among many components of this extract aliphatic aldehyde, monoterpenes and
nutcatone dominate. Discussed substances can show synergetic effect in limitation of
defined factor development or stimulate spores’ germination. Protective Biosept 33 SL
activity is connected with endogenous flavonoids, glycosides, citrate and limone
(Saniewska, 2002) existence in the mentioned preparation.
In plant protection against fungal and bacterial diseases Bioczos BR based on
garlic pulp has a great weight. Antibiotic garlic activity is assigned of alliacin existence,
which under the influence of lyase enzyme and after mechanical tissue damage is
born from allina. The allicina is an acetyl-Co A synthetase inhibitor and in this way it
blocks production of many different substances as fatty acid, sterols and the others. It
was found that the aioen, product of allicin transformation, shows stronger antifungal
activity than allicin (Wolski, Ludwiczuk, 2002).
Biochikol 020 PC preparation contains chitosan as biological active substance
and may show antivirus, antibacterial and antifungal effect. Followed to Poъpieszny
(1997), chitosan induces immunity of many plants through cell wall lignification,
phytoalexins production and synthesis of proteinase inhibitors. The treatment of the
cells with chitosan stimulates them to intensification through production of additional
structures and accumulation of phenol substances, which are harmful to fungi.
The aims of presented research work was determination of fungi occurred on
diseased Stewartia pseudocamellia seedlings and the in vitro effect of Bioczos BR
(B. A. S. garlic extract), Biochikol 020 PC (B. A. S. chitosan), Biosept 33 SL
(33 % extract from grapefruit seeds and pulp) on the mycelium linear growth of
fungi pathogenic to stewartia plants. As standard fungicide Bravo 500 SC (B. A. S.
chlorotalonil) was applied.
Object, methods and conditions. The experiment was carried out on material
from dr. hab. Muras collection in Tomaszkowice-Wielickie Foot hils. The samples of
one-year-old diseased stewartia seedlings were taken for mycological analysis at second
decade of October 2006. After washing under running water and soaking in distilled,
sterile water, diseased root and stem bases were desinfected in 75 % ethyl alcohol
solution for 10 min, then again rinsed in sterile, destilled water. After cutting them to
0.5 cm fragments they were dried in sterile blotting paper and put on Petri dishes with
solidified PDA medium (5 pieces for one dish). Inoculation was in room temperature.
Colonies, which grew, where inoculated on PDA slands. Isolated fungi were identiffied
by using mycological keys and monographs (Domsch et al., 1980; Gerlach, Nirenberg,
1982; Nelson et al., 1983). The pathogenicity of Alternaria alternata, Cylindrocarpon
radicicola, Fusarium oxysporum, F. avenaceum and Phomopsis thea in relation to
stewartia seedling were investigated in seperate examination.
The Kowalik and Krechniak (1961) method was used to evaluate the in vitro
effect of discussed preparations on tested pathogens mycelium linear growth. Each
of examined preparations were applied at three different concentrations: Bioczos BR
at concetrations 1.0 %, 2.0 %, 3.0 %; Biochikol 020 PC at concentrations: 2.0 %
428

4.0 %, 6.0 %; Biosept 33 SL: 0.05 %, 0.1 %, 0.2 %; Bravo 500 SC in doses: 0.025 %,
0.05 %, 0.1 %.
During the test preparations were introduced directly into liquid and slightly
cooled PDA agar. After thorough mixing with the agar, obtained suspension was poured
into Petrie dishes of 90 mm in diameter. Afterwards, an agar disk with mycelium of
examined fungi was placed centrally into each dish for each combination. A medium
without amendments and with a disk of appropriate fungus was used as the control for
each combination. The test was carried out in five repetitions for each combination.
Measurement started when first increase of mycelium in control dishes was observed
and was conducted till the complete overgrowth of mentioned dishes was noted down
(2–3 weeks). The percent of inhibition of mycelium linear growth on medium with
amendments of appropriate preparation compared to the growth of fungi on control
Petri dish was used to measure the preparations activity (Kowalik and Krechniak,
1961). Received results were subjected to statistical analysis using analysis of variance.
