Influence of days of culture on cryoprotectant-supplemented medium and of terminal freezing temperature on the survival of cryopreserved pea shoot tips

Abstract

Germplasm preservation, via shoot tip cryopreservation, is one method used to ensure access in the future to a broad genetic base. A method commonly used to achieve this goal is slow cooling in the presence of a cryoprotectant down to −30 or −40 °C followed by immersion in liquid nitrogen. Two factors that influence survival are days of culture on cryoprotectant-supplemented medium and the terminal temperature prior to immersion in liquid nitrogen. Pea ( Pisum sativum L. cv Early Alaska) shoot tips were cultured on solid B5 pea shoot medium containing 5% Me 2SO. The time that the shoot tips remained on the cryoprotectant-supplemented medium prior to freezing was varied (0 to 4 days). Percentage survival was evaluated at −10, −20, −30, and −40 °C, with and without immersion in liquid nitrogen, using three-way factorial analyses of variance. For shoot tips not immersed in liquid nitrogen, number of days of culture did not influence survival, whereas terminal freezing temperature did have an effect. Optimum survival was at −10 and −20 °C. For shoot tips immersed in liquid nitrogen, terminal freezing temperature had no effect, whereas number of days of culture did influence survival. The largest number of survivors was obtained after 2 days of culture. In addition, the interaction between terminal freezing temperature and exposure to liquid nitrogen was significant, indicating additional damage as a result of this step alone. That is, there was decreased survival as a result of transfer from the terminal freezing temperature to liquid nitrogen, and this damage was greater at −10 and −20 °C than at −30 and −40 °C. These results suggest that the dehydration levels at −10 and −20 °C might be very close to those at −30 or −40 °C.