Analele Universităţii din Craiova, seria Agricultură – Montanologie – Cadastru Vol. XXXIX 2009 CAPACITATEA DE MULTIPLICARE IN VITRO A CĂPȘUNULUI ORNAMENTAL “SERENATA” ŞI EVALUAREA STABILITĂŢII GENETICE UTILIZÂND MARKERI RAPD IN VITRO MICROPROPAGATION OF ORNAMENTAL STRAWBERRY CVS. “SERENATA” AND THE ASSESSMENT OF GENETIC STABILITY BY RAPD MARKERS 1 A.N. SUTAN 1, A. POPESCU 1, R. GHEORGHE 2, C.F. POPESCU 2, V. ISAC 3 University of Pitesti, Faculty of Sciences, Department of Biology and Horticulture, 110040 Pitesti, Romania, ancasutan@yahoo.com 2 National Research and Development Institute for Biotechnology in Horticulture, Laboratory of Molecular Genetics, 117713 Stefanesti, Arges, Romania 3 Research Institute for Fruit Growing, 011464 Maracineni, Romania Key words: intergeneric hybrid, in vitro, micropropagation, genetic stability. REZUMAT Pentru determinarea capacitătii de multiplicare in vitro a hibridului intergeneric Fragaria x Potentilla, varietatea “Serenataˮ, mediile nutritive MS si LF au fost suplimentate cu BAP, Kin, AIA, AIB și GA3, în concentratii si combinatii diferite. O rată înaltă de multiplicare asociată cu o vigoare ridicată a lăstarilor a fost obtinută pe mediile de cultură continând 1.0 mg/l BAP, 1.0 mg/l AIA si 0.1 mg/l GA3. Pentru determinarea stabilitătii genetice a microlăstarilor au fost folositi 10 primeri (selectati din 48 de primeri, testati initial), iar similaritatea dintre profilele RAPD corespunzătoare plantei martor, respectiv microlăstarilor obținuți după 4 subculturi in vitro, a fost interpretată ca sugerând stabilitatea genetică. ABSTRACT The feasibility of in vitro propagation of intergeneric hybrid Fragaria x Potentilla, named “Serenata -based proliferation media supplemented with different combinations and concentrations of benzylaminopurine (BAP), kinetin (Kin), indolylacetic acid (IAA), 3-indolylbutiric acid (IBA), and giberellic acid (GA3). A high multiplication rate combined with good quality of proliferated shoots and in vitro rooting potential was induced by media containing 1.0 mg/l BAP, 1.0 mg/l IAA and 0.1 mg/l GA 3. Ten primers (from 48 previously tested) were selected and used in RAPD analysis to assess the genetic stability of these shoots. The lack of polymorphisms in micropropagated plants screened through molecular markers was proved by identity of the banding patterns. INTRODUCTION Sexual compatibility of Potentilla palustris with some Fragaria species (NiemirowiczSzcytt, 1984; Sayegh and Hennerty, 1993), allowed the obtaining of a large range of Fragaria x Potentilla intergeneric hybrids (“Serenata”, “Pink Panda”, “Lipstick”, “Red Ruby” and “Vivarosa”), combining the ornamental value given by the beauty of their flowers and prolonged blossoming season (May - October) with production of edible fruits. Because the genetic limitations associated with high heterozygosity and polyploidy, which hamper the traditional breeding methods, the clonal propagation of intergeneric hybrids Fragaria x Potentilla provides an advantage for the multiplication of these elite plants without sexual recombination. 366 Analele Universităţii din Craiova, seria Agricultură – Montanologie – Cadastru Vol. XXXIX 2009 Growth and regeneration in vitro is a complex phenomenon and is influenced by a number of genetic and environmental factors (Husaini and Abdin, 2007). As every species seems to have its own specific requirements, there are several reports about the substances and conditions which help cells to differentiate (Sen et al., 2002). Auxins and cytokinines are the two main growth regulators in plants involved in the regulation of division and differentiation (Fehér et al., 2003). Propagation through in vitro regeneration of plants has many disavantages (Scowcroft and Larkin, 1988), the