The multiple t-Duncan test was used for estimation of the differences between mean
values at significance level a = 0.05.
Results. From Stewartia pseudocamellia Max. root and stem bases with observed
disease symptoms the 55 isolates belonging to 14 fungi species and 12 genera and 15
bacteria were isolated (Table).
Table. Fungi isolated from root and stem base of diseased Stewartia seedlings
Lentelė. Grybai išskirti iš ligotų Stewartia pseudocamellia daigų šaknų ir stiebų
The dominant fungi were: Alternaria alternata – 12.7 %, Cylindrocarpon radicicola,
Fusarium oxysporum – each 10,9 %, Fusarium avenaceum – 9.0 %, Phomopsis
429

thea, Phoma eupyrena, Humicola fuscoatra var. fuscostra and Cladosporium
cladosporioides – each 7.3 % (Table). Besides, above-mentioned fungi Phytophthora
cactorum, Rhizoctonia solani and Verticillium albo-atrum (potentially pathogenic
fungi) in slightly less percentage settled stewartia seedlings (each 3.6 %) – Table.
According to the received results it was found that all investigated preparations
(with exception of Biochikol 020 PC applied at concentrations 2.0 % in relation
to F. avenaceum) significantly inhibited mycelium linear growth of tested fungi in
comparison to the control (Fig. 1–5).
Fig. 1. Percent of inhibition of Alternaria alternata mycelium linear growth
1 pav. Alternaria alternate grybienos linijinio augimo inhibicijos procentas
Figure 1 represents the percent of inhibition of A. alternata mycelium linear growth.
Among preparations under examination, Biosept 33 SL in all applied doses have
shown the best inhibitory effect of mycelium linear growth of mentioned species. The
percent of inhibition of mycelium linear growth appropriately amounted from 83.4 %
to 90.0 % (Fig. 1).
Similar Biospet 33 SL reaction was observed in combination with C. radicicola
(Fig. 2) and P. thea (Fig. 5). The percent of inhibition of mycelium linear growth of
above-mentioned fungi appropriately amounted from 60.1 % to 78.8 % (Fig. 2) and
from 92.9 % to 96.0 % (Fig. 5). Biospet 33 SL in all tested concentrations showed the
best effect of mycelium linear growth inhibition also in combination with F. oxysporum
(from 80.0 % to 85.0 %) – Fig. 3. Similar result in relation to F. oxysporum was found
in combination where Bravo 500 SC was used. The percent of inhibition of mycelium
linear growth appropriately amounted from 80.9 % to 85.0 % – Fig. 3.
In case of F. avenaceum, it was found that, the most important effect on mycelium
linear growth inhibition of discussed pathogen was obtained after Bravo 500 SC
application (from 69.5 % to 75.6 %) – Fig. 4. Under in vitro condition bio-preparation
430

Bioczos has also demonstated comparable hight efficiency. Mentioned preparation
limited tested fungi mucelium linear growth at slightly less percentage than
Biosept 33 SL – Fig. 1–5.
Fig. 2. Percent of inhibition of Cylindrocarpon radicicola mycelium linear growth
2 pav. Cylindrocarpon radicicola grybienos linijinio augimo inhibicijos procentas
Fig. 3. Percent of inhibition of Fusarium oxysporum mycelium linear growth
3 pav. Fusarium oxysporum grybienos linijinio augimo inhibicijos procentas
431

Fig. 4. Percent of inhibition of Fusarium avenaceum mycelium linear growth
4 pav. Fusarium avenaceum grybienos linijinio augimo inhibicijos procentas
Fig. 5. Percent of inhibition of Phomopsis thea mycelium linear growth
5 pav. Phomopsis thea grybienos linijinio augimo inhibicijos procentas
The least effect on mycelium linear growth inhibition of tested pathogens was
found in combinations where Biochikol 020 PC was applied. However, the activity
of this preparation (with the exception of the lowest concentration in relation to
