生長素誘導玫瑰花小葉器內培養之體胚發生

For increasing embryogenic calli formation rate and reducing time of somatic embryogenesis therefore. A procedure for plant regeneration leaflets of roses (Rosa hybrida L.) via in vitro culture is described. Two auxins, NAA and picloram alone, at various concentrations(0.25, 0.5, 1 or 2 mg/l) were tested for its capacity to induce somatic embryogenesis from in vitro- derived leaflet of five cultivars (Christian Dior, Tiffany, Oklahome, Laser, White Queen Elizabeth). Calli could be initiated from in vitro leaflet of five rose cultivars on Murashige and Skoog (MS) medium with NAA or picloram. Although all rose cultivars could from calli, but only ‘Tiffany'could produce embryogenic calli on MS medium including NAA(1 or 2 mg/l).The influence of genetype on embryogenic differentiation is remarkable. In auxins experiment, leaflet explants were precultured on MS medium with 1 mg/l NAA for four weeks followed by subculture on MS medium with 2 mg/l BA. ‘Tiffany'showed the highest amount of regeneration plants.為了提高胚性癒傷組織的形成率並縮短體胚發生的時間，因此描述一種從玫瑰花器內小葉經由體胚發生再生植株的步驟。測試NAA和picloram兩種生長素在不同濃度(0.2L0.5,1 or 2 mg/l)下對五個品種玫瑰花(克利斯汀 迪奧、鐵凡尼、奧克拉荷馬、雷射、白英國女王)的器內小葉培植體誘導體胚發生的能力。在含NAA 或 picloram的MS培養基中，癒傷組織可從五種玫瑰花品種的器內小葉中被產生。雖然所有品種都能產生癒傷組織，但是僅'鐵凡尼'能在含NAA的MS培番基產生胚性癒傷組織。基因型在胚性分化上的影響顯著。'鐵凡尼'小葉培植體培養在含lmg/l NAA的MS培養基4週後，再繼代培養到含2mg/l BA的MS培養基，再生株數量最高

