Abstract
Germplasm preservation plays an important role in current breeding programs. A simple vitrification procedure that allows for the reproducible cryopreservation of two alder embryogenic lines is presented for the first time. Somatic embryos clumps (1–2 mm) were precultured in hormone-free medium (Murashige and Skoog half-strength macronutrients, MS1/2) supplemented with 0.3 M sucrose for 3 days, and then treated with a mixture of 2 M glycerol plus 0.4 M sucrose for 20 min at 25 °C. Osmoprotected somatic embryos were dehydrated using plant vitrification solution 2 (PVS2). The effect of different PVS2 incubation times was evaluated, and 60 min at 0 °C was considered to be the optimum period. After changing the solution with fresh PVS2, the somatic embryos were directly immersed in liquid nitrogen. Following rapid thawing in a water bath at 40 °C for 2 min, the somatic embryos were transferred onto MS1/2 supplemented with 0.1 mg l−1 benzyladenine, 30 g l−1 sucrose and 6 g l−1 Vitro agar. The cultures were kept in the dark for 1 week prior to exposure to light (16 h/8 h light/dark cycle). The recovery rate of vitrified somatic embryos reached over 90 % in both embryogenic lines. Cryopreservation did not affect the plant regeneration potential of Alnus glutinosa through somatic embryogenesis. The ploidy stability of the regenerated material was assessed by flow cytometry. Analysis of DNA ploidy stability of the control, PVS2 treated, cryopreserved somatic embryos and plantlets showed no significant differences.
Similar content being viewed by others
References
Adu-Gyambi R, Wetten A (2012) Cryopreservation of cocoa (Theobroma cacao L.) somatic embryos by vitrification. CryoLetters 33:494–505
Ai P-F, Lu L-P, Song J-J (2012) Cryopreservation of in vitro-grown shoot-tips of Rhabdosia rubescens by encapsulation-dehydration and evaluation of their genetic stability. Plant Cell Tissue Organ Cult 108:381–387
Brasier CM, Kirk SA, Delcan J, Cooke DL, Jung T, Man In’t Veld WA (2004) Phytophthora alni sp nova and its variants: designation of a group of emerging heteroploid hybrid pathogens. Mycol Res 108:1172–1184
Chmielarz P (2010) Cryopreservation of orthodox seeds of Alnus glutinosa. CryoLetters 31:139–146
Corredoira E, San José MC, Ballester A, Vieitez AM (2004) Cryopreservation of zygotic embryo axes and somatic embryos of European chestnut. CryoLetters 25:33–42
Corredoira E, San José MC, Vieitez AM, Ballester A (2007) Improving genetic transformation of European chestnut and cryopreservation of transgenic lines. Plant Cell Tissue Organ Cult 91:281–288
Corredoira E, Valladares S, Martínez MT, Vieitez AM, San José MC (2013) Somatic embryogenesis in Alnus glutinosa (L.) Gaertn. Trees 27:1597–1608
Corredoira E, Toribio M, Vieitez AM (2014) Clonal propagation via somatic embryogenesis in Quercus spp. In: Ramawat KG, Mérillon J-M, Ahuja MR (eds) Tree biotechnology. CRC Press, Boca Raton, pp 264–302
Crowe JH, Crowe LM, Carpenter JF, Aurell-Wistrom C (1984) Stabilization of dry phospholipid bilayers and proteins by sugars. Plant Cell Rep 12:89–94
Crowe LM, Womersley C, Crowe JH, Reid D, Appel L, Rudolph A (1986) Prevention of fusion and leakage in freeze-dried liposomes by carbohydrates. Biochim Biophys Acta 861:131–140
Dolezel J, Bartos J, Voglmayr H, Greilhuber J (2003) Nuclear DNA content and genome size of trout and human. Cytometry 51A:127–128
Endemann M, Hristoforoglu K, Stauber T, Wilhelm E (2001) Assessment of age-related polyploidy in Quercus robur L. somatic embryos and regenerated plants using DNA flow cytometry. Biol Plant 44:339–345