F. avenaceum) was statistically important in comparison to control Fig. 1–5.
432

Discussion. Among fungi isolated from root and stem bases of diseased stewartia
seedlings the most numerous group constituted species considered as pathogenic for
plants. The most dominating species (over 50 % of isolated colonies) were: Alternaria
alternata, Cylindrocarpon radicicola, Fusarium oxysporum, F. avenaceum, Phomopsis
thea and fungi from genera: Phytophthora, Rhizoctonia and Verticillium. Similar results
were obtained by Gajda et al. (2002). Accoridng to Werner (1998) fungi C. radicicola,
F. oxysporum and F. avenaceum may cause wilting and decay of plants. C. radicicola
affect roots and root neck and Fusarium oxysporum destroy conductive wisps. Infected
young plants die quickly. In case of older planst, disease develops slowly, and shrups
die gradually. Moreover, followed to Orlikowski (2000), fungi from Phytophthora
genera, wilting and dying of ornamental leafed shrubs can cause other fungi – Fusarium
avenaceum and F. oxysporum.
Findings from conducted investigations under in vitro conditions showed, that
all applied preparations (with the exception of Biochikol 020 PC at concentration
2.0 % in relation to F. avenaceum) significantly reduced mycelium linear growth of
the tested Stewartia pathogens. Among preparations under consideration, the strongest
inhibition of examined pathogens mycelium linear growth (with the exception of
F. avenaceum) was found in combination where Biosept 33 SL was used. Similar
results were received by co-author in previous examinations (Kurzawińska et al.,
2007). According to Orlikowski et al. (2001) Biosept 33 SL added to medium where
pathogenic fungi grow, strongly inhibited mycelium expansion. High efficiency of
discussed preparation was confirmed other investigations conducted by Orlikowski
et al. (2001, 2002), Wojdyła (2004). Biosept 33 SL added to the ground significantly
reduced number of Phytophthora spp., Pythium spp., F. oxysporum (Orlikowski,
Skrzypczak, 2003; Orlikowski et al., 2001, 2002). Bioczos BR preparation was also
efficient in inhibiting mycelium linear growth of the tested fungi. Mentioned preparation
limited mycelium linear growth at slightly less percent than Biosept 33 SL. Followed
to Saniewska (2002) garlic extract has an effect on over 100 fungi species. Besides,
more of them show total growth inhibition. Author claims that content of 0.75 %
garlic extract in medium strongly limiting growth of Phoma narcisii mycelium and
some form specials of F. oxysporum Phytophthora cryptogea, Phyllosticta antirrhini,
Rhizoctonia solani, Botrytis cinerea, Botrytis tulipae (Saniewska, 2000). The least
in vitro effect on mycelium linear growth inhibition of fungi under consideration was
found in combinations where Biochikol 020 PC was used. The effect of the mentioned
preparation was statistically important in comparison with control (with the exception
of concentration 2,0 % in combination with F. avenaceum). Followed to Orlikowski
et al. (1998) in vitro activity of chitosan is rather poor. Results obtained from later
studies conducted by this author have shown that discussed substance does not inhibit
mycelium growth and spore germination under in vitro conditions. However, according
to Poъpieszny (1997), chitosan inhibits growth of several fungi and bacteria but not
all of them. Chitosan activity to pathogens mostly takes place through direct and longlasting
contact of both factors.
Conclusions. 1. The root and stem bases of diseased Stewartia seedlings were
colonized mostly by species: Alternaria alternata, Cylindrocarpon radicicola,
Fusarium oxysporum, Fusarium avenaceum and Phomopsis thea.
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2. Among all the tested preparations under in vitro conditions the most effective in
reduction of above mentioned pathogens mycelium linear growth (with the exception of
F. avenacum) turned to be Biosept 33 SL bio-preparation. The in vitro response of tested
pathogens depended on the fungi species, type of preparation and its concentration.
4. Bravo 500 SC, chemical, standard preparation, showed the most effective
influence on F. avenaceum mycelium linear growth inhibition.
Acknowledgements. The studies were financed by The Ministry of Science and
Information within grant No N 310377633.
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SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Bio-preparatų veiksmingumas prieš Stewartia pseudocamellia
(Max.) patogenus in vitro
H. Kurzawińska, J. Duda-Surman
Santrauka
Tyrimų tikslas buvo nustatyti tipinius grybus, gautus nuo sergančių Stewartia
pseudocamellia daigų ir biologinių preparatų, susidedančių iš natūralių medžiagų, poveikį
potencialiai šiems augalams patogeniрkų grybų micelio linijiniam augimui.