most important being the associated genetic instability. The detection by phenotypic identification of off-types among micropropagateed plants is cumbersome and time-consuming, and is more difficult in perenial crops, were many observation have to be made until maturity. Moreover, all the genetic changes may not be reflected in the observed phenotypic changes (Rani and Raina, 2000). In one of the first studies, Swartz et al., (1981) listed a divers range of variations in meristem culture (shoot tip)-derived strawberry plants. The abnormal features noted were hyperflowering, fruit malformation, small plants, lower yields, change in bloom date and runnerless female sterile plants (Swartz et al., 1981; Martinelli, 1992). Brown et al., (1993) suggested the use of PCR for assessing the genetic fidelity of tissue culture-derived plants, RAPD analysis being used successfully for genetic analysis of in vitro-raised plant materials (Rani and Raina, 2000). MATERIAL AND METHODS Plant material: An ornamental strawberry variety (Fragaria x Potentilla), named ”Serenata”, was established in vitro culture starting from meristems and then subcultured succesively on Murashige and Skoog (MS) and respectively Lee and Fossard (LF) media supplemented with various combinations of growth regulators (Table 1). For the initiation of shoot cultures, meristems with 2-3 leaf primordia, of 0.1- 0.3 mm in size, excised from runners formed by field plants of ”Serenata” variety (with red flowers), was used. Six treatments with different combinations and concentration of BAP, Kin, IAA, IBA and GA3, added to both MS and LF basic culture media, were used in order to find an adequate medium for obtaining a high rate of micropropagation while maintaining a good vigor of micropropagated shoots (Table 1). Table 1. The combinations and concentration of growth regulators added to MS and LF culture media Culture medium code MM1 MM2 MM3 MM4 MM5 MM6 Basic medium MS, or LF MS, or LF MS, or LF MS, or LF MS, or LF MS, or LF Concentration of growth regulators in the culture medium (mg/l) BAP 0.5 1.0 0.5 1.0 2.0 1.0 IBA 0.1 0.2 - IAA 0.5 1.0 1.0 - GA3 0.1 0.1 0.1 0.1 2.0 Kin 0.5 The observations were carried out at every 4 weeks, respectively at the moment of subculturing the micropropagated shoots. The micropropagation rate was calculated as the average number of shoots regenerated on each primary explant cultured in vitro on each of the media tested. After four subcultures, shoots which regenerated from Fragaria x Potentilla explants were separated from the micropropagation basal media, when they were approximately 23 cm long, and placed on a medium suitable for root growth. Root growth was stimulated by supplementing the solidified basal medium, containing half strenght Murashige -Skoog 367 Analele Universităţii din Craiova, seria Agricultură – Montanologie – Cadastru Vol. XXXIX 2009 macroelements and half Lee-Fossard microelements, with the auxins IBA and IAA at different concentrations. In all treatments, 0.1 mg/l of GA3 was also added to the basal medium (Table 2). In vitro rooting was followed by acclimatization to ex vitro conditions, plantlets being transfered in perlite in greenhouse conditions. Table 2. The combinations and concentration of growth regulators added to rooting medium Culture medium code Basic medium RM1 Macroelements MS 1/2 n, Microelements LF 1/2n, Vitamins MS n Macroelements MS 1/2 n, Microelements LF 1/2n, Vitamins MS n Macroelements MS 1/2 n, Microelements LF 1/2n, Vitamins MS n RM2 RM3 Concentration of growth regulators in the culture medium for in vitro rooting (mg/l) IBA IAA GA3 0.25 0.1 0.5 - 0.1 - 0.5 0.1 To avoid major statistical errors, at least 5 conical flasks (each with 30 ml of culture medium and closed with