麻花秦艽休眠芽的玻璃化超低温保存及植株再生

Cryopreservation and Plantlet Regeneration of Dormant Buds of Gentiana straminea by Vitrification

目的:采用玻璃化超低温技术保存濒危药用植物麻花秦艽的休眠芽。方法:以休眠芽作试材,研究休眠芽大小、不同的预处理方法、装载时间和玻璃化保护液等因素对休眠芽超低温保存效果的影响。结果:10～11 mm大小的休眠芽在含有0.3 mol/L、0.5 mol/L、0.7 mol/L蔗糖的MS培养基中暗培养各1 d,在装载液(2 mol/L甘油+0.4 mol/L蔗糖)中20℃处理20 min,于玻璃化保护液PVS2(30%甘油+15%乙二醇+15%二甲基亚砜+0.4mol/L蔗糖)中0℃处理40 min,换新鲜PVS2保护液并迅速投入液氮,液氮中保存24 h后,在40℃水浴中解冻2～3 min,用含1.2 mol/L蔗糖的1/2MS无机盐液体培养基洗涤2次,每次10 min,无菌滤纸吸干后接种到恢复培养基中,在22℃条件下暗培养1周后转入正常条件培养,再生率高达83.3%。结论:建立了高效的麻花秦艽休眠芽玻璃化超低温保存技术体系。

Objective： To investigate the detailed techniques for cryopreservation of Gentiana straminea dormant buds by vitrifica- tion. Methods ： Dormant buds as an experimental material, the influence of the different size of dormant buds, preeuhure and PVS etc. on eryopreservation of C, entiana straminea were studied. Results： The optimal procedures were as follows： 10 - 11 mm long dormant buds which were cultured on MS medium supplemented with different sucrose concentration （0. 3,0. 5 or 0. 7 tool/L） for 1 day respective- ly. The buds were immersed in loading solution for 20 rain at 20 ~C, and then treated in PVS2 solution for 40 rain at 0 ~C and finally plunged into liquid nitrogen quickly. After 24 hours, the buds were rapidly thawed in a water bath at 40 ~C for 2 ~ 3 min and washed twice with MS medium supplemented with 1/2 MS liquid medium containing 1.2 mol/L sucrose. Finally the buds Were transferred to re- generation medium（ MS ＋ 0. 5 mg/L 6-BA ＋ 0. lmg/L NAA ＋ 3% sucrose ＋ 0. 7% agar）, the survival rate was up to 83.3%. Conclusion： A high-efficiency cryopreservation protocol of Gentiana straminea is set up.