Buvo tirti šie preparatai: Bioczos BR (B. A. S. česnako ekstraktas), Biochikol 020 PC
(B. A. S. chitosanas), Biosept 33 SL (33 % ekstraktas iš greipfrutų sėklų ir minkštimo).
Kiekvieno preparato tirtos 3 koncentracijos. Fungicidas Bravo 500 SC (B. A. S. chlorotalonilas)
buvo panaudotas kaip kontrolė. Preparatų poveikis linijiniam micelio augimui buvo įvertintas
naudojant Kowalik ir Krechniak metodа (1961).
Laboratorinio eksperimento sąlygomis nuo šaknų ir stiebo, remiantis pastebėtais ligų
simptomais, Išskirta 14 rūšių grybų ir 15 rūšių bakterijų. Dominavo šie grybai: Alternaria
alternata, Cylindrocarpon radicicola, Fusarium oxysporum, Fusarium avenaceum, Phomopsis
thea.
Gauti rezultatai rodo, kad visi naudoti preparatai žymiai sumažino testuotų grybų micelio
linijinį augimа. Preparatų poveikis buvo įvairus ir priklausė nuo koncentracijos ir preparatų
tipo.
Reikšminiai žodžiai: chitosanas, chlorotalonilas, česnakų ekstraktas, daigai, greipfrutų
ekstraktas, Stewartia pseudocamellia.
435

Contents – Turinys
Pavelas Duchovskis
The vicennial of scientific searches in the field of plant physiology...................3
Dvidešimt metų mokslinių ieškojimų augalų fiziologijos srityje......................16
Giedrė Samuolienė, Akvilė Urbonavičiūtė, Gintarė Šabajevienė,
Pavelas Duchovskis
Flowering initiation in carrot and caraway........................................................17
Žydėjimo iniciacija morkose ir kmynuose.........................................................25
Jūratė Darginavičienė, Sigita Jurkonienė, Nijolė Bareikienė,
Vaidevutis Šveikauskas
H + -ATPase functional activity in plant cell plasma membrane.........................27
Funkcinis augalų ląstelių plazmolemos H + -ATPazės aktyvumas......................37
Anželika Kurilčik, Stasė Dapkūnienė, Genadij Kurilčik,
Silva Žilinskaitė, Artūras Žukauskas, Pavelas Duchovskis
Effect of the photoperiod duration on the growth of Chrysanthemum plantlets
in vitro................................................................................................................39
Fotoperiodo trukmės poveikis chrizantemų eksplantų augimui in vitro............46
Regina Losinska, Danguolė Raklevičienė, Danguolė Švegždienė
Light and gravity-related tropistic responses of garden cress leaves.................47
Tropinės sėjamosios pipirinės lapų reakcijos į šviesą ir gravitaciją..................55
Laima Česonienė
Effect of 2-chlorethylphosphonic acid application on the growth and
development of Actinidia kolomikta..................................................................57
2-chloretilfosfoninės rūgšties poveikio Actinidia kolomikta augimui ir vystymuisi
tyrimai......................................................................................................63
Danguolė Raklevičienė, Danguolė Švegždienė, Regina Losinska
Photomorphogenic responses of garden cress to light in altered gravity................65
Fotomorfogenetinės sėjamosios pipirinės reakcijos į šviesą pakeisto svarumo
sąlygomis...........................................................................................................74
Danguolė Švegždienė, Dalia Koryznienė, Danguolė Raklevičienė
Gravisensing of garden cress roots under varying g-magnitude........................75
Gravitacijos jutimas sėjamosios pipirinės šaknyse esant skirtingiems
g-dydžiams.........................................................................................................82

Akvilė Urbonavičiūtė, Giedrė Samuolienė, Aušra Brazaitytė,
Raimonda Ulinskaitė, Julė Jankauskienė, Pavelas Duchovskis,
Artūras Žukauskas
The possibility to control the metabolism of green vegetables and sprouts using
light emitting diode illumination.......................................................................83
Galimybė kontroliuoti žalumyninių daržovių ir želmenų metabolizmа
kietakūnės šviesos pagalba................................................................................92
Gabriela Wyżgolik, Joanna Nawara, Maria Leja
Photosynthesis and some growth parameters of sweet pepper grown under different
light conditions........................................................................................93
Saldžiosios paprikos fotosintezė ir kai kurie augimo parametrai auginant
skirtingomis apšvietimo sąlygomis....................................................................98
Nijolė Anisimovienė, Jurga Jankauskienė, Leonida Novickienė
Actualities in plant cold acclimation..................................................................99
Augalų grūdinimosi problemos........................................................................109
Jurga Sakalauskaitė, Eugenija Kupčinskienė, Darius Kviklys,
Laisvunė Duchovskienė, Akvilė Urbonavičiūtė, Gintarė Šabajevienė,
Aida Stiklienė, Juratė Bronė Šikšnianienė, Ričardas Taraškevičius,
Alfredas Radzevičius, Rimantė Zinkutė, Pavelas Duchovskis
Nutritional diagnosis of apple-tree growing in the nitrogen fertilizer factory
region...............................................................................................................111
Obelų, augančių azoto trąšų gamyklos poveikyje, mitybinės būklės įvertinimas.
