Cryopreservation of coconut plumule using droplet vitrification

Abstract

In the present investigation, four types of explants from mature zygotic embryos of coconut, viz., whole upper cotyledonary region without haustorium, half of the upper cotyledonary region without haustorium, plumule with a portion of radicle and exclusively plumular tissue, were cultured in 12 different media combinations to find a suitable explant which could be regenerated after cryopreservation. Explants were pre-cultured in medium with 0.4 and 0.5 M sucrose for three days followed by dehydration in PVS3 solution for different durations on a sterile aluminum strip after treating with loading solution. Strips were treated with liquid nitrogen inside a cryoflask until bubbling stopped and quickly transferred to a cryovial and stored for a minimum period of 24 hours in liquid nitrogen. It was observed that plumule alone or with a small portion of outer tissue was ideal for fast in vitro growth and recovery of whole plantlets of coconut in a medium supplemented with NAA alone. Addition of glutamine (5 mg L-1), TDZ (1 mg L-1) and NAA (18 mg L-1) aided the vigorous growth of plantlets. In control, the survival rate ranged from 60 to 90 per cent in plumule pre-grown in media containing 0.5 M sucrose after dehydration with PVS3 for various durations, whereas it was 14 to 75 per cent in cryopreserved ones. Considering the high survival (75%) and regrowth (35%) of cryopreserved plumule in the present study, there is much scope for further improvement of the procedure to find the right combination of factors so as to enhance complete recovery of plantlets without much injury to plumules during cooling and rewarming.