臺灣博碩士論文加值系統

本研究利用''台農17號''鳳梨組培苗為材料，探討以EMS誘變之最適條件。隨著處理濃度與處理時間增加，植株存活率隨之降低；以0.4% EMS處理12小時之植株存活率為97.5%，但處理時間增加至24小時存活率則降至25%，顯示處理濃度與時間兩者對植株存活率均有顯著影響。在調整EMS溶液pH值方面，在相同處理時間條件下，其中溶液pH值為6.0之處理組存活率為最高，且與未經誘變之對照組相比其遺傳相似係數均為最低。
　　利用不同的培養基配方測試''台農17號''鳳梨組培苗之白化苗誘導率，結果顯示，種植於含有NAA之全量MSMO培養基之培植體經暗期培養8週後其白化苗誘導率皆顯著高於未添加NAA之對照組。而以 EMS溶液處理白化苗後其側芽萌芽率為依據，0.01% 1hr為接近LD50之處理條件。白化苗增殖系統方面，頂芽扦插苗之新生白化苗誘導率較側芽扦插苗需要較短時間即可達到最大新生白化苗誘導率；前者培養5週後白化苗誘導率為96.9%，後者培養11週後誘導率只有65.1%。頂芽扦插苗平均節位數為8.1節，而側芽扦插苗僅有6節。將白化苗莖段種於含有全量MSMO培養基並進行照光處理2週後側芽陸續萌發，並發育成一新生植株。此白化苗繼代增殖系統，可用於鳳梨誘變育種。
　　經EMS處理之鳳梨組培苗培養於不同低溫環境下，隨著處理溫度降低，對照組及誘變處理組之植株存活率均隨之降低，EMS處理後植株先於室溫下恢復2週再進行低溫處理者之存活率較高，其中誘變後先於室溫恢復2週，再置於8℃低溫下2週之處理組其植株存活率為56.7%，顯著高於對照組之36.7%。以RAPD分子標誌分析經過不同低溫逆境及恢復時間搭配EMS誘變之M1V2後代，對照組與處理組之相似度係數介於0.953~0.985之間，顯示這些後代均與對照組植株有一定之歧異度。將處理組之存活植株之M1V2後代再次進行低溫逆境篩選處理，結果顯示，於第一次低溫處理後存活之植株後代有較高之低溫逆境耐受性。
　　調查''台農17號''鳳梨EMS誘變株於田間生長情形，可觀察到許多具有畸形果實、葉刺及裔芽過度生長等異常生長之植株，顯示離體誘變可增加鳳梨植株生長情況之歧異度。進一步分析誘變株成熟果實之基本性狀與品質，除果重、冠芽數、冠芽重、可滴定酸及糖酸比之變異係數值較大外，果重、果長、果目數、果心及可溶性固形物等項目其變異係數均不高。