..........................................................................................................................118
Valeriy Popov, Olga Antipina, Tamara Trunova
Oxidative stress in the tobacco plants at hypothermia.....................................121
Oksidacinis stresas tabako augaluose hipotermijos sąlygomis........................127
Vida Rančelienė, Regina Vyšniauskienė
Reaction of model plant Crepis capillaris to stress-inducing factors ozone and
UV-B................................................................................................................129
Modelinio augalo Crepis capillaris reakcija į stresą sukeliančius veiksnius
ozonа ir UV-B..................................................................................................137
Nina Astakhova, Alexander Deryabin, Maxim Sinkevich,
Stanislav Klimov, Tamara Trunova
Alteration of source-sink relations in the leaves of in vitro plants of two Solanum
tuberosum L. genotypes under hypothermia............................................139
Asimiliacinių ir sandėlinių audinių ryšio kitimas dviejuose in vitro Solanomum
tuberosum L. genotipų lapuose hipotermijos metu..........................................148

Jurga Sakalauskaitė, Aušra Brazaitytė, Akvilė Urbonavičiūtė,
Giedrė Samuolienė, Gintarė Šabajevienė, Sandra Sakalauskienė,
Pavelas Duchovskis
Radish response to distinct ozone exposure and to its interaction with elevated
CO 2
concentration and temperature.................................................................151
Kompleksinis ozono ir didėjančios anglies dioksido koncentracijos bei
temperatūros poveikis valgomajam ridikėliui..................................................159
Jūratė Darginavičienė, Virgilija Gavelienė, Donatas Butkus,
Benedikta Lukšienė, Sigita Jurkonienė
Response reactions to the complex influence of radionuclides and heavy metals.
