In vitro culture medium and explant type effect on callogenesis and ...
In vitro culture medium and explant type effect on callogenesis and ...
In vitro culture medium and explant type effect on callogenesis and ...
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Romanian Biotechnological Letters Vol. 15, No.2, Supplement, 2010<br />
Copyright © 2010 University of Bucharest Printed in Romania. All rights reserved<br />
ORIGINAL PAPER<br />
<str<strong>on</strong>g>In</str<strong>on</strong>g> <str<strong>on</strong>g>vitro</str<strong>on</strong>g> <str<strong>on</strong>g>culture</str<strong>on</strong>g> <str<strong>on</strong>g>medium</str<strong>on</strong>g> <str<strong>on</strong>g>and</str<strong>on</strong>g> <str<strong>on</strong>g>explant</str<strong>on</strong>g> <str<strong>on</strong>g>type</str<strong>on</strong>g> <str<strong>on</strong>g>effect</str<strong>on</strong>g> <strong>on</strong> <strong>callogenesis</strong> <str<strong>on</strong>g>and</str<strong>on</strong>g> shoot<br />
regenerati<strong>on</strong> in two geno<str<strong>on</strong>g>type</str<strong>on</strong>g>s of ornamental strawberry<br />
Abstract<br />
12<br />
Received for publicati<strong>on</strong>, December 20, 2009<br />
Accepted, February 5, 2010<br />
ANCA NICOLETA SUTAN * , AUREL POPESCU * , VALENTINA ISAC **<br />
*<br />
University of Pitesti, Faculty of Sciences, Department of Biology <str<strong>on</strong>g>and</str<strong>on</strong>g> Horti<str<strong>on</strong>g>culture</str<strong>on</strong>g>,<br />
Pitesti, Romania<br />
**<br />
Research <str<strong>on</strong>g>In</str<strong>on</strong>g>stitute for Fruit Growing, Pitesti, Romania<br />
Corresp<strong>on</strong>ding author: Anca Nicoleta Sutan, University of Pitesti, Faculty of Sciences,<br />
Department of Biology <str<strong>on</strong>g>and</str<strong>on</strong>g> Horti<str<strong>on</strong>g>culture</str<strong>on</strong>g>, Targul din Vale, no. 1, 110040 Pitesti,<br />
Romania, tel.: 0040 348 453100, fax: 0040 348 453123, e-mail: ancasutan@yahoo.com<br />
As some of the ornamental varieties of strawberry obtained from Fragaria x Potentilla crosses<br />
are lacking the ability to form runners, their in <str<strong>on</strong>g>vitro</str<strong>on</strong>g> propagati<strong>on</strong> is dependent <strong>on</strong> either direct or<br />
indirect organogenesis. The influence of <str<strong>on</strong>g>culture</str<strong>on</strong>g> <str<strong>on</strong>g>medium</str<strong>on</strong>g> compositi<strong>on</strong> <str<strong>on</strong>g>and</str<strong>on</strong>g> <str<strong>on</strong>g>explant</str<strong>on</strong>g> <str<strong>on</strong>g>type</str<strong>on</strong>g> were<br />
investigated in two geno<str<strong>on</strong>g>type</str<strong>on</strong>g>s of ornamental strawberry, “Pink P<str<strong>on</strong>g>and</str<strong>on</strong>g>a” <str<strong>on</strong>g>and</str<strong>on</strong>g> “Serenata”, respectively,<br />
in order to establish an efficient protocol for regenerati<strong>on</strong> by indirect organogenesis. Aiming to a<br />
good rate of <strong>callogenesis</strong> <str<strong>on</strong>g>and</str<strong>on</strong>g> shoot regenerati<strong>on</strong>, the <str<strong>on</strong>g>effect</str<strong>on</strong>g> of different combinati<strong>on</strong>s <str<strong>on</strong>g>and</str<strong>on</strong>g><br />
c<strong>on</strong>centrati<strong>on</strong> of growth regulators (2,4-D, IBA, <str<strong>on</strong>g>and</str<strong>on</strong>g> BAP) added in <str<strong>on</strong>g>culture</str<strong>on</strong>g> media (either MS or LF)<br />
were evaluated with leaf <str<strong>on</strong>g>and</str<strong>on</strong>g> petiole <str<strong>on</strong>g>explant</str<strong>on</strong>g>s. It was found that the highest frequency of <str<strong>on</strong>g>explant</str<strong>on</strong>g>s<br />
forming callus have been induced in both varieties investigated <strong>on</strong> the LF basal <str<strong>on</strong>g>medium</str<strong>on</strong>g> c<strong>on</strong>taining<br />