Somatic Embryogenesis via In Vitro Culture of Leaflet Explants of Roses

為了提高胚性癒傷組織的形成率並縮短體胚發生的時間，因此描述一種從玫瑰花器內小葉經由器內培養再生植株的步驟。 測試NAA 和picloram兩種生長素在不同濃度（0.25,0.5,1or 2mg/l）下對五個品種玫瑰花（克利斯汀 迪奧、鐵凡尼、奧克拉荷馬、雷射、白英國女王）的器內小葉培植體誘導體胚發生的能力。在含NAA或picloram的MS培養基中，癒傷組織可從五種玫瑰花品種的器內小葉中被產生。雖然所有品種都能產生癒傷組織，但是僅‘鐵凡尼’能在含NAA的MS培養基產生胚性癒傷組織。基因型在胚性分化上的影響顯著。在生長素實驗中，‘鐵凡尼’小葉培植體培養在含1mg/lNAA的MS培養基4週後，再繼代培養到含2mg/l BA的MS培養基，再生株數量最高。暗培養可縮短誘導體胚發生的時間和增加胚性癒傷組織形成率。光照下培養或暗培養1週、4週或5週都能形成胚性癒傷組織。暗培養1週能產生最多胚性癒傷組織和再生株。玫瑰花‘鐵凡尼’需要光線以誘導體胚發生。在4500lux光照下，胚性癒傷組織的形成率高於其它兩種光強度處理且可縮短體胚發生的時間。雖然小葉的所有位置都能形成癒傷組織，但是僅小葉中間位置和兩側葉緣有胚性癒傷組織產生。小葉中間位置的胚性癒傷組織形成率比兩側葉緣者高。For increasing embryogenic calli formation rate and reducing time of somatic embryogenesis therefore,A procedure for plant regeneration leaflets of roses(Rosa hybrida L.)via in vitro culture is described. Two auxins, NAA and picloram alone , at various concentrations(0.25,0.5,1or 2mg/l)were tested for its capacity to induce somatic embryogenesis from in vitro-derived leaflet of rose of five cultivars(Christian Dior,Tiffany,Oklahoma,Laser,White Queen Elizabeth). Calli could be initiated from in vitro leaflet of five rose cultivars on Murashige and Skoog(MS)medium with NAA or picloram. Although all rose cultivars could form calli but, only ‘Tiffany'could produce embryogenic calli on MS medium including NAA(1or2 mg/l). the influence of genetype on embryogenic differentiation is remarkable. In auxins experiment, leaflet explants were precultured on MS medium with 1mg/l NAA for four weeks followed by subsculture on MS medium with 2mg/l BA.‘Tiffany'showed the highest amount of regeneration plants. Dark incubation periods could reduce time of rose‘Tiffany'inducing somatic embryogenesis and increasing embryogenic callus formation rate.Culture under light or one, four and five of darkness result in embryogenic calli. Dark incubation period of one could produces the highest embryogenic callus and regeneration plantlet. Rose‘Tiffany'needed light to induce somatic embryogenesis. Under 4500lux, embryogenic calli formation rate was higher than other two light intensity treatments and reducing time of somatic embryogenesis. Diferent positions of leaflet might result in callus production, but only middle of leaflet and edge of leaflet could produce embryogenic callus. Middle of leaflet produced higher embryogenic callus rate than that of edge of leaflet.壹、中文摘要…………………………………………………………..i 貳、英文摘要………………………………………………………….ii 参、前言……………………………………………………………....1 肆、前人研究 一、體胚發生…………………………………………………....2 （一）體胚發生的方式…………………………….........2 （二）與胚性能力有關的內在因子…………………….....2 1.構造因子…………………..…………………..............2 2.生理因子…………………………...…………………….....3 3.基因表現…………………………………………………………4 4.分子標記…………………………………………...….......5 （三）內生荷爾蒙…………………………….............5 1.培植體內生荷爾蒙濃度………………............6 2.胚性和非胚性培養物的內生植物荷爾蒙濃度………6 3.體胚發生期間任一荷爾蒙群的作用…………………7 4.荷爾蒙濃度VS.感受性..………………………………………8 （四）植物生長調節劑 1.器官形成…………………………………….……….8 2.體胚發生…………………………………………...11 （五）其它因子 1.無機鹽成分………………………………………….11 2.礦物元素…………………………………………...12 3.碳水化合物………………………………………….13 4.溫度………………….……………….…………...13 5.滲調物質………………………………………….…14 6.光…………………………………………………….14 7.凝膠物質…………………………………….......15 8.冷處理……………………………………………….15 伍、材料方法 一、試驗材料………………………………………………………...16 二、培養基成份……………………………………………………...16 三、培養基之配製…………………………………………………….17 四、培養室環境條件………………………………...……………..17 五、試驗方法 （一）初代培養 1.無菌材料之建立………………………………….…17 （二）體胚形成 1.生長調節劑試驗 （1）小葉癒傷組織誘導…………………………….18 （2）小葉癒傷組織增殖…………………………….19 2.暗培養週數對誘導胚性癒傷組織的影響 （1）暗期誘導……………………………………….19 （2）明期增殖及再生……………………………….19 3. 不同光強度下的體胚發生癒傷組織增殖…………19 4.小葉不同位置的胚性誘導反應…………………...20 5.組織學的檢定 （1）冷凍組織切片及染色………………………….20 （2）胚性細胞觀察………………………………….20 六、再生株馴化………………………………………………….20 七、統計分析………………………………………….........20 陸、結果 一、體胚形成 （一）癒傷組織誘導及體胚發生 1.生長素種類與癒傷組織誘導……………………...………......22 2.癒傷組織增殖與植株再生…………………………………......23 二、暗培養時間與體胚發生 （一）暗期癒傷組織誘導……………………………………24 （二）明期癒傷組織增殖與植株再生………………………25 三、光強度與體胚發生 （一）癒傷組織誘導……………………….……….......25 （二）癒傷組織增殖與植株再生………………………....26 四、小葉不同位置的體胚發生 （一）癒傷組織誘導…………………………………......26 （二）癒傷組織增殖與植株再生…………………………..27 五、組織學觀察 （一）冷凍組織切片及染色………………………………..27 （二）胚性細胞觀察………………………………………..27 柒、討論 一、生長素和體胚發生………………………………….......58 二、暗培養時間與體胚發生……………………………….....60 三、光強度與體胚發生…………………………………….....61 四、小葉不同位置與體胚發生………………………………...62 五、玫瑰花體胚再生植株困難的因素…………………….....63 捌、參考文獻…………………………………….................64 玖、附錄……………………………………………………….......7