目的为桔梗Platycodongrandiflorum种质资源的保存提供一条新途径。方法采用玻璃化法研究了桔梗试管苗茎尖超低温保存及植株再生。结果桔梗嫩梢在培养基(MS+5%DMSO+103g/L蔗糖)上培养3d,切取2~3mm茎尖,室温(20℃)下装载液(60%PVS2)过渡20min,0℃下玻璃化液(PVS2)处理90min,投入液氮保存1d后,40℃水浴化冻,含410g/L蔗糖的MS培养基洗涤20min,接种于含0.6mg/LKT、0.2mg/LBA、0.05mg/LNAA的MS培养基表面的滤纸上,暗处理1d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,80%以上成活,植株生长正常。结论桔梗种质资源的玻璃化法超低温保存操作简单、成活率高、再生植株正常,可用于生产实践。

Objective To get a new approach to conserve the germplasm of Platycodon grandiflorum. Methods A method of vitrification was studied to cryopreserve in vitro shoot-tips of P. grandiflorum, and the regenerated plantlets were observed subsequently. Results Shoot-tips, 2—3 mm length, gotten from in vitro shoots of P. grandiflorum precultured on MS medium supplemented with 5% DMSO and 103 g/L sucrose for 3 d, were loaded with the 60% PVS₂ for 20 min at 20 ℃, and incubated in PVS₂ for 90 min at 0 ℃ prior to a direct plunging into liquid nitrogen (LN) and keeping for 1 d. After rapid thawing in water at 40 ℃, the shoot-tips were rinsed in the MS medium supplemented with 410 g/L sucrose for 20 min, and plated on the filter paper sustained by the MS regeneration medium supplemented with 0.6 mg/L KT, 0.2 mg/L BA, and 0.05 mg/L NAA for 1 d in dark and subcultured on the above regeneration medium for one week in dark prior to exposure to the light. The survival of shoot-tips was up to 80%, and they grew normally. Conclusion The method of vitrification to cryopreserve the germplasm of P. grandiflorum is simple in handling with high in survival and normal in regeneration and can be applied in practice.

河北科技大学博士基金项目(QD200309)