0.5 mg/l or 1.0 mg/l 2.4-D <str<strong>on</strong>g>and</str<strong>on</strong>g>, respectively, 3.0 mg/l BAP. A maximum of 100% leaf <str<strong>on</strong>g>explant</str<strong>on</strong>g>s, <str<strong>on</strong>g>and</str<strong>on</strong>g><br />
92% petiole <str<strong>on</strong>g>explant</str<strong>on</strong>g>s formed calli having characteristics of those regenerating shoots in “Serenata”<br />
variety. Similarly, a maximum of 92% petiole <str<strong>on</strong>g>explant</str<strong>on</strong>g>s formed callus in “Pink P<str<strong>on</strong>g>and</str<strong>on</strong>g>a” intergeneric<br />
variety.<br />
Key words: callus inducti<strong>on</strong>, geno<str<strong>on</strong>g>type</str<strong>on</strong>g>, <str<strong>on</strong>g>explant</str<strong>on</strong>g> <str<strong>on</strong>g>type</str<strong>on</strong>g>, <str<strong>on</strong>g>culture</str<strong>on</strong>g> <str<strong>on</strong>g>medium</str<strong>on</strong>g>, plant growth regulators.<br />
<str<strong>on</strong>g>In</str<strong>on</strong>g>troducti<strong>on</strong><br />
Sexual compatibility of Potentilla palustris with some Fragaria species<br />
(Niemirowicz-Szczytt, [13]; Sayegh <str<strong>on</strong>g>and</str<strong>on</strong>g> Hennerty, [17]), allowed the occurence of a large<br />
range of Fragaria x Potentilla intergeneric hybrids, combining the ornamental value given by<br />
the beauty of their flowers <str<strong>on</strong>g>and</str<strong>on</strong>g> prol<strong>on</strong>ged blossoming seas<strong>on</strong> (May - October) with producti<strong>on</strong><br />
of edible fruits. Because the genetic limitati<strong>on</strong>s associated with high heterozygosity <str<strong>on</strong>g>and</str<strong>on</strong>g><br />
polyploidy which hamper the traditi<strong>on</strong>al breeding methods, the cl<strong>on</strong>al propagati<strong>on</strong> of<br />
intergeneric hybrids Fragaria x Potentilla provides an advantage for the multiplicati<strong>on</strong> of<br />
these elite plants without sexual recombinati<strong>on</strong>.<br />
Callogenesis <str<strong>on</strong>g>and</str<strong>on</strong>g> regenerati<strong>on</strong> of callus <str<strong>on</strong>g>culture</str<strong>on</strong>g> via shoot organogenesis seem to be highly<br />
dependent of a number of factors, such as ratio of auxins <str<strong>on</strong>g>and</str<strong>on</strong>g> cytokinins in the <str<strong>on</strong>g>culture</str<strong>on</strong>g> <str<strong>on</strong>g>medium</str<strong>on</strong>g><br />
(Dewitte <str<strong>on</strong>g>and</str<strong>on</strong>g> Murray [4]), the geno<str<strong>on</strong>g>type</str<strong>on</strong>g> (L<str<strong>on</strong>g>and</str<strong>on</strong>g>i <str<strong>on</strong>g>and</str<strong>on</strong>g> Mezzetti [7]), <str<strong>on</strong>g>and</str<strong>on</strong>g> the source of the <str<strong>on</strong>g>explant</str<strong>on</strong>g>s<br />
used. Different cultivars of strawberry have shown large variability in the differentiati<strong>on</strong><br />
competence of their somatic tissue, <str<strong>on</strong>g>and</str<strong>on</strong>g> many workers have reported the use of different<br />
combinati<strong>on</strong>s of growth regulators for shoot regenerati<strong>on</strong> from different <str<strong>on</strong>g>type</str<strong>on</strong>g>s of <str<strong>on</strong>g>explant</str<strong>on</strong>g>s (Schaart<br />
et al. [18]; Passey et al. [14]; Zhao et al. [23] ; Y<strong>on</strong>ghua et al. [21]; Biswas [2]).<br />
The objective of this study was to find the most appropriate <str<strong>on</strong>g>type</str<strong>on</strong>g> of <str<strong>on</strong>g>explant</str<strong>on</strong>g> <str<strong>on</strong>g>and</str<strong>on</strong>g> to<br />
investigate the specific nutrient requirements for obtaining callus tissue from leaf <str<strong>on</strong>g>and</str<strong>on</strong>g> petiole<br />
