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对红芽芋(Colocasia esculenta var.cormosus ‘Hongyayu’)茎尖的包埋玻璃化法超低温保存技术进行了研究。茎尖从培养8周的试管苗上切下并包埋成海藻酸钙凝胶珠,并在MS+3.5 mg·L~(-1)6-BA+0.5 mg·L~(-1)IBA+0.1 mg·L~(-1)GA_3+0.3 mol·L~(-1)蔗糖的液体培养基中预培养24 h,随后用2 mol·L~(-1)甘油+0.4 mol·L~(-1)蔗糖的混合物在25℃下装载30 min,并用PVS2在25℃脱水20 min后将包埋的茎尖直接投入液氮保存。保存1 d后取出材料在40℃水浴快速复温3 min后,吸去冷冻管中PVS2,并用MS+3.5 mg·L~(-1)6-BA+0.5mg·L~(-1)IBA+0.1 mg·L~(-1)GA_3+1.2 mol·L~(-1)蔗糖的液体培养基在25℃洗涤3次,每次10 min。最后将茎尖接种于MS+3.5 mg·L~(-1)6-BA+0.5 mg·L~(-1)IBA+0.1 mg·L~(-1)GA_3的固体培养基上,暗培养3 d后转入正常的光周期中培养。红芽芋茎尖冻后成活率约为80%,其再生植株没有发生形态学的变化。这种包埋玻璃化法程序有望成为红芽芽茎尖超低温保存的常规方法。
The technology of cryopreservation of the shoot tip of Colocasia esculenta var.cormosus ’Hongyayu’ was studied. The shoot tips were excised from 8-week-old test-tube seedlings and embedded in calcium alginate gel beads. MSCs were cultured in MS + 3.5 mg · L -1 6-BA + 0.5 mg · L -1 IBA Medium supplemented with 0.1 mol·L -1 GA_3 + 0.3 mol·L -1 sucrose for 24 h, then treated with 2 mol·L -1 glycerol +0.4 mol·L ~ (-1) sucrose was loaded at 25 ° C for 30 min and dehydrated with PVS2 at 25 ° C for 20 min. The embedded stem tips were directly stored in liquid nitrogen. After 1 d of storage, the material was removed and the tubes were snap frozen for 3 min in a water bath at 40 ° C. The PVS2 was aspirated off in the cryostat and the cells were treated with MS + 3.5 mg · L -1 6-BA + 0.5 mg · L -1 IBA +0.1 mg · L -1 GA 3 + 1.2 mol·L -1 sucrose was washed three times for 10 min at 25 ℃. Finally, shoot tips were inoculated on solid medium with MS + 3.5 mg · L -1 6-BA + 0.5 mg · L -1 IBA + 0.1 mg · L -1 GA 3, After 3 d into normal photoperiod culture. The survival rate of red bud taro shoots was about 80%, and the regenerated plants did not change morphologically. This embedding vitrification procedure is expected to be the conventional method for the cryopreservation of the shoot tips of red bud buds.