Cryopreservation of germplasm through encapsulation-dehydration technique
2014
Gupta, S.
A range of cryopreservation techniques for conserving plant cells and tissues have been developed and tested in the last four decades and thus significantly progressed routine storage of plant germplasm in liquid nitrogen. Protocols are available to cryopreserve in vitro derived explants such as shoot tips, meristems, somatic embryos, hairy roots, cell suspensions in various plant species. Amongst the available protocols, cryopreservation of shoot apices is the most common method for long-term ex situ conservation of clonally propagated plants, which is increasingly being used to conserve germplasm of various herbaceous and woody plant species. The commonly applied vitrification-based cryopreservation techniques include vitrification, encapsulation-dehydration, encapsulation-vitrification and droplet-vitrification. Vitrification involves the application of cryoprotectant solutions that increase cell viscosity to a critical point at which water forms a meta-stable glass on exposure to ultra low temperatures while encapsulation-dehydration involves removing cell water through osmotic and evaporative dehydration to achieve the same state. The encapsulation-dehydration procedure is based on the technology developed for the production of artificial seeds and established to cryopreserve potato shoot tips for the first time. Explants are encapsulated in alginate beads, pregrown in sucrose-enriched medium for 1-7 days, partially desiccated in the air current of a laminar airflow cabinet or with silica gel to get moisture content around 20% on the fresh weight basis, then frozen rapidly. Recovery of cryopreserved samples is generally rapid and direct, without callus formation. This technique has been applied to apices of numerous species from temperate and tropical origin for example, dicots: apple, pear, blackberry, raspberry, mulberry, Eucalyptus, Melia, Robinia, mint; monocots: Cynodon, Zoysia and Lolium grasses, sugarcane, yam, lily and banana; and to cell suspensions and somatic embryos of several species. Using encapsulation-dehydration technique is advantageous because no special equipment is needed, and because of the use of nontoxic cryoprotectant. The disadvantage include requirement of handling each bead several times, and some plants may not tolerate the high sucrose concentrations. We have cryopreserved wide range of germplasm of Morus, Pyrus, Rubus using encapsulation-dehydration technique in the In Vitro Genebank at National Bureau of Plant Genetic Resources, New Delhi, India. The paper discusses various aspects of cryobanking of in vitro-grown shoot tip explants using encapsulation-dehydration technique.
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