Agricultural and Food Science, vol. 18 (2009): 136-143 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 18 (2009): 136–143. 136 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 18 (2009): 136–143. 137 © Agricultural and Food Science Manuscript received April 2008 Genetic stability of wild pear (Pyrus pyraster, Burgsd) after cryopreservation by encapsulation dehydration Emiliano Condello, Maria Antonietta Palombi, Maria Grazia Tonelli, Carmine Damiano and Emilia Caboni* CRA – Fruit Tree Research Center, Via di Fioranello, 52 00134 Rome – ITALY *email: e.caboni@propag.org Shoot tips of Pyrus pyraster were successfully cryopreserved by encapsulation-dehydration. Na--alginate beads each containing one shoot tip, dehydrated for 2 days in 0.75M sucrose and desiccated to 20% mois- ture content (fresh weight basis), gave 60% recovery after exposure to liquid nitrogen. Regenerated shoots showed no differences in length and leaf shape compared to the mother plant. Multiplication rate and root- ing ability of cryopreserved shoots were lower than those of untreated controls after one subculture, but were completely restored following the third subculture. Fifteen cryopreserved lines derived from single buds were used for genetic analyses by RAPDs and SSRs, in comparison with the mother plant. In RAPD analysis, of a total of 24 primers, only 15 showed reproducible and well resolved bands and were further used. These primers produced a total of 66 fragments ranging from about 500 to 2500 base pair size. SSR (microsatellite) marker amplification was performed using 19 primers which produced 57 reproducible frag- ments. Microsatellites fragments ranged from 60 to 600 base pairs. Both RAPDs and SSRs did not reveal any polymorphism between cryopreserved lines and the original genotype, suggesting that cryopreservation, using encapsulation-dehydration, does not affect genetic stability of wild pear. Key-words: germplasm preservation, multiplication ability, RAPDs, rooting, somaclonal variation, SSRs, tissue culture. A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 18 (2009): 136–143. 136 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 18 (2009): 136–143. 137 Introduction In situ plant germplasm preservation plays an impor- tant role in the maintenance of biodiversity and the avoidance of genetic erosion, but the preservation of woody species in field gene banks requires huge land areas and it is expensive (Panis and Lambardi 2005). Cryopreservation is an alternative choice for the long-term conservation of germplasm including woody fruit species and their wild relatives (Engel- man 2004). In recent years, several new techniques have been developed for the cryopreservation of shoot tips of tropical and temperate plant species. The encapsulation-dehydration method, originally described for cryopreservation of Solanum shoot tips (Fabre and Dereuddre 1990), has been also success- fully applied to cryopreserve shoot tips of several fruit tree species (Gonzalez-Arnao and Engelmann 2006), including Pyrus (Dereuddre et al. 1990, Niino and Sakai 1992), Prunus (Shatnawi et al. 1999), Malus (Niino and Sakai 1992, Paul et al. 2000, Wu et al. 2001b), Vitis and Actinidia (Plessis et al. 1993, Wu et al. 2001a). Pyrus pyraster is considered an important wild relative of cultivated pear (Pyrus communis L.). The tree is considerable in size and diameter and its high quality wood makes this species interesting for reforestation of marginal farmland and for the production of highly valued timber (Kleinschmitt et al. 1998). In cultivated regions with calcareous soils, where Fe-chlorosis is a serious problem, wild pear can be also preferred as rootstock for pear cul- tivars. The species is indigenous in nearly all Eu- rope, except in the northern countries, but it is now seriously endangered (Kleinschmitt et al. 1998) and cryopreservation, being less labour requiring, could represent an alternative and/or complemen- tary method to the in