Vitrification-based shoot tip cryopreservation of Carica papaya and a wild relative Vasconcellea pubescens

Abstract

This paper reports on the effects of pre- and post-liquid nitrogen modifications to a previously published protocol for vitrification-based cryopreservation of papaya (Carica papaya L.) shoot tips. The aim was to improve the protocol for application across a wider range of papaya genotypes. Results showed that recovery from cryopreservation is genotype dependent, but the post-subculture age of the shoot tips was not significant in the two genotypes tested. Pre-culture for 2 days gave greater recovery than that for 0, 1 or 4 days. The duration and temperature of exposure to plant vitrification solution 2 (PVS2) had the most significant impact, with optimal recovery of 60 and 64% with 10 min at room temperature or 20 min at 0°C, respectively. Exposure to PVS2 for greater than 30 min reduced recovery to below 20%. Post-cryopreservation recovery was highest in media containing 1.0 µm 6-benzylaminopurine (BAP) or a combination of BAP and gibberellic acid (GA3) (1.0 and 0.5 µm, respectively). Incubation in the dark for the first 24–48 h had no significant effect on recovery. A refined protocol was developed based on these results and application of this protocol proved to be effective across seven papaya genotypes and one related species, Vasconcellea pubescens V.M.Badillo. Genotypes previously showing no survival on an unrefined protocol recovered using this revised protocol.