论文部分内容阅读
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 preserve the dormant buds of Gentiana macrophylla, an endangered medicinal plant, by vitrification cryogenic technique. Methods: The dormant buds were used as test materials to study the effects of sleeping bud size, different pretreatment methods, loading time and vitrification solution on cryopreservation of dormant buds. Results: The dormant buds of 10 ~ 11 mm in diameter were cultured in MS medium containing 0.3 mol / L, 0.5 mol / L and 0.7 mol / L sucrose for 1 d each in the medium (2 mol / L glycerol + 0.4 mol / L sucrose) at 20 ℃ for 20 min and treated with PVS2 (30% glycerol + 15% ethylene glycol + 15% dimethylsulfoxide + 0.4 mol / L sucrose) Fresh PVS2 protective solution and quickly put into liquid nitrogen, stored in liquid nitrogen for 24 h, thawed in a water bath at 40 ℃ for 2 ~ 3 min, washed twice with 1 / 2MS inorganic salt liquid medium containing 1.2 mol / L sucrose, 10 min, sterile filter paper was inoculated into the recovery medium, dark culture at 22 ℃ for 1 week and then transferred to normal conditions, the regeneration rate as high as 83.3%. Conclusion: An efficient vitrification cryopreservation technique system for dormant bud of Gentiana macrophylla was established.