怀山药种质资源的包埋玻璃化超低温保存与植株再生

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0.5mol·L-1CaCl2包埋,包埋珠用MS＋0.2mol·L-1蔗糖＋2mol·L-1甘油＋50g·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＋0.5mol·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%。

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

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, 2 mol·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.