Optimization of Method for Potato Meristem Slow Freezing

Abstract

Cryopreservation methods of rare plant clones with vegetative propagation comprise the techniques of procurement, cooling, cryopreservation in containers with temperature of liquid nitrogen, thawing and growth renewalin nutrient solution of plant material. Cryopreservation methods for potato meristems of different species under 10% DMSO protection and freezing rate of 0.3÷0.5°C/min down to –35÷–40°C/min with following immersion into liquid nitrogen are known. Viability results for different potato species (when applying one protocol of cryopreservation) vary from 0 to 80%. Data about acclimation under low temperature conditions for potato sprouts before meristem freezing with slow freezing rates, as well as about applying cryoprotectant of propanediol series are absent. Research was aimed to evaluate the possibility for cold acclimation and propanediol application (1,2-PD) when cryopreserving “Kosyn” potato meristems. Cryopreservation object was Solanum tuberosum potato meristems of “Kosyn” species, obtained from in vitro cultured plants. Some plants in vials were exposed for 16 hrs per day without light at 8÷10°C and 8 hrs at normal light conditions and temperature for 14 days. Non-acclimated plants were exposed under normal light conditions (3000 lux), photoperiod (16 hrs day/8 hrs night) and 24-26°C temperature. Before experiment meristems were isolated and exposed in nutrient medium for 48 hrs, then for 1 hr in 1M DMSO or 1,2-PD solutions at 22±2°C. Afterwards meristems on filter paper slips, moistened with cryoprotectant solution were placed into the 1 cm3 polyethylene containers for freezing. Cooling rate at the first stage (0÷15°C) was 0.3°C/min and 1.5÷2.5°C/min for the second one (–15÷ –40°C). Initiation of crystallisation occurred at -7°C. Containers with meristems were then directly immersed into liquid nitrogen. Thawing was carried-out by immersing containers with meristems in 40°C water bath. Integrity of cryopreserved meristems was estimated to the 5th day of culturing taking into account green colour and positive dynamics in their growth. Freezing of acclimated potato meristems under DMSO protection was established to result in augmentation of their integrity in comparison with the same index in nonacclimated ones. Potato meristem acclimation when applying propanediol is not expedient. Observation during 30 days for development of cryopreserved meristems demonstrated that treatment with cryoprotectant and freezing caused a slowing down in their development in comparison with the control. Propanediol occurred to be less toxic. DMSO treatment can cause callus development.