Droplet-vitrification cryopreservation of in vitro-cultured shoot tips of apple rootstock
The orthogonal experiment was designed to obtain the optimal cryopreservation system for in vi-tro-cultured shoot tips of apple rootstock'54-118'based on the main factors involved in the process of cryopres-ervation by droplet-vitriflication.The results showed that cold acclimation and the treatment time of plant vitrifi-cation protection solution 2(PVS2)were key factors affecting the survival rate and regeneration rate of cryopreserved shoot tips of apple rootstock.An optimized droplet-vitrifiication cryopreservation system was de-veloped with the following parameters:use of 30 d in vitro plants,10 d acclimatization at 4℃,2 d preculture,20 min LS treatment,60 min PVS2 treatment,exposure to liquid nitrogen by droplet-vitrification.Frozen shoot-tips were treated in unloading solution(ULS)for 20 min(room temperature)and then post-cultured on a shoot tip re-generation medium for shoot regrowth under the normal culture condition after 7 d in the dark.Inter-simple se-quence repeats(ISSR)analyses showed no any polymorphic loci in the plants from cryopreserved shoot tips compared to the original cultures.This protocol was successfully applied to three other apple rootstocks and the average survival rate and regeneration rate of the four apple rootstocks were74.58％and 64.81％,respectively.
apple rootstockshoot tipscryopreservationdroplet-vitrification
NETL
NSTL
万方数据
