Propagation of endangered Thermopsis turcica Kit Tan, Vural - Tübitak

Propagation of endangered Thermopsis turcica Kit Tan, Vural &
Küçüködük using conventional and in vitro techniques
Süleyman CENKCİ, Mehmet TEMEL, Mustafa KARGIOĞLU, Sergun DAYAN
Department of Biology, Faculty of Arts and Science, Afyon Kocatepe University, 03200, Afyonkarahisar - TURKEY
Received: 03.11.2008
Abstract: This report deals with the successful clonal propagation of endangered T. turcica using rhizome cuttings and
epicotyl explants. Rhizome cuttings were treated with α-naphthaleneacetic acid (NAA) or indole-3-butyric acid (IBA)
before planting for vegetative multiplication. Rhizome cuttings pretreated with NAA (10 mg/L) were both rooted and
sprouted (66.6%) after 100 days. Application of NAA induced callus and adventitious root formation in epicotyl explants
and 6-benzyladenine (BA) induced production of microshoots. Low levels of NAA (0.5-1 μM) together with BApromoted
shoot initiation and development. The highest regeneration rate (86.6%), with a mean number of shoots (3.05) and a mean
length of shoots (2.3 cm) per epicotyl, was achieved at 10 μM BA and 0.5 μM NAA. About 83% of in vitro regenerated
shoots rooted on a ½ Murashige and Skoog (MS) medium supplemented with 0.3 μM NAA. In vitro plantlets were
morphologically normal and a uniform chromosome complement of 2n = 18 was detected in root tips. The study
demonstrated that both conventional and in vitro techniques could be useful for large scale multiplication and
propagation of this endangered plant species.
Key words: Conservation, rhizome cuttings, epicotyl, propagation, Thermopsis turcica
Tehlike altındaki Thermopsis turcica’nın Kit Tan, Vural & Küçüködük geleneksel ve
in vitro teknikler kullanılarak çoğaltımı
Özet: Bu rapor, tehlike altındaki T. turcica’nın rizom çelikleri ve epikotil eksplantları kullanılarak klonal çoğaltmasını
kapsamaktadır. Rizom çelikler dikilmeden önce vejetatif çoğaltma için α-naftalenasetik asit (NAA) veya indol-3-butrik
asit (IBA) ile ön muameleye tutulmuştur. NAA (10 mg/L) ile ön muamele edilmiş rizom çelikler 100 gün sonunda hem
köklenmiş hem de filizlenmiştir (% 66,6). Epikotil eksplanlarında, NAA uygulaması kallus ve adventif kök oluşumunu
ve 6-benziladenin (BA) uygulaması mikro-fide üretimini teşvik etmiştir. BA ile birlikte düşük NAA değerleri (0,5-1 μM)
fidelenmeye uyarımı ve fide gelişimini arttırmıştır. En yüksek rejenerasyon oranı (% 86,6), explant başına ortalama fide
sayısı (3,05) ve ortalama fide uzunluğu (2,3 cm) 10 μM BA ve 0,5 μM NAA ile başarılmıştır. In vitro rejenere fidelerin
yaklaşık % 83’ü 0,3 μM NAA ile desteklenmiş ½ Murashige ve Skoog (MS) besininde köklenmiştir. In vitro bitkicikler
morfolojik olarak normal ve kök uçlarındaki sabit kromozom sayısı 2n = 18 olarak tespit edilmiştir. Çalışma, hem
geleneksel hem de in vitro tekniklerin tehlike altındaki bu bitki türünün seri üretilmesinde ve çoğaltılmasında kullanışlı
olabileceğini göstermiştir.
