Unifying principles of cryopreservation protocols for new plant materials based on alternative cryoprotective agents (cpas) and a systematic approach

This review addresses a frequently encountered problem of designing an effective cryopreservation procedure for new (not previously cryopreserved) or difficult plant materials. This problem hinders worldwide efforts of applying cryopreservation across a wide genetic base of wild and a number of cultivated plants. We review recent advances in modifications of routinely applied cryoprotective solutions (CPAs) and suggest a practical approach to protocol development which embraces the physiological complexity of plant tissues as well as a wide spectrum of behaviours under CPA treatment. We suggest that vegetative plant materials are classified into four categories based on their size, structure, and the response to osmotic and chemical stresses provoked by CPA mixtures of varied composition and concentration, including alternative osmoprotection and vitrification solutions. A number of up to 15 preset protocols designed specifically for each category is then applied to the material. The protocols resulting in the best regrowth are then combined into the optimized procedure. The main advantage of this system over a conventional "trial-and-error" search for working cryopreservation protocol is a minimal amount of starting materials required for the tests and a relatively accurate prediction of material behaviour under cryopreservation stress provided by the relatively few CPAs treatments. The unifying principles revealed by this approach could broaden a spectrum of wild species and materials which can be safely conserved by cryopreservation. Also anticipated is application of this approach to plant materials of biotechnological value as well as cultivars of agricultural and horticultural crops which do not respond well to standard protocols developed for their kind.