field collection and to the in vitro slow-growth conservation of this species. The applicability of cryogenic protocols de- pends not only on the ability to survive, but also on the assumption of obtaining recovered material without any genetic modification with respect to the starting genotype. Various studies with molecular markers have been performed to asses somaclonal variation in cryopreserved plant material (Hard- ing 2004, Harding et al. 2005, Helliot et al. 2002). Among the DNA analysis techniques used, random- ly amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) analysis have also been ap- plied to evaluate genetic stability of the material surviving cryopreservation and in most of the cases have not provided evidence for genetic variation. When DNA polymorphisms were detected, they were considered as results of the culture-cryopro- tection-regeneration process and not of the cryop- reservation by itself (Harding 2004). However, in a recent work the encapsulation-dehydration method was shown, even at very low rate, to induce soma- clonal variability in chrysanthemum (Martín and Gonzáles-Benito 2005). The present study evaluates the genetic stability in wild pear cryopreserved by the encapsulation- dehydration method through RAPD and SSR analy- ses. Morphological (shoot length and leaf shape) and physiological parameters (multiplication and rooting ability) were also studied to characterize the cryopreserved cultures. Materials and methods Plant material In vitro propagated cultures, obtained from a single axillary bud of a wild pear (Pyrus pyraster, Burgsd) genotype, were established according to Caboni et al. (1999). Shoots in the proliferation phase were sub- cultured every 21 days on a medium (“LPmod” medium) consisting of LP (Quoirin and Lepoivre 1977) salts and the following organics: 0.5 mg L-1 nicotinic acid, 0.5 mg L-1 pyridoxine, 2.0 mg L-1 glycine, 0.5 mg L -1 thiamine-HCl, 150 mg L-1 myo-inositol, 1.0 mg L-1 Ca-pantothenate, 0.1 mg L-1 biotin and 0.5 mg L-1 riboflavin, according to Caboni et al. (1999). The medium was also sup- plemented with 1.78 µM benzyladenine (BA), 0.25 µM indole-3-butyric acid (IBA), 20 g L -1 sucrose and 6 g L-1 agar (B & V - Italy). The pH was ad- justed to 5.7 before sterilization and cultures were A G R I C U L T U R A L A N D F O O D S C I E N C E Condello, E. et al. Cryopreservation and genetic stability in wild pear 138 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 18 (2009): 136–143. 139 maintained at 25° C under a 16 h photoperiod and a light intensity of 40 µmol m-2 .s-1 provided by cool white fluorescent tubes (Philips TLD - France). Cryopreservation protocol Mother plants were cold acclimatized for 2 weeks in darkness at 5 °C. Subsequently, shoot tips (2–4 mm in length) were excised and sub-cultured for 24 h on the LPmod medium containing 0.3M sucrose. Apices were then transferred to MS (Murashige and Skoog 1962) calcium free liquid medium, supplemented with 3% alginate (Sigma). Beads were prepared by dispensing drops of alginate medium, each one containing one shoot tip, in a 100 mM CaCl2 MS liquid medium. The beads formed were cultured in liquid LPmod containing 0.75M sucrose for 2 days, desiccated in vessel containing silica gel (5 beads in 18g) to a bead moisture content of 20% fresh weight, placed in 1ml cryo-vials (Nalgene, 10 beads in each cryovial) and immersed in liquid nitrogen where they were kept for 1 week. Beads were then directly transferred to Petri dishes containing 0.3M sucrose enriched LPmod medium in darkness at room temperature and finally transferred to the standard multiplication medium and sub-cultured regularly, as reported above. Morphological observations, multiplication and rooting performance evaluation Thirty days after recovery, necessary to overcome an initial lag phase in growth after storage in liq- uid nitrogen, 30 single shoots were transferred to standard proliferation conditions reported above for morphological