The effects of using EMS (ethyl methanesulphonate) as a mutagen on the survival rate of ''Tainung No.17'' pineapple plantlets cultured in vitro were examined. The survival rate of plantlets decreased when the EMS concentration and treatment duration increased; The survival rate in the treatment of 0.4% EMS 12hr was 97.5% which decreased to 25% in the treatment of 0.4% EMS 24hr. In the same concentration and duration treatments, adjusting the pH value of EMS solution to 6 had the highest survival rate of plantlets and had the lowest genetic similarity with the control.
The influence of different mediums on the induction rate of etiolated ''Tainung No.17'' pineapple shoots was tested. Plantlets cultured on the MSMO medium contained NAA had higher induction rate of etiolated shoots than those cultured on the MSMO medium without NAA after 8 weeks of culture in darkness. Base on the regeneration rate of lateral buds of the EMS treated etiolated plantlets, the treatment of 0.01% EMS 1hr was closed to the LD50. In the study of the proliferation of etiolated shoots, apical bud cuttings had an induction rate of 96.9% in 5 weeks of culture and lateral bud cutting had only 65.1% in 11 weeks of culture. The average number of nodes per shoot regenerated from apical bud cutting was 8.1, while lateral bud cutting had only 6 nodes per shoot. Segments of etiolated shoots were placed on MSMO medium and incubated in lightness. The lateral buds started to regenerate after 2 weeks of culture and then grew into new plantlets. This proliferation system of etiolated shoots could be utilized in the mutation breeding of pineapple.
''Tainung No.17'' pineapple plantlets were exposed to different low temperatures and the results showed that the survival rates of control plantlets and EMS treated plantlets decreased when the temperature was reduced. However, before exposure to low temperature, a recovery period of 2 weeks at room temperature after EMS treatment could increase the survival rate of plantlets. When the EMS treated plantlets were incubated at room temperature for 2 weeks before transfered to 8℃for another 2 weeks, the survival rate increased to 56.7%, compared with 36.7% in the control. The genetic similarities between the control plants and the EMS treated plants were 0.953~0.985. The M1V2 progenies from the plantlets survived after the first low temperature treatment had higher cold tolerance.
Growth of EMS treated ''Tainung No.17'' pineapple plants in field was also investigated. Some plants had morphological distinctions such as abnormal fruits, over proliferation of slips, and leaves with more spines. Analysis of the characteristics of the mature fruit of EMS treated ''Tainung No.17'' pineapple plants indicated that fruit weight, the number of slips, crown weight, titratable acid, and the ratio between total soluble solids and titratable acid had high coefficient of variance. Nevertheless, fruit weight, fruit length, the number of fruitlets, core diameter, and total soluble solids had low coefficient of variance.

中文摘要 i
Abstract ii
表目次 vi
圖目次 vii
一、前言 1
二、前人研究 3
（一）鳳梨組織培養 3
（二）人為誘導突變 4
（三）EMS（ethyl methanesulfonate）誘變 5
1.EMS誘變機制 5
2.影響EMS誘變率之因子 6
3.EMS在作物育種上之應用 7
（四）誘變後代之篩選 9
（五）分子標誌 9
1.種類及原理 9
2.分子標誌之應用 11
（六）鳳梨誘變育種 12
三、材料與方法 14
試驗一、EMS誘變''台農17號''鳳梨組培苗之最適條件 14
（一）不同濃度EMS與處理時間組合之誘變處理 14
（二）不同pH值對EMS溶液誘變能力之影響 17
試驗二、''台農17號''白化苗誘導及增殖系統 18
（一）白化苗誘導 18
（二）白化苗EMS誘變 18
（三）白化苗繼代增殖系統之建立 19
試驗三、低溫逆境搭配EMS離體誘變 20
（一）試驗材料 20
（二）低溫逆境處理 20
（三）誘變處理後存活率調查 20
（四）誘變後代低溫逆境篩選 20
（五）RAPD分子標誌分析 20
（六）統計分析 21
試驗四、''台農17號''誘變植株其果實外觀性狀及品質調查 22
（一）試驗材料 22
（二）試驗方法 22
四、結果 23
（一）EMS 誘變''台農17號''鳳梨組培苗之最適條件 23
1.不同濃度EMS與處理時間組合之誘變處理 23
2.不同pH值對EMS溶液誘變能力之影響 23
（二）''台農17號''鳳梨白化苗誘變 35
1. 白化苗誘導 35
2. 白化苗EMS誘變 35
3. 白化苗繼代增殖系統建立 35
（三）低溫逆境搭配EMS離體誘變 43
1.不同低溫逆境搭配EMS誘變處理 43
2.EMS誘變後代低溫逆境篩選 44
3.誘變後代之RAPD分子標誌分析 46
4.誘變後代之植株性狀調查 46
（四）台農17號鳳梨誘變植株其果實外觀性狀及品質調查 59
五、討論 73
（一）EMS 誘變條件之探討 73
（二）鳳梨白化苗增殖系統及誘變處理 74
（三）低溫逆境搭配離體誘變 75
（四）鳳梨誘變株於田間之生長情形 77
（五）總結 78
六、參考文獻 79

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