Engelmann F (1992) Cryopreservation of embryos. In: Dattée Y, Dumas C, Gallais A (eds) Reproductive biology and plant breeding. Springer, Berlin, pp 281–290
Engelmann F (2000) Importance of the cryopreservation for the conservation of plant genetic resources. In: Engelmann F, Takagi H (eds) Cryopreservation of tropical germplasm. Current research progress and application. JIRCAS/IPGRI, Tsukuba, pp 8–20
Fernandes P, Rodríguez E, Pinto G, Roldán-Ruíz I, De Loose M, Santos C (2008) Cryopreservation of Quercus suber somatic embryos by encapsulation–dehydration and evaluation of genetic stability. Tree Physiol 28:1841–1850
Galbraith DW, Harkins KR, Maddox JM, Ayres NM, Sharma DP, Firoozabady E (1983) Rapid flow cytometric analysis of the cell-cycle in intact plant tissues. Science 220:1049–1051
Galbraith DW, Lambert G, Macas J, Dolezel J (2002) Analysis of nuclear DNA content and ploidy in higher plants. In: Robinson J, Darzynkiewicz Z, Dean P, Hibbs A, Orfao A, Rabinovitch P, Wheeless L (eds) Current protocols in cytometry. Wiley, New York, pp 7.6.1–7.6.22
Gibbs JN, van Dijk C, Webber JF (2003) Phytophthora disease of alder in Europe. For Comm Bull 126. Forestry Commission, Edinburgh
Guzman-García E, Bradaï F, Sánchez-Romero C (2013) Cryopreservation of avocado embryogenic cultures using droplet-vitrification method. Acta Physiol Plant 35:183–193
Harding K (2004) Genetic integrity of cryopreserved plant cells: a review. CryoLetters 25:3–22
Hernández I, Cuenca B, Carneros E et al (2011) Application of plant regeneration of selected cork oak trees by somatic embryogenesis to implement multivarietal forestry for cork production. In: Nageswara-Rao M, Soneji JR (eds) Focus on tree micropropagation and tissue culture. Tree and forestry science and biotechnology 5 (special issue 1). Global Science Books, Isleworth, pp 19–26
Jitsuyama Y, Suzuki T, Harada T, Fujikawa S (2002) Sucrose incubation increases freezing tolerance of asparagus (Asparagus officinalis L.) embryogenic cell suspensions. CryoLetters 23:103–112
Kaviani B (2011) Conservation of plant genetic resources by cryopreservation. Aust J Crop Sci 5:778–800
Krajnákova J, Sutela S, Aronen T, Gömory D, Vianello A, Häggman H (2011) Long-term cryopreservation of Greek fir embryogenic cell lines: recovery, maturation and genetic fidelity. Cryobiology 63:17–25
Krajnákova J, Bertolini A, Gömöry D, Vianello A, Häggman H (2013) Initiation, long-term cryopreservation, and recovery of Abies alba Mill. embryogenic cell lines. In Vitro Cell Dev Biol-Plant 49:560–571
Lambardi M, De Carlo A (2003) Application of tissue culture to the germplasm conservation of temperature broad-leaf trees. In: Jain JM, Ishii K (eds) Micropropagation of woody trees and fruits. Kluwer Academic Publishers, Dordrecht, pp 815–840
Lambardi M, De Carlo A, Capuana M (2005) Cryopreservation of embryogenic callus of Aesculus hippocastanum L. by vitrification. CryoLetters 26:185–192
Lelu-Walter M-A, Thompson D, Harvengt L, Sánchez L, Toribio M, Páques LE (2013) Somatic embryogenesis in forestry with a focus on Europe: state-of-the-art, benefits, challenges and future direction. Tree Genet Genomes 9:883–899
Lloyd G, McCown B (1980) Commercially feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot tip cultures. Comb Proc Int Soc 30:421–427
Loureiro J, Pinto G, Lopes T, Dolezel J, Santos C (2005) Assessment of ploidy stability of the somatic embryogenesis process in Quercus suber L. using flow cytometry. Planta 221:815–822
