Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23668
標題: 臺灣原生植物牛樟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 Plant of Cinnamomum kanehirae Hay
作者: 黃敬涵
Huang, Jing-Han
關鍵字: Cinnamomum kanehirae Hay.;牛樟;vitrification;cryopreservation;osmotic adjustment;玻璃化法;超低溫冷凍保存;滲透調節
出版社: 生命科學系所
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摘要: 
本研究以牛樟(Cinnamomum kanehirae Hay.) 組培苗為試驗材料,切取頂芽及腋芽進行玻璃化法超低溫冷凍保存,探討不同高醣 (0.09 M~0.6 M) 預培養處理及冷凍保護劑處理時間,對牛樟頂芽及腋芽存活率之影響,並探討預培養對芽體生理狀態的影響。
以牛樟株齡80-90天植株進行預培養,切取1-1.5 mm 頂芽及腋芽,依序測試PVS2不同處理時間、LS不同處理時間、不同蔗糖濃度培養基及不同預培養天數,研究結果顯示,牛樟頂芽以0.4 M蔗糖培養基預培養21天後,以LS處理90 min,PVS2冰浴處理120 min,有最佳存活率86.7%;牛樟腋芽以0.4 M蔗糖培養基預培養21天,再以LS處理150 min,PVS2處理120 min,有最佳存活率47%。腋芽存活率較頂芽低,且易長成癒傷組織,因此將牛樟莖段培養於增殖培養基,先誘導腋芽生長3週,再移植到0.4 M蔗糖預培養7天後切取頂端分生組織,以LS處理90 min,PVS2處理120 min,可得存活率53.3%,與株齡3個月植株經0.4 M蔗糖預培養7天,同處理條件之頂芽存活率 (55.9%) 無顯著差異,顯示誘導腋芽生長能提升腋芽之存活率。
牛樟經預培養後,分析芽體水分生理及內含物累積變化,頂芽及腋芽變化趨勢相同,隨蔗糖濃度及預培養天數增加,芽體相對含水量、水分潛勢及滲透潛勢皆有下降趨勢,可溶性糖及可溶蛋白含量明顯增加,顯示牛樟經高醣 (0.09 M~0.6 M) 預培養,透過滲透調節改變芽體生理狀態,增加芽體對冷凍保護劑的忍受力,進而提升存活率。
總結以上,牛樟以玻璃化法超低溫冷凍保存最適處理流程為株齡80-90天,以0.4 M蔗糖預培養21天,切取1-1.5 mm 頂芽,以LS處理90 min,PVS2處理120 min有最佳存活率。

This study used in vitro-grown of Cinnamomum kanehirae Hay. as experimental material. Excised apical buds and axillary buds were cryopreserved by vitrification, and investigated for the effect of pretreatment of different precultures with sucrose medium and cryoprotectant duration on survival rate.
1-1.5 mm apical buds and axillary buds excised from 80-90 days old plants were precultured, and then treated with different duration of PVS2, LS solution and precultures. The results demonstrated that the apical buds precultured on 0.4 M sucrose medium for 21 days, treated with LS solution for 90 minutes and dehydrated with PVS2 for 120 minutes at 0℃ reached a survival rate of 86.7%.
The axillary buds, on the other hand, were precultured on 0.4 M sucrose medium for 21 days, treated with LS solution for 150 minutes and dehydrated with PVS2 for 120 minutes, and the best survival rate achieved 46.97%. The survival rate of axillary buds was lower than that of the apical buds. Also, the axillary buds had a higher tendency of producing callus. To solve the problem, the nodal segments of Cinnamomum kanehirae Hay. were cultured on proliferation medium for 3 weeks to induce axillary buds and then transferred to 0.4 M sucrose medium for 7 days. Then the axillary shoot-tip meristems were then excised from segments. Shoot tips were treated with LS solution for 90 minutes and dehydrated with PVS2 for 120 minutes. The survival rate of the shoot tips amounted to 53.3%, which was not significantly different from the survival rate of the apical buds excised from 3-month-old plants precultured on 0.4 M sucrose for 21 days with the same treatments (55.9%). This revision showed that inducing axillary buds can elevate the survival rate of axillary buds.
Physiology analysis revealed that the preculture treatment of Cinnamomum kanehirae Hay. decreased relatively water content, water potential and osmotic potential, and increased the content of soluble sugar and soluble protein of both apical buds and axillary buds. The result indicated that the plantlets of Cinnamomum kanehirae Hay. regulated the physiology state of buds with osmotic adjustment during the preculture, which increased the ability tolerate the cryoprotectant solution, and thus enhanced the survival rate.
In conclusion, the most optimal protocol for cryopreservation of Cinnamomum kanehirae Hay. would employ 80-90 day old planlets precultured on 0.4 M sucrose medium for 21days; after the preculture, 1-1.5 mm apical buds are to be excised, treated with LS for 90 minutes and PVS2 for 120 minutes. This procedure would achieve the highest survival rate.
URI: http://hdl.handle.net/11455/23668
其他識別: U0005-0808201113372700
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