臺灣博碩士論文加值系統

番石榴(Psidium guajava L.)為桃金孃科(Myrtaceae)番石榴屬(Psidium)熱帶果樹，為台灣重要的經濟果樹之ㄧ。為建立番石榴微體繁殖有效率的再生系統，本試驗以結合子胚誘導體胚發生和利用下胚軸進行器官發生。在不同週齡的結合子胚中，以夏果10週齡結合子胚有最好的體胚誘導率。而不論結合子胚週齡為何，又以先於含3%蔗糖和2.3或4.5 μM 2,4-D及含5%蔗糖和2.3 μM 2,4-D的MS基本培養基處理10天後轉換到不含生長調節劑的MS基本培養基培養30天，誘導體胚形成的效果最佳。另季節和基因型也會影響體胚的形成率。下胚軸器官發生則以NAA和BA二者的組合效果較單獨使用者好，且延長培養時間可提高下胚軸培植體產生不定枝梢的比例。此外，在番石榴的枝梢增殖上，BA的處理效果優於TDZ處理者，枝梢增殖後轉換至不含BA的培養基或提高MS大量鹽類濃度，可改善枝梢節間短縮的問題，在本試驗所用含有6.66 μM BA的1/2 MS培養基也可應用於其他番石榴基因型的枝梢增殖。番石榴高效率的微體繁殖方法，不僅提供優良基因型快速增殖的方法，也可能藉由基因轉殖改善基因型。

Guava (Psidium guajava L.) is a tropical fruit which belongs to the family Myrtaceae and the genus Psidium. It is an important economical fruit crop in Taiwan. To establish an efficient regeneration system for guava micropropagation, zygotic embryos for somatic embryogenesis and hypocotyls for organogenesis were used in this study. Among zygotic embryos with different week-old ages, the best induction rate of somatic embryogenesis was achieved with 10 week-old zygotic embryos from summer fruits. Regardless the week-old ages of zygotic embryos, the best induction rate occurred on MS medium supplemented with 3% sucrose and 2.3 or 4.5 μM 2,4-D, or MS medium supplemented with 5% sucrose and 2.3 μM 2,4-D for 10 day then transferring to PGR-free full-strength MS basal medium. Their somatic embryo induction rates were all above 60%. Furthermore, somatic embryo formation rate was also affected by seasons and genotypes. For hypocotyl organogenesis, the combined effect of NAA and BA was more efficient than either of them used alone. Extending the incubation time can further increase the proportion of hypocotyl producing adventitious shoots. In addition, BA treatment exhibited better effect on shoot proliferation of guava than of TDZ. Transferring proliferated shoots to MS medium without BA or with increased MS macro salt concentration can alleviate the problem of short shoot internodes. The 1/2 MS medium supplemented with 6.66 μM BA used in this experiment can also be applied to other guava genotypes for shoot proliferation. A high efficiency micropropagation system of guava not only provides a way for rapid proliferation of elite genotypes, but also offers a possibility to improve the genotype by genetic transformation.

中文摘要 ……………………………………………………………………Ⅰ
Abstract …………………………………………………………………Ⅱ
謝誌 …………………………………………………………………………Ⅳ
目錄 …………………………………………………………………………Ⅴ
圖表目錄 ……………………………………………………………………Ⅷ
略字解 ………………………………………………………………………Ⅹ
壹、前言 ……………………………………………………………………1
貳、前人研究 ………………………………………………………………3
一、植物細胞的全能性與形態發生 ………………………………………3
(一)器官發生 ………………………………………………………………3
1.影響器官發生的因素 ……………………………………………………3
(1)培植體的來源和生理年齡 ……………………………………………3
(2)基本培養基的組成 ……………………………………………………4
(3)植物生長調節物質 ……………………………………………………4
(4)其他添加成分 …………………………………………………………5
(5)培養條件 ………………………………………………………………6
(二)體胚發生 ………………………………………………………………7
1. 影響體胚發生的因素 …………………………………………………8
(1)培植體的來源和生理年齡 …………………………………………8
(2)生長素(auxins)和細胞分裂素(cytokinins) ………………9
(3)離層酸(abscisic acid、ABA) ………………………………10
(4)培養基和培養條件 ……………………………………………………10
二、番石榴微體繁殖 ………………………………………………………12
(一)以種子或結合子胚為培植體 …………………………………………12
(二)以果肉為培植體 ……………………………………………………13
(三)以實生苗為培植體 ……………………………………………………13
(四)成年植株培植體 ……………………………………………………14
三、番石榴組織培養的應用與遠景 ………………………………………15
參、材料與方法 …………………………………………………………………16
一、植物材料和消毒方法 …………………………………………………16
(一)結合子胚體胚發生試驗 …………………………………………………16
(二)下胚軸培養試驗 ……………………………………………………16
(三)枝梢增殖試驗 ……………………………………………………………16
二、基本培養基與培養方法 …………………………………………………17
三、試驗方法 …………………………………………………………………17
(一)結合子胚體胚發生試驗 …………………………………………………17
1. 2,4-D和蔗糖濃度組合、週齡和季節的影響 …………………………17
2. 基因型的影響 ……………………………………………………………17
3. 體胚組織學觀察 …………………………………………………………18
(二)下胚軸培養試驗 …………………………………………………………18
(三)枝梢增殖試驗 ……………………………………………………………18
1. BA和TDZ對枝梢增殖的影響 ……………………………………………19
2. MS大量鹽類濃度對枝梢增殖和植株高度的影響 ………………………19
3. 移除生長調節劑對植株高度的影響 ……………………………………19
4.基因型的影響 ………………………………………………………………19
四、統計方法 …………………………………………………………………19
肆、結果 ……………………………………………………………………21
一、番石榴結合子胚的培養 …………………………………………………21
(一)不同週齡結合子胚的發育 ………………………………………………21
(二)夏果結合子胚癒傷組織誘導 …………………………………………21
(三)夏果結合子胚體胚形成 ………………………………………………21
(四)冬果結合子胚癒傷組織誘導和體胚形成 ……………………………22
(五)基因型對番石榴體胚發生的影響 ……………………………………23
(六)體胚的組織切片分析 …………………………………………………23
二、下胚軸植株再生 ………………………………………………………23
三、枝梢增殖 ………………………………………………………………24
(一) BA和TDZ濃度的影響 ………………………………………………24
(二)移除生長節調劑對增殖枝梢植株高度的影響 ………………………24
(三) MS大量鹽類濃度對枝梢增殖和植株高度的影 ……………………25
(四)基因型對枝梢增殖的影響 ……………………………………………25
伍、討論 ……………………………………………………………………27
一、培植體類型對體胚發生的影響 ……………………………………27
二、生長素和蔗糖濃度對體胚誘導的影響 ……………………………27
三、生長素和細胞分裂素對器官發生的影響 …………………………28
四、細胞分裂素對枝梢增殖的影響 ……………………………………28
陸、結論 …………………………………………………………………30
柒、參考文獻 ……………………………………………………………59
附表一、基本培養基之組成 ……………………………………………69
附表二、不同週齡夏果番石榴果實結合子胚體胚發生之試驗總表 …70
作者簡介 …………………………………………………………………71

