Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22631
標題: 台灣藥用植物食茱萸與牛樟玻璃化法超低溫冷凍保存前處理流程之探討
Investigation of Pretreatment Protocols on the Cryopreservation of Taiwan Medicinal Plants- Zanthoxylum ailanthoides Sieb. & Zucc. and Cinnamomum kanehirai Hay. by Vitrification.
作者: 李坤紘
Li, Kuen-Hung
關鍵字: 食茱萸;Zanthoxylum ailanthoides Sieb. & Zucc.;牛樟;超低溫冷凍保存;Cinnamomum kanehirai Hay.;Cryopreservation
出版社: 生命科學系所
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摘要: 
本研究以食茱萸與牛樟組織培養苗為試驗材料,探討高濃度蔗糖預培養、loading solution、冷凍保護劑(PVS2)以及回復生長培養基對頂芽與腋芽經冷凍保存後回復生長存活率與再生率之影響。經由試驗結果發現食茱萸頂芽以LS處理30分鐘,PVS2處理時間140分鐘,可獲得最佳保護效果,回復生長培養基以WPM+BA 0.1 mg/L+NAA 0.01 mg/L可促進頂芽再生。未經預培養的食茱萸頂芽以LS處理30分鐘,PVS2處理140分鐘,經冷凍保存後以WPM+BA 0.1 mg/L+NAA 0.01 mg/L回復生長存活率與再生率分別為39.4%與12.4%,經由0.3 M蔗糖培養基預培養1~5週,頂芽存活率可提升至72.7%~93.9%,再生率可達39.4%~81.5%。其中以0.3 M預培養3週,頂芽存活率與再生率分別達到93.9%與81.5%。經由生理分析的測定,發現食茱萸以0.3 M蔗糖預培養後,頂芽相對含水量減少、水勢與滲透潛勢降低、可溶性糖類以及蔗糖含量增加,顯示0.3 M蔗糖預培養有助於頂芽適應冷凍保護劑所形成的滲透逆境及增加頂芽的抗凍性,因此能有效提高冷凍保存後的存活率與再生率。食茱萸經由0.3 M蔗糖預培養3週,增加PVS2處理時間雖可提升腋芽存活率,但最高存活率僅有21.8%,再生率也只有3.3%。推測主因為休眠腋芽較不易恢復生長。
牛樟頂芽與腋芽以0.2 M蔗糖培養基預培養3天,僅有頂芽以LS處理60分鐘,PVS2處理100與180分鐘約有3%的存活率。提高預培養時間與蔗糖濃度,頂芽存活率仍在3%~10%之間,而腋芽存活率也僅約3%。在本試驗中,即使回復生長培養基添加2 g/L的活性碳,但仍可觀察到芽體出現酚酸化合物氧化累積所形成的褐化現象,可能是因為細胞遭遇逆境而產生自由基,進而增加酚酸化合物的氧化,因此推測酚酸化合物的毒害可能是牛樟冷凍保存回復生長的限制因子。

In this study, apical buds and axillary buds of Zanthoxylum ailanthoides Sieb. & Zucc. and Cinnamomum kanehirai Hay. were used for experiment materials, to investigate effect of preculture, loading solution (LS), cryoprotection agent (PVS2), and recovery medium on survival rates and regeneration rates after cryopreservation. The results demonstrated that LS treatment for 30 min and PVS2 for 140 min obtained the best cryoprotection on apical buds and recovery medium WPM containing 0.1 mg/L BA and 0.01 mg/L NAA was the most appropriate for regeneration. Plantlets without preculture treated with LS treatment for 30 min, followed by PVS2 for 140 min and recovered in WPM containing 0.1 mg/L BA and 0.01 mg/L NAA obtained 39.4% of survival rates and 12.4% of regeneration rates. While plantlets precultured on 0.3 M sucrose for 1 to 5 weeks, survival rates increased to 72.7% to 93.9% and regeneration rates increased to 39.4% to 81.5%. The survival rates and regeneration rates reached 93.9% and 81.5%, respectively, in plantlets preculturing for 3 weeks. Physiological analysis revealed that apical buds preclturing with 0.3 M sucrose had lower relative water content, water potential and osmotic potential, and had higher soluble sugar and sucrose content. The results indicated that apical buds preculturing with 0.3 M sucrose had positive effect on not only the adaptation to osmotic stress caused by cryoprotection agent but also freezing tolerance, and as the results, the survival and regeneration rates were increased after cryopreservation. Axillary buds of Zanthoxylum ailanthoides Sieb. & Zucc. were precultured with 0.3 M sucrose for 3 weeks, and the survival rates increased as increasing PVS2 treatment duration. The highest survival rate only reached 21.8% and the highest regeneration rate only reached 3.3%. The possible explanation was suggested, dormancy of axillary buds were hard to recover.
Only 3% survival rate was found in the apical buds of Cinnamomum kanehirai Hay. precultured with 0.2 M sucrose for 3 days, then loaded with LS for 60 min, and followed by PVS2 treatment for 100 and 180 min. Increasing preculturing duration and sucrose concentration, the survival rate of apical buds was still between 3% to 10%, and the survival rate of axillary buds was about 3%. Addition of 2 g/L activated charcoal in the recovery medium can not prevent the browning of shoot tips caused by the accumulation of oxidated polyphenolic compounds. The increased oxidation of polyphenolic compounds might be result from the release of free radical in stressed cells, suggesting that toxic polyphenolic compounds may be a limiting factor for recovery after cryopreservation.
URI: http://hdl.handle.net/11455/22631
其他識別: U0005-0708200819422000
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