Anahtar sözcükler: Koruma, rizom çelik, epikotil, propagasyon, Thermopsis turcica
Introduction
Fabaceae belongs to one of the largest families of
dicotyledons. It is a large, diverse family ranging from
Turk J Biol
33 (2009) 327-333
© TÜBİTAK
doi:10.3906/biy-0811-1
herbaceous annuals to woody perennials that, because
of their capacity to fix nitrogen, are essential
components in natural and managed terrestrial
327

Propagation of endangered Thermopsis turcica Kit Tan, Vural & Küçüködük using conventional and in vitro techniques
ecosystems (1). Thermopsis turcica Kit Tan, Vural &
Küçüködük is the sole endemic representative of the
genus Thermopsis R. Br. in Turkey (2). The occurrence
of 2-3 free carpellate-ovary is the main morphological
character of this rare plant, first recorded in the
Papilionoideae subfamily of Fabaceae (2,3). It spreads
on the south shores of Eber Lake, and the south and
south-west shores of Akşehir Lake, in west-central
Anatolia. Destruction of marshy habitats by excessive
drainage of lake water, causing consequent water
withdrawals from Eber and Akşehir Lakes, and the
utilization by the locals of the best habitats containing
native vegetation are the major threats for the region
(4). Therefore, almost all populations of T. turcica are
in agricultural lands and under heavy destruction. In
addition, it is not possible to obtain viable and
germinable seeds from the plants growing in
farmlands because unidentified seed predators
(probably Coleoptera) utilize T. turcica seeds for larval
development. Therefore, the major regeneration
mode of this endangered species is through rhizomes.
One of the most appropriate actions for
safeguarding over-exploited species is to improve
propagation techniques and to encourage cultivation
(5). Seeds are preferred to vegetative material as the
source of propagation material because a wider
genetic base can be maintained (6). However, in some
species such as Delphinium malabaricum (7),
Rauvolfia micrantha (8), and Thermopsis turcica (9)
seed is not readily available and therefore vegetative
material has to be used (5,6). Although in vitro
preservation methods have been traditionally used for
crop species, these techniques have also been
increasingly used in the conservation of rare or
endangered plants in recent years, and this trend is
likely to continue as more species face the risk of
extinction (10). In vitro multiplications of some
threatened plant species native to Turkey, e.g.
Anthemis xylopoda O.Schwarz (11), Sternbergia
fisheriana (Herbert) Rupr. (12), and Centaurea
tchihatcheffii Fisch. et Mey. (13) have been reported.
This work is part of a project that aims at the
propagation and in vitro conservation of endangered
T. turcica. The present investigation was conducted to
achieve vegetative propagation using rhizome
cuttings, and to develop an effective in vitro
propagation protocol using epicotyl segments of T.
turcica. Cytological analyses were also performed to
328
determine number of chromosomes and to check the
clonal fidelity of regenerants.
Materials and methods
Plant material
Mature pods containing viable and germinable
seeds were collected from a small (less than 50
individuals) and isolated (naturally protected against
pest infestation) T. turcica population near Eber Lake
(38°36´N, 31°14´E) in September 2005 and 2006.
Rhizomes obtained from a widespread Akşehir
population were brought to the laboratory in plastic
bags and used immediately for vegetative propagation
studies.
Rhizome propagation
Rhizomes were cut into small segments of 4-5 cm
in length (with 2-3 nodes) and treated with auxins (24
h, dark) by dipping the basal portion (1-1.5 cm) of
rhizome cuttings in treatment solutions kept in
separate glass beakers. The auxin treatments
contained IBA or NAA (10, 100, and 250 mg L -1 ).
Subsequently, the control (treated with water) and
treated rhizome cuttings were planted in an open field
(50 × 300 cm) enriched with farmyard manure on the
Campus of Afyon Kocatepe University (38°49´N,
30°32´E; 1000 m altitude). Each treatment consisted
of 8 rhizome cuttings, each with 3 replicates. After 100
days, the rhizome cuttings were carefully removed
from the soil, and rooting/sprouting data were
recorded.
In vitro propagation
Hard-coated T. turcica seeds were both scarified
and sterilized by immersing them into concentrated
H2SO4 for 90 min, and subsequently rinsed 6 times
with sterile and distilled water. Scarified and sterilized
seeds were then sown on solid agar for germination.
Epicotyls dissected from 15-day-old seedlings were
cut into 7-10 mm long segments and placed on a
medium consisting of salts and vitamins of MS (14)
and 3% sucrose. Plant growth regulators were
incorporated into the medium at varying
concentrations and combinations. The pH of the
medium was adjusted to 5.8 ± 0.1 before adding 0.7%
agar and autoclaved at 1.5 kg cm -2 and 121 °C for 15
min. Thirty epicotyl explants were cultured for each of
the 20 treatments on the MS solid medium

supplemented with 0, 1, 2, 5, and 10 μM of BA in
combination with 0, 0.5, 1, and 2 μM of NAA. The
cultures were started in sterile Petri dishes, and they
were then, after 3 weeks, transferred into in vitro vent
culture boxes (DUCHEFA, Netherlands) containing
the same medium composition. The percentage of
shoot formation, the mean number and length of
shoots per epicotyl explant were recorded after 6
weeks of culture. All the cultures were grown at 25 ±
1 °C under 16 h photoperiods with an irradiance of
75 μmol m -2 s -1 and using Grolux fluorescent tubes
(Sylvania).