圖1. J×W番石榴8-12週週齡夏果的外觀 ……………………………31
圖2. J×W番石榴8-12週齡夏果果實結合子胚的觀察 ………………32
圖3. J×W番石榴結合子胚的體胚發生 ………………………………33
圖4. J×W番石榴體胚組織學切片 ……………………………………34
圖5. 番石榴品系6M(6)下胚軸器官發生 ……………………………35
圖6. BA和TDZ對番石榴品系J(×)18枝梢增殖的影響 ………………36
圖7. 番石榴枝梢培養於含有TDZ的培養基，植株生長情形 …………37
圖8. 移除TDZ或BA對番石榴品系J(×)18枝梢生長的影響 …………38
圖9. BA的有無與MS大量鹽類濃度對番石榴品系J(×)18枝梢增殖和株高的影響……………………………………………………………………………39
圖10. 番石榴基因型對枝梢增殖和株高的影響 ………………………40
表1. 週齡、蔗糖和2,4-D對J×W番石榴夏果癒傷組織形成影響之ANOVA分析表 ………………………………………………………………………………42
表2. 週齡、蔗糖和2,4-D對J×W番石榴夏果結合子胚癒傷組織形成率(%)影響之交感分析 ………………………………………………………………………43
表3. 週齡、蔗糖和2,4-D對J×W番石榴夏果結合子胚癒傷組織形成率的影響 ………………………………………………………………………………44
表4. 週齡、蔗糖和2,4-D對J×W番石榴夏果結合子胚體胚形成影響之ANOVA分析表 ……………………………………………………………………………………45
表5. 週齡、蔗糖和2,4-D對J×W番石榴夏果結合子胚體胚形成率(%)影響之交感分析 ………………………………………………………………………………46
表6. 週齡、蔗糖和2,4-D對J×W番石榴夏果結合子胚體胚形成率(%)的影響 ……………………………………………………………………………………48
表7. 週齡、蔗糖和2,4-D對J×W番石榴冬果癒傷組織形成影響之ANOVA分析表 ……………………………………………………………………………………49
表8. 週齡、蔗糖和2,4-D對J×W番石榴冬果結合子胚癒傷組織形成率(%)的影響 ………………………………………………………………………………………50
表9. 週齡、蔗糖和2,4-D對J×W番石榴冬果結合子胚體胚形成影響之ANOVA分析表 ………………………………………………………………………………………51
表10. 週齡、蔗糖和2,4-D對J×W番石榴冬果結合子胚體胚形成率(%)的影響 ………………………………………………………………………………………52
表11. 基因型、培養基和週齡對番石榴體胚形成的影響 ……………………53
表12. BA和NAA濃度組合對番石榴品系6M(6)下胚軸培植體於培養30和60天後癒傷組織、枝梢和根形成的影響 ………………………………………………………54
表13. BA與TDZ對番石榴品系J(×)18枝梢增殖和株高的影響 ………………56
表14. MS大量鹽類濃度對番石榴品系J(×)18枝梢增殖和株高的影響 ………57
表15. 基因型對番石榴枝梢增殖和株高的影響 …………………………………58

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