and multiplication ability evaluation, in comparison with 30 shoots obtained from shoot tips of the mother plant. Multiplication rates (final number of shoots – initial number of shoots divided by initial number of shoots) were calculated and shoot length and leaf shape were evaluated in newly formed shoots. Data were collected at the end of the 1st and 3rd subculture. For rooting experiments, 30 microcuttings were immersed for 5 days in a 2 mg L-1 IBA solution plus 20 g L-1 sucrose in the darkness and then transferred to a hormone free LPmod medium and to the light. Data were collected 30 days after the beginning of the root induction treatment. The experiment was repeated after three subcultures. Data (percentages were transformed to arc-sin root before analysis) were subjected to analysis of variance and differenc- es among means were compared by Fisher’s test. RAPD and SSR analysis Fifteen of the recovered shoots were cultured separately and these lines were used for molecular analyses. Genetic stability of the 15 lines was tested, in comparison with the mother plant, using 24 RAPD primers, designed according to Williams et al. (1990) and 19 SSR primer pairs, previously selected in Yamamoto et al. (2002) for giving unambiguous and reproducible fragment patterns in pear. Total DNA was extracted from 100 mg of plant tissue with DNeasy Plant Mini Kit (Quiagen). Two independent extractions were performed for each line and for the control (mother plant). RAPD reactions were car- ried out in a volume of 30 µl containing 25 ng total DNA, 1X PCR buffer (Qiagen), 1.5 mM MgCl2, 200 µM dNTPs, 0.4 µM 10–mer oligonucleotide primer (Invitrogen) and 1U Taq polymerase (Qiagen). SSR amplifications were performed in a volume of 30 µl containing 25 ng total DNA, 1X PCR buffer (Qiagen), 2.3 mM MgCl2, 200 µM dNTPs, 0.3 µM oligodeoxynucleotide primers (Invitrogen) and 1U Taq polymerase (Qiagen). DNA amplifications were performed in a Biometra T thermal cycler with a preliminary step of 5 min at 94 °C, 45 cycles of 60 s at 94 °C, 60 s at 36 °C and 2 min at 72 °C and a final step of 5 min at 72 °C for RAPDs. For SSRs, an initial step of 5 min at 94 °C, followed by 35 cycles of 1 min at 94 °C, 1 min at 55–58 °C and 2 min at 72 °C and a final 5 min extension at 72 °C were performed. In order to obtain reproducible and clear DNA fragment patterns, each amplification was repeated twice. RAPD amplification products were separated in a 1.5 % agarose (Duchefa – NL) gel using A G R I C U L T U R A L A N D F O O D S C I E N C E Condello, E. et al. Cryopreservation and genetic stability in wild pear 138 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 18 (2009): 136–143. 139 1X TBE buffer and stained with ethidium bromide. SSR fragments were analysed on 3.5% MetaPhor agarose (Cambrex Bio Science - USA) gel in 1X TBE buffer and stained with ethidium bromide. Results After cryopreservation the survival rate of the explants was 60% (Fig. 1), they grew well, devel- oped normally and, compared with the control, no morphological differences were observed (Fig. 2; Table 1). Multiplication rate and rooting ability of the cryopreserved shoots were lower than those of the control after one subculture, but were completely restored after three subcultures (Table 1). In order to evaluate if the encapsulation dehy- dration method preserves genetic integrity in wild pear, we used RAPD and SSR markers for the molecular analysis. To increase the confidence of the analysis, we selected only those primers which gave very reproducible bands. In RAPD analysis, a total of 24 primers were firstly used to amplify DNA of all genotypes and with 15 of them we ob- tained reproducible and well resolved bands and they were selected for further use (Table 2). These primers produced a total of 66 fragments ranging from about 500 to 2500 base pairs in size. The high- est number of analysable bands was obtained with the primers 70.13 and 70.20 (six fragments each), the lowest