In vitro rooting
In vitro differentiated shoots (1-5 cm long) were
excised and transferred to ½ MS medium
supplemented with various concentrations (0, 0.1, 0.3,
0.5, and 1 μM) of NAA. Twenty regenerates were used
in each trail and each experiment was repeated at least
twice. Data were collected 35 days after culture.
Acclimatization
In vitro raised plantlets were removed from culture
tubes and their roots washed under running tap water.
They were then transferred to cups containing
autoclaved vermiculite mixed with sterile garden soil
using a ratio of 1:1. The cups were kept in a mist
chamber for hardening at 80%-90% relative humidity
for 4 weeks. The regenerates were finally transferred
to greenhouse conditions and watered with tap water.
S. CENKCİ, M. TEMEL, M. KARGIOĞLU, S. DAYAN
Cytological analysis
For chromosome counts, root tips obtained from
in vitro plantlets were incubated at 0 °C for 16 h in
saturated α-bromonaphthalene, fixed in glacial acetic
acid for 30 min, stained by the aceto-orcein reaction,
and squashed with 45% acetic acid on a microscope
slide for examination using a light microscope at 100×
magnifications.
Statistical analysis
The effect of different treatments was quantified
and the data were analyzed using one-way analysis of
variance (ANOVA). The means were compared using
Duncan’s test at a P < 0.05 significance level.
Results and discussion
Rhizome propagation
In general, root and sprout formation occurred in
the rhizome cuttings within 100 days, irrespective of
the treatment regime. Table 1 summarizes the effect of
IBA and NAA on the rooting and sprouting of the
rhizome cuttings. Among the various treatments, 250
mg L -1 IBA or 10 mg L -1 NAA induced rooting
significantly more than the water treated group. The
IBA treatment (250 mg L -1 ) on cuttings resulted in the
highest rooting rate (83.3%), a mean root number of
3.2, and a mean root length of 4.2 cm after 100 days;
however, this treatment did not promote sprouting
Table 1. Effect of IBA and NAA on rooting and sprouting of rhizome cuttings of T. turcica.
Treatment Percentage of Percentage of Mean number Mean root
(mg L -1 ) rooting rooting and sprouting of roots length (cm)
Control 16.6 a
IBA
10 50.0 ab
100 58.3 ab
250 83.3 b
NAA
10 75.0 b
100 58.3 ab
250 58.3 ab
16.6 a
33.3 ab
41.6 ab
8.3 a
66.6 b
25.0 a
16.6 a
0.33 a
1.08 a
2.83 bc
3.25 c
1.42 ab
0.75 a
1.17 a
0.56 a
0.73 ab
2.23 abc
4.22 c
2.70 bc
2.39 abc
2.35 abc
Data were recorded after 100 days of planting; n = 24; Means within each column followed by the same superscripts are not significantly
different according to Duncan’s test at 0.05% probability level.
329

Propagation of endangered Thermopsis turcica Kit Tan, Vural & Küçüködük using conventional and in vitro techniques
(8.3%) (Figure 1a) compared to the water treated
group (25%). In contrast, a treatment of 10 mg L -1
NAA on the cuttings resulted in the highest sprouting
and rooting ratio (66.6%) (Figure 1b). Vegetative
propagation via rhizome cuttings has also been
reported to be effective in the multiplication of
330
a b c
d e f
g h i
endangered plant species, such as Aconitum atrox
(Bruhl) Muk. (15), Picrorhiza kurrooa Royle ex Benth.
