Author: Dr. Vartika Srivastava
Scientist, Tissue Culture and Cryopreservation Unit
ICAR-NBPGR, Pusa Campus, New Delhi
Conservation of plant genetic resources involves two basic approaches viz. ex situ and in situ conservation. Ex situ conservation includes seed storage, in vitro storage, DNA storage, pollen storage, field genebanks and botanical gardens, while in situ approach encompasses genetic reserves, on farm and home garden conservation. The ex-situ conservation is the most widely adopted method for the preservation of germplasm obtained from cultivated and wild plant materials. The genetic materials in the form of seeds or from in vitro cultures (plant cells, tissues or organs) can be preserved either in seed gene banks, in vitro genebanks or in cryobanks for medium to long term storage under suitable conditions. For successful establishment of gene banks, adequate knowledge of genetic structure of plant populations, and the techniques involved in sampling, regeneration, maintenance of gene pools etc. are essential.
Short and medium term conservation technique:
Those plant species which produce orthodox seeds, i.e which can withstand extensive dehydration and stored dry at low temperature (Roberts, 1973), the emphasis is given to seed storage in genebank with appropriate moisture levels at lower temperature. However, three categories of crop viz., plants not producing seeds or vegetatively propagated, plants producing recalcitrant seeds and intermediate types show problem with seed storage and need to be conserved through alternative strategy. Recalcitrant and intermediate seeds, together termed non-orthodox, cannot be dried below critical moisture content (10 to 40%) and cannot withstand chilling temperatures without substantial loss in viability. Yet another method to define them is in relation to the seed’s tolerance of dehydration across water sorption regions and appropriate temperature for storage. Seed longevity of intermediate seeds ranges from few months to years while recalcitrant seeds from weeks to months. Cryopreservation currently offers the only safe and cost-effective option for the long-term conservation of genetic resources of these problem species.
In vitro genebanks:
Tissue culture technique is mainly deployed for short and medium term storage of vegetatively propagated plant germplasm. It allows propagating plant material with high multiplication rates aseptically. Various viruses and seed borne diseases can be eliminated through meristem culture in combination with thermotherapy, thus ensuring the production of disease-free stocks. In vitro propagation protocols have been established for several thousand plant species. Different in vitro conservation methods are employed, depending on the storage duration. For short- and medium-term storage, the aim is to reduce growth and to increase the intervals between subcultures. For long-term storage, cryopreservation, i.e. storage at ultra-low temperature, usually that of liquid nitrogen (-196°C), is the only current method. At this temperature, all cellular divisions and metabolic processes are stopped.
Long term conservation technique:
Cryopreservation, is a technique in which the biological material is stored at ultra-low temperature of liquid nitrogen (-196˚C). The principle involved in cryopreservation is to bring the plant cell and tissue cultures to a zero metabolism or non-dividing state at ultra low temperature. It is the only method currently available to ensure the safe and cost-effective long-term conservation of genetic resources especially that have recalcitrant and intermediate seeds or are vegetatively propagated.
Cryopreservation of vegetatively propagated species:
Most of the experimental systems viz., cell suspensions, calluses, shoot-tips, embryos etc which employed in cryopreservation, contain high amount of cellular water and are thus extremely sensitive to freezing injury. Such tissues must be dehydrated artificially to protect them from the damages caused by the crystallization of intracellular water in to ice (Meryman and Williams, 1985; Mazur, 1984). Most of the vegetatively propagated species are cryopreserved using vitrification technique. Vitrification involves treatment of biological tissues with cryoprotective substances, dehydration with highly concentrated vitrification solutions, rapid freezing and thawing followed by removal of cryoprotectants and recovery. The shoot-apices are the most widely used explants for vitrification as these can be regenerated directly in to a whole plant without any significant loss in viability. The survival rates obtained are generally high to very high up to 100% viz., in Allium, yam and potato.
Cryopreservation of recalcitrant seed species:
Seeds of many fruit species are of very large size (e.g. mango, jackfruit, litchi) to be frozen directly, and hence embryos or embryonic axes and plumules are successfully employed for cryopreservation (Chaudhury, 2000). However, embryos are often of very complex tissue composition and are extremely sensitive to desiccation. There are various options to consider for improving storage of non-orthodox seeds. Precise controlled desiccation (e.g. using saturated salt solutions) and cooling conditions allow to freeze whole seeds, e.g. papaya, grape and guava. Pregrowth on media containing cryoprotective substances may confer the tissues increased tolerance to further desiccation and to reduce the heterogeneity of the material. Flash fast drying, followed by ultra-rapid freezing, has also been very effective for cryopreservation of several species. It is highly important to select embryos at the right developmental stage for the success of any cryopreservation experiment.
Retesting of viability
Post-cryopreservation handling procedures and regeneration protocols need also be fully standardized for seeds, embryos, embryonic axes, in vitro meristems and readily available to ensure high survival rates. It is essential to monitor the viability of explants after regular intervals to ensure that no deterioration is there over the time.
Additional uses of cryopreservation
Recently, the benefit of cryopreservation has been identified in elimination of many plant diseases caused by viruses. In cryotherapy, many pathogens infecting plants, such as viruses, phytoplasmas and bacteria are eradicated by brief exposure of the shoot tips to liquid nitrogen. Thermotherapy followed by cryotherapy of shoot tips can be used to enhance virus eradication. Cryotherapy of shoot tips is easy to implement. It allows treatment of large samples and results in a high frequency of pathogen-free regenerants. Difficulties related to excision and regeneration of small meristems is largely circumvented in shoot tip cryotherapy. To date, severe pathogens in banana (Musa spp.), Citrus spp., grapevine (Vitis vinifera), Prunus spp., raspberry (Rubus idaeus), potato (Solanum tuberosum ) and sweet potato (Ipomoea batatas) have been eradicated using cryotherapy.
Suggested readings / References
- Chaudhury, R. 2000. Cryopreservation of seeds, embryos, embryonic axes and pollen at National cryobank of NBPGR. In: Engelmann F and Takagi H (eds) Cryopreservation of tropical Plant Germplasm – Current Research Progress and Application. IPGRI, Rome and JIRCAS, Tsukuba, Japan. Pp. 457-459.
- Engelmann, F. (2004). Plant cryopreservation: progress & prospects. In Vitro Cell. Dev. Biol.- Plant 40: 4: 27-43.
- Mazur, P. 1984. Freezing of living cells: mechanisms and applications. American Journal of Physiology 247, Cell Physiology 16, C125-142.
- Meryman, H.T. and R.J. Williams. 1985. Basic principles of freezing injury to plant cells: Natural tolerance and approaches to cryopreservation. Pp. 13-47 in Kartha, K.K. (Ed.) Cryopreservation of Plant Cells and Organs. CRC Press, Boca Ration, Florida.
- Roberts,H.F. 1973. Predicting the viability of seeds. Seed Science and Technology, 499-514.
About Author / Additional Info:
Currently working as Scientist in Tissue Culture and Cryopreservation Unit of ICAR-NBPGR