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  • 學位論文

延緩生長與超低溫對於艷斑苣苔人工種子保存之影響

Effect of slow growth and ultra-low temperature on the storage of Kohleria synseed

指導教授 : 方中宜

摘要


對於食品安全及維持農業生物多樣性而言,離體保存為一項種原保存的重要技術,此技術可有效利用於已瀕臨滅絕的植物種原長期保存上。 在過去幾年內,已有超過1,000種之熱帶植物被成功保存,其中延緩生長為短期保存植物材料之技術,而超低溫保存則為長期保存植物種原之策略。本研究以豔斑苦苣之頂芽進行人工種子製作並建立短期及長期保存之技術。首先將頂芽利用3%海藻酸鈉及100 mM氯化鈣進行人工種子備製,延緩生長是採用2-16%之滲透壓溶液 (甘露醇和山梨醇)、溫度 (4、15及25°C)以及不同MS鹽類濃度來處理。研究結果顯示,以2%甘露醇及山梨糖醇保存人工種子一個月後,其種子可貯藏性和活性可達36.7%和100%;另外,當人工種子貯藏於15℃下84天後,其可貯藏性可高達100%,然而,隨者貯藏時間增加,種子活性逐漸遞減 (分別為68.5%、47.8%及37.7%) . 當人工種子以 ¼ MS鹽類濃度貯藏1和2個月後,活性仍可維持於87.7%及70.8%。另外,在冰凍保存實驗上,許多研究指出需以預措處理延長人工種子貯藏時間,本試驗以0.3 M蔗糖預處理9天後,風乾6小時後種子含水量降為23.6%,但人工種子仍無法在液態氮中生存。另外玻璃質化之實驗中,人工種子先以PVS2溶液處理後再風乾0 - 6小時,隨後快速放置於液態氮中,依然無任何頂芽可成功再生。未來將持續測試不同抗凍劑對於提升人工種子抗凍性之能力。

並列摘要


In vitro conservation of plant genetic resources is important for food security and agro-biodiversity. It has the potential to guarantee the long-term preservation of germplasm of species threatened with extinction. During the past years, in vitro techniques have been extensively developed and applied to more than 1,000 species, including many tropical species. In vitro conservation via slow growth technique has been considered a useful short-term storage system for the in vitro cultured plant materials, whereas cryopreservation has been used for the long-term preservation of plant germplasm. This study aims at establishing a short- and long-term conservation protocol for Kohleria using shoot tip-based synseed. Synseeds were produced when the shoot tips were encapsulated with 3% sodium alginate in combination with 100 mM calcium chloride. For the short-term storage, preservation techniques included the use of osmoticum (mannitol and sorbitol) at concentrations ranging from 2 to 16%, reduced temperature (4, 15 and 25°C) and reduced nutrient availability (full, ½ and ¼ Murashige and Skoog (MS) salts and vitamins). In the osmoticum experiment, best results were obtained with the 2% mannitol and 2% sorbitol combination treatment which resulted in a storability of 36.7% and a viability of 100% after the first month of storage. At temperature of 15°C synseeds maintained a high storability percentage of 100% throughout the 84 days period with a fairly good viability percentage after each month (68.5, 47.8 and 37.7%). Kohleria synseeds can be stored for up to 60 days at ¼ MS nutrient strength while still maintaining high viability percentage of 87.7 and 68.5% after the first and second month storage respectively. For the long-term storage the encapsulation-dehydration technique was used. Nine days preculture with 0.3 M sucrose followed by 6 hour dehydration resulted in a low moisture content of 23.6%. For encapsulation-vitrification, precultured synseeds were dehydrated for 0 to 6 hours and treated with PVS2 before rapid immersion in liquid nitrogen. Under these conditions, regrowth of explants was not successful. Despite several attempts in modifying the cryogenic conditions, no synseed survived the liquid nitrogen storage.

參考文獻


Sharma, S. K. and M. Sharma. 2013. Improved Protocol for In Vitro Propagation of Gloxinia (Sinningia sp.). Journal of Cell and Tissue Research, 13: 3545-3548.
Scherwinski-Pereira, J. E. and F. H. S. Costa. 2010. Conservation In Vitro of Plant Genetic Resources: Strategies, Principles and Applications, In Vitro Culture of Plants. Brasilia. Embrapa Information Technology, 15: 177-234.
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Ai, P. F. and Z. R. Luo. 2004. Conservation of In Vitro Shoots of Persimmon and Date Palm by Slow Growth and Genetic Stability of Recovered Planlets. Acta Horticultural Science, 31: 41-46.

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