with the primer 70.15 (one fragment). SSR marker amplification was performed us- ing 19 primers that produced 57 reproducible frag- ments (Table 3). Fragments ranged from 60 to 600 base pairs; the highest number of analyzable bands was obtained with the primer pair NH020 (six frag- ments), the minimum with primer pair NB103 (one fragment). A summary of the results of RAPD and SSR marker analysis is given in Tables 2 and 3. The total number of fragments scored for the whole plant material analysed was 1056 (66 fragments Shoot length Leaf shape MR Rooting % 1sc 3sc 1sc 3sc 1sc 3sc 1sc 3sc Cryo-shoots 3.1a 3.3a stand* stand 2.4a 5.6a 24.4a 66.6a Control 3.4a 3.2a stand stand 5.4b 5.6a 62.4b 68.7a *Stand, expanded leaves of standard obvoidal form. Means on the column followed by the same letters are not significantly different at p=0.05. Percentage data were transformed to arc-sin root before statistical analysis. Table 1. Shoot length, leaf shape, proliferation (MR, multiplication rate) and rooting (% of rooted explants) ability in cryopreserved and control (mother plant) shoots after one or three sub-cultures (sc) Fig. 1. Wild pear shoots encapsulated in alginate beads and recovered after cryopreservation. Fig. 2. Wild pear shoot, 30 days after recovery from cry- opreservation (left) and mother plant shoot A G R I C U L T U R A L A N D F O O D S C I E N C E Condello, E. et al. Cryopreservation and genetic stability in wild pear 140 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 18 (2009): 136–143. 141 × 16 samples, included the control) and 912 (57 fragments × 16 samples, included the control) for RAPD and SSR markers, respectively. Identical patterns were obtained with both markers when cryopreserved plantlets were compared with the mother plant (control plant) (Fig. 3 and 4). RAPD primers Sequence 5’–3’ Total fragments scored 70.2 CACAGGCGGA 4 70.4 CCCGCTACAC 5 70.5 CAAAGGGCGG 5 70.7 AAGTGCACGG 4 70.9 AACGGGCGTC 4 70.12 GGCCTACTCG 3 70.13 GTGTCGCGAG 6 70.15 GCCCTCTTCG 1 70.17 GAGACCTCCG 5 70.19 GCTCTCACCG 5 70.20 TGCACGGACG 6 70.22 GTCGCCGTCA 5 70.23 TTGGCACGGG 3 70.24 GTGTGCCCCA 5 70.30 CGCGCTACGT 5 Table 2. RAPD primers used for DNA amplification and total fragments scored. SSR primer couples Annealing Temperature (°C) Total of scored fragments NB102 55 2 NB103 55 1 NB105 55 5 NB106 55 3 NB109 55 4 NB110 55 2 NB111 55 4 NB113 58 2 NH019 55 2 NH020 55 6 NH021 55 3 NH022 58 2 NH023 55 4 NH024 55 3 NH025 58 3 NH026 55 3 NH027 55 2 NH029 58 3 NH030 58 3 Table 3. SSR primers, annealing temperatures used for DNA amplification and the total number of fragments scored. Fig. 3. RAPD banding profiles of DNA samples from mother plant (control, C) and cryopreserved shoots (1–15) of Pyrus pyraster. Amplification products were generated by primer 70.8. M: HyperLadder II (Bioline) marker. Fig. 4. SSR profiles of DNA samples from mother plant (control, C) and cryopreserved shoots (1–15) of Pyrus pyraster. Amplification products were generated by primer couple NH030 FW-NH030 RW. Ma: 50 bp ladder marker and Mb: 100 bp ladder (Amersham-Pharmacia)marker. A G R I C U L T U R A L A N D F O O D S C I E N C E Condello, E. et al. Cryopreservation and genetic stability in wild pear 140 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 18 (2009): 136–143. 141 Discussion Conservation of plant genetic resources has to rely on methods that not only allow to obtain good sur- vival, but also to guarantee that material remains genetically stable after conservation. Some of the steps involved in cryopreservation, putatively caus- ing stress responses, may induce genetic instability (Engelmann 2004). For this reason, evaluation of genetic variation in cryopreserved material is an essential step before the large scale use of the established storage protocols. In this study we used morphological, physi- ological and molecular markers to evaluate sta- bility of preserved material. Similarly to Liu and co-workers (2004), who evaluated cryopreserved