大叶桉以玻璃质化法进行超低温保存之研究-NationalTaiwan.PDF

臺大實驗林研究報告 Jour. Exp. For. Nat. Taiwan Univ. 20(2): 125-131 (2006) 研究論文 大葉桉以玻璃質化法進行超低溫保存之研究 1 2 蔡國書 、王亞男 【摘要】本試驗嘗試利用大葉桉（Eucalyptus robusta ）的芽體與懸浮培養細胞為材料，利用玻 璃質化法進行超低溫保存之研究，檢測冰凍保護劑 PVS2 （plant vitrification solution 2 ）的毒性， 並進行抗凍劑 LS （loading solution ）試驗及不同濃度PVS2處理的效果，嘗試找出較佳的配方 與處理方式，以提高芽體與懸浮培養細胞冰凍後的活力。試驗結果顯示，浸泡 PVS2溶液超過 15 分鐘將使大葉桉懸浮細胞活性降低至 35% 以下；LS 處理可以降低 PVS2 對大葉桉懸浮細胞 的毒性，使懸浮細胞在 PVS2處理 15 分鐘後，仍可維持將近 60%的活性。以 50% PVS2處理大 葉桉懸浮細胞 5 分鐘，再以 100% PVS2處理 10 分鐘，細胞活力可達 68% ，解凍後能誘導出癒 合組織。芽體經 50% PVS2處理 60 分鐘，再以 100% PVS2處理 60 分鐘，細胞活力可達 82% 。 【關鍵詞】大葉桉、超低溫保存、玻璃質化法 Cryopreservation of Eucalyptus robusta by Vitrification Technique 1 2 Kuo-Shu Tasi Ya-Nan Wang 【Abstract 】 This study was to investigate the cryopreservation by vitrification technique; Eucalyptus robusta buds and suspension cultured cell were used in this study. The toxicity of plant vitrification solution 2 (PVS2) and treatment by different concentrations of PVS2 were also studied in order to find out the optimal formula for the improvement of cell viability. After soaking in PVS2 more then 15 minutes, the viability of the suspension cultured cells of Eucalyptus robusta decreased to less than 35%. The loading solution (LS) treatment reduce the toxicity of PVS2 to the suspension cultured cells of Eucalyptus robusta, after soaking in PVS2 more then 15 minutes, with 60% cell viability main- tained. Placing the suspension cultured cells of Eucalyptus robusta into 50% PVS2 for 5 minutes, then into 100% PVS2 for 10 minutes, then cell viability