Cryobiology, as field of science, has evolved over the years, aided by technological and methodical advances, mainstreaming cryopreservation as the preferred method of germplasm conservation in a large number of genebanks worldwide. Cryopreservation had found its roots in the 17th century (Boyle, 1665), however this field gained momentum in the 1940s and beyond. Storage of biological material for extended lengths of time at ultra low temperatures (−196°C) in liquid nitrogen, in a state of suspended animation, such that all metabolic and cellular processes cease, is known as cryopreservation. Conventional method of plant germplasm storage involves storing desiccated seeds at low temperature (−18°C to −20°C) in seed banks. However, seeds of some plant species are sensitive to drying and do not survive on moisture reduction. Such seeds are known as 'non-orthodox seeds' and cannot be stored by the conventional methods of seed storage. Storage of these species requires the application of alternate strategies that involve biotechnological techniques of micropropagation along with cryopreservation to conserve them in liquid nitrogen. These techniques have been collectively referred to as cryobiotechnology (Pritchard, 2018; Pence et al., 2020).  

Most of the tropical crops, trees as well as recalcitrant fruit species, produce recalcitrant seeds which cannot be stored at −20°C after desiccation. Along with these, there are several other plant species which produce seeds that withstand drying but donot survive for long at −20°C.  Apart from these, there are plant species which are propagated vegetatively and not by seeds. Also, valuable crop wild relatives needed robust conservation strategies. All of these plant species can be stored and conserved for long term using the approaches of cryobiotechnology. Cryobiotechnology has emerged as a cost-effective, safe and workable tool for seeded and non-seed tissues.

Cryopreservation techniques work on three basic principles : 

1) initial partial drying to reduce the water content of tissues, 

2) using cryoprotectants to remove water from cells osmotically and depressing the freezing properties of the remaining essential water in the cells and 

3) ultra rapid cooling and warming of cells to avoid ice crystallization. 

The aim is to avoid ice crystal formation in the meristematic cells and is achieved by forming an amorphous glassy state of water on cooling by a process called as vitrification. Various cryopreservation techniques have evolved over the course of time, each one with some technolgical advancement over the previous. The techniques include -

   i. Controlled rate freezing

   ii. Encapsulation-dehydration

  iii. Vitrification

  iv. Encapsulation-vitrification

  v. Droplet-vitrification

 vi. V-cryo plate method

vii. D-cryo plate method

Each of these methods have their own pros and cons and together they have been used to conserve different propagules, ranging from dormant buds, seeds, embryos, embryonic axes, meristems, shoot tips and pollen of a large of plant species.

The recent developments in systems biology and omics technologies are now being used to reveal the underlying mechanisms of cold stress tolerance along with the mechanisms of survival of tissues on desiccation and low temperature exposure. Changes in patterns of expression of cold responsive genes and variation in protein profiles of cryopreserved and normal plants are being explored to better understand the molecular regulation of survival and growth of conserved plants (González-Arnao et al., 2011; Volk et al., 2011; Gross et al., 2017). Also, efforts are being made to understand the structural changes that occur in cells on dehydration and low temperature exposure, in a bid to better condition the cells for low temperature storage.

Cryobiotechnology is being applied by researchers the world over for conserving valuable plant germplasm. This is important for long term ex situ conservation of plants, so as to secure threatened plant species and ensure the creation of a back up of the global plant diversity.

References

Boyle R (1665) New experiments and observations touching cold. Royal Society of London.

González-Arnao MT, Durán-Sánchez B, Jiménez- Francisco B, Lázaro-Vallejo CE, Valdés-Rodríguez SE and Guerrero A (2011) Cryopreservation and proteomic analysis of vanilla (V. planifolia A.) apices treated with osmoprotectants. Acta Horticulturae 908: 67–72.

Gross BL, Henk AD, Bonnart R and Volk GM (2017) Changes in transcript expression patterns as a result of cryoprotectant treatment and liquid nitrogen exposure in Arabidopsis shoot tips. Plant Cell Reports 36: 459–470.

Pence VC, Ballesteros D, Walters C, Reed BM, Philpott M, Dixon KW, Pritchard HW, Culley TM, Vanhovea AC (2020) Cryobiotechnologies: tools for expanding long-term ex situ conservation to all plant species. Biological Conservation 250: 108736.

Pritchard HW. 2018. The rise of plant cryobiotechnology and demise of plant cryopreservation? Cryobiology 85: 160-161

Volk GM, Henk AD and Chhandak B (2011) Gene expression in response to cryoprotectant and liquid nitrogen exposure in Arabidopsis shoot tips. Acta Horticulturae 908: 55–66.