臺灣博碩士論文加值系統

滲透壓處理有助於水稻癒傷組織誘導體胚形成，並發育為芽體，相關機制仍不清楚，誘導過程中可能牽涉許多基因的調控。本論文目的即是要探討主要在水稻胚部表現且受ABA及不同逆境誘導之Aldose Reductase 基因 (OsARs)，在滲透壓逆境誘導水稻癒傷組織再生植株過程中，可能扮演的角色。本研究以水稻台農67號(TNG67; OsAR5 野生型)、OsAR5過量表現轉殖系(OxAR5)，及OsAR5 T-DNA插入突變系（KoAR5）未熟胚為材料，分別於N6D2基礎培養基中添加不同濃度sorbitol（0、0.3M、0.6M），分別以N6D2S0、N6D2S3及N6D2S6表示，觀察癒傷組織誘導情形與移至N6K4N2再生培養基後，植株再生情形。此外，也利用反轉錄酶聚合酶連鎖反應（RT-PCR）分析培養過程中5個OsARs之基因表現，並進一步以OsAR5多株抗體（polyclonal antibody）進行酵素免疫定位分析。試驗結果顯示三種水稻材料，會隨著滲透壓處理濃度的提高，癒傷組織誘導率、鮮重、及水分含量均會顯著下降，尤其是KoAR5，除生長受阻外，癒傷組織褐化率大幅提高，顯示OsAR5與滲透壓逆境下細胞之生長有關。此外，TNG67與OxAR5會隨著高濃度sorbitol處理顯著提高植株再生率（再生率分別為13％與26％），而KoAR5則完全沒有植株再生，顯示OsAR5與滲透壓誘導之水稻植株再生有密切相關。RT-PCR結果顯示5個OsARs基因均可在水稻癒傷組織中偵測到，不過以OsAR1與OsAR2表現量較為明顯，唯兩者的表現較不受滲透壓誘導，反之，除了在KoAR5細胞中偵測不到OsAR5表現外，sorbitol處理可提高TNG67與OxAR5中OsAR5之表現。進一步由免疫組織化學分析結果顯示，在癒傷組織誘導與植株再生初期，在表皮（epidermis）或細胞外圍組織（peripheral layer）、原生質濃密的細胞以及管胞分子（tracheary element），可觀察到OsAR5之分佈。上述結果均顯示OsAR5與滲透壓誘導之水稻癒傷組織植株再生能力有關，推測可能與其影響醣醇之生合成與降低細胞之氧化逆境傷害（褐化）有關。本論文是第一篇探討AR與植物生長與分化有關之研究，值得進一步深入研究，以釐清其可能扮演之角色。

It’s well known that osmotic stress can induce somatic embryogenesis and further shoot regeneration in rice callus. The mechanisms of totipotency are less understood so far even though plentiful research has been discussed with respect to cell differentiation. The purpose of this study was explored the possible role of Aldose Reductase gene (OsARs), which is subjected to ABA and stresses regulation, during callus induction and shoot regeneration in rice. The immature embryos from three genotypes of rice relevant to OsAR5, Tainung 67 (TNG67; OsAR5 wild-type), OsAR5 overexpression transgenic lines (OxAR5), and OsAR5 T-DNA insertion mutant (KoAR5) were used in present study. The callus were derived from N6D2 (N6 basal medium containing 2 mgL-1 2,4-D) induction medium supplemented with different concentration of sorbitol (0, 0.3M, 0.6M), represented as N6D2S0, N6D2S3, and N6D2S6, respectively. It showed the callus induction rate, fresh weight, and water content were decreased significantly accompany with sorbitol concentration in all genotypes. In KoAR5 has the highest browning rate than the others. Besides, the 14-days-old of callus were transferred to N6K4N2 (N6 basal medium containing 4 mgL-1 kinetin and 2 mgL-1 NAA) regeneration medium. The result showed that has no plantlets regenerated from TNG67 in N6D2S0 and N6D2S3 treatment. It can be increased to 13% in N6D2S6 treatment. Besides, the shoot regeneration frequency in N6D2S3 and N6D2S6 from OxAR5 enhanced to 13% and 26% respectively. In opposite, all three treatments in KoAR5 have no shoot regenerated. It suggested that the expression of OsAR5 is relevant to shoot regeneration in rice callus.
The reverse transcriptase polymerase chain reaction (RT-PCR) analysis and immunohistochemical localization of OsARs were further determined. The result showed that five OsARs genes can be detected in rice callus especially OsAR1 and OsAR2. The expression, except of OsAR5 is up-regulated by osmotic stress in TNG67 and OxAR5, the others seem have no significant difference no matter of with / without sorbitol treatment. In addition, it cannot detect any expression of OsAR5 in KoAR5 either callus induction or regeneration stage. According to the immunohistochemical analysis, the OsARs can be observed in the epidermis, peripheral layer, and tracheary elements during callus induction and shoot regeneration. The regenerable callus from N6D2S6 in TNG67 has more obviously distribution of OsARs.
The result is consistent with our previous study that shoot regeneration frequency is increased significantly by osmotic stress treatment in rice callus. We suggested that OsAR5 is closely related to shoot regeneration induced by high concentration of sorbitol treatment. It may have roles on detoxification of reactive aldehyde compounds and osmotic adjustment under osmotic stress treatment. This is the first study to mention the correlation between plant differentiation and AR. More morphological, physiological, and gene expression analysis is necessary to clarify the possible role of OsARs during shoot regeneration in rice callus under osmotic stress treatment.

內容......................................................................................................頁次
中文摘要..................................................................................................Ι
英文摘要................................................................................................III
謝誌...........................................................................................................V
目錄.........................................................................................................VI
表目錄....................................................................................................VII
圖目錄.....................................................................................................IX
一、前言....................................................................................................1
二、前人研究............................................................................................3
三、材料與方法.......................................................................................11
四、結果....................................................................................................25
五、討論....................................................................................................31
六、參考文獻............................................................................................35
附錄一......................................................................................................61
附錄二…………………………………………………………….….…62
附錄三………………………………………………………………..…63

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