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標題: 不同前處理對番木瓜莖頂超低溫保存存活率之影響
Survival of cryopreserved shoot tips of papaya (Carica papaya L.) exposed to various pretreatments
作者: 朱麗淑
Ju, Lih-Shuwn
關鍵字: Papaya
出版社: 植物學系
摘要: 本實驗以番木瓜台農二號(Carica papaya L. cv. Tainung No.2)組織 培養苗之莖頂為材料,主要目的係探討不同前處理對番木瓜莖頂超低溫保 存存活率之影響。同時探討不同前處理對番木瓜莖頂MDA( malondialdehyde )生成量變化情形及冷凍保護劑(cryoprotectant)對 番木瓜葉片光合成率之影響,以了解超低溫保存過程中對番木瓜莖頂細胞 膜及生理機能之傷害程度。番木瓜組織培養苗以abscisic acid (ABA)及 低溫(20℃、15℃、10℃)前處理之後,進行超低溫保存,無法有效提升 番木瓜莖頂超低溫保存存活率。利用含0.5M蔗糖之健化培養基前處理培 養一週之組織培養苗,配合乾燥劑脫水20分鐘,直接置入液態氮桶中,回 復生長後,可達60%之存活率。或組織培養苗以健化培養基前處理一週, 配合乾燥劑脫水40分鐘後,利用冷凍保護劑震盪一小時後,將莖頂移入冷 凍管(cryotube)中,經遞降溫系統以1℃/min降溫速率,降溫至 -40℃之 後,置入液態氮桶中,回復生長後,可達77.8%之存活率。分析超低溫保 存各處理步驟對莖頂MDA生成量之影響,可得知番木瓜細胞膜受傷害之情 形。結果發現使用冷凍保護劑後,其MDA生成量較其他處理步驟之生成量 高,顯示冷凍保護劑對細胞膜脂質過氧化影響較大。而冷凍保護劑之組成 中,又以蔗糖的影響最大。由此可知,本實驗中冷凍保護劑之蔗糖濃度可 能過高,而造成番木瓜莖頂細胞之滲透逆境,導致細胞膜產生脂質過氧化 。冷凍保護劑組成對番木瓜葉片光合成率之影響中﹐E.G.(ethylene glycol)、DMSO(dimethyl sulfoxide)及glycerol等皆會隨濃度增加而 導致光合成率下降,而sorbitol、PEG( polyethylenglycol )、sucrose 及proline等,在一定濃度範圍內會提高光合成率。因此,在番木瓜超低 溫保存過程中,冷凍保護劑組成及濃度之選擇為一重要影響因子。
Abstract This study used tissue cultured plantlets of papaya ( Carica papaya L. cv. Tainung No2. ) as materials for investigation. The main purpose is to investigate the survival ratio of the shoot tips under various pretreatments after cryopreservation. To understand the degree of damage to the overall physiology performance of papaya, MDA production of shoot tips wasmeasured to indicate the effect of cryopreservation on cell membrane, as well as the photosynthetic rate of the leaves. Survival of cryopreserved shoot tips of papaya did not increase when tissue cultured papayas was pretreated with ABA ( 100mm abscisic acid ) and different low temperatures treatment ( 20℃、15℃、10℃ ). After plantlets were grown in hardening media supplemented with 0.5 M sucrose for a week, the shoot tips were excised, dehydrated for 20 mins, transferred to cryotubes and plunged directly into liquid nitrogen for storage. After 24 hr, frozen shoot tips were thawed at 40℃for 1 min and then transferred to regrowth media. The survival of shoot tips was up to 60%. In another experiment, tissue cultured plantlets were grown in standard hardening media for a week, the shoot tips were excised, dehydrated for 40 mins, treated with different cryoprotectants by shaking for 1 hr, transferred to cryotubes and cooled at 1℃per minute to -40℃, and then plunged into liquid nitrogen. After 24hr, frozen shoot tips were thawed at 40℃for 1min and then transferred to regrowth media. The survival of shoot tips was up to 77.8%. The damage of membrane in shoot tips was measured as MDA production from various procedures of cryopreservation. Application of cryoprotectants increased the amounts of MDA synthesized,which is higher than other treatments. These results suggest that cryoprotectants have positive effect on lipid peroxidation of cell membrane. Moreover, sucrose was the most effective one among those cryoprotectants. As the results show, the concentration of sucrose used in this experiment may be too high for papaya shoot tips, therefore it caused osmotic stress, resulting in lipid peroxidation of cell membrane. The effects of various cryoprotectants on leaf photosynthetic rates were also measured. Photosynthetic rates decreased with increasing the concentrations of ethylene glycol, dimethyl sulfoxide and glycerol. However, certain ranges of sorbitol, polyethylenglycol, sucrose and proline caused increased photosynthetic rates. Here we concluded that selecting of suitable compositions and concentrations of cryoprotectants is a major factor for cryopreserving papaya shoot tips.
Appears in Collections:生命科學系所



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