Optimization of Droplet-Vitrification Protocol for the Endemic Endangered Species Amygdalus ledebouriana and Confirmation of Genetic Stability

Abstract

Amygdalus ledebouriana (Schltdl.) Y.Y. Yao is a rare, endangered wild almond species native to Eastern Kazakhstan, listed in both Kazakhstan’s Red Book and the IUCN Red List, and valued as a source of drought- and frost-tolerance genes for stone fruit breeding. This study developed an efficient droplet-vitrification cryopreservation protocol for A. ledebouriana shoot tips and assessed regenerant genetic stability using simple sequence repeat (SSR) markers. The protocol comprised five stages: preculture, osmotic conditioning (loading), vitrification with plant vitrification solutions 2 and 3 (PVS2/PVS3), liquid nitrogen storage, and unloading/regeneration. Each stage was optimized by stepwise variation of a single factor while holding the remaining conditions constant, using generalized linear models (quasi-binomial distribution, Holm-corrected post hoc comparisons). Optimal conditions included preculture on 0.75 M sucrose for 3 days (31.1% survival), 30 min loading (38.9%), and 120 min dehydration with PVS3, which significantly outperformed PVS2 in terms of shoot tip survival after cryopreservation (50.0% vs. 30.0%). Unloading with 0.8 M sucrose under mild conditions (22 °C, 20 min) and recovery medium supplemented with ascorbic acid achieved the highest survival (61.1%) and regrowth (60.0%). SSR analysis at five loci revealed no genetic changes in regenerants following cryopreservation. The developed protocol is the first reported for A. ledebouriana and can be recommended for the long-term ex situ conservation of its genetic resources.