Cryopreservation and genetic stability assessment of regenerants of the critically endangered medicinal plant Dioscorea deltoidea Wall. ex Griseb. for cryobanking of germplasm

Dioscorea deltoidea Wall. ex Griseb. is a critically endangered, commercially important crop of high medicinal value. Several accessions of this valuable plant are conserved as slow growth cultures in the in vitro gene bank of Indian Council of Agricultural Research-National Bureau of Plant Genetic Resources (ICAR-NBPGR), New Delhi, India. The purpose of this investigation was to develop a cryopreservation protocol that can be used for long-term conservation of 15 different D. deltoidea accessions. The vitrification technique of cryopreservation was compared to the droplet-vitrification technique. Shoot meristems excised from 4-wk-old in vitro plantlets were precultured on Murashige and Skoog (MS) medium containing 0.3 M sucrose for 16 h, treated with a loading solution for 20 min, dehydrated with plant vitrification solution (PVS2) for 90 min, and cryoconserved using both vitrification and droplet-vitrification protocols. Two-way ANOVA data subjected to post hoc Sidak's multiple comparisons test (P <= 0.05) indicated that shoot regrowth after LN treatment, using vitrification (30 to 54%) and droplet-vitrification (21 to 51%) techniques, was statistically comparable in 66.7% accessions, while in 33.3% accessions vitrification gave significantly better results. Genetic stability of plants cryopreserved using both the protocols was confirmed using 39 Inter-Simple Sequence Repeat (ISSR) and 30 Expressed Sequence Tag-derived Simple Sequence Repeat (EST-SSR) markers. Results showed that there was no significant difference between mother plants and cryopreserved samples. The vitrification protocol has been implemented for long-term cryobanking of D. deltoidea germplasm (15 accessions), due to improved regrowth and ease of sample handling as compared to droplet-vitrification.

Automated summary

A cryopreservation protocol was developed for long-term conservation of D. deltoidea germplasm, and vitrification technique showed better results compared to droplet-vitrification. Genetic stability of cryopreserved plants using both protocols was confirmed.