apple, we did not observe any morphological dif- ference between leaf shape of the mother plant and of the cryopreserved wild pear shoots and, while multiplication and rooting ability was lower after one sub-culture, they were fully restored after three subcultures. Rooted microcuttings were acclima- tised and they are now under further observation in the greenhouse. Most of the studies previously performed have shown that the cryopreservation process does not affect genetic stability of the stored lines. Nev- ertheless, genetic variation was recently shown to be induceable by the encapsulation-dehydration method in Dendrathema grandiflora (Martin and González-Benito 2005) suggesting that attention should be paid to the evaluation of the genetic stability in cryopreserved lines. In this study, we used RAPDs and SSRs to evaluate the genetic stability of the cryopreserved lines. These markers, both visualised by PCR (Polymerase Chain Reaction) and agarose-based electrophoresis, offer the advantage of being less expensive and quicker to be performed than RFLP or AFLP (Lanham and Brennan 1999, Powell et al. 1996). RAPDs, in particular, have been widely used to evaluate genetic stability in tissue cultures (Carvalho et al. 2004, Palombi et al. 2007 and references therein) and they have been also adopted to evaluate stability of cryopreserved material in various species (Hao et al. 2002, Ryynanen and Aronen, 2005, Zhai et al. 2003, Ventkatachalam et al. 2007). SSR markers allow screening of dif- ferent regions of the genome than RAPDs, includ- ing repetitive and hypervariable DNA regions and they were shown to be valuable molecular tools for determining somaclonal variation in tissue cul- ture (Rahman and Rajora 2001) and for genetic fingerprinting of fruit tree species, pear included (Yamamoto et al. 2002). Thus, RAPDs and SSRs, showing a different polymorphism capability, can be conveniently used, in combination, to evaluate somaclonal variability induced by tissue culture (Palombi and Damiano 2002). In our study we used 15 RAPD primers to am- plify DNA of 15 cryopreserved lines and of the mother plant. These primers produced a total of 66 fragments and the total number of bands con- sidering all the plant material analysed was 1056. No differences were found between the wild pear lines and the mother plant in the number of frag- ments obtained, as also found in most of the works performed on genetic stability of cryopreserved material (Dixit et al. 2003, Sales et al. 2001, Zhai et al. 2003). SSR marker amplification was also performed using 19 primer pairs that produced 57 reproducible bands corresponding to a total number of 912 frag- ments in the analysed material. Also this method showed no differences between the cryopreserved lines and the mother plant. This is, to our knowledge, the first report on the combined application of RAPDs and SSRs for evaluation of genetic stability in cryopreserved lines of fruit trees. We analysed a total of 1968 fragments (from RAPD and SSR markers) without observing any genetic variation. This number of analysed fragments, can be considered to be in- formative, as reported in other studies performed on genetic stability of cryopreserved materials (Dixit et al. 2003, Sales et al. 2001, Zhai et al. 2003) and also in micropropagation (Kawiak and Lojkowska 2004). However, due to the relatively low fraction of the genome screened with the molecular markers and the moderate sized test population used, these results cannot be interpreted as a final proof that no somaclonal variation has occurred. However, they A G R I C U L T U R A L A N D F O O D S C I E N C E Condello, E. et al. Cryopreservation and genetic stability in wild pear 142 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 18 (2009): 136–143. 