..........................................................................................................................161
Atsako reakcijos į kompleksinį radionuklidų ir sunkiųjų metalų poveikį.......168
Oleg Ilnitsky, Ivan Paliy, Tatiana Bystrova, Sergey Radchenko,
Nikolay Radchenko
Investigation of water regime and drought resistance of various kinds of plants
applying phytomonitoring methods.................................................................169
Įvairių rūšių augalų vandens režimo ir atsparumo sausrai tyrimai, naudojant
fitomonitoringo metodus..................................................................................177
Rima Juozaitytė, Asta Ramaškevičienė, Algirdas Sliesaravičius,
Natalija Burbulis, Ramunė Kuprienė, Vytautas Liakas,
Aušra Blinstrubienė
Effects of UVB radiation on photosynthesis pigment system and growth of pea
(Pisum sativum L.)...........................................................................................177
UV-B spinduliuotės poveikis sėjamojo žirnio (Pisum sativum L.) augimui ir
fotosintezės pigmentų sistemai........................................................................186
Asta Ramaškevičienė, Rima Juozaitytė, Algirdas Sliesaravičius,
Egidija Venskutonienė, Liuda Žilėnaitė
Ozone influence on photosynthesis pigments system and growth of Soya (Glycine
max (L.) Merr.) under warming climate conditions.................................187
Pažemio ozono įtaka sojos fotosintetiniams pigmentams bei augimui (Glicine
max (L.) Merr.) šylančio klimato sąlygomis ...................................................196
Sandra Sakalauskienė, Gintarė Šabajevienė, Sigitas Lazauskas,
Aušra Brazaitytė, Giedrė Samuolienė, Akvilė Urbonavičiūtė,
Jurga Sakalauskaitė, Raimonda Ulinskaitė, Pavelas Duchovskis
Complex influence of different humidity and temperature regime on pea photosynthetic
indices in VI–VII organogenesis stages...........................................199
Skirtingo drėgmės ir temperatūros režimo kompleksinis poveikis žirnių fotosintetiniams
rodikliams VI–VII organogenezės etapuose.................................207

Regina Vyšniauskienė, Vida Rančelienė
Changes in the activity of antioxidant enzyme superoxide dismutase in Crepis
capillaris plants after the impact of UV-B and ozone......................................209
Antioksidacinio fermento superoksido dismutazės aktyvumo pokyčiai Crepis
capillaris augaluose po UVB ir ozono poveikio..............................................214
Peter Ferus, Mariįn Brestič, Katarķna Olšovskį, Anna Kubovį
Photosystem II thermostability of apple tree leaves: effect of rootstock, crown
shape and leaf topology...................................................................................215
Vaismedžio vainiko formos, poskiepio ir topologijos įtaka obelų lapų II fotosistemos
termostabilumui.....................................................................................234
Marina Rubinskienė, Pranas Viškelis, Vidmantas Stanys,
Tadeušas Šikšnianas, Audrius Sasnauskas
Quality changes in black currant berries during ripening...............................235
Juodųjų serbentų uogų kokybės pokyčiai nokimo metu..................................242
Ona Bundinienė, Pavelas Duchovskis, Aušra Brazaitytė
The influence of fertilizers with nitrification inhibitor on edible carrot
photosynthesis parameters and productivity....................................................245
Trąšų su nitrifikacijos inhibitoriumi įtaka valgomosios morkos fotosintezės
rodikliams irproduktyvumui............................................................................257
Elena Jakienė, Virginijus Venskutonis, Vytautas Mickevičius
The effect of additional fertilisation with liquid complex fertilisers and growth
regulators on potato productivity.....................................................................259
Papildomo tręšimo skystosiomis kompleksinėmis trąšomis ir augimo reguliatoriais
įtaka bulvių produktyvumui.....................................................................267
Vytautas Šlapakauskas, Vidmantas Stanys, Judita Varkulevičienė
Correlation between chlorophyll fluorescence of primrose (Primula malacoides
Franch.) and DNA polymorphic bands.................................................269
Koreliacija tarp raktažolės (Primula malacoides Franch.) chlorofilo fluorescencijos
ir polimorfinių DNR žymenų..................................................................275
Maria Leja, Gabriela Wyżgolik, Iwona Kamińska
Changes of some biochemical parameters during the development of sweet
pepper fruits.....................................................................................................277
Kai kurių biocheminių parametrų kitimai saldžiosios paprikos vaisių vystimosi
metu..................................................................................................................283
Julė Jankauskienė, Aušra Brazaitytė
The influence of various substratum on the quality of cucumber seedlings and
photosynthesis parameters...............................................................................285
Įvairių substratų įtaka agurkų daigų kokybei bei fotosintetiniams rodikliams..294

Nobertas Uselis, Juozas Lanauskas, Vytautas Zalatorius,
Pavelas Duchovskis, Aušra Brazaitytė, Akvilė Urbonavičiūtė
Evaluation of the methods of soil cultivation growing dessert strawberries in
