Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23022
標題: 臺灣原生藥用植物艾納香玻璃化法超低溫冷凍保存流程之探討
Investigation on Vitrification Protocols of Cryopreservation of Taiwan Native Medicinal Plant of Blumea balsamifera (L.) DC.
作者: 詹金鳳
Chan, Chin-Feng
關鍵字: Blumea balsamifera (L.) DC.;艾納香;vitrification;cryopreservation;osmotic adjustment;ion leakage;玻璃化法;超低溫冷凍保存;滲透調節;離子滲漏
出版社: 生命科學系所
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摘要: 
本試驗材料為臺灣原生藥用植物艾納香( Blumea balsamifera (L.) DC. )組織培養苗,以頂芽進行玻璃化法超低溫冷凍保存,並藉由各項生理分析,瞭解高醣預培養和冷凍保護劑處理之傷害,藉以改進冷凍保存技術,期達種原保存和永續利用的目的。
以株齡30–40天植株,切取1–2 mm頂芽,依序進行PVS2不同時間處理、LS不同時間處理與預培養蔗糖濃度及預培養天數試驗。試驗結果得知,較適宜冷凍保存流程為0.5 M蔗糖培養基預培養3天,於25℃進行LS處理60 min,PVS2冰浴處理90 min後進行長期保存。保存1週後回溫,以1/2 MS添加BA 0.2 mg L-1,IBA 0.04 mg L-1,活性碳3 g L-1回復生長1個月,其存活率為50 %。並觀察到隨預培養蔗糖濃度或天數之增加,芽體相對含水量、水分潛勢及滲透潛勢均逐漸降低,可溶性蛋白及可溶性醣類含量逐漸提高,可知艾納香具滲透調節能力,以適應高醣預培養之脫水逆境。以艾納香長期預培養處理,將相對含水量、滲透潛勢、可溶性蛋白含量及蔗糖含量,與冷凍保存後存活率進行迴歸分析,得知適當蔗糖含量可提高存活率。
進一步分析冷凍保存各流程之離子滲漏結果,結果顯示芽體經冷凍保護劑處理後,離子滲漏達95%,又PVS2處理後芽體再生率不到20 %,由此可知冷凍保護劑處理為造成冷凍保存後低存活率之主因。另以PVS3為冷凍保護劑,冷凍保存後芽體存活率低於PVS2,且以無菌風乾燥脫水進行冷凍保存處理,芽體存活率均為0%,因此艾納香不適用PVS3或乾燥脫水法進行冷凍保存。芽體回復生長研究,觀察到以1/2 MS為基底培養基,添加BA 0.2 mg L-1,IBA 0.04 mg L-1的回復培養基,存活率可達60 %,且芽體葉片展開,建議以此為艾納香回復生長培養基。
總結上述,艾納香超低溫冷凍保存適宜處理流程為0.5 M蔗糖預培養3天,LS處理60 min,PVS2處理90 min,經液態氮保存後,回復生長培養基為1/2 MS為基底培養基,添加BA 0.2 mg L-1,IBA 0.04 mg L-1,可達60%之存活率。

This study is on cryopreservation of micropropagation of Blumea balsamifera (L.) DC., a Taiwan native medicinal plant, by vitrification the shoot tips. This thesis investigates the treatment of different precultures on sucrose medium and the injury of explants by plant vitrification solution. The goal of this thesis is to achieve germplasm conservation and sustainable use.
B. balsamifera (L.) DC. was cultured on the proliferation medium for 30 to 40 days. Excised 1-2 mm buds were placed on different treatments of PVS2, LS solution, and preculture. The established protocol was that buds were treated with 0.5 M sucrose medium for 3 days, loaded to LS 60 min at 25℃, and dehydrated to PVS2 90 min at 0℃ for long-term storage. After one week, buds were rewarmed rapidly, and then transferred to petri dishes containing 1/2 MS medium, supplemented with 0.2 mg L-1 BA, 0.04 mg L-1 IBA, and activated carbon 3g L-1, as a recovery medium for 1 month. The protocol provides the survival rate of 50%. The preculture treatment decreased the relative water content, water potential, and osmotic potential and increased soluble sugar and soluble protein content of the buds. The result indicated that plantlets of B .balsamifera (L.) DC. underwent osmotic adjustment during preculture. Regression analysis of the relative water content, osmotic potential, soluble protein, and sucrose content of buds to the survival rate after cryopreservation showed that the suitable sucrose content improved survival rate of B.balsamifera (L.) DC. shoot tips.
The result of ion leakage assay of buds for cryopreservation process showed the rate achieved 95% and the regeneration rate was lower than 20% after plant vitrification solution. The treatment of PVS2 caused low survival rate of B. balsamifera (L.) DC. buds for cryopreservation. The survival rate of buds by PVS3, as a plant vitrification solution, were lower than by PVS2. The survival rate of buds were 0% by the treatment of air drying for cryopreservation. Thus cryopreservation of B.balsamifera (L.) DC. of shoot tips was not suitable by PVS3 or dessciation treatments. On recovery treatment of buds, the result showed 60 % recovery rate of buds grown in the recovery medium containing 1/2 MS medium, supplemented with 0.2 mg L-1 BA and 0.04 mg L-1 IBA. The composition of recovery medium is another key factor of survival after cryopreservation.
In conclusion, the optimal protocol for cryopreservation of B. balsamifera (L.) DC. in this study was precultured on 0.5 M sucrose medium for 3 days, treated with LS for 60 min and PVS2 for 90 min, and recovered in 1/2 MS medium containing 0.2 mg L-1 BA and 0.04 mg L-1 IBA. This protocol could achieve 60% survival rate.
URI: http://hdl.handle.net/11455/23022
其他識別: U0005-0608201001123200
Appears in Collections:生命科學系所

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