cotton-wool bungs and tinfoil) with 6 shoots per flask were used as repetitions in each of the experimental treatment investigated. In order to establish the efficiency of each treatment, the rooting rate, average root number and root length, were determined. Statistical analysis of the data obtained on basal media containing different concentrations of citokinins and/or auxins for in vitro micropropagation and in vitro rooting, respectively, were performed using Windows SPSS 16.0 program (SPSS, 2007) at p < 0.05. The cultures have been incubated in a growth chamber at the temperature of 2224°C, with a photoperiod of 16 hours light/8 hours darkness, and a light intensity of about 40 µmol m-2 s-1. DNA extraction: Total genomic DNA was extracted from both tissue culture-derived plants and field grown mother plant using DNEasy Plant Mini Kit (Qiagen), following the manufacturers protocol.To check the degradation of DNA, samples were loaded into 0.8% agarose gel in 0.5 × TBE submitted to electrophoresis, stained with ethidium bromide and observed under ultraviolet (UV). The DNA used for the amplification was of high molecular weight with little degradation and free of RNA as revealed by UV and gel electrophoresis. PCR conditions: RAPD amplification was performed in a reaction volume of 25 µl containing 4 mM MgCl2, 0.4 mM of each dNTP, 0.6 units Taq DNA polymerase, 0.8 µM primer and 100-200 ng genomic DNA. The amplification reactions were carried out in an TC-512 Gradient Thermocycler (Bibby Scientific Ltd) programmed as following: preliminary denaturation of DNA at 95°C for 2 min, 45 cycles of 92°C for 30 s, 36°C for 25 s and 72°C for 74 s, and a final extension step at 72°C for 7 min. The PCR products were separated by gel electrophoresis on a 1.2 % agarose gel, in 0.5 x TBE buffer during 1 h at 90 V and stained with ethidium bromide (10 mg/ml). RAPD analysis using each primer was repeated at least twice in order to establish the reproducibility of banding pattern of the DNA sample studied. Electrophoretic DNA bands of low visual intensity that could not be readily distinguished as present or absent were considered ambiguous markers and were not scored. Photographs of the gels were obtained with a Gene Flash Syngene Bio Imaging. The 100 bp DNA Ladder (Fermentas) was used as a molecular size standard and the size of 368 Analele Universităţii din Craiova, seria Agricultură – Montanologie – Cadastru Vol. XXXIX 2009 the amplification products was estimated using LabImage software from the photographs of the gels. RESULTS AND DISCUSSION After the first subculture, the “Serenata” variety of Fragaria x Potentilla responded by a good rate of micropropagation during the first subculture on all the six variants of MS culture media, irrespective of combinations of growth regulators. Thus, an average number of 20.6 shoots formed per primary explant was calculated for the treatment with 0.5 mg/l BAP, 0.1 mg/l IBA and 0.1 mg/l GA 3 added to the MS (Fig. 1). A significantly lower rate of micropropagation was obtained with this Fragaria x Potentilla variety when the same combination of growth regulators was added to the LF medium. Excepting the treatment with 0.5 mg/l BAP, 0.1 mg/l IBA and 0.1 mg/l GA 3, no other combinations of growth regulators resulted in significantly different rate of shoot micropropagation on the two basic culture media tested (Fig. 1). The micropropagation rate maintained higher over the subsequent subculture on any of the culture media tested, reflecting a superior genetic potential of in vitro multiplication. It is relevant the fact that at the end of the second subculture, on the LF medium, a rate of micropropagation as high as 24.3 was calculated in treatment with 1.0 mg/l BAP, 0.2 mg/l IBA, and 0.1 mg/l GA3 (Fig. 1). 