<str<strong>on</strong>g>explant</str<strong>on</strong>g>s in “Pink P<str<strong>on</strong>g>and</str<strong>on</strong>g>a” <str<strong>on</strong>g>and</str<strong>on</strong>g> “Serenata” intergeneric hybrids derived from Fragaria x
ANCA NICOLETA SUTAN, AUREL POPESCU, VALENTINA ISAC<br />
Potentilla crosses, in order to establish an highly efficient protocol for large scale propagati<strong>on</strong><br />
of new varieties of ornamental strawberry developed through breeding <str<strong>on</strong>g>and</str<strong>on</strong>g> selecti<strong>on</strong>.<br />
Materials <str<strong>on</strong>g>and</str<strong>on</strong>g> methods<br />
Plant material. “Pink P<str<strong>on</strong>g>and</str<strong>on</strong>g>a” <str<strong>on</strong>g>and</str<strong>on</strong>g> “Serenata” geno<str<strong>on</strong>g>type</str<strong>on</strong>g>s from the Fragaria<br />
Germplasm Collecti<strong>on</strong> of the <str<strong>on</strong>g>In</str<strong>on</strong>g>stitute for Fruit Growing, Pitesti, Maracineni, Romania were<br />
<str<strong>on</strong>g>culture</str<strong>on</strong>g>d in <str<strong>on</strong>g>vitro</str<strong>on</strong>g> starting from either meristems or self-pollinati<strong>on</strong> derived seeds, <strong>on</strong> LF basal<br />
<str<strong>on</strong>g>medium</str<strong>on</strong>g>. Leaf disks <str<strong>on</strong>g>and</str<strong>on</strong>g> petiole segments collected from six weeks old in <str<strong>on</strong>g>vitro</str<strong>on</strong>g> plantlets,<br />
pre<str<strong>on</strong>g>culture</str<strong>on</strong>g>d for two weeks <strong>on</strong> LF basal <str<strong>on</strong>g>medium</str<strong>on</strong>g> c<strong>on</strong>taining 0.5 mg/l BAP, 0.5 mg/l IBA <str<strong>on</strong>g>and</str<strong>on</strong>g><br />
0.2 mg/l GA3 (Sorvari et al. [19]), were used as <str<strong>on</strong>g>explant</str<strong>on</strong>g>s.<br />
Callus inducti<strong>on</strong> experiments. Callus inducti<strong>on</strong> media c<strong>on</strong>sisted of Murashige <str<strong>on</strong>g>and</str<strong>on</strong>g><br />
Skoog [11] <str<strong>on</strong>g>and</str<strong>on</strong>g> Lee <str<strong>on</strong>g>and</str<strong>on</strong>g> Fossard [8] basal media, each of them supplemented with various<br />
combinati<strong>on</strong>s of plant growth regulators, a total of six different combinati<strong>on</strong> for each variety<br />
<str<strong>on</strong>g>and</str<strong>on</strong>g> <str<strong>on</strong>g>explant</str<strong>on</strong>g> <str<strong>on</strong>g>type</str<strong>on</strong>g>, respectively, being tested (Table 1). Dextrose, at a c<strong>on</strong>centrati<strong>on</strong> of 40 g/l,<br />
was used as carb<strong>on</strong> source in all <str<strong>on</strong>g>culture</str<strong>on</strong>g> media.<br />
Table 1. Combinati<strong>on</strong> <str<strong>on</strong>g>and</str<strong>on</strong>g> c<strong>on</strong>centrati<strong>on</strong> of plant growth regulators added in basal media in order to induce<br />
callus formati<strong>on</strong>.<br />
Callus inducti<strong>on</strong><br />
media code<br />
Basal <str<strong>on</strong>g>culture</str<strong>on</strong>g><br />
media<br />
Combinati<strong>on</strong> <str<strong>on</strong>g>and</str<strong>on</strong>g> c<strong>on</strong>centrati<strong>on</strong><br />
of plant growth regulators (µM)<br />
2,4 - D IBA BAP<br />
CIM 1 MS, LF 2,7 - 1.3<br />
CIM 2 MS, LF 4.5 - 1.3<br />
CIM 3 MS, LF 4.5 - 2.2<br />
CIM 4 MS, LF - 2.5 1.3<br />
CIM 5 MS, LF - 4.9 1.3<br />
CIM 6 MS, LF - 4.9 2.2<br />
Both the leaf <str<strong>on</strong>g>explant</str<strong>on</strong>g>s (0.3-0.5 mm diameter) <str<strong>on</strong>g>and</str<strong>on</strong>g> petiole segments (0.3-0.5 mm) were<br />
