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Surivival of Cryopreserved Axillary Buds of Yam (Dioscorea L.)Exposed to Various Pretreaments
本研究以三種山藥:大陸淮山（D.doryophora Hance）、血薯(D.alata L.var.purea(Roxb.)M.Pouch）及北投（D.alata L.）的組織培養無菌苗為供試材料，主要目的為探討各種預培養條件(高糖、低溫配合短日照、ABA及生長抑制劑)及不同冷凍保護法(玻璃質化法、乾燥脫水法及藻膠包埋脫水法)對山藥超低溫冷凍保存存活率的影響。並藉由測量供試材料的相對含水量及滲透壓的變化，以了解山藥組織含水狀況對超低溫冷凍保存之影響。
研究結果顯示，三品種中以血薯對抗凍保護劑之忍受力最強，大陸淮山較耐高糖脫水。比較各種前處理發現低溫(20、15及10℃)配合短日照預培養1-3周、100μM ABA預培養1-3天、生長抑制劑CCC (chlorocholine chloride) 0.025、0.05及0.1μM預培養2周及生長抑制劑B995 (succinic acid 2，2-dimethylhydrazine) 1、2及4μM預培養2周均無法有效誘導山藥的抗凍耐性。只有以0.75M高糖預培養10天能誘導大陸淮山的抗凍耐性。
Abstract In vitro plantlets of three yam species were subjected to a wide range of preculture condition (high sucrose , cold hardening ,short photoperiod , ABA and growth retarding agents) and different cryopreservative treatments (vitrification, cryoprotective-dehydration and encapsulation-dehydration) were used for cryopreservation technique. According to the results, among these three species, D.alata L.var.purea (Roxb.) M. Pouch was the most tolerant one to cryoprotectant and D.doryophora Hance had higher dehydration tolerance than other two species. Several pre-treatments tested, such as cold hardening and short photoperiod preculture for 1-3 weeks, 100μM ABA preculture for 1-3 days, growth retarding agent CCC at 0.025μM,0.05μM and 0.1μM or B995 at 1μM,2μM and 4μM preculture for 2 weeks, did not enhance the tolerance to freezing in three yams. Preculturing in the medium containing 0.75M sucrose for 10 days can induce the resistance to desiccation and to freezing in the liquid nitrogen of D.doryophora Hance . All three species of yam were cryopreserved by vitrification protocol, but they did not survive after freezing- thawing. D.doryophora Hance was precultured with 0.75M sucrose and dehydrated in silica gels for 90 min and the results showed 30% of survival rate. The highest survival rate (33.33%) was achieved by using encapsulation-dehydration protocol. We found that after one to three months subculturing, yam can survive after cryopreserved. Besides, we also found if alginate beads were shaken in medium with high sucrose for 3 days, the chemical properties of alginate beads changed. We suggested that the change occurred in alginate beads can reduce serious dehydration damage caused by freezing — thawing process on yam. Therefore, yams can survive after cryopreservation.
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