柿和君迁子试管苗茎尖玻璃化法超低温保存及再生植株遗传稳定性研究

Cryopreservation of in vitro Shoot-Tips of Persimmon and Date Plum by Vitrification and Genetic Stability of Regenerated Plantlets

对柿(Diospyros kaki Thunb.)和君迁子(D. lotus L.)试管苗茎尖玻璃化法超低温保存及再生植株遗传稳定性进行了研究。结果表明,试管苗在添加有蔗糖0.3 molL-1的改良MS培养基 (KNO3和NH4NO3减半)上,于低温6±1℃下锻炼6周后,切取侧芽茎尖1.5～2.0 mm在含蔗糖0.7 molL-1的改良MS培养基上预培养2 d,室温(20℃)下装载液(2.0 molL-1甘油+0.4 molL-1蔗糖)过渡20 min,0℃下玻璃化液PVS2(30%甘油+15%乙二醇+15%二甲基亚砜+0.4 molL-1蔗糖)处理80 min,换成新鲜的PVS2后投入液氮保存1 d,40℃水浴化冻1 min,含蔗糖1.2 molL-1的改良MS培养基洗涤20 min,接种于含0.2 mgL-1 CPPU、1.0 mgL-1 BA、0.05 mgL-1 IAA、500 mgL-1 可溶性PVP、30 gL-1蔗糖和7 gL-1琼脂的改良MS培养基(再生培养基)的滤纸上,暗处理1 d后转移到新鲜的上述再生培养基中,暗培养1周后转到正常光下,再生率超过30%;再生植株通过细胞学和AFLP标记检测,没有发现核DNA含量、染色体数目和AFLP谱带的改变。该结果为柿属植物种质资源的长期保存提供了理论依据。

Shoot-tips, 1.5–2.0 mm in size, excised from axillary buds of in vitro shoots of persimmon (Diospyros kaki Thunb.) and date plum (D. lotus L.) cold-hardened on the modified MS medium with nitrates reduced to half strength [MS(1/2N)]added with 0.3 molL-1 sucrose at 6±1℃, that were precultured on MS(1/2N) medium supplemented with 0.7molL-1 sucrose for 2 days, were loaded with MS(1/2N)medium supplemented with 2 mol稬-1 glycerol and 0.4 mol稬-1 sucrose for 20 min at 20℃, and incubated in PVS2 for 80 min at 0℃ prior to direct plunge into liquid nitrogen. After rapid thawing for 1 min in water at 40℃, the shoot-tips were rinsed in MS(1/2N)medium supplemented with 1.2 mol稬-1 sucrose for 20 min,plated on the filter paper sustained by the MS(1/2N)regeneration medium supplemented with 0.2 mg稬-1 CPPU,1.0 mg稬-1 BA, 0.05 mg稬-1IAA , 500 mg稬-1soluble PVP, 30 g稬-1sucrose and 7 g稬-1agar for 1 day in dark and subcultured on the above regeneration medium for one week in dark prior to exposure to the light. The shoots formation was over 30% after 6 weeks. And the genetic stability of regenerated plantlets examined on cytological and AFLP-based molecular levels. As a result, there were no variations in DNA content, chromosome number and AFLP patterns. The result provided a feasible proof of conservation Diospyros spp. through cryopreservation of in vitro shoot-tips by vitrification for long term.