143 give us a preliminary information on the genetic stability of the wild pear cryopreserved material and let us to be confident of the possibility of using routinely the encapsulation-dehydration method for long term conservation of Pyrus pyraster. Acknowledgements: The authors thank Loreta Marinac- cio for the skilful assistance in sub-culturing the in vitro material. Research supported by the Italian Ministry of Agriculture. Project RGV-FAO. Public. N. 142 References Caboni, E., Tonelli, M.G., Lauri, P., D’Angeli, S. & Damiano, C. 1999. In vitro shoot regeneration from leaves of wild pear. Plant Cell Tissue Organ Culture 59: 1–7. Carvalho, L.C., Goulao, L., Oliveira, C., Gonçalves, J.C. & Amancio, S. 2004. RAPD Assessment for Identifica- tion of Clonal Identity and Genetic Stability of in vitro Propagated Chestnut Hybrids. Plant Cell Tissue Organ Culture 77: 23–27. Dereuddre, J., Scottez, C., Arnaud, Y. & Duron, M., 1990. Resistance of alginate-coated axillary shoot tips of pear tree (Pyrus communis L., Beurre Hardy) in vitro plantlets to dehydration and subsequent freezing in liquid nitro- gen: effect of previous cold hardening Comptes Rendus de l’Académie des Sciences, Paris 310: 317–323. Dixit, S., Mandal, B.B., Ahuja, S., Srivastava, P.S. 2003. Genetic stability assessment of plants regenerated from cryopreserved embryogenic tissues of Dioscorea bul- bifera l. Using RAPD, biochemical and morphological analysis. CryoLetters 24: 77–84. Engelmann, F. 2004. Plant cryopreservation: progress and prospects. In Vitro Cell Developmental Biology Plant 40: 427–433. Fabre, J. & Dereuddre, J. 1990. Encapsulation-dehydra- tion:. A new approach to cryopreservation of Solanum shoot tips. CryoLetters 11: 413–426. Gonzalez-Arnao, M.T. & Engelmann, F. 2006. Cryopreser- vation of plant germplasm using the encapsulation-de- hydration technique: review and case study on sugar- cane. CryoLetters. 27: 155–68 Hao, Y.J., You, C.X. & Deng, X.X. 2002. Effects of cryopreservation on devel- opmental competence, cytological and molecular stabil- ity of citrus callus. CryoLetters 23: 27–35. Harding, K. 2004. Genetic integrity of cryopreserved plant cells: a review. CryoLetters 25: 3–22. Harding, K., Johnston, J. & Benson, E.E. 2005. Plant and algal cell cryopreservation: issues in genetic integrity, concepts in ‘Cryobionomics’ and current Europe- an applications:. In: Contributing to a Sustainable Future (eds. I.J. Benett, E. Bunn, H. Clarke & J.A. McComb) Proc. Australian Branch of the IATPT & B, Perth, West- ern Australia: 112–119 Helliot, B., Madur, D., Dirlewanger, E. & De Boucaud, M.T. 2002. Evaluation of genetic stability in cryopreserved prunus. In Vitro Cell Developmental Biology Plant 38: 493–500. Kawiak, A. & Lojkowska, E. 2004. Application of RAPDs in the determination of genetic fidelity in micropropagated Drosera plantlets. In Vitro Cell Developmental Biology Plant. 40: 592–595. Kleinschmit, J., Stephan, R. & Wagner, I. 1998. Wild fruit trees (Prunus avium, Malus sylvestris and Pyrus pyraster) genetic resources conservation strategy. In: Noble Hardwoods EUFORGEN. 1996. Updated 2006. Available on the Internet: www.bioversityinternation- al.org Lanham, P.G. & Brennan, R.M. 1999. Genetic characteri- zation of gooseberry (Ribes grossularia subgenus Gros- sularia) germplasm using RAPD, ISSR and AFLP mark- ers. Journal of Horticultural Science and Biotechnology 74: 361–366. Liu, Y.G., Wang, X.Y. & Liu, L.X. 2004. Analysis of genetic variation in surviving apple shoots following cryopreser- vation by vitrification. Plant Science 166: 677–685. Martín, C. & González-Benito, M.E. 2005. Survival and genetic stability of Dendranthema grandiflora, Tzvelev shoot apices after cryopreservation by vitrification and encapsulation-dehydration. Cryobiology 51: 281–289. Murashige, T. & Skoog, F. 1962. Revised Medium for rapid growth and bioassay with Tobacco Tissue Culture Phys- iologia Plantarum 15: 473–497. Niino, T. & Sakai, A. 1992. Cryopreservation of alginate- coated in vitro-grown shoot tips of apple, pear and mul- berry. Plant Science 87: 199–206. Palombi, M.A. & Damiano, C. 2002. Comparison between RAPDs and SSRs molecular