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標題: 台農69號品種甘藷之超低溫冷凍保存研究
Cryopreservation of Tai-nung 69 sweet potato(Ipomoea batatas [L.] Lam)
作者: 楊忠祐
Yang, Chung-Yu
關鍵字: cryopreservation
sweet potato
出版社: 植物學系
摘要: 本實驗以甘藷(Ipomoea batatas (L.) Lam)台農69號組織培養苗莖頂為材料,探討高濃度蔗糖培養基預培養及冷凍保護前處理對甘藷超低溫冷凍保存之貢獻,同時探討高濃度蔗糖預培養下莖頂可溶性糖類與蛋白質累積情形,以了解溶質累積與超低溫冷凍保存存活率間的關係。 台農69號甘藷經0.3 M高濃度蔗糖預培養7天後,分別以LS處理20-60分鐘、PVS2冷凍保護劑脫水10-80分鐘後直接置入液態氮中保存可得到存活成果。以LS處理20分鐘,PVS2脫水70分鐘可達80﹪存活。以相同冷凍保護流程進行徐薯超低溫冷凍保存,結果以LS處理40分鐘,配合PVS2脫水70分鐘可達最高存活率25﹪,明顯低於台農69號。 經0.3 M高濃度蔗糖預培養之台農69號甘藷與徐藷滲透潛勢均有下降的現象,以台農69號甘藷為例,發現滲透潛勢下降原因為:可溶性醣類累積量增加了3.6倍、可溶性蛋白質增加了3倍;經由SDS-PAGE電泳分析發現蛋白質大量累積的現象,以小分子蛋白質:13.1、8.8及7.7 kDa之蛋白質累積情形較為特殊。 經超低溫冷凍保存後之莖頂明顯發生冷凍保護劑毒害及冰晶形成之物理性傷害。因此,高濃度蔗糖培養基與冷凍保護劑處理時間為甘藷超低溫冷凍保存成功與否的決定關鍵。
This study investigates effects of preculturing high concentration of sucrose and effects of cryoprotectant pretreatment on sweet potato (Ipomoea batatas (L.) Lam) cryopreservation, and changes of solute content after precultured in high concentration sucrose medium in order to reveal the importance of solutes in cryopreservation. After 7 days precultured in 0.3 M sucrose medium, shoot tips of TN69 sweet potato pretreated with LS for 20 to 60 minutes, followed by PVS2 dehydration for 10 to 80 minutes, and immersed in liquid nitrogen directly. 80% survival rate was obtained by pretreatment with LS 20 minutes and PVS2 70 minutes. When the same methods were applied to different clones, the survival rates were obviously much lower than that of TN69. The osmotic potential decreased after preculturing with 0.3 M sucrose in TN69. It was because the soluble sugars increased 3.6 fold, and soluble proteins increased 3 fold than those of untreated ones. Using SDS-PAGE electrophoresis, we find that proteins contents increase tremendously, especially in low molecular weight proteins with MW of 13.1, 8.8 and 7.7 kDa. Obviously, shoot tips were suffered from cryoprotectant toxicity and ice crystal injury that made meristems can't be recovered well after cryopreservation. The conclusion is that preculture with high concentration of sucrose, time of cryoprotectant pretreatment are key factors leading to successful cryopreservation.
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