30 a 20 ab abc abc abc average number of shoots/explant average number of shoots/explant 25 ab ab abc abc abc 15 bc c 10 5 0 MM1 MM2 MM3 MM4 LF MS MM5 a 25 ab 20 ab ab 15 ab ab 10 ab ab ab ab b 5 0 MM1 MM6 a MM2 MM3 LF MM4 MS MM5 MM6 Figure 1. The micropropagation rate of the “Serenata” variety after the first subculture(left) and second subculture (right) on either LF or MS media. The statistical analysis have revealed that, “Serenata” variety responded by a decreased ability of shoot micropropagation during the third and fourth subcultures. Thus, irrespective of the basic culture medium, the best rates of micropropagation does not exceeds 16 shoots formed per primary explant. However, good rates of shoot micropropagation were promoted in treatments with 1.0 mg/l BAP, combined with either 0.2 mg/l IBA or 1.0 mg/l IAA. After four weeks in culture medium for in vitro rooting, the percentage of microshoots rooted, number of roots and length of roots per culture was influenced by the different types and concentrations of auxins added in the rooting media and hormonal composition of the basal media used for explants micropropagation. Rooting induction of the shoots started about 16 days after the initiation of culture. Excepting the shoots obtained by treatment with 1.0 mg/l BAP, 1.0 mg/l IAA and 0.1 mg/l GA3, no other combinations of growth regulators added to the LF basic medium resulted in significantly different rate of shoots rooting. The highest rooting rate (96.6%) was obtained on rooting medium with IBA at 0.25 mg/l and GA3 at 0.1 mg/l concentration (Table 3). 369 Analele Universităţii din Craiova, seria Agricultură – Montanologie – Cadastru Vol. XXXIX 2009 Table. 3 Effect of different types and concentration of growth regulators on root development from primary expants, after four weeks. Number of roots per Length of the roots (mm) shoot RM1 RM2 RM3 RM1 RM2 RM3 RM1 RM2 RM3 5.91 1.66 2.76 3,73 1,1 1,49 46,6 20 40 -LF abcd bcd abcd abc bc abc abcd bcd abcd MM1 4.61 9.96 1.43 40 20 20 2,13 6,19 0,93 -MS bc a bc ab ab ab abc a bc 6.63 1.83 3.96 1,4 2,7 60 20 20 -LF 5,7ab abcd bcd bcd abc bc abc abcd abcd MM2 7.22 4.56 5.79 80 50 60 5,19 3,53 3,59 -MS ab abc abc a ab ab ab abc abc 1.71 6.16 2.83 1,61 5,27 0,4 33,2 80 26,6 -LF abcd abc bcd bc abc abc abcd abc cd MM3 0 0 0 -MS 0 0 0 0 0 0 7.49 9.53 2.56 5,66 6,49 2,39 96,6 86,6 40 -LF a ab abcd ab a abc ab a abcd MM4 1.7 1.7 20 20 2,4 1,46 0 -MS 0 0 bc bc ab ab abc abc 4.44 4.76 2.28 4,69 2,79 2,63 59,8 40 16,6 -LF abcd b cd abc abc bc abcd abcd abcd MM5 4.83 1.25 1.33 53,2 13,2 20 4,26 0,7 1,93 -MS abc bc bc ab b ab abc bc abc 5.36 5.5 5,16 4,06 60 66,6 0 0 -LF 0 abcd abcd abc abc abc abcd MM6 1.33 4.46 3.38 20 40 33,2 0,63 3,26 2,87 -MS bc abc bc ab ab ab bc abc abc Mean comparison: values in each column on the same rooting medium followed by different letters are significantly different, p<0.05 Shoots provenance Rooting rate (%) Shoots obtained on MM6-LF failed to induce rhizogenesis. A significantly lower value of the rooting rate was obtained in treatment with IAA at 0.5 mg/l and GA 3 at 0.1 mg/l concentration (RM3), irrespective of the basal medium used for explant micropropagation. The statistical analysis revealed that the highest mean root number per shoot (6.49) and the highest average length of the roots (9.53 mm) were induced in this variety by the combination 0.5 mg/l IBA and 0.1 mg/l GA 3, for those shoots cultured on MM4-LF variant of medium (Table 3). During fourth successively subcultures, micropropagation rate values on MM4-MS and MM4-LF did not varied extremely (18.45 – 8.35, respectively 15.55 – 15.35) and shoots obtained were more vigorous, with a better in vitro rooting potential than those developed on other culture medium