Vitrification-cryopreservation of dormant buds of Fritillaria przewalskii Maxim. and evaluation of its histology injuries and genetic fidelity of cryo-derived plants

Abstract

Abstract Germplasm protection of an endangered plant Fritillaria przewalskii Maxim. is important to preserve genetic diversity, to store material for breeding. In the current study, A vitrifcation method was developed for cryopreserving dormant buds of F. przewalskii. Bulblets collected at July were stored at 4℃ for 3-4 months cold acclimation, 2-5 mm dormant buds dissected from the bulblets were pre-cultured on 0.5M sucrose 1/2 MS medium for 3 days. Then the dormant buds were treated with loading solution for 20 min at room temperature, dehydrated with vitrification solution 2 (PVS2) for 60 min, and finally directly plunged into liquid nitrogen. After rapid warming in water at 38°C, the dormant buds were directly plated on recovery medium without unloading. The recovery rate reached up to 93%. Successfully vitrified dormant buds developed bulblets within 5 weeks without intermediary callus formation. Freezing-thawing steps caused severe damage to the buds axis whereas cells in shoot apical meristem and leaf primordium were still intact and normal. Loading and appropriate PVS2 treatment resulted in hyperosmotic pressure leading to progressively cell plasmolysis which is beneficial to cell alive suffered ultra-low temperature. According to the morphology and the RAPD profiles of regenerated plants, no variation was found. As an superior cryopreserved material, dormant buds can assist in a faster and efficient development of new protocols or even in the creation of easy-to-use procedures.