r<str<strong>on</strong>g>and</str<strong>on</strong>g>omly placed <strong>on</strong>to the <str<strong>on</strong>g>culture</str<strong>on</strong>g> <str<strong>on</strong>g>medium</str<strong>on</strong>g>. The leaf <str<strong>on</strong>g>explant</str<strong>on</strong>g>s were placed with their abaxial<br />
surface in c<strong>on</strong>tact with the <str<strong>on</strong>g>medium</str<strong>on</strong>g>. After two weeks of incubati<strong>on</strong> in the dark, in the growth<br />
chamber at the temperature of 22-24°C, the <str<strong>on</strong>g>culture</str<strong>on</strong>g>s were maintained under a photoperiod of<br />
16 hours light/8 hours darkness <str<strong>on</strong>g>and</str<strong>on</strong>g> a light intensity of about 40 µmol m -2 s -1 . The number of<br />
<str<strong>on</strong>g>explant</str<strong>on</strong>g>s forming calli was scored after 45 days of <str<strong>on</strong>g>culture</str<strong>on</strong>g> for both varieties.<br />
Shoot organogenesis. Callus <str<strong>on</strong>g>culture</str<strong>on</strong>g>s initiated from the leaf <str<strong>on</strong>g>and</str<strong>on</strong>g> petiole-derived calli<br />
were maintained <strong>on</strong> the same <str<strong>on</strong>g>medium</str<strong>on</strong>g> whithout subculturing them, until the shoots inducti<strong>on</strong>.<br />
The number of shoots formed per callus was determined after five m<strong>on</strong>ths in <str<strong>on</strong>g>culture</str<strong>on</strong>g> under a<br />
16 hour light photoperiod.<br />
Experimental design <str<strong>on</strong>g>and</str<strong>on</strong>g> statistical analysis. The rate of <strong>callogenesis</strong> (%) was<br />
determined as the ratio of the number of <str<strong>on</strong>g>explant</str<strong>on</strong>g>s that developed calli to the total number of<br />
<str<strong>on</strong>g>explant</str<strong>on</strong>g>s. To avoid major statistical errors, all of the experimental treatment (two geno<str<strong>on</strong>g>type</str<strong>on</strong>g>s x<br />
two <str<strong>on</strong>g>type</str<strong>on</strong>g> of <str<strong>on</strong>g>explant</str<strong>on</strong>g>s x two <str<strong>on</strong>g>type</str<strong>on</strong>g> of basal media x six growth regulators combinati<strong>on</strong>s) was<br />
performed with five replicates (in c<strong>on</strong>ical flasks with 30 ml of <str<strong>on</strong>g>culture</str<strong>on</strong>g> <str<strong>on</strong>g>medium</str<strong>on</strong>g>, closed with<br />
cott<strong>on</strong>-wool bungs <str<strong>on</strong>g>and</str<strong>on</strong>g> tinfoil) of six somatic <str<strong>on</strong>g>explant</str<strong>on</strong>g>s. Analysis of variance was used to<br />
analyse the <str<strong>on</strong>g>effect</str<strong>on</strong>g>s <str<strong>on</strong>g>and</str<strong>on</strong>g> interacti<strong>on</strong>s of the four main factors (geno<str<strong>on</strong>g>type</str<strong>on</strong>g>, basal <str<strong>on</strong>g>medium</str<strong>on</strong>g>,<br />
combinati<strong>on</strong> <str<strong>on</strong>g>and</str<strong>on</strong>g> c<strong>on</strong>centrati<strong>on</strong> of PGR <str<strong>on</strong>g>and</str<strong>on</strong>g> <str<strong>on</strong>g>explant</str<strong>on</strong>g> <str<strong>on</strong>g>type</str<strong>on</strong>g>s) <strong>on</strong> inducti<strong>on</strong> of <strong>callogenesis</strong>.<br />
Statistical analysis of the data obtained with “Pink P<str<strong>on</strong>g>and</str<strong>on</strong>g>a” <str<strong>on</strong>g>and</str<strong>on</strong>g> “Serenata” varieties<br />
respectively, <strong>on</strong> basal media c<strong>on</strong>taining different c<strong>on</strong>centrati<strong>on</strong>s of auxins for callus inducti<strong>on</strong>,<br />
were performed using Duncan’s Multiple Range test, at p < 0.05 <str<strong>on</strong>g>and</str<strong>on</strong>g> <str<strong>on</strong>g>In</str<strong>on</strong>g>dependent Sample T-<br />
Romanian Biotechnological Letters, Vol. 15, No. 2, Supplement (2010) 13
14<br />
