The word ‘cryo’ is a derivative of cryogenic which is defined as very low temperatures. Cryopreservation refers to stabilizing cells at cryogenic temperatures, usually below -100°C. Cryopreservation reduces cell and tissue deterioration in storage by virtually halting metabolism and hence is an important tool for non-lethal storage of biological materials. It ensures theoretically ‘infinite’ periods of storage (long-term conservation), and reportedly causes no change in viability, vigour and genetic makeup of the conserved materials. The low temperatures are created by use of cryogen like liquid nitrogen in liquid (-196°C) or in vapour (-150°C to -180°C) phase in double walled Cryotanks without depending on mechanical system and electricity.
Seeds
Based on the seed storage characteristics, seeds have been classified as orthodox, intermediate and recalcitrant. Orthodox/desiccation tolerant are those seeds which are capable of retaining viability after being dried to less that about 5% moisture content. Seeds of most common agricultural species belong to this category and can be easily cryopreserved.
Difficult-to-store seeds
Non-orthodox seed species comprising intermediate and recalcitrant seeds are large sized and are shed with high moisture levels and are desiccation- and freeze-intolerant. They lose viability after being dried below a critical limit usually between 12-30% moisture. With advancement in research it has been possible to successfully cryopreserve zygotic embryos and embryonic axes of intermediate and recalcitrant seeds using new techniques of cryopreservation. The embryonic axes excised from the large seed is the preferred explant because of its organized small structure, independent identity and the presence of appreciable proportion of meristematic tissues. The aseptically excised embryonic axes are desiccated to around 11-16% moisture level using air drying (desiccation method) or by addition of cryoprotectants (pregrowth-desiccation and vitrification method) before exposing to temperatures of liquid nitrogen (-196°C). The axes are later thawed rapidly in a water bath maintained at +38°C and cultured on a defined media to obtain complete plants.
At National Cryogenebank at NBPGR cryopreservation of intermediate and recalcitrant seed species using zygotic embryos and embryonic axes has been successfully achieved for almond (Prunus amygdalus), Citrus sp., neem (Azadirachta indica), jackfruit (Artocarpus heterophyllus), Litchi (Litchi chinensis), trifoliate orange (Poncirus trifoliata), black pepper (Piper nigrum), cardamom (Elettaria cardamomum) and Wild banana (Musa balbisiana) with high percentage of recovery. In addition, cryopreservation of pollen from various crop species of importance to plant breeders has been carried out. Cryopreservation of orthodox seed species representing diverse group of crops and having special attributes and of dormant buds of mulberry has been carried out.
The goal of a Cryogenebank is to collect, store and disseminate specimens and related data. In order to provide high quality plant germplasm/ materials with well-characterized data, the management team must insure that they follow ‘best practices’. Several factors have been considered in the design and development of a Cryobank which are published in a Manual.