马铃薯离体茎尖玻璃化法超低温保存

摘要

以马铃薯(Solanum tuberosum)试管苗为材料,利用玻璃化法保存其离体茎尖,研究影响马铃薯超低温保存的主要因素和再生植株的遗传稳定性,优化马铃薯茎尖玻璃化法超低温保存的体系.结果表明:2.1～3.0 mm(含有2个叶原基)的马铃薯茎尖预培养4h,装载液(MS+2 mol/L甘油+0.4 mol/L蔗糖)装载40 min,0℃下PVS2脱水处理35 min,加少量新鲜的PVS2后迅速注入液氮保存至少24h;室温下浸入含有1.2 mol/L蔗糖的MS恢复培养液中90s进行解冻,并洗涤25 min,最后转至恢复培养基(MS+30 g/L蔗糖+0.05 g/L EDTA+0.50 mg/L IAA+0.04 mg/L KT+0.10 mg/L GA3)上,暗培养10 d后,转入弱光条件下培养,20 d后转入正常光照下培养,存活率和再生率最高,分别为86.93％和77.05％.通过形态学和SSR标记检测,再生植株生长和分化正常,SSR谱带未发生改变.且液氮冻存时间不同,超低温保存后的成活率和再生率均无显著差异.表明茎尖玻璃法超低温保存对马铃薯的种质资源保存具有较强的实用意义.%Orthogonal test was designed to study the main factors affecting cryopreservation of in vitro shoot tips of potato by vitrification, and the genetic stability of regenerated plant was assessed. The experiment combination favoring the survival and regeneration of potato shoot tip is to use 4. 0-h pretreated shoot tip with the length of 2. 1-3. 0 mm followed by loading for 40 min at room temperature, dehydration by PVS2 for 35 min at 0 ℃ , cryopreservation in liquid nitrogen for o-ver 24 h, thawing in MS medium containing 1. 2 mol/L sucrose and culturing in recovery medium. In such a cryopreservation system, the survival rate and regeneration rate reached the highest, 86. 93% and 77. 05% , respectively, and were independent of the time duration of liquid nitrogen freezing. The morphologic observation revealed that the regenerated plants grew and differentiated normally. SSR markers presented consistent profile of the regenerated plants before and after cryopreservation. The vitrification of potato shoot tip by appears promising for cryopreservation of potato germplasm.