Cryopreservation of rhizome buds of Asparagus officinalis L. (cv. Morado de Huétor) and evaluation of their genetic stability
We describe an encapsulation–dehydration procedure with prefreezing steps for the cryopreservation of rhizome bud explants of Asparagus officinalis L. cv. Morado de Huétor. With this procedure, survival of Rhizome buds was at least 84 and 42% developed to complete plantlets at 8 weeks. Flow cytometry and EST-SSR molecular markers were used to assess genetic stability of the regenerated material. Effects of preculture time in a medium rich in sucrose and prefreezing treatments (0 °C or/and − 20 °C) on plant recovery were evaluated. Rhizome Buds of the “Morado de Huétor” landrace were incubated in preculture medium (MS + 0.3 M sucrose) for 48 h, encapsulated in alginate beads and desiccated until a water content of 35%, prefrozen for one hour at 0 °C plus one hour at − 20 °C, followed by cryopreservation in liquid nitrogen, and then were rewarmed and recovered in ARBM medium for 6 weeks and finally incubated in ARBM-0 for 4 weeks. Analyses of ploidy and molecular stability of plantlets recovered from cryopreserved rhizome buds of two selected genotypes showed no differences compared with the mother plants. Cryopreservation of RB explants of A. officinalis with this Encapsulation–Dehydration procedure will be useful in long-term preservation programs.
KeywordsEncapsulation–dehydration Asparagus Rhizome bud Cryopreservation Molecular markers Flow cytometry
All authors conceived and planned the experiments. E.C.M. performed the experiments and wrote the manuscript with input from all authors.
Compliance with ethical standards
Conflict of interest
The authors declare that the research review was conducted in the abscense of any commercial or financial relationships that could be construed as a potential conflict of interest.
- Dereuddre J, Scottez C, Arnaud Y, Duron M (1990) Effects of cold hardening on cryopreservation of axillary pear (Pyrus communisL. cv. Beurre Hardy) in vitroplantlets to dehydration and subsequent freezing in liquid nitrogen: effects of previous cold hardening. CR Acad Sci Paris 310, Ser III: 317–323Google Scholar
- Fabre J, Dereuddre J (1990) Encapsulation–dehydration: a new approach to cryopreservation of Solanum shoots tips. Cryo Lett 11:413–426Google Scholar
- Harding K (1997) Stability of the ribosomal RNA genes in Solanum tuberosum L. plants recovered from cryopreservation. Cryo Lett 18:217–230Google Scholar
- Harding K (2004) Genetic integrity of cryopreserved plant cells: a review. Cryo Lett 25:3–22Google Scholar
- Jitsuyama Y, Suzuki T, Harada T, Fujikawa S (2002) Sucrose incubation increases freezing tolerance of Asparagus (Asparagus officinalisL.) embryogenic cell suspensions. Cryo Lett 23:103–112Google Scholar
- Kumu Y, Harada T, Yakuwa T (1983) Development of a whole plant from a shoot tip of Asparagus officinalisL. frozen down to –196 °C. J Fac Agric Hokkaido Univ 61(3):285–294Google Scholar
- Liu YG, Liu LX, Wang L, Gao AY (2008) Determination of genetic stability in surviving apple shoots following cryopreservation by vitrification. Cryo Lett 29:7–14Google Scholar
- Nishizawa S, Sakai A, Amano Y, Matsuzawa T (1992) Cryopreservation of Asparagus(Asparagus officinalisL.) embryogenic cells and subsequent plant regeneration by a simple freezing method. Cryo Lett 13:379–388Google Scholar
- Panis B, Lambardi M (2005) Status of cryopreservation technologies in plants (crops and forest trees). In: The role of biotechnology for the characterization and conservation of crop, forest,animal and fishery genetic resources in developing countries. FAO, Turin, Italy, pp 43–54Google Scholar
- Reed BM (1996) Pretreatment strategies for the cryopreservation of plant tissues. In: Normah MN, Narimah MK, Clyde NM (eds) In Vitroconservation of plant genetic resources. Plant Biotechnology Laboratory, Faculty of Life Sciences, University Kebangsaan, BangiGoogle Scholar
- Regalado JJ, Carmona-Martín E, Castro P, Moreno R, Gil J, Encina CL (2015a) Micropropagation of wild species of the genus AsparagusL. and their interspecific hybrids with cultivated A. officinalisL., and verification of genetic stability using EST-SSRs. Plant Cell Tissue Organ Cult 121:501–510CrossRefGoogle Scholar
- Sakai A, Matsumoto T, Hirai D, Niino T (2000) Newly developed encapsulation-dehydration protocol for plant cryopreservation. Cryo Lett 21:53–62Google Scholar
- Yang HJ, Cloré WJ (1974) Development of complet plantlets from moderately vigorous shoot of stocks of Asparagusin vitro. HortScience 9:138–139Google Scholar