臺灣扁柏與大葉桉組織培養與玻璃質化法超低溫保存之研究

Studies on Cryopreservation and Tissue Culture of Chamaecyparis obtusa var. formosana and Eucalyptus robusta by Vitrification Technique.

蔡國書(Kuo-Shu Tasi)

指導教授 ： 王亞男

國立臺灣大學/生物資源暨農學院/森林學研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.10235

摘要

本試驗以台灣扁柏Chamaecyparis obtusa var. formosana與大葉桉Eucalyptus robusta 的芽體與懸浮培養細胞為材料，利用玻璃質化法進行超低溫保存之研究，並檢測PVS2的毒性、LN(loading solution)試驗及不同濃度的PVS2處理，以找出最佳的配方與處理方式，提高冰凍後細胞之活力。

大葉桉以MS培養基添加0.5ppm BA與1ppm NAA誘導多芽體，單一芽體平均產生10個芽，並以5ppm 2,4-D誘導癒合組織得到最佳結果；台灣扁柏以WPM培養基添加0.5-1 ppm BA時多芽體誘導率最高，以NAA 5ppm誘導癒合組織得到最佳結果。

將大葉桉懸浮細胞浸置PVS2溶液15分鐘以上會使活性降低至35％以下，但台灣扁柏懸浮細胞浸置30分鐘以上卻依然有60％以上的細胞活力；LN(loading solution)試驗則可降低PVS2對其懸浮細胞的毒性，使細胞活性經PVS2處理15分鐘後仍維持將近60％的活性。

先以50％PVS2處理大葉桉懸浮細胞5分鐘，再以100％PVS2處理10分鐘，細胞活力可以高達68％，並能誘導出癒合組織生長，而芽體經50％PVS2處理60分鐘再以100％PVS2處理60分鐘，可使細胞活力高達82％。而臺灣扁柏多芽體先以50％PVS2處理，再以100％PVS2處理60分鐘，細胞活力可以高達76％，並有33％的芽體能誘導出癒合組織。

關鍵字

玻璃質化法 ； 台灣扁柏 ； 大葉桉 ； 超低溫保存

並列摘要

This study try to use cryopreservation by vitrification technique to experiment on Chamaecyparis obtusa var. formosana and Eucalyptus robusta buds and suspension cell culture. Then we test PVS2 toxicity、LS(loading solution) test and different concentration of PVS2 to find out the best formula to enhance of the cell viability after freezed.

The best result to induce buds of Eucalyptus robusta in MS medium with 0.5 ppm BA was to use 1 ppm NAA. The results showed that single bud could most effectly induce 10 buds and to induce callus with 5 ppm 2,4-D. And the best results to induce buds of Chamaecyparis obtusa var. formosana in WPM medium with 0.5-1 ppm BA. And callus induction were to use 5 ppm NAA.

The Eucalyptus robusta suspension culture cell load in PVS2 solution over 15 minutes will decrease cell viability below 35％, but the Chamaecyparis obtusa var. formosana suspension culture cell load in PVS2 solution will still preserve cell viability over 60％ after 30 minutes. The Eucalyptus robusta suspension culture cell can preserve about 60％ viability of cell by LS(loading solution) test and to decrease PVS2 toxicity by soaking in PVS2 solution after 15 minutes.

Take Eucalyptus robusta suspension culture cell with 50％PVS2 solution after 5 minutes and then with 100％PVS2 after 10 minutes will preserve the viability of cell over 68% and to induce the growth of callus. Take Eucalyptus robusta buds with 50％PVS2 solution after 60 minutes and then with 100％PVS2 after 60 minutes will preserve the viability of cell over 82％. Take Chamaecyparis obtusa var. formosana buds with 50％PVS2 solution after 60 minutes and then with 100％PVS2 after 60 minutes will preserve the viability of cell over 76％ and make 33％buds induce callus.

並列關鍵字

Chamaecyparis obtusa var. formosana ； Eucalyptus robusta ； Cryopreservation ； Vitrification

參考文獻

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