<str<strong>on</strong>g>In</str<strong>on</strong>g> <str<strong>on</strong>g>vitro</str<strong>on</strong>g> <str<strong>on</strong>g>culture</str<strong>on</strong>g> <str<strong>on</strong>g>medium</str<strong>on</strong>g> <str<strong>on</strong>g>and</str<strong>on</strong>g> <str<strong>on</strong>g>explant</str<strong>on</strong>g> <str<strong>on</strong>g>type</str<strong>on</strong>g> <str<strong>on</strong>g>effect</str<strong>on</strong>g> <strong>on</strong> <strong>callogenesis</strong><br />
<str<strong>on</strong>g>and</str<strong>on</strong>g> shoot regenerati<strong>on</strong> in two geno<str<strong>on</strong>g>type</str<strong>on</strong>g>s of ornamental strawberry<br />
Test, working with Statistical Package for the Social Science (SPSS) statistical software<br />
(versi<strong>on</strong>. 16.0).<br />
Results<br />
The callogenic resp<strong>on</strong>se was induced after 3 weeks of in <str<strong>on</strong>g>vitro</str<strong>on</strong>g> <str<strong>on</strong>g>culture</str<strong>on</strong>g>, in resp<strong>on</strong>se to<br />
IBA or 2.4-D <str<strong>on</strong>g>and</str<strong>on</strong>g> BAP added in MS <str<strong>on</strong>g>and</str<strong>on</strong>g> LF basic media. These calli were formed all over the<br />
surface of the leaf <str<strong>on</strong>g>explant</str<strong>on</strong>g>s <str<strong>on</strong>g>and</str<strong>on</strong>g> were predominantly initiated at the cut edges of the petiole<br />
segments, directly in c<strong>on</strong>tact with the <str<strong>on</strong>g>culture</str<strong>on</strong>g> <str<strong>on</strong>g>medium</str<strong>on</strong>g>. It is known that plant horm<strong>on</strong>es are<br />
present in higher quantities after wounding <str<strong>on</strong>g>and</str<strong>on</strong>g> are involved in cell proliferati<strong>on</strong> at the wound<br />
site (Khal [6]). Callus <str<strong>on</strong>g>culture</str<strong>on</strong>g>s exhibited a pink pigmentati<strong>on</strong> of anthocyanins, in both leaf <str<strong>on</strong>g>and</str<strong>on</strong>g><br />
petiole <str<strong>on</strong>g>explant</str<strong>on</strong>g>s of “Serenata” variety.<br />
Effect of geno<str<strong>on</strong>g>type</str<strong>on</strong>g>. Significant differences <strong>on</strong> callus inducti<strong>on</strong> <str<strong>on</strong>g>and</str<strong>on</strong>g> producti<strong>on</strong> was<br />
observed between the two geno<str<strong>on</strong>g>type</str<strong>on</strong>g>s of ornamental strawberry. Irrespective of the basic<br />
<str<strong>on</strong>g>culture</str<strong>on</strong>g> <str<strong>on</strong>g>medium</str<strong>on</strong>g>, plant growth regulators combinati<strong>on</strong> <str<strong>on</strong>g>and</str<strong>on</strong>g> c<strong>on</strong>centrati<strong>on</strong>, or <str<strong>on</strong>g>explant</str<strong>on</strong>g> <str<strong>on</strong>g>type</str<strong>on</strong>g>, the<br />
rate of <strong>callogenesis</strong> was found to be, generally, higher in “Serenata” than in “Pink P<str<strong>on</strong>g>and</str<strong>on</strong>g>a”<br />
variety. Differences was less pr<strong>on</strong>ounced when petiole <str<strong>on</strong>g>explant</str<strong>on</strong>g>s were <str<strong>on</strong>g>culture</str<strong>on</strong>g>d <strong>on</strong> MS basic<br />
<str<strong>on</strong>g>medium</str<strong>on</strong>g> or indicated a better performance of “Pink P<str<strong>on</strong>g>and</str<strong>on</strong>g>a” variety <strong>on</strong> LF <str<strong>on</strong>g>medium</str<strong>on</strong>g><br />
supplemented with IBA <str<strong>on</strong>g>and</str<strong>on</strong>g> BAP (Fig.1).<br />
Number of <str<strong>on</strong>g>explant</str<strong>on</strong>g>s forming callus / total<br />
number of <str<strong>on</strong>g>explant</str<strong>on</strong>g>s (%)<br />
140<br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
b<br />
a<br />
b<br />
a<br />
b<br />
a<br />
b<br />
a<br />
b<br />
a<br />
a a<br />