Loureiro J, Capelo A, Brito G, Rodríguez E, Silva S, Pinto G, Santos C (2007a) Micropropagation of Juniperus phoenicea from adult plant explants and analysis of ploidy stability using flow cytometry. Biol Plant 51:7–14
Loureiro J, Rodríguez E, Dolezel J, Santos C (2007b) Two new nuclear isolation buffers for plant DNA flow cytometry. A test with 37 species. Ann Bot 100:875–888
Martínez MT, Ballester A, Vieitez AM (2003) Cryopreservation of embryogenic cultures of Quercus robur using desiccation and vitrification procedures. Cryobiology 46:182–189
Marum L, Loureiro J, Rodriguez E, Santos C, Oliveira MM, Miguel C (2009) Flow cytometry and morphological analyses of Pinus pinaster somatic embryogenesis. J Biotechnol 25:288–295
Matsumoto T, Sakai A, Yamada K (1994) Cryopreservation of in vitro grown apical meristems of wasabi (Wasabia japonica) by vitrification and subsequent high plant regeneration. Plant Cell Rep 13:442–446
Merkle S, Cunningham M (2011) Southern hardwood varietal forestry: a new approach to short-rotation woody crops for biomass energy. J For 109:7–14
Mikula A, Olas M, Sliwinska E, Rybczynski JJ (2008) Cryopreservation by encapsulation of Gentiana spp cell suspensions maintains regrowth, embryogenic competence and DNA content. CryoLetters 29:409–418
Mikula A, Tomiczak K, Rybczynski JJ (2011) Cryopreservation enhances embryogenic capacity of Gentiana cruciata (L.) suspension culture and maintain (epi) genetic uniformity of regenerants. Plant Cell Rep 30:565–574
Murashige T, Skoog F (1962) A revised medium for the rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497
Niino T, Sakai A, Enomoto S, Magosi J, Kato S (1992) Cryopreservation of in vitro-grown shoot tips of mulberry by vitrification. CryoLetters 13:303–312
Panis B, Piette B, Swennen R (2005) Droplet vitrification of apical meristems: a cryopreservation protocol applicable to all Musaceae. Plant Sci 168:45–55
Park YS, Bonga JM (2011) Application of somatic embryogenesis in forest management and research. In: Park Y-S, Bonga JM, Park SY, Moon HK (eds) Advances in somatic embryogenesis of trees and its applications for the future forest and plantations. Proc IUFRO working party 2.09.02 conference, Suwon, Republic of Korea, pp 3–8
Park YS, Lelu-Walter MA, Harvengt L, Trontin JF, Maccacheron I, Klimaszewska K, Bonga JM (2006) Initiation of somatic embryogenesis in Pinus banksiana, P. strobus, P. pinaster, and P. sylvestris at three laboratories in Canada and France. Plant Cell Tissue Organ Cult 86:87–101
Pence VC (2014) Tissue cryopreservation for plant conservation: potential and challenges. Int J Plant Sci 175:40–45
Pinto G, Loureiro J, Lopes T, Santos C (2004) Analysis of the genetic stability of Eucalyptus globulus Labill. somatic embryos by flow cytometry. Theor Appl Genet 109:580–587
Ramon M, Geuns JMC, Swennen R, Panis B (2002) Polyamines and fatty acids in sucrose precultured banana meristems and correlation with survival rate after cryopreservation. CryoLetters 23:345–352
Reed BM (2008) Plant cryopreservation: a practical guide. Springer, New York
Ryynämen L, Aronen T (2005) Vitrification, a complementary cryopreservation method for Betula pendula Roth. Cryobiology 51:208–219
Sakai A (2000) Development of cryopreservation techniques. In: Engelmann F, Takagi H (eds) Cryopreservation of tropical plant germplasm. Int Plant Genet Resour Inst, Rome, pp 1–7
Sakai A, Engelmann F (2007) Vitrification, encapsulation-vitrification and droplet-vitrification: a review. CryoLetters 28:151–172
Sakai A, Kobayashi S, Oiyama I (1990) Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tnaka) by vitrification. Plant Cell Rep 9:30–33
