马铃薯茎尖超低温保存流程TTC活力响应

Study on Potato Shoot Tips Vitality Responses during Cryopreservation by TTC Staining Method

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。

TTC（ 2,3,5-Triphenyl tetrazolium chloride） vitality staining conditions for potato shoot tips of Huzi83-213 cultivar were optimized in this study. The optimal staining temperature and duration were determined as40 ℃ and 2 h,respectively. The viability of potato shoot tips was investigated during droplet- vitrificaion cryopreservation by TTC staining under the optimal conditions. The fresh shoot tips and precutured（ Murashige and Skoog liquid medium with 0. 3 mol / L and 0. 5 mol / L sucrose） shoot tips showed high vitality. However,after cryoprotection with plant vitrification solution No. 2（ PVS2 solution）,the shoot tips had the temporal and spatial specificity of viality loss and survival,and the inner meristem and the middle part of the leaf primordium kept high vitalities. The results between TTC staining viality and survival percentages after recovery showed extremely significant positive correlation when the area ratio of the red- stained shoot tips was higher than 0. 4.