a a<br />
b b<br />
b<br />
a<br />
a<br />
b<br />
a<br />
a<br />
a b<br />
b<br />
CIM CIM CIM CIM CIM CIM CIM CIM CIM CIM CIM CIM CIM CIM CIM CIM CIM CIM CIM CIM CIM CIM CIM CIM<br />
1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6<br />
Leaf <str<strong>on</strong>g>explant</str<strong>on</strong>g> Petiole <str<strong>on</strong>g>explant</str<strong>on</strong>g> Leaf <str<strong>on</strong>g>explant</str<strong>on</strong>g> Petiole <str<strong>on</strong>g>explant</str<strong>on</strong>g><br />
M&S L&F<br />
“Pink P<str<strong>on</strong>g>and</str<strong>on</strong>g>a" “Serenata"<br />
Fig. 1. The influence of geno<str<strong>on</strong>g>type</str<strong>on</strong>g> <strong>on</strong> the <strong>callogenesis</strong> ability of leaf <str<strong>on</strong>g>and</str<strong>on</strong>g> petiole <str<strong>on</strong>g>explant</str<strong>on</strong>g>s<br />
(bars represent st<str<strong>on</strong>g>and</str<strong>on</strong>g>ard deviati<strong>on</strong>; letters above the columns indicate percentages significantly different from each other, p
ANCA NICOLETA SUTAN, AUREL POPESCU, VALENTINA ISAC<br />
c<strong>on</strong>taining low c<strong>on</strong>centrati<strong>on</strong>s of 2,4-D <str<strong>on</strong>g>and</str<strong>on</strong>g> BAP. As shown in Figure 1, an inhibiti<strong>on</strong> of<br />
<strong>callogenesis</strong> occurred for both <str<strong>on</strong>g>type</str<strong>on</strong>g>s of <str<strong>on</strong>g>explant</str<strong>on</strong>g>s <strong>on</strong> media with an increased BAP<br />
c<strong>on</strong>centrati<strong>on</strong> (2.2 µM, instead of 1.3 µM).<br />
The plant growth regulators had significant <str<strong>on</strong>g>effect</str<strong>on</strong>g>s <strong>on</strong> the inducti<strong>on</strong> of callus<br />
formati<strong>on</strong> in “Pink P<str<strong>on</strong>g>and</str<strong>on</strong>g>a” variety. The highest overall percentage of leaf <str<strong>on</strong>g>explant</str<strong>on</strong>g>s forming<br />
callus (92%) was induced in LF basal <str<strong>on</strong>g>medium</str<strong>on</strong>g> with low c<strong>on</strong>centrati<strong>on</strong>s of IBA (2.5 µM) <str<strong>on</strong>g>and</str<strong>on</strong>g><br />
BAP (1.3 µM). Surprisingly, the leaf <str<strong>on</strong>g>explant</str<strong>on</strong>g>s failed to form callus <strong>on</strong> MS basal <str<strong>on</strong>g>medium</str<strong>on</strong>g><br />
supplemented with the above menti<strong>on</strong>ed combinati<strong>on</strong> of growth regulators. However, the<br />
same combinati<strong>on</strong> of growth regulators promoted callus formati<strong>on</strong> from the petiole <str<strong>on</strong>g>explant</str<strong>on</strong>g>s<br />
<strong>on</strong> both LF <str<strong>on</strong>g>and</str<strong>on</strong>g> MS media, with a frequency of 84% <str<strong>on</strong>g>and</str<strong>on</strong>g> 80%, respectively.<br />
When added to the MS basal <str<strong>on</strong>g>medium</str<strong>on</strong>g>, the combinati<strong>on</strong> of 2.2 µM BAP <str<strong>on</strong>g>and</str<strong>on</strong>g> 4.9 µM<br />
IBA (Fig. 2) promoted callus proliferati<strong>on</strong> in both leaf <str<strong>on</strong>g>and</str<strong>on</strong>g> petiole <str<strong>on</strong>g>explant</str<strong>on</strong>g>s from “Serenata”<br />
variety, while <strong>on</strong>ly petiole <str<strong>on</strong>g>explant</str<strong>on</strong>g>s were induced to form callus in “Pink P<str<strong>on</strong>g>and</str<strong>on</strong>g>a” variety.<br />
Number of <str<strong>on</strong>g>explant</str<strong>on</strong>g>s forming callus / total number of<br />
<str<strong>on</strong>g>explant</str<strong>on</strong>g>s (%)<br />
140<br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
a a<br />
b b<br />
ab<br />
b<br />
a<br />
a<br />
ab<br />
ab<br />
b<br />
Leaf <str<strong>on</strong>g>explant</str<strong>on</strong>g> Petiole<br />
