Evaluation of Cryogenic Procedures for Cryopreservation of Cassava Genotypes.

Cassava (Manihot esculent Crantz) is a perennial plant widely grown in many tropical countries as one of the most important staple foods. Being a vegetatively propagated crop, the application of innovative germplasm conservation is primordial. The cryopreservation in liquid nitrogen (LN2) is the best choice for long-term storage of plant germplasm. However, availability and development of simple and effective micropropagation protocol, which can supply a large number of uniform and ideal apices required for successful cryopreservation are the basic requirements. In this study, shoot tips of in vitro plantlets of two cassava genotypes were cryopreserved using the vitrification method. Mononodal microcuttings of 5 mm length with an axillary bud were taken from 2-month-old stock cultures and densely cultured on 2 different fresh media (Maintenance x Wild media) in a Petri dish 90 mm × 20 mm. After 14 days, shoot tips of 1 mm from in vitro plantlets were excised, placed on pre-culture media, supplemented with 0.3 M sucrose, and kept in growth room (protected from direct light) for 16 h. The osmo-protected shoot tips were sufficiently dehydrated with a highly concentrated vitrification solution (designated PVS2) in different time periods prior to plunging into LN2. Successfully vitrified shoot tips were rewarmed rapidly for 1 min in water bath at 45 ̊C and then plated on culture medium. These vitrified shoot tips developed shoots within 4-6 weeks after being recultured. The results showed that the best culture media, considering the growth speed and uniformity, is the Wild Cassava media, and that the exposure of shoot tips to PVS2 for more than 30 minutes makes the regeneration of the material very difficult. Preliminary results on the adjustments of vitrification and encapsulation methods are reported.