variants. On the basis of the number, intensity and reproductibility of RAPD bands, ten random primers (Table 4) were selected out of the 48 previously tested and used in RAPD analysis to prove the clonal fidelity (i.e. genetic stability) of the tissue culture-derived microshoots obtained on MM4-LF and MM4-MS. The primers selected for this study gave rise to a total of 60 scorable bands ranging from 312 bp to 2774 bp in size. From the selected markers, two (OPA11 and OPB10) yielded 20 bands across the shoots tested, and another one (OPC09) yielded 7 bands. The other primers were not as efficient in generating succesive PCR products. A total of 180 bands (number of plantlets analyzed X number of bands with all primers) were generated by the RAPD technique, giving rise to monomorphic patterns across all microshoots and control plant studied. All RAPD profiles from the tissue culturederived ornamental strawberry plants were found to be analogous to those of the control plant, indicating either identity or similarity. Examples of RAPD patterns amplified with primers OPC09 and OPB09 are shown in Figure 2 (a) and (b). 370 Analele Universităţii din Craiova, seria Agricultură – Montanologie – Cadastru Vol. XXXIX 2009 Table 4. List of primers, their sequence and size of the amplified fragments generated by 10 RAPD primers No. Primer Sequence No of scorable bands 1 2 3 4 5 6 7 8 9 10 OPA02 OPA11 OPA19 OPA20 OPB09 OPB10 OPB15 OPC05 OPC06 OPC09 TGCCGAGCTG CAATCGCCGT CCAACGTCGG GTTGCGATCC TGGGGGACTC CTGCTGGGAC GGAGGGTGTG GATGACCGCC GAACGGACTC CTCACCGTCC 5 10 5 5 4 10 5 4 5 7 MM4 -LF MM4 -MS control plant L MM4 -LF No of monomorphic bands 5 10 5 5 4 10 5 4 5 7 MM4 -MS Size in bp 2248-810 2667-312 2356-1138 1881-986 1713-710 2600-480 2351-973 1755-750 2140-598 2774-647 control plant Figure 2. RAPD profiles of the plants investigated with primers OPC05 (a) and OPC06 (b). L= 100 bp DNA ladder. CONCLUSIONS In “Serenata” variety of Fragaria x Potentilla, the average number of shoots formed per primary explant was higher when the explants were subcultivated on the MS medium, rather than on LF medium (currently used for the micropropagation of the octoploid cultivated strawberry), indicating a more adequate composition of nutrients to the in vitro growth requirements of these intergeneric hybrids. Irrespective of the basic culture media, the micropropagation rate of “Serenata” variety was demonstrated to be generally higher when combinations of 1.0 mg/l BAP with either 0.2 mg/l IBA or 1.0 mg/l IAA are used. In “Serenata” variety, a fairly good rate of rooting was promoted in treatment with 0.25 mg/l IBA and 0.1 mg/l GA3, but average root number and root elongation were singnificantly higher when IBA concentration was higher. The shoots cultured on MM4-LF responded by higher values for all three rooting caracteristics analysed. Because there were no changes in the banding patterns observed in the tissue culture plants as compared with that of the control plant, we conclude that our micropropagation protocol can be followed without much risk of genetic instability. The lack of polymorphic bands among the RAPD profiles of microshoots regenerated in basal media supplemented with low concentration of growth regulators, and control plant, denote that no genetic 371 Analele Universităţii din Craiova, seria Agricultură – Montanologie – Cadastru Vol. XXXIX 2009 variation had occurred. Also, the correspondence between RAPD profiles can be associated with a higher genetic stability of this genotype. REFERNECES 1. Brown, P.T.H., Lange, F.D., Kranz, E., Lorz, H., 1993 - Analysis of single protoplasts and regenerants plants by PCR and RAPD technology, Molecular and General Genetics, 237: 311-317. 2. 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