Studies on cell suspension culture and somatic embryogenesisi of Musa formosana

台灣芭蕉(Musa formosana)幼雄花序接種於含有IAA 1 mg/L及NAA 1 mg/L之逆分化培養基，另參試組合Auxin 2,4-D 1 mg/L及Picloram 3 mg/L能誘導較高的癒傷組織生成率。幼雄花序第13-17節是較能誘導癒傷組織生成的段節，誘導率皆有58%以上。合子胚培植體當單獨始使用Dicamba或Picloram做為強效培養基，培植體褐化程度較低，並且癒傷組織可以形成較多的亮黃色、粒狀癒傷組織的胚性癒傷組織及擬胚，其癒傷組織誘導率皆有60%以上。繼代培養幼雄花序衍生癒傷組織，培養於黑暗環境下，添加Picloram 1 mg/L或Dicamba 1 mg/L搭配gelrite的培養基，癒傷組織有較佳的表現。雖然將2,4-D做為主要的Auxin來誘導逆分化時，隨著濃度增加，培植體褐化程度增加。但是癒傷組織可以在含有2,4-D的液體培養基中增生，幼雄花序及合子胚衍生癒傷組織懸浮培養2個月後可建立均質的胚性懸浮細胞系。酸化處理(pH 4.0-4.5)會促進球體相的細胞游離。酸化處理7和14天後，促進球狀體解體，釋放游離單細胞，並朝對稱分裂為主，加速胚性細胞增生。SH3培養基及SH3+MES可將pH提高誘導細胞不對稱分裂，培養於SH3+MES可以加速前胚性細胞提早極化、形成前胚及球狀體。SH3添加PIPES同樣也可以誘導極化和球體胚形成完整個原表皮。胚性懸浮細胞經TB5、SH3及搭配MES 10 g/L預處理誘導極化後平板培養，其中由 SH3預處理的細胞能誘導再生較多的體胚，並且以濾紙棉花墊做為培養介質，較能誘導出正常發育的體胚。將懸浮細胞利用<60或介於30-60的篩網過篩分級，稀釋成1/30 c.c PCV，平板培養於添加SH3 8 ml液體培養基的濾紙棉花墊上，可以發育成較多的正常體胚。體胚接種於1/2 MS 培養基添加BA 10 mg/L可誘導69%的發芽率。培養基添加NAA無法增加小植株的發根率，然而BA組合NAA可提升小植株的發芽率。培養基中添加GA可增加發芽率及葉長，但是植株會異常脫色及突長。Summaryhe young male inflorescence of Taiwan native diploid banana (Musa formosana) was used as explant, and cultured in modified MS medium supplemented with complex auxin including IAA 1 mg/L, NAA 1 mg/L, and 2,4-D 1 mg/L and Picloram 3 mg/L suitable for callus formation. The top male hand portions ranged from the 13th to 17th sections were capable to give rise 64-82% callus formation on the medium as above. The zygote embryos were aptly to generate embryogenic callus on MS medium added with NAA 1 mg/L, IAA 1 mg/L combined Picloram 1 mg/L or Dicamba 1 mg/L in 2-3 months after inoculation. In further, the callus proliferation could be subcultured on the above medium but supplemented Picloram or Dicamba as the sole auxin. The male inflorescence-derived callus was subcultured in liquid TB5 medium ( Ma, 1988), and obtained homogenous cell population about 2 month after suspension culture. he extracellular pH level in TB5 medium of the cell suspension culture was varied in the range pH 4.45±0.37, and also concordantly associated with growth phase change. The addition of MES 10 g/L could upgrade the extracellular pH in the stable level 4.51±0.17, and also induce the preembryogenic cells destine to polar growth depicted by the asymmetric division. Controlling extracellular pH at 5.7 and culture in SH3、SH3 with MES led up to the polar growth. The cells culture in SH3 with MES were polarized earlier, and remain extracellular pH over 5.46. After polarization treatment for 7 days, the embryogenic cells could formed proembryo and globoids. Used PIPES with SH3 medium could also be induced to polarization and formed globoids with complete protoderm Acidic treatment (pH 4.0-4.5) promoted globoids to disunite small mass and release single cells. After acidic treatment for 7 and 14 days, the bicellular trended to do symmetric division. Controlling extracellular pH at 5.7 and culture in SH3、SH3 with MES led up to the polar growth. The cells culture in SH3 with MES were polarized earlier, and kept extracellular pH over 5.0. After polarization treatment for 7 days, the embryogenic cells could formed proembryo and globoids. Used PIPES with SH3 medium could also be induced to polarization and formed globoids with complete protoderm Before plating, the suspension prembryogenic cells were pretreated on TB5, SH3 medium with or without was consistent 10 g/L MES. The SH3 pretreated cells regenerated higher quantity of somatic embryos on plating medium than those on TB5, TB5+MES and SH3+MES pretreated. The size of suspension cell cluster was separated with <60 or 30-60 meshes and were diluted 1/30 c.c PCV proceeding to plat on 8ml SH3 medium with filter paper and cotton as the culture bridge, could development normal somatic embryos than agar, gelrite and 4 ml SH3 addition. Somatic embryo inoculated on 1/2 MS medium supplement with 10 mg/L BA induced 69% of shooting. The rooting rate did not increase, when medium supplement NAA. However, the shooting rate increase of plantlets on medium complex added NAA with BA. The increase of shooting rate and leave length in medium GA, but the leaves had exceptional discoloration and elongation.目 錄試委員會審定書…………………………….…...…………...……………………….i謝…………………………………………..….……..………..…...………………….ii寫字……………………………………………………..