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以马铃薯栽培种呼自83-213无菌试管苗茎尖为材料,通过开展2,3,5-氯化三苯基四氮唑(TTC,2,3,5-Triphenyl tetrazolium chloride)茎尖活力染色关键因素研究,优化了马铃薯茎尖TTC活力染色条件,确定了适合的染色温度为40℃,染色时间为2 h。利用优化的TTC活力染色条件,对马铃薯茎尖小滴玻璃化超低温保存关键步骤处理茎尖进行TTC活力观察。研究发现:经蔗糖预培养(MS培养液添加0.3 mol/L和0.5 mol/L蔗糖)的茎尖与新鲜茎尖均保持高活力;经PVS2处理后茎尖表现时空特异性活力丧失和存活,分生组织和叶原基中间区域仍保持较高活力。通过对茎尖TTC活力染色面积测定,发现当茎尖TTC活力染色面积比≥0.4时,TTC活力染色与恢复培养存活率呈极显著正相关。
In this study, the tip of potato germplasm from 83-213 sterile plantlets was used as material, and the stem tip activity of 2,3,5-Triphenyl tetrazolium chloride (TTC) Dyeing key factors to optimize the potato shoot tip TTC vitality dyeing conditions, identified a suitable dyeing temperature is 40 ℃, dyeing time is 2 h. TTC vitality was observed with the optimized TTC vitality staining conditions on the key steps of vitrification and cryopreservation of potato shoot tips. The results showed that the stem tips and fresh shoot tips of sucrose pre-culture (MS medium supplemented with 0.3 mol / L and 0.5 mol / L sucrose) both maintained high viability. After treatment with PVS2, the shoot tips showed the spatiotemporal-specific loss of vitality and survival, Mesenteric and leaf primordium in the middle of the area still maintain high vitality. By measuring the viable area of TTC, we found that there was a significant positive correlation between TTC viability staining and recovery culture survival when the ratio of TTC viable stained area ≥0.4.