论文部分内容阅读
玻璃化法作为简单方便的超低温保存方法之一 ,目前已成功地应用于植物茎尖、胚性细胞、原生质体、悬浮细胞等多种单一组织或细胞培养体系 ,但尚未见对幼苗这种复合组织的保存报道 .本文研究了拟南芥 (Arabidopsisthaliana ,Landsbergerecta)幼苗的玻璃化保存程序并发现了一些有价值的研究现象 .幼苗来自萌发的种子 ,种子消毒后播种在MS培养基上 ,4℃处理 4 8h后转入 2 5℃ ,10h光周期 ,萌发不同天数为实验材料 .玻璃化程序包括 :1)预处理———用 2M甘油 +0 4M蔗糖在 2 0℃处理2 0min ;2 )脱水处理———用玻璃液PVS2 (30 %w/v蔗糖 +15%w/v乙二醇 +15%w/v二甲基亚砜 +含 0 4M蔗糖的MS) 0℃处理 50min ;3)冷冻融化———玻璃液及幼苗一起投入液氮 (LN2 )保存 ,2 0℃室温自然融化 ;4 )置换———用MS +1 2M蔗糖 2 0℃处理 30min ,成活力检查 ;幼苗转到MS培养基培养5天 ,观察再生长状况 .10天后转到培养土中 ,在生长箱中长到结实 .结果表明 :1)使用完整的程序 ,冻融后成活率最高 ,为 76 5% ;玻璃液处理是成功的关键 ,省去此处理成活率为 0 ;冻融是造成玻璃化保存成活率降低的主要因子 ;2 )由萌发 0～ 2天的种子形成的幼苗便使用该技术 ,冻存融化后成活率分别为 88% ,83 8%和 76 5% ,结实正常 ;3)
As one of the simple and convenient cryopreservation methods, vitrification has been successfully applied to a variety of single tissue or cell culture systems such as plant shoot tips, embryogenic cells, protoplasts and suspension cells, In this paper, we studied the vitrification of Arabidopsis thaliana (Landsbergerecta) seedlings and found some valuable researches.These seedlings were from germinated seeds, and the seeds were sterilized and sown on MS medium at 4 ℃ After 48 h of treatment, the samples were transferred to 25 ℃ and 10h photoperiod and germinated for different days for the experimental materials.The vitrification program consisted of: 1) Pretreatment --- treated with 2M glycerol + 04M sucrose at 20 ℃ for 20min; 2) Dehydration treatment --- glass solution PVS2 (30% w / v sucrose + 15% w / v ethylene glycol + 15% w / v dimethyl sulfoxide + MS containing 0 4M sucrose) 0 ℃ 50min; 3 ) Freeze-thaw --- glass and seedlings with liquid nitrogen (LN2) preservation, natural melting at room temperature 20 ℃; 4) Replacement --- with MS +1 2M sucrose 2 0 ℃ for 30min, vitality check; seedling turn Cultivate MS medium for 5 days and observe the regrowth condition And then transferred to cultivate soil and grew to fruiting in the growth chamber.The results showed that: 1) the highest survival rate was 76.5% after freeze-thaw using the complete program; the treatment of glass liquid was the key to success, Freezing and thawing are the main factors that reduce the survival rate of vitrification; 2) The seedlings formed by seed germination of 0 to 2 days will use this technology, the survival rates after thawing are 88%, 83 8% And 76 5%, strong and normal; 3)