beds..................................................................................................................295
Dirvos priežiūros būdų įvertinimas auginant desertines braškes lysvėse........304
Zdzisław Kawecki, Anna Bieniek
Influence of climatic conditions of northeastern Poland on growth of bower
actinidia ...........................................................................................................307
Šiaurės rytų Lenkijos klimato sąlygų įtaka smailialapės aktinidijos augimui.318
Brigita Čapukoitienė, Vidmantas Karalius, Elena Servienė,
Juozas Proscevičius, Vytautas Melvydas
Expression of yeast Saccharomyces cerevisiae K2 preprotoxin gene in transgenic
plants......................................................................................................319
Mielių Saccharomyces cerevisiae K2 preprotoksino geno raiška transgeniniuose
augaluose.................................................................................................327
Bogumił Markuszewski, Jan Kopytowski
Transformations of chemical compounds during apple storage......................329
Cheminių junginių pokyčiai laikant obuolius..................................................338
Nomeda Kviklienė, Alma Valiuškaitė, Pranas Viškelis
Effect of harvest maturity on quality and storage ability of apple
cv. ‘Ligol’.........................................................................................................339
Skynimo laiko įtaka ‘Ligol’ obuolių kokybei vaisiams nokstant ir juos
laikant...............................................................................................................346
Oksana Urbanovich, Zoya Kazlouskaya
Identification of scab resistance genesin apple trees by molecular
markers.............................................................................................................347
Rauplėms atsparių genų nustatymas obelyse naudojant molekulinius
žymenis............................................................................................................357
Alena Biruk, Zoya Kazlouskaya
Prospects for using of isozyme markers in identification of apple
cultivars............................................................................................................359
Izozimų žymenų naudojimas obelų veislių identifikavui.................................364
Edita Dambrauskienė, Pranas Viškelis, Audrius Sasnauskas
The use of black currant buds for the production of essential oils..................365
Juodųjų serbentų pumpurų panaudojimas eterinių aliejų gavybai...................370

Beatrice Denoyes-Rothan, Audrius Sasnauskas, Rytis Rugienius,
Philippe Chartier, Aurelie Petit, Stuart Gordon, Julie Graham,
Alison Dolan, Monika Hцfer, Walther Faedi, Maria Luigia Maltoni,
Gianluca Baruzzi, Bruno Mezzetti, Jose F. Sanchez Sevilla,
Edward Zurawicz, Margaret Korbin, Mihail Coman, Paulina Mladin
Genetic resources of European small berries according to GENBERRY
project..............................................................................................................371
Europos uoginių augalų genetiniai resursai pagal GENBERRY projektа ......377
Anna Kołton, Agnieszka Baran
Effect of different mineral nitrogen and compost nutrition on some
compounds of corn salad (Valerianella locusta (L.) Latter.)...........................379
Skirtingo mineralinio azoto ir kompostinių Trąšų poveikis poveikis Salotinės
sultenės (Valerianella locusta (L.) Latter.) biocheminei sudėčiai....................386
Audrius Sasnauskas, Rytis Rugienius, Tadeušas Šikšnianas,
Nobertas Uselis, Laimutis Raudonis, Alma Valiuškatė,
Aušra Brazaitytė, Pranas Viškelis, Marina Rubinskienė
Small berry research according to COST 863 Action......................................389
Uogininkystės tyrimai pagal COST 863 programą..........................................400
Rasa Karklelienė, Pranas Viškelis, Marina Rubinskienė
Growing, yielding and quality of different ecologically grown pumpkin cultivars...................................................................................................................401
Skirtingų, ekologiрkai auginamų, moliūgų veislių augimas, derėjimas ir
kokybė..............................................................................................................409
Laimutis Raudonis, Alma Valiuškaitė, Elena Survilienė
Effect of abiotic factors on risk of Venturia inaequalis infection depending on
apple tree growth stages...................................................................................411
Abiotinių faktorių įtaka Venturia inaequalis infekcijos rizikai priklausomai nuo
obelų augimo tarpsnių......................................................................................418
Halina Kurzawińska, Stanisław Mazur
Biological control of potato against Rhizoctonia solani (Kühn).....................419
Bulvių biologinė kontrolė prieš Rhizoctonia solani (Kühn)............................425
Halina Kurzawińska, Joanna Duda-Surman
In vitro efficiency of bio-preparations against Stewartia pseudocamellia (Max.)
pathogens.........................................................................................................427
Bio-preparatų veiksmingumas prieš Stewartia pseudocamellia (Max.) patogenus
in vitro.......................................................................................................435