(16), and Podophyllum hexandrum Royle (17). In this
study, propagation through rhizome cuttings was
found to be quite successful with 10 mg L -1 NAA. Seed
availability, seed germination, and seedling survival
Figure 1. Conventional and in vitro propagation in T. turcica. (a) Rooted rhizome cuttings at 100 days after the 250 mg L -1 IBA
pretreatment. (b) Rooted and sprouted rhizome cuttings at 100 days after the 10 mg L -1 NAA. (c) Callus induction in epicotyl
explants with 0.5 μM NAA. (d) Simultaneous root development in an epicotyl explant with 2 μM NAA. Induction (e) and
development (f) of in vitro shoots with 10 μM BA and 0.5 μM NAA after 2 and 6 weeks of culture. (g) Rooted in vitro raised
shoots on ½ MS medium supplemented with 0.3 mg L -1 NAA after 5 weeks of culture. (h) Six-week-old in vitro raised plantlets.
(i) A chromosome complement of 2n = 18 from the root-tip cell of a regenerated plant; the bar represents 10 μm.

problems for T. turcica had been reported previously
(9). Therefore, raising the crop through rhizome
cuttings is an additional method of obtaining plantlets
and is advantageous because it eliminates some of the
difficulties with seed germination and seedling
survival. Nadeem et al. (17) suggested that
propagation through rhizome cuttings would also
reduce the length of the cultivation cycle and being a
clonal method of propagation should avoid variation
found in the seedling populations.
In vitro propagation
Legumes are notoriously recalcitrant to tissue
culture and are difficult to regenerate in vitro (18).
However, during the last decade, it has been achieved
with different kinds of legumes such as Astragalus
condensatus Ledeb (19), Vigna mungo (20), Phaseolus
vulgaris, P. acutifolius (21), and Cajanus cajan (22).
Successful regeneration of legumes has been aided by
S. CENKCİ, M. TEMEL, M. KARGIOĞLU, S. DAYAN
species specific determination of critical regeneration
parameters such as explant source, genotype, and
media constituents (18,23).
No morphogenetic response was observed when
epicotyl explants were cultured on the MS basal
medium alone. On the medium with NAA alone, only
callus (Figure 1c) or simultaneous roots were formed
(Table 2, Figure 1d). In general, callusing and
simultaneous root formation were also observed in
the MS medium supplemented with high levels of
NAA and low levels of BA. Shoot induction was
observed from the cut ends of epicotyl explants after
2 weeks in the culture (Figure 1e). BA alone induced
a large frequency of shoots, but high levels of BA and
low levels of NAA were more effective. The optimum
concentrations of BA and NAA were 10 μM and 0.5
μM, respectively, at which 86.6% of explants
responded; the number of shoots per explant was 3.05
Table 2. Effects of BA/NAA combinations in MS medium on morphogenetic responses from epicotyl explants of T. turcica after 8
weeks of culture.
BA NAA Morphogenetic Percentage of Mean number Mean length
(μM) (μM) response †
shooting of shoots of shoots (cm)
0.0 0.0 - 0.0 a
0.0 0.5 c;sr 0.0 a
0.0 1.0 c;sr 0.0 a
0.0 2.0 c;sr 0.0 a
1.0 0.0 s 26.6 ab
1.0 0.5 s 33.3 ab
1.0 1.0 c;sr;s 26.6 ab
1.0 2.0 c;sr;s 26.6 ab
2.0 0.0 s 40.0 abc
2.0 0.5 s 50.0 bcd
2.0 1.0 c;sr;s 46.6 bcd
2.0 2.0 c;sr;s 46.6 bcd
5.0 0.0 s 60.0 bcd
5.0 0.5 s 76.6 cd
5.0 1.0 c;s 66.6 bcd
5.0 2.0 c;s 60.0 bcd
10.0 0.0 s 60.0 bcd
10.0 0.5 s 86.6 d
10.0 1.0 s 66.6 bcd
10.0 2.0 c;s 56.6 bcd
0.0 a
0.0 a
0.0 a
0.0 a
0.26 ab
0.53 abc
0.41 abc
0.29 ab
0.56 abc
1.30 cd
1.20 cd
1.21 cd
2.20 ef
2.61 f
1.73 de
1.53 de
0.90 abcd
3.05 f
1.16 bcd
1.12 bcd
0.0 a
0.0 a
0.0 a
0.0 a
0.88 ab
1.75 ab
1.77 ab
1.86 ab
1.88 ab
2.07 ab
1.71 ab
1.85 ab
1.26 ab
1.91 ab
1.10 abc
1.31 ab
1.68 ab
2.30 c
2.13 c
1.61 ab
(†)c, callusing; sr, simultaneous rooting; s, shooting. Means within each column followed by the same superscripts are not significantly
different according to Duncan’s test at 0.05% probability level.
