刺梨茎尖的玻璃化超低温保存及植株再生

Cryopreservation of in vitro Rosa roxburghii shoot-tips by vitrification and its regeneration

【目的】建立刺梨茎尖超低温保存技术体系,为刺梨种质资源的长期稳定保存提供新途径。【方法】以刺梨为材料,研究了适合茎尖超低温保存的蔗糖预培养时间与浓度、预处理时间、玻璃化液处理时间等影响因素。【结果】适合超低温保存芽诱导的适宜培养基为MS+0.5 mg·L-16-BA+30 g·L-1蔗糖+7 g·L-1琼脂。刺梨茎尖玻璃化法超低温保存体系为:约0.5 cm长的腋芽茎尖在4℃条件下于培养基MS+0.5 mg·L-16-BA+0.05 mg·L-1IAA+0.3 mo1·L-1蔗糖+7 g·L-1琼脂上预培养3 d,切取约2 mm长的茎尖,室温下LS装载液处理40～50 min,然后用玻璃化液PVS2于0℃下处理40 min,换入新鲜的PVS2,迅速投入液氮中。保存24 h后,在40℃水浴中化冻,用MS+1.2 mol·L-1蔗糖+100 mg·L-1Vc+0.5 mg·L-1GA3培养基洗涤2次,接种到MS+0.3 mol·L-1蔗糖的培养基上暗培养1 d,再转接到MS+0.1mg·L-16-BA+0.05 mg·L-1IAA+30 g·L-1蔗糖+7.0 g·L-1琼脂上暗培养7 d后转入正常光下培养,成活率可达44.3%。【结论】建立了刺梨茎尖超低温保存技术体系。

【Objective】This research aims at developing a cryopreservation system of in vitro Rosa roxburghii Tratt shoot-tips,and thereby establishing a foundation for long-term and stable preservation of Rosa roxburghii Tratt germplasm resources.【Method】Taking Rosa roxburghii Tratt as materials,factors for cryopreservation of in vitro Rosa roxburghii Tratt shoot-tips such as sucrose preculture,LS pretreatment time,and PVS2 treatment time were investigated.【Result】 The suitable medium for bud break and growth was MS ＋ 0.5 mg·L-1 6-BA ＋ 30 g·L-1 sucrose ＋ 7 g·L-1 agar.A system of cryopreservation for in vitro Rosa roxburghii Tratt shoot-tips was established： the axillary shoot tips which were about 0.5 cm in length were precultured on MS medium containing 0.5 mg·L-1 6-BA,0.05 mg·L-1 IAA,0.3 mol·L-1 sucrose and 7.0 g·L-1 agar for 3 days at 4 ℃.The shoot tips which were about 2 mm in length were cut and loaded in a solution containing 2 mol·L-1 glycerol and 0.4 mol·L-1 sucrose for 40 to 50 minutes at room temperature.The shoot tips were sufficiently dehydrated with vitrification solution for 40 min at 0 ℃.Then the shoot tips were transferred into the tube with fresh PVS2 and immediately put into liquid nitrogen.24 h later,the tips were thawed in a water bath at 40 ℃,and then rinsed twice with MS medium containg 1.2 mol·L-1 sucrose,100 mg·L-1 Vc and 0.5 mg·L-1 GA3.Then the shoot tips were plated on MS medium supplemented with 0.3 mol·L-1 sucrose under darkness for 1 day and subcultured on MS medium supplemented with 0.1 mg·L-1 6-BA,0.05 mg·L-1 IAA,30 g·L-1 sucrose and 7 g·L-1 agar under darkness for one week prior to exposure to the light.The survival rate of shoot-tips after cryopreservation was 44.3%.【Conclusion】 Cryopreservation system of in Vitro Rosa roxburghii Tratt Shoot-tips was developed successfully.