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標題: 草莓玻璃化法超低溫冷凍保存流程之探討
Investigation of the Protocol on the Cryopreservation of Strawberry (Fragaria x ananassa Duch.) Treated by Vitrification
作者: 黃坤陽
Hwang, Kun-Yang
關鍵字: cryopresevation
cold acclimation
出版社: 生命科學系所
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摘要: 草莓(Fragaria x ananassa Duch.)為溫帶果樹,適合以低溫冷馴化預培養後進行超低溫冷凍保存。本研究期以桃園一號與桃園三號兩種品系草莓經10℃低溫冷馴化7-60天後,進行玻璃化法超低溫冷凍保存能提升存活率。結果顯示,兩種草莓經過冷馴化30天預培養能明顯提升存活率。桃園一號經冷馴化預培養後以LS處理20分鐘、PVS2處理150分鐘,獲最佳存活率約97%;桃園三號是以LS處理20分鐘、PVS2處理240-270分鐘,存活率可達96%;桃園三號預培養於0.2 M培養基30天後,再以LS處理60分鐘、PVS2處理240分鐘,存活率達87%;兩種草莓未經預培養之對照組存活率最高僅為80%。草莓在預培養期間,頂芽細胞之相對含水量、水分潛勢與滲透潛勢亦下降,顯示有輕微脫水現象;可溶性糖類總量增加,果糖、葡萄糖與蔗糖含量產生變化,其中蔗糖大量累積,以上生理變化皆可提升頂芽細胞之抗凍性,進而反應在超低溫冷凍保存存活率上。因此細胞之水分生理變化與滲透調節作用,可使草莓頂芽適應超低溫冷凍保存前處理,進而進入液態氮保存。而回溫生長後的草莓頂芽組織,外觀與一般培養無異,表示冷凍保存處理並未造成組織變異。綜合以上所述,此研究建立之處理流程極適合桃園一號與桃園三號草莓進行之玻璃化法超低溫冷凍保存。
Strawberry (Fragaria x ananassa Duch.) is one kind of temperate plants; therefore, it could be cryopreserved after preculture with cold acclimation. In this study, it is expected that the survival rates of two cultivars of strawberry cv. ''TY-1'' and ''TY-3'', could be maximized through the process of cold acclimation for 7 to 60 days and vitrificated cryopreservation. The results shows that the survival rates of these two kinds of strawberry were obviously promoted after cold acclimation for 30 days and cryopreservation. At first, the survival rate of these two cultivars without preculture was only 80%. However, the highest sur-vival rate of ''TY-1'' became 97% after the shoot tips were cryopreserved following loaded with loading solution (LS) (20 minutes), dehydrated with plant vitrification solution 2 (PVS2) (150 minutes). As for TY-3, the highest survival rate of TY-3 shoot tips was 96% after the procedure of LS (20 minutes), PVS2 (240-270 minutes) and cryopreservation. In addition, after a 30-day preculture on MS medium with 0.2 M sucrose, the TY-3 shoot tips were loaded for 60 minutes and then dehydrated for 240 minutes. After these treatments and cryopreservation, the survival rate of TY-3 shoot tips was 87%. Moreover, during the process of preculture, there had been some decrease in the relative water content, water potential, and osmotic potential of the shoot tips, but there was an increase in the amount of soluble sugar content within apex tissue. All of the content of simple sugar, fructose, glucose and sucrose changed. Among which, sucrose was accumulated in shoot tips.As a result, the water status, osmore-gulation and enhance the strawberry shoot tips to adapt to the pretreatment of cryopreservation protocols and then being directly plugged into LN for cryopreservation. The morphology of the rewarmed shoot tips are the same with the normal ones . This outcome means that the regrowth of cryopreserved strawberry shoot tips were not variable after the protocols. In conclusion, the protocol established in this study could be adapted to the cryopreservaion of two cultivars strawberry TY-1 and TY-3 by vitrifyication.
其他識別: U0005-1807200720493900
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