331

Propagation of endangered Thermopsis turcica Kit Tan, Vural & Küçüködük using conventional and in vitro techniques
and the mean length of shoots per explant was 2.3 cm
(Figure 1f). Similar micropropagation values were
reported for BA plus NAA treated epicotyl explants of
some legumes such as Vigna mungo L. (20) and
Lathyrus sativus L. (24). On the other hand, a very low
regeneration rate (< 29%) via indirect organogenesis
from calli initiated from the epicotyl explants of
Macroptilium atropurpureum (DC.) Urb. (Fabaceae)
(25) was observed after cytokinin/auxin treatments.
The results indicated that the morphogenetic
response of epicotyl explants was determined by the
amounts of plant growth regulators used in the study.
NAA alone promoted callusing or simultaneous
rooting; such a situation was also reported for
Rudbeckia bicolor Nutt. (26) and Thermopsis
lupinoides L. (27). BA is considered one of the most
useful cytokinins for achieving the multiplication and
micropropagation of plants (28). However, in the
present study with T. turcica, an optimized
combination of NAA with BA gave the best response.
Existing reports suggest that NAA at low
concentrations along with BA have a critical role in
plant regeneration for some leguminous plants, such
as Acacia catechu Willd. (29), Clitoria ternatea L. (30),
Mucuna pruriens L. (31), Psoralea corylifolia (L.)
Medik. (32), and Vigna mungo L. (20).
There was no visible root induction until 5 weeks
after transfer to hormone free ½ MS medium. A high
frequency of rooting was induced on ½ MS medium
supplemented with 0.1-1.0 μM NAA (Table 3, Figure
1g). The maximum frequency of root formation was
achieved on ½ MS medium supplemented with 0.3
μM NAA. The success of NAA for efficient root
induction was also reported in Clitoria ternatea (30)
332
and Cercis canadensis var. mexicana (33). In vitro
raised plantlets were subsequently transferred to
plastic cups filled with sterile vermiculate and garden
soil, and then successfully acclimatized (80%) in a
mist house (Figure 1h). None of these plantlets
showed any phenotypic variation when compared
with the mother plant after 10 months. Root tip
squashes of the randomly selected micropropagated
plants revealed a uniform chromosome complement
of 2n = 18 (Figure 1i). The diploid chromosome
numbers found for T. turcica are similar to other
species of Thermopsis R.Br. (3,34). Chromosomal
analyses showed that the regenerates were
cytologically stable.
Conclusion
In this study, it has been shown that rhizome
cuttings and epicotyl explants can be successfully used
to propagate endangered T. turcica. Endangered plants
can be regenerated in a short span of time (< a year)
using both conventional and in vitro procedures; the
mother plant can be conserved too. The efficient plant
regeneration system developed in this study will be
useful for the ex situ conservation of T. turcica.
Corresponding Author:
Süleyman CENKCİ
Department of Biology,
Afyon Kocatepe University, ANS Campus, 03200,
Afyonkarahisar – TURKEY
E-mail: scenkci@aku.edu.tr
Table 3. Effects of ½ MS medium supplemented with different concentrations of NAA on root induction in the shoots regenerated
from epicotyl explants of T. turcica after 35 days of culture.
NAA (μM) Rooting percentage Number of roots Root length (cm)
0.0 0.0 a
0.1 67.5 ± 7.5 bc
0.3 82.5 ± 6.1 c
0.5 77.5 ± 6.5 bc
0.7 52.5 ± 7.9 b
1.0 17.5 ± 6.8 a
0.0 a
2.5 ± 0.27 de
2.72 ± 0.30 e
1.45 ± 0.26 cd
1.18 ± 0.19 bc
0.50 ± 0.19 ab
0.0 a
2.37 ± 0.38 c
4.27 ± 0.49 d
2.48 ± 0.35 c
1.83 ± 0.33 bc
0.62 ± 0.23 ab
Means (±SE) within each column followed by the same superscripts are not significantly different according to Duncan’s test at 0.05%
probability level.

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