markers to detect genetic variation in kiwifruit (Actinidia deliciosa A., Chev). Plant Cell Report 20: 1061–1066. Palombi, M.A., Lombardo, B. & Caboni, E. 2007. In vitro regeneration of wild pear (Pyrus pyraster, Burgsd) clones tolerant to Fe-chlorosis and somaclonal variation analy- sis by RAPD markers. Plant Cell Report 26: 489–496. Panis, B. & Lambardi, M. 2005. Status of cryopreserva- tion technologies in plants (crops and forest trees). In- ternational Workshop “The Role of Biotechnology for the Characterisation and Conservation of Crop For- estry Animal and Fishery Genetic Resources”. Turin. March 5–7 p. 43–54. Paul, H., Daigny, G. & Sangwan-Norreel, B.S. 2000. Cryo- preservation of apple (Malus x domestica Borkh.) shoot tips following encapsulation-dehydration or encapsula- tion-vitrification Plant Cell Report 19: 768–774. Plessis, P.C., Leddet, C., Colas, A. & Dereuddre, J. 1993. Cryopreservation of Vitis vinifera L., cv. Chardonnay shoot tips by encapsulation-dehydration: effect of pre- treatment, cooling and postculture conditions. CryoLet- ters 14: 309–320. Powell, W., Morgante, M., Doyle, J.J., McNicol, J.W., Tin- gey, S.V. & Rafalski, A.J. 1996. Gene pool variation in genus Glycine subgenus Soja revealed by polymor- phic nuclear and chloroplast microsatellites. Genetics 144: 793–803. Quoirin, M. & Lepoivre, P. 1977. Improved media for in vitro culture of Prunus sp. Acta Horticolturae 78: 437–442. Rahman, M. & Rajora O. 2001. Microsatellite DNA soma- clonal variation in micropropagated trembling aspen. A G R I C U L T U R A L A N D F O O D S C I E N C E Condello, E. et al. Cryopreservation and genetic stability in wild pear 142 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 18 (2009): 136–143. 143 Plant Cell Reports 20: 531–536 Ryynänen, L. & Aronen, T. 2005. Vitrification, a comple- mentary cryopreservation method for Betula pendula Roth. Cryobiology 51: 208–19. Sales, E., Nebauer, S.G., Arrillaga, I. & Segura, J. 2001. Cryopreservation of Digitalis obscura selected geno- types by encapsulation-dehydration. Planta Medica 67: 833–8. Shatnawi, M., Engelmann, F., Frattarelli, A., Damiano, C. 1999. Cryopreservation of apices of in vitro plantlets of almond (Prunus dulcis Mill) CryoLetters 20: 13–20. Venkatachalam, L., Sreedhar, R.V. & Bhagyalakshmi, N. 2007. Molecular analysis of genetic stability in long- term micropropagated shoots of banana using RAPD and ISSR markers. Electronic Journal of Biotechnolo- gy vol. 10, no. 1. Available on the Internet: http://www.ejbiotechnology.info/content/vol10/issue1/ full/12/index.html. Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A. & Tingey, S.V. 1990. DNA polymorphisms amplified by ar- bitrary primers are useful as genetic markers . Nucleic Acids Research 18: 6531–5. Yamamoto, T., Kimura, T., Shoda, M., Imai, T., Saito, T., Sawamura, Y., Kotobuki, K., Hayashi, T. & Matsuta, N. 2002. Genetic linkage maps constructed by using an in- terspecific cross between Japanese and European pear. Theoretical and Applied Genetics. 106: 9–18. Wu, Y., Zhao, Y., Engelmann, F., Zhou M. 2001a. Cryo- preservation of kiwi shoot tips. CryoLetters 22: 277– 284. Wu, Y., Zhao, Y., Engelmann, F., Zhou M., Zhang, D., & Chen, S. 2001b. Cryopreservation of apple dormant buds and shoot tips. CryoLetters 22: 375–380 Zhai, Z, Wu, Y., Engelmann, F., Chen, R. & Zhao, Y. 2003. Genetic stability assessments of plantlets regenerat- ed from cryopreserved in vitro cultured grape and kiwi shoot-tips using RAPD. CryoLetters 24: 315–22 Zhao, Y., Wu, Y., Engelmann, F. & Zhou, M. 2001. Cryo- preservation of axillary buds of grape (Vitis vinifera) in vitro plantlets. CryoLetters 22: 321–328. Genetic stability of wild pear (Pyrus pyraster, Burgsd) after cryopreservation by encapsulation dehydration Introduction Materials and methods Plant material Cryopreservation protocol Morphological observations, multiplication and rooting performance evaluation RAPD and SSR analysis Results Discussion References