<str<strong>on</strong>g>explant</str<strong>on</strong>g><br />
a<br />
a<br />
ab<br />
bc<br />
a<br />
a<br />
a<br />
bc b<br />
c<br />
b<br />
Romanian Biotechnological Letters, Vol. 15, No. 2, Supplement (2010) 15<br />
a<br />
Leaf <str<strong>on</strong>g>explant</str<strong>on</strong>g> Petiole<br />
<str<strong>on</strong>g>explant</str<strong>on</strong>g><br />
b<br />
b<br />
c<br />
c<br />
c<br />
c<br />
b<br />
c c<br />
b<br />
Leaf <str<strong>on</strong>g>explant</str<strong>on</strong>g> Petiole<br />
<str<strong>on</strong>g>explant</str<strong>on</strong>g><br />
a<br />
a<br />
ab<br />
bcc<br />
b<br />
bc<br />
ab ab<br />
c<br />
a a<br />
Leaf <str<strong>on</strong>g>explant</str<strong>on</strong>g> Petiole<br />
<str<strong>on</strong>g>explant</str<strong>on</strong>g><br />
LF MS LF MS<br />
“Serenata” “Pink P<str<strong>on</strong>g>and</str<strong>on</strong>g>a”<br />
CIM 1 CIM2 CIM3 CIM4 CIM5 CIM6<br />
Figure 2. The <str<strong>on</strong>g>effect</str<strong>on</strong>g> of <str<strong>on</strong>g>culture</str<strong>on</strong>g> <str<strong>on</strong>g>medium</str<strong>on</strong>g> <strong>on</strong> the <strong>callogenesis</strong> ability of leaf <str<strong>on</strong>g>and</str<strong>on</strong>g> petiole <str<strong>on</strong>g>explant</str<strong>on</strong>g>s<br />
(bars represent st<str<strong>on</strong>g>and</str<strong>on</strong>g>ard deviati<strong>on</strong>; letters above the columns indicate percentages significantly different from each other, p
<str<strong>on</strong>g>In</str<strong>on</strong>g> <str<strong>on</strong>g>vitro</str<strong>on</strong>g> <str<strong>on</strong>g>culture</str<strong>on</strong>g> <str<strong>on</strong>g>medium</str<strong>on</strong>g> <str<strong>on</strong>g>and</str<strong>on</strong>g> <str<strong>on</strong>g>explant</str<strong>on</strong>g> <str<strong>on</strong>g>type</str<strong>on</strong>g> <str<strong>on</strong>g>effect</str<strong>on</strong>g> <strong>on</strong> <strong>callogenesis</strong><br />
<str<strong>on</strong>g>and</str<strong>on</strong>g> shoot regenerati<strong>on</strong> in two geno<str<strong>on</strong>g>type</str<strong>on</strong>g>s of ornamental strawberry<br />
basal <str<strong>on</strong>g>medium</str<strong>on</strong>g>, supplemented with 2.5 µM IBA <str<strong>on</strong>g>and</str<strong>on</strong>g> 1.3 µM BAP. For this geno<str<strong>on</strong>g>type</str<strong>on</strong>g>, the callus<br />
inducti<strong>on</strong> frequency <str<strong>on</strong>g>and</str<strong>on</strong>g> plant regenerati<strong>on</strong> ability appeared to be in an inverse relati<strong>on</strong>ship.<br />
Table 2. Percentages of leaf <str<strong>on</strong>g>and</str<strong>on</strong>g> petiole <str<strong>on</strong>g>explant</str<strong>on</strong>g>s showing shoot formati<strong>on</strong> in “Serenata” variety of intergeneric<br />
hybrid Fragaria x Potentilla<br />
Cv. “Serenata”<br />
Basal <str<strong>on</strong>g>culture</str<strong>on</strong>g><br />
<str<strong>on</strong>g>medium</str<strong>on</strong>g><br />
PGR combinati<strong>on</strong>s<br />
Explant <str<strong>on</strong>g>type</str<strong>on</strong>g> forming shoots (%)<br />
Leaf <str<strong>on</strong>g>explant</str<strong>on</strong>g>s Petiole <str<strong>on</strong>g>explant</str<strong>on</strong>g>s<br />
LF 2.5 µM IBA + 1.3 µM BAP 36 ± 2.3 a 10 ± 1.1 a<br />
MS 4.9 µM IBA + 1.3 µM BAP 22 ± 1.6 b -<br />
Data are expressed as means ± SE. Means in the same column that are followed by different letters are<br />
significantly different (p
ANCA NICOLETA SUTAN, AUREL POPESCU, VALENTINA ISAC<br />
organogenesis. Callus inducti<strong>on</strong> frequency was affected by c<strong>on</strong>centrati<strong>on</strong> of BAP <str<strong>on</strong>g>and</str<strong>on</strong>g> the<br />
