玻璃化法超低温保存柑桔茎尖及植株再生

Cryopreservation of Citrus Shoot-tips by Vitrification and Regeneration

采用玻璃化法对柑桔茎尖的离体超低温保存进行了研究。约 10mm长的柑桔茎尖于含 5 %二甲基亚砜 (DMSO)的培养基上预培养 3d ,切取 2～ 2 .5mm长的茎尖 ,室温下6 0 %玻璃化溶液 2 (PVS2 )装载 2 0～ 30min ,然后用PVS2 于 0℃处理 5 0～ 6 0min ,换入新鲜的PVS2 ,迅速投入液氮中 ,2 4h后在 4 0℃水浴中迅速化冻 ,再用 1.2mol/L蔗糖培养基洗涤 2次 ,接种于含BA 1.0mg/L的MT培养基上 ,2 6℃暗培养 1周后转于正常光下培养。枳壳茎尖超低温保存后用氯化三苯基四氮唑 (TTC)法检测 ,成活率为 10 0 % ,培养再生率达到 90 % ,再生后的苗能正常生根 ,与对照没有形态上的变异 。

A procedure had been studied on cryopreservation of citrus shoot tips in vitro by vitrification.Shoot tips of citrus about 10mm were precultured for 3 days in MT medium supplemented with 5% DMSO.The excised shoot tips,2-2.5mm in length,were loaded with 60% PVS 2 for 20-30min at room temperature,and were exposed to PVS 2 at 0℃ for 50-60min.Followed by changing the solution with fresh PVS 2,the shoot tips were immersed into LN directly,and kept for 24h.After rapid thawing in a water bath at 40℃,the shoot tips were washed with 1.2mol/L sucrose solution for twice and transferred onto MT medium supplemented with BA 1.0mg/L.The cultures were kept in dark for one week prior to exposure to the light.Survival rate of shoot tips of Poncirus trifoliata was 100% by TTC examination,and regeneration rate reached to 90%.The plantlets rooted normally and survived after transplantion.No morphological difference between the regenerated plantlets and the control was observed.