Title

腎葉山螞蝗腋芽及油菜花粉超低溫冷凍保存流程之探討

Translated Titles

Investigation on Protocols of Cryopreservation in the Axillary Buds of Desmodium renifolium (L.) Schindler and Pollen of Brassica campestris L. var. amplexicaulis Makino

Authors

廖郁欣

Key Words

腎葉山螞蝗 ; 油菜花粉 ; 超低溫冷凍保存 ; Desmodium renifolium (L.) Schindler ; Pollen of Brassica campestris L. var. amplexicaulis Makino ; Cryopreservation

PublicationName

中興大學生命科學系所學位論文

Volume or Term/Year and Month of Publication

2013年

Academic Degree Category

碩士

Advisor

廖松淵

Content Language

繁體中文

Chinese Abstract

本研究材料為腎葉山螞蝗 [Desmodium renifolium (L.) Schindler] 腋芽及油菜(Brassica campestris L. var. amplexicaulis Makino)’阿茵’及’秀珍’花粉,依不同植物器官特性,分別探討玻璃化法及乾燥脫水法超低溫保存最佳處理流程。使用玻璃化法冷凍保存藥用植物腎葉山螞蝗腋芽,探討預培養處理及冷凍保護劑對存活率之影響,並以生理分析探討預培養對培植體之影響。以矽膠脫水乾燥法,進行油菜花粉超低溫冷凍保存,探討花粉含水率與保存後存活率、授粉、結莢率相互關係。 以株齡35~42天腎葉山螞蝗植株,切取1~2 mm含腋芽之培植體進行實驗,依序測試不同PVS2處理時間、不同LS處理時間、不同預培養蔗糖濃度及預培養天數。研究結果顯示,腎葉山螞蝗培植體以0.3 M蔗糖預培養處理3天,於室溫下 (25℃) 處理LS 60 min,冰浴 (0℃) 下處理PVS2 150 min,接著快速置入液態氮中進行冷凍保存,回溫後置於以WPM為基底,添加BA 0.1 mgL-1,NAA 0.01 mgL-1之回復培養基中,進行回復生長,可得最佳存活率75 %~ 80%。腎葉山螞蝗經預培養後,分析培植體內水分生理及內含物之變化,隨著預培養蔗糖濃度及天數增加,培植體相對含水量、水分潛勢及滲透潛勢皆有下降趨勢,可溶性糖和可溶性蛋白含量增加,可知腎葉山螞蝗具滲透調節能力,改變培植體生理狀態,增加培植體耐旱及耐凍性,以提升存活率。 油菜花粉使用21%蔗糖濃度之B&K培養基,以體外萌發法測試冷凍保存前後花粉活力。將當天採集花苞內花藥取下,放於塑膠小盒內,置入矽膠乾燥劑中進行乾燥脫水,經冷凍保存後,油菜(‘阿茵’及’秀珍’)花粉經乾燥80 ~100 min,含水量約為25%~30%,有較高的體外萌發率。經最佳條件處理之花粉,回溫後進行授粉,結莢率亦最佳,且其冷凍保存一年以上之花粉,仍具有受精力,具有高結莢率。

English Abstract

This study used in vitro grown of Desmodium renifolium (L.) Schindler and pollen of Brassica campestris L. var. amplexicaulis Makino (‘All-in’ and ‘Show-jean’). According to the specialty of different organs, this study investigated individually the best cryopreservation protocols of vitrification and desiccation dehydration by silica gel. Excised explants of Desmodium renifolium (L.) Schindler were cryopreseved by vitrification approach. Furthermore, the buds was explored for the effect of preculture and cryprotectant and analyzed for physiological state after preculture treatments. Otherwise, pollen of Brassica campestris L. var. amplexicaulis Makino was cryopreseved by desiccation dehydration approach and explored the relationship among water content, survival rate and podding. Excised the explants including axillary bud from 35~42 day-old Desmodium renifolium (L.) Schindler were precultured on medium for different tests, as follows, loaded with different duration of PVS2, LS and the plantlet were precultured on medium of different sucrose concentration for different duration.The result demonstrated the explants from plantlet precultured on 0.3M sucrose medium for 3 days, treated with LS for 60 min at room temperature and PVS2 for 150 min at 0℃ and then immerged in liquid nitrogen directly for cryostorage. Buds were rewarmed rapidly after one week, then plated on petri dishes containing WPM medium supplemented with BA 0.1 mgL-1,NAA 0.01 mgL-1 for the growth of recovery. The survival rate following this protocol could reach to 75~80%. Physiological analysis of explants shows that preculture decreased realative water content, water potential and osmotic potential, and increased soluble sugar and protein concentration. This result indicated that the physiological state of explants was regulated with osmotic adjustment during preculture treatment, which influenced tolerance of dehydrated and frozen, hence increasing survival rate. All in vitro experiment of pollen germination test on B&K medium supplemented with 21% sucrose concentration. Remove the anthers from the buds of rape collected on the day, put them into the small plastic bowl which diameter is 2.2 cm, and dehydrated the anthers by silica desiccant, lastly immerged into liquid nitrogen. Pollen of rape, ‘All-in’ and ‘Show-jean’, when dehydrated for 80 ~ 100 min reached the better germination after cryopreservation, at the time, the water content is between 25%~30%. The result of podding from the pollination of the best treatment confirmed that the viability of cryostoraged pollen. Moreover, the pollen of rape which storaged in liquid nitrogen for one year still keeped the germinability and ability of podding.

Topic Category 生命科學院 > 生命科學系所
生物農學 > 生物科學
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Times Cited
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  2. 楊凱植(2015)。青脆枝超低溫冷凍保存流程及蝴蝶蘭去病毒技術之探討。中興大學生命科學系所學位論文。2015。1-57。