<str<strong>on</strong>g>effect</str<strong>on</strong>g> of auxins <strong>on</strong> callus inducti<strong>on</strong> was geno<str<strong>on</strong>g>type</str<strong>on</strong>g> – specific. <str<strong>on</strong>g>In</str<strong>on</strong>g> low c<strong>on</strong>centrati<strong>on</strong> 2,4-D has<br />
been shown to be the most <str<strong>on</strong>g>effect</str<strong>on</strong>g>ive for callus inducti<strong>on</strong> in “Serenata” variety, irrespective of<br />
<str<strong>on</strong>g>explant</str<strong>on</strong>g> <str<strong>on</strong>g>type</str<strong>on</strong>g> <str<strong>on</strong>g>culture</str<strong>on</strong>g>d. As have been reported, 2,4-D induce callus formati<strong>on</strong> in a variety of<br />
species (Ma <str<strong>on</strong>g>and</str<strong>on</strong>g> Xu [9], Thao et al. [20]). <str<strong>on</strong>g>In</str<strong>on</strong>g> c<strong>on</strong>trast, IBA added in low c<strong>on</strong>centrati<strong>on</strong>s in<br />
<str<strong>on</strong>g>culture</str<strong>on</strong>g> <str<strong>on</strong>g>medium</str<strong>on</strong>g>, was most <str<strong>on</strong>g>effect</str<strong>on</strong>g>ive in stimulating callus formati<strong>on</strong>, in “Pink P<str<strong>on</strong>g>and</str<strong>on</strong>g>a” variety.<br />
These observati<strong>on</strong>s were similar to those noted by other autors (Nehra et al. [12] ; Barcelo et<br />
al. [1] 1998; Gruchala et al. [5])<br />
Presumably, maintainance of tissue-derived calli in <str<strong>on</strong>g>culture</str<strong>on</strong>g> without their transfer to the<br />
fresh <str<strong>on</strong>g>medium</str<strong>on</strong>g> may be the reas<strong>on</strong> for the low regenerati<strong>on</strong> frequency in “Pink P<str<strong>on</strong>g>and</str<strong>on</strong>g>a” <str<strong>on</strong>g>and</str<strong>on</strong>g><br />
“Serenata” varieties, respectively, or even the lack of regenerati<strong>on</strong> in the case of leaf-derived<br />
calli. On the other h<str<strong>on</strong>g>and</str<strong>on</strong>g>, while IBA in combinati<strong>on</strong> with BAP stimulated shoot organogenesis<br />
in “Serenata” intergeneric hybrid of Fragaria x Potentilla, 2,4-D has proven inappropriate for<br />
shoots regenerati<strong>on</strong> in both investigated geno<str<strong>on</strong>g>type</str<strong>on</strong>g>s of ornamental strawberry. Moreover,<br />
anthocyanin producti<strong>on</strong> does not influence callus inducti<strong>on</strong> in “Serenata” variety, according<br />
with Mori et al. [10].<br />
Geno<str<strong>on</strong>g>type</str<strong>on</strong>g> was proven to be a critical factor for indirect shoot organogenesis in<br />
intergeneric hybrids Fragaria x Potentilla. There were distinct differences in callus formati<strong>on</strong><br />
ability <str<strong>on</strong>g>and</str<strong>on</strong>g> shoots regenerati<strong>on</strong> frequency between the two ornamental strawberry varieties<br />
investigated. Significant variati<strong>on</strong>s in callus formati<strong>on</strong> <str<strong>on</strong>g>and</str<strong>on</strong>g> shoot regenerati<strong>on</strong> ability of the<br />
different geno<str<strong>on</strong>g>type</str<strong>on</strong>g>s have been previously reported in strawberry (Barcelo et al. [1]; Passey et<br />
al. [14]; L<str<strong>on</strong>g>and</str<strong>on</strong>g>i <str<strong>on</strong>g>and</str<strong>on</strong>g> Mezzetti [7]). C<strong>on</strong>sidering that specific genes are involved in shoot<br />
organogenesis (dedifferentiati<strong>on</strong>, acquisiti<strong>on</strong> of competence <str<strong>on</strong>g>and</str<strong>on</strong>g> inducti<strong>on</strong>), as reported by<br />
Phillips [15], it is likely that, in some geno<str<strong>on</strong>g>type</str<strong>on</strong>g>s, genes involved in shoot organogenesis may<br />
be suppressed due to inappropriate <str<strong>on</strong>g>culture</str<strong>on</strong>g> c<strong>on</strong>diti<strong>on</strong>.<br />
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