San José MC, Janeiro LV, Corredoira E (2013) Micropropagation of threatened black alder. Silva Fenn 47:1–12
San José MC, Valladares S, Janeiro LV, Corredoira E (2014) Cryopreservation of in vitro-grown shoot tips of Alnus glutinosa (L.) Gaertn. Acta Physiol Plant 36:109–116
Sánchez C, Martínez MT, Vidal N, San José MC, Valladares S, Vieitez AM (2008) Preservation of Quercus robur germplasm by cryostorage of embryogenic cultures derived from mature trees and RAPD analysis of genetic stability. CryoLetters 29:493–504
Sánchez-Romero C, Sweenen R, Panis B (2009) Cryopreservation of olive embryogenic cultures. CryoLetters 30:359–372
Sant R, Panis B, Taylor M, Tyagi A (2008) Cryopreservation of shoot tips by droplet vitrification applicable to all taro (Colocasia esculenta var. esculenta) accessions. Plant Cell Tissue Organ Cult 92:107–111
Schoenweiss K, Meieer-Dinkel A, Grotha R (2005) Comparison of cryopreservation techniques for long-term storage of ash (Fraxinus excelsior L.). CryoLetters 26:201–212
Sen-Rong H, Ming-Hua Y (2012) A simple and efficient protocol for cryopreservation of embryogenic calli of the medicinal plant Anemarrhena asphodeloides Bunge by vitrification. Plant Cell Tissue Organ Cult 109:287–296
Takagi H (2000) Recent development in cryopreservation of shoot apices of tropical species. In: Engelmann F, Takagi H (eds) Cryopreservation of tropical plant germplasm. Current research progress and applications. JIRCAS/IPGRI, Rome, pp 178–193
Takagi H, Tien TN, Islam OM, Senboku T, Sakai A (1997) Cryopreservation of in vitro-grown shoot tips of taro (Colocasia esculenta (L.) Schott) by vitrification. I. Investigation of basic conditions of the vitrification procedure. Plant Cell Rep 16:594–599
Touchell DH, Chiang VL, Tsai CJ (2002) Cryopreservation of embryogenic cultures of Picea mariana (black spruce) using vitrification. Plant Cell Rep 21:118–124
Valladares S, Toribio M, Celestino C, Vieitez AM (2004) Cryopreservation of embryogenic cultures from mature Quercus robur trees using vitrification. CryoLetters 25:177–186
Vieitez AM, San José MC, Corredoira E (2011) Cryopreservation of zygotic embryo axes and somatic embryos of European chestnut. In: Thorpe TA, Yeung EC (eds) Plant embryo culture: methods and protocols, methods in molecular biology, vol 710. Springer Science + Business Media, New York, pp 201–213
Webber J, Gibbs J, Hendry S (2004) Phytophthora disease of alder. For Comm Inf Note 6. Forestry Commission, Edinburgh
Zhu GY, Geuns JMC, Dussert S, Swennen R, Panis B (2006) Change in sugar, sterol and fatty acid composition in banana meristems caused by sucrose-induced acclimation and its effect on cryopreservation. Physiol Plant 128:80–94
Acknowledgments
The authors thank Carlos Súarez and Mª José Cernadas for their technical assistance.
Author contribution statement
M.C. San José and E Corredoira designed the research, analyzed the data and wrote the paper. H. Oliveira and C. Santos performed the ploidy fidelity studies, analyzed and wrote their results. All authors have read and approved the final manuscript.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflicts of interest.
Rights and permissions
About this article
Cite this article
San José, M.d.C., Corredoira, E., Oliveira, H. et al. Cryopreservation of somatic embryos of Alnus glutinosa (L.) Gaertn. and confirmation of ploidy stability by flow cytometry. Plant Cell Tiss Organ Cult 123, 489–499 (2015). https://doi.org/10.1007/s11240-015-0853-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11240-015-0853-9