Vitrification-cryopreservation of Shoot Tips Excised from Dormant Bulbs in Fritillaria przewalskii Maxim: Assessing Histological lnjuries and Evaluating Genetic Fidelity in Cryo-Derived Plants

Fritillaria przewalskii Maxim. an essential medicinal plant in the Fritillaria genus of the Liliaceae family, is widely used in traditional medicine. This study focused on developing a cryopreservation protocol for F. przewalskii , an endangered and endemic species in China. We aimed to determine if ex vitro bulbs from field-grown F. przewalskii plants could serve a source of shoot tips for cryopreservation. Key factors influencing cryopreservation outcomes were studied using a vitrification-based approach. Prior to cryopreservation, cold hardening and sucrose preculture conditions were optimized. The best results were achieved by hardening bulbs at 4℃ for 3–4 months,followed by surface disinfection and isolation of 2–5 mm Shoot tips. The tips were then then precultured on a medium with 0.5M sucrose for 3 days. Cryopreservation steps included a 20-minute loading treatment, 60-minute PVS2 dehydration at room temperature, and storage in liquid nitrogen more than one hour. After thawing, this protocol resulted in 93% shoot survival rate, with tips regenerating into small bulbs within 5 weeks without intermediary callus formation. Histological analysis showed that while cooling and rewarming caused severe bud damage, apical meristem cells are the primary survival sites. Although loading and PVS2 treatment induced cell plasmolysis, it was not lethal. Optimal PVS2 treatment enhanced cellular dehydration resistance, promoting cell viability at ultralow temperature. RAPD analysis of regenepercentaged plants showed no genetic variation, confirming strong genetic stability. The direct use of ex vitro shoot tips provides a straightforward and effective cryopreservation method for F. przewalskii , combining convenience, efficiency, and genetic stability.