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植物学报 >

2010 , Vol. 45 >Issue 03: 379 - 383

DOI: https://doi.org/10.3969/j.issn.1674-3466.2010.03.010

技术方法

怀山药种质资源的包埋玻璃化超低温保存与植株再生

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  • 1河南师范大学生命科学学院2河南师范大学体育学院3河南高校道地中药材保育及利用工程技术研究中心

收稿日期: 2010-01-04

  网络出版日期: 2010-05-01

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Cryopreservation and Plantlet Regeneration of Germplasm Resources of Dioscorea opposita by Encapsulation-vitrification

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  • 1Henan Normal University, Xinxiang 453007, China
    2Henan Normal University, Xinxiang 453007, China
    3University of Henan Province, Xinxiang 453007, China

Received date: 2010-01-04

  Online published: 2010-05-01

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摘要

通过对怀山药(Dioscorea opposita)种质资源的包埋玻璃化超低温保存与植株再生进行研究,结果表明: 将低温下锻炼7天的怀山药试管苗带芽茎段放入预培养基中,低温下培养3天,然后在室温下用3%的海藻酸钠和0.5 mol·L–1 CaCl2包埋, 包埋珠用MS+0.2 mol·L–1蔗糖+2 mol·L–1甘油+50 g·L–1二甲基亚砜在0°C下装载60分钟, 用30%甘油+15%乙二醇+10%二甲基亚砜+15%聚乙二醇+0.4mol·L–1蔗糖在0°C下脱水60分钟, 迅速投入液氮, 24小时后立即用40°C水浴快速化冻, 再用MS+0.5 mol·L–1蔗糖溶液洗涤2次, 转入再生培养基中培养, 可获得再生植株。再生植株成活率因基因型而异, 铁棍山药、太谷山药、怀庆1号山药和B号山药的成活率分别为64.29%、49.21%、13.11%和39.81%。

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李海兵;周娜Δ;赵姣;李翔;冯秋妍;赵喜亭;李明军;* . 怀山药种质资源的包埋玻璃化超低温保存与植株再生[J]. 植物学报, 2010 , 45(03) : 379 -383 . DOI: 10.3969/j.issn.1674-3466.2010.03.010

Abstract

We studied the cryopreservation of germplasm resources of Dioscorea opposita Thunb. by encapsulation-vitrification. D. opposita plantlets of different cultivars were cold-hardened for 1 week at low temperature, then stems with one axillary bud were excised and pre-cultured at low temperature for 3 days. Stems with axillary buds suspended in Murishige and Skoog (MS) inorganic medium supplemented with 3% Na-alginate were placed in MS inorganic medium supplemented with 0.5 mol·L–1 CaCl2 solution. Beads were loaded with MS medium co-mixed with 0.2 mol·L–1 sucrose, 2mol·L–1 glycerol and 50 g·L–1 dimethyl sulfoxide (DMSO) for 60 min at 0°C,then were dehydrated with a highly concentrated vitrification solution (30% glycerol+ 15% ethylene glycol+10% DMSO+ 15% polyethylene glycol+0.4 mol·L–1 sucrose) for 60 min at 0°C and plunged into liquid nitrogen quickly After 24 hours, the beads were rapidly thawed in a water bath at 40°C for 1-3 min. The beads were washed twice with MS medium supplemented with 0.5 mol·L–1 sucrose, then transferred to regeneration medium (MS+2 mg·L–1 KT +0.02 mg·L–1 NAA +3% sucrose+0.5% agar). Stems with one axillary bud could lead to regenerated plantlets with regeneration culture, but the survival rates of cultivars differed: 64.29%, 49.21%, 13.11% and 39.81% for D. opposita 'Tiegun', D. opposita 'Taigu' D. opposita 'Huaiqing No.1' and D. opposita 'No.B', respectively.
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