目的 对影响黄独Dioscorea bulbifera微型块茎胚性愈伤组织小滴玻璃化法超低温保存的多种因素进行探讨,并从形态学、生理学、DNA量以及光合特性参数和叶绿素荧光参数等方面对其冻后再生苗的遗传稳定性进行检测。方法 采用微型块茎及其胚性愈伤组织进行诱导,小滴玻璃化法超低温保存,通过检测包括总叶绿素量、可溶性蛋白量、可溶性总糖量以及超氧化物歧化酶和过氧化物酶活性等植物生理指标,并采用流式细胞术的方法对遗传稳定性进行考察。结果 最佳的黄独微型块茎胚性愈伤组织超低温保存条件:室温下将黄独微型块茎胚性愈伤组织块转入MS+2 mg/L KT+0.5 mg/L NAA+0.5 mg/L 2,4-D+0.3 mol/L蔗糖的培养基中预培养1 d。预培养后的胚性愈伤组织块在(25±1)℃下转入装载液(MS+2 mol/L甘油+0.4 mol/L蔗糖,pH 5.8)中处理20 min,再用100% PVS2于0 ℃脱水40 min。脱水后将材料转入铝箔条上的PVS2小滴中,液氮冰冻后迅速将材料转入冷冻管中(装满液氮)。投入液氮罐保持1 d后,取出铝箔条,浸入37 ℃洗涤液(MS+2 mg/L KT+0.5 mg/L NAA+0.5 mg/L 2,4-D+1.2 mol/L蔗糖,pH 5.8)中,胚性愈伤组织块脱落后,室温下再用新鲜洗涤液对其洗涤3次,每次10 min。洗涤后的材料接入分化培养基(MS+2 mg/L KT+0.5 mg/L NAA+30 g/L蔗糖+5 g/L琼脂粉)中,暗培养2 d后转到12 h/d的光周期中培养,细胞存活率达89%以上。黄独微型块茎胚性愈伤组织小滴玻璃化法超低温保存后的再生苗与胚性愈伤组织常温再生苗在形态指标和生理指标等方面均无显著性差异(P> 0.05),两者的DNA量也未发生显著变化(P> 0.05)。结论 建立了黄独微型块茎胚性愈伤组织小滴玻璃化法超低温保存的技术体系,其再生植株经检测无遗传变异,为薯蓣属植物种质资源的长期保存提供了理论依据和技术基础。

Objective To discuss the influence of several factors on the cryopreservation of embryogenic calli induced from Dioscorea bulbifera microtuber by droplet-vitrification and to test the genetic stability of the regenerated plantlets after freezing from the aspects of morphology, physiology, DNA content, as well as the photosynthetic characteristics and chlorophyll fluorescence parameters in this paper. Methods Plant tissue culture (including microtuber induction and embryogenic callus induction), plant physiology index detection (including total chlorophyll, soluble protein, soluble sugar and superoxide dismutase enzyme and peroxide enzyme activity), and cell flow cytometry were applied. Results The best cryopreservation conditions of embryogenic callus of D. bulbifera microtuber were as following: Embryogenic calli were precultured in liquid media of MS + KT 2 mg/L + NAA 0.5 mg/L + 2,4-D 0.5 mg/L + 0.3 mol/L sucrose for 1 d and then treated in loading liquid (MS + 2 mol/L glycerol + 0.4 mol/L sucrose, pH 5.8) for 20 min. In order to dehydrate, embryogenic calli were transferred in 100% PVS2 at 0 ℃ for 40 min. After dehydration, the embryogenic calli were inoculated to PVS2 small drops in the aluminum foil strips and then dipped in liquid nitrogen (LN). Finally the aluminum foil strips were quickly transferred to freezing tube that filled with LN and then put into LN tank. After conserving for 1 d in LN, the aluminum foil strips were removed and the embryogenic calli were immersed into liquid washing media (MS + KT 2 mg/L + NAA 0.5 mg/L + 2,4-D 0.5 mg/L + 1.2 mol/L sucrose, pH 5.8) preheated in 37 ℃ warm water. After separated from the aluminum foil strips, the embryogenic calli were washed with fresh liquid washing media at room temperature for there times, 10 min each time. After washing, the embryogenic calli were transferred onto differentiation medium (MS + KT 2 mg/L + NAA 0.5 mg/L + 30 g/L sucrose + 5g/L agar), and cultured in dark for 2 d and then cultured in 12 h/d photoperiod, the cell survival rate reaches above 89%. The morphological and physiological indexes and the content of DNA of two kinds of plantlets, which regenerated from cryopreserved and non-cryopreserved embryogenic calli induced from D. bulbifera microtuber by droplet-vitrification, showed no significant difference (P > 0.05). Conclusion Cryopreservation technology system of embryogenic calli induced from D. bulbifera microtuber by droplet-vitrification is established and the regeneration plants have no genetic variation, which provides the theoretical basis and technical basis for the long-term preservation of germplasm resources in the plants of Dioscorea L.

国家自然科学基金项目(31360072);江西省教育厅2014年度科学技术研究一般项目(GJJ14713)