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Investigation of pretreatments protocol on the cryopreservation of sweet potato (Impomoea batatas(L.)Lam.) treated by vitrification
甘藷(Ipomoea batatas (L.) Lam.)為熱帶作物，在冷馴化期間對低溫敏感且對PVS2之脫水忍受度低，故較不易利用玻璃化法進行冷凍保存。為達到成功的超低溫冷凍保存，以高濃度蔗糖(0.3－1.0M)進行預培養3天，針對不同甘藷品種，選擇較佳的滲透逆境，並誘導甘藷莖頂產生對PVS2滲透與冷凍之忍受能力。
Sweet potato (Ipomoea batatas (L.) Lam.) is a tropical plant which it has sensitivity to low temperature during cold-hardening and it has low tolerance to dehydration with PVS2. Cryopreservation of sweet potato using vitrification has been shown difficult. For successful cryopreservation by vitrification of this plant, we have precultured for 3 days on different high concentrations sucrose (0.3－1.0M) media and investigated suitable osmotic stress induced the osmotolerance to PVS2 solution and tolerance to subsequent freezing by different species of sweet potato.
The shoot tips from in vitro-grown plantlets of sweet potato TN25, TN64 and TN66 (Ipomoea batatas (L.) Lam. cv. Tainung No. 25, 64 and 66) were precultured for 3 days on hormone-free 1/2MS medium containing different sucrose concentrations. The precultured shoot tips were treated with a loading solution (LS) for 60 minutes and then dehydrated with a vitrification solution (PVS2) for 75 minutes, and following cryopreservation. The best survival rates of 50%, 76.6% and 86.6% were obtained when shoot tips were precultured on 0.8M, 0.5M and 0.4M sucrose for sweet potato TN25, TN64 and TN66, respectively.
After preculturing three species of sweet potato shoot tips on different sucrose concentrations for 3 days, we analyzed the hydric and biochemical modification. The results indicated that sucrose concentration affected the accumulation of soluble sugar, and indirect affected osmotic potential change. Especially in sweet potato TN25 that the soluble sugar content and osmotic potential were significantly increased when were precultuered on 0.6－0.8M sucrose. We obtained that proteins content increase in the different high sucrose concentrations, and accumulation of the 26.2kDa, 25.3 kDa and 23.8 kDa protein increased as detected by SDS-PAGE protein analysis, especially the 26.2 kDa protein was significantly accumulated. The N-terminal amino acid sequence of the 26.2 kDa protein was determined and exhibited 83% homology to the cation-chloride cotransporter.
This study pretreatment samples on the optimal high concentration sucrose which showed increase the cation-chloride cotransporter accumulated was induced by osmotic stress. Ion transporters selectively transport ions and maintain them at physiologically relevant concentrations, permitting plant survival and growth under osmotic stress. Thus preculture with high concentration sucrose and pretreatment with cryoprotectant time were achieved through investigating two key steps of the vitrification protocol.
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