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The Application of Cryopreservation in Germplasm Conservation
由於番木瓜及部份甘藷品種之莖頂超低溫冷凍保存已獲穩定存活率,因此本年度研究目標除繼續改進冷凍保存處理技術外,並朝實際應用邁進,主要目標如下: (1) 繼續探討改進超低溫保存之前處理方法、冷凍保存處理過程及方法,希望進一步提昇保存材料之生命力與存活率. (2) 將已建立之超低溫冷凍保存技術與經驗,實際應用到種原之長期超低溫保存. (3) 繼續搜集並建立台灣重要民俗作物、瀕臨絕種原生種原及營養繁殖作物之營養增殖系,供日後進行冷凍保存所需.本研究擬透過下列各項處理方法及步驟進行研究: (1) 莖頂(shoot tip)來源:利用已建立營養增殖系之無菌苗莖頂供試.並繼續建立重要作物之營養增殖系. (2) 莖頂之前處理:進行超低溫保存前,先進行低溫處理或高濃度(0.3-0.5 M)蔗糖處理,模擬植物之冷健化(cold-hardening). (3) 莖頂之脫水處理:利用乾燥劑脫水,並監控莖頂滲透潛勢及相對含水量變化,以掌握最適含水量. (4) 冷凍保護劑(cryoprotectants):分別添加各種保護劑(DMSO、sucrose、sorbitol、glycerol等),探討最適濃度及處理方式. (5) 冷卻方法(cooling):利用快速降溫法直接投入液態氮中保存或利用two step cooling method (Towill, 1991)進行材料之冷卻處理.遞降溫系統進行冷卻之降溫速率為1 ℃/min,待降到-30 ℃- -40 ℃後,直接置入液態氮桶保存(-196 ℃).長期保存之sample vials改為玻璃材質. (6) 加溫(回溫):經液態氮超低溫保存後,從液態氮桶中取出sample vials,直接置入40 ℃ water bath中,快速回溫. (7) 回溫後之處理:直接加入液體培養基,稀釋冷凍保護劑.經更換培養基,排除保護劑後,將莖頂移植於培養基中恢復生長. (8) 生存能力分析:為評估超低溫保存處理過程對存活率之影響,經培養後,估算能恢復生長之莖頂百分比.預期效益: (1) 嘗試以低溫處理模擬冷健化之過程,產生有利超低溫保存之條件.透過此項研究,可瞭解超低溫保存前處理與超低溫保存材料生命力之關係. (2) 探討不同冷凍保護劑及處理方法對超低溫保存之貢獻,希藉此獲知最佳保護劑及處理方法,提高超低溫保存材料之生命力. (3) 測定超低溫保存前處理(高糖培養基、脫水處理)材料之滲透潛勢及相對含水量變化,將有助於瞭解溶質累積、含水量與超低溫保存活力間關係,更有助於建立可信賴之超低溫保存前處理方法與技術. (4) 上述處理材料經超低溫保存後之存活率為評估指標,並可據此經驗改進技術,達到最終建立簡單、可信賴之超低溫保存技術. (5) 建立之超低溫保存技術與經驗,可直接應用於大量基本材料種質之長期保存.將可達到節省人力、物力與空間且遺傳穩定性又高之種原長期保存目的,應用於台灣重要作物之種原保存極具經濟效益.
The viability of cryopreserved shoot tips of papaya and some varieties of sweet potato has getting high and stable. Project for this year is to apply the established technology to routine practice in germplasm conservation in addition to continue to improve the cryopreservation protocols. The aims of this project are as the following. (1) Continue to improve the pretreatments and procedures of cryopreservation to increase the viability of preserved shoot tips. (2) Apply the established cryopreservation protocols to routine practice in long-term germplasm conservation. (3) Continue to collect and establish the mericlones of important ethnobotanical plants, endanger native plants and vegetatively propagated crops to supply to cryopreservation in the future. We propose the treatment methods and procedures as the following. (1) Shoot tip sources: Utilize the established mericlones for sterilized shoot tips, and continue to establish the mericlones of important crops. (2) Pretreatment of shoot tips: Before being preserved in ultra-low temperature, samples are pretreated in low temp, or in high-concentrated (0.3-0.5 M) sucrose solution, to simulate plants' cold-hardening. (3) Dehydration treatment of shoot tips: Dehydrate the samples with desiccants, also monitoring the osmotic potential and relative water content, to obtain the optimum water content. (4) Cryoprotectants: By applying various cryoprotectants, such as DMSO, sucrose, sorbitol and glycerol, test for the optimum concentration of cryoprotectants and treating methods. (5) Cooling: Utilize two-step-cooling method to cool down the samples. By controlled cooling equipment, shoot tips in cryotubes are cooled at 1 ℃ per minute to -30 ℃- -40 ℃ and then preserved directly in liquid nitrogen tank (-196 ℃). The plastic cryotubes are changed to glass vials. (6) Thawing: After preserved in liquid nitrogen, the sample vials are taken from the tank directly to the 40 ℃ water bath to recover the temperature immediately. (7) Recovery after thawing: Add liquid medium to dilute the cryoprotectants for several changes to completely remove the cryoprotectants. Then, the shoot tips can be cultivated in recovery medium. (8) Viability analysis: To assess the effect of cryopreservation protocols on viability of the samples, we measure the percentage for the recovered growth of the cultivated shoot tips. There are five objectives for the above treatments and method: (1) Treating the samples with low temperature and ABA to simulate the cold-hardening process, we will be able to find the optimum conditions for cryopreservation. By this study, we should be able to understand the relationship between the pretreatment and viability for samples preserved in ultra-low temp. (2) By testing various cryoprotectants and treatment methods for preserving samples, we hope to find the best condition to increase the viability of the cryopreserved samples. (3) By measuring the changes of osmotic potential and relative water content for the samples which are pretreated with high-sucrose medium or dehydration, it will help to understand the relationships among solute accumulation, water content and viability for cryopreserved samples. It also helps to establish a more reliable method and technique of pretreatment for cryopreservation. (4) The ratio of viabilities of cryopreserved samples can be used as an indicator for the technique. Based on experiences to improve the technique with time, we will establish a simple and reliable cryopreservation technique ultimately in the future. (5) The established cryopreservation protocols can be used directly in long-term conservation of massive routine germplasm conservation. This will cost less in labor, finance and space and get a high genetic stabilit
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