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目的:建立皖贝母茎尖超低温保存体系。方法:采用玻璃化法超低温保存技术,4℃低温预处理1周,2～3 mm的茎尖经0.4 mol.L-1蔗糖MS液体培养液预处理3 d,60%PVS2装载20 min,100%PVS2冰浴脱水60 min,换入新鲜的0℃PVS2溶液后迅速投入液氮保存,40℃水浴解冻1 min后用1.2 mol.L-1蔗糖培养液洗涤2次,每次10 min,接种在恢复培养基上,20℃暗培养2周,转入正常光下培养,1个月后统计再生率。利用PCR技术检测再生植株的遗传稳定性。结果与结论:TTC法检测茎尖冻存后存活率为79.9%,在恢复培养基上茎尖再生率为52.3%。从基因组DNA检测结果表明,再生植株其遗传稳定性未发生改变。
Objective: To establish a cryopreservation system of shoot tip of Fritillary Methods: Cryopreservation with vitrification method was used. After pretreatment at 4 ℃ for 1 week, 2 ~ 3 mm stems were pretreated with 0.4 mol·L -1 sucrose MS broth for 3 days, 60% PVS2 for 20 min, 100 % PVS2 ice bath dehydration 60 min, into the fresh 0 ℃ PVS2 solution quickly put into liquid nitrogen preservation, 40 ℃ water bath thawed 1 min later with 1.2 mol.L-1 sucrose culture medium washed twice, each 10 min, inoculation On the recovery medium, dark culture was carried out at 20 ° C for 2 weeks and then transferred to normal light for culture. After 1 month, the regeneration rate was calculated. Detection of genetic stability of regenerated plants by PCR. RESULTS AND CONCLUSION: The survival rate of TTC method after cryopreservation was 79.9%. The regeneration rate of shoot tip on the recovery medium was 52.3%. The results of genomic DNA test showed that the genetic stability of regenerated plants did not change.