……………………………iii文摘要………………………………………….…….……..…...…...………………iv文摘要……………………………………………..….…..………..…………………v、 前言 .................................................................................................................1、 前人研究 .........................................................................................................2、體胚發生.............................................................................................................2一)基因型.........................................................................................................2 (二)培植體.........................................................................................................3三)植物生長調節劑.........................................................................................4 四)培養基pH....................................................................................................5五)光照.............................................................................................................6 (六)氣體……………….....................................................................................7 二、 pH緩衝劑對細胞之影響..................................................................................8、體胚發生相關之基因.........................................................................................8amp;#21441;、 材料方法 .......................................................................................................10、參試材料...........................................................................................................10 二、胚性癒傷組織誘導...........................................................................................10一)、幼雄花序培植體節位對癒傷組織形成之影響.....................................10二)、強活性生長素對幼雄花序培植體誘導癒傷組織之影響.....................10三)、幼雄花序癒傷組織之繼代培養…………………………………….....11四)、強活性生長素對合子胚培植體誘導癒傷組織之影響.........................11五)、癒合組織之切片解剖觀察……………….............................................12、細胞懸浮培養...................................................................................................12一)、建立胚性懸浮細胞系……………………….........................................12二)、酸化(pH 4.0-4.5)處理球胚相細胞….....................................................13、體胚誘導...........................................................................................................13一)、TB5增生培養基及SH3再生培養基及MES緩衝液預處理試驗........13二)、TB5及SH3與PIPES緩衝液處理誘導極化生長試驗……….............14三)、培養介質對幼雄花序衍生胚性細胞體胚發生之影響……….............15四)、台灣芭蕉懸浮細胞大小及濾紙棉花墊溼度對體胚發生之影響.........15、胚苗轉換………………………………...........................................................15一)、固化劑及PGR組合對胚苗轉換之影響…………................................15二)、BA及NAA對台灣芭蕉幼雄花序衍生體胚胚苗轉換之影.................15三)、IBA及活性碳對臺灣芭蕉芽體發根之影響………………………….16、小植株建立………….......................................................................................16、 結果 ...............................................................................................................17、胚性懸浮細胞之建立…………………………...............................................17一)、幼雄花序培植體誘導癒傷組織………………….................................17二)、幼雄花序節位對癒傷組織誘導之影響.................................................17三)、結合子胚培植體誘導胚性癒傷組織…………….................................18四)、幼雄花序衍生癒合組織增生繼代培養之生長情形….........................19、懸浮細胞系建立………………………………...............................................19、極化與非極化生長調控………………………………...................................20一)、球體相生長型以pH 4.0-4.5調控之生長之影響….…......................20二)、pH緩衝劑及再生培養基預處理對胚性細胞極性誘導.......................21三)、PIPES對胞外pH 變化及細胞極化生長之關係...................................22、平板培養與擬胚誘導……………...................................................................22一)、台灣芭蕉體胚發育過程…………………….........................................22二)、預處理對平板培養後體胚形成之影響…………….............................22三)、不同基質對幼雄花序衍生胚性細胞分化之影響………………….....23四)、台灣芭蕉懸浮細胞群大小及濾紙棉花墊溼度對體胚發生之影響….24、生長調節劑組合對胚苗轉換之影響...............................................................24 (一)、植物生長調節劑及培養介質對幼雄花序衍生體胚胚苗轉換之影響.24二)、BA及NAA處理對幼雄花序衍生體胚苗生長發育之影響………….25三)、IBA及活性碳對臺灣芭蕉芽體發根之影響………………………….25、台灣芭蕉小植株建立………………………………………………...………25、 討論.................................................................................................................73、胚性懸浮細胞之建立………………………...................................................73一)、幼雄花序及合子胚培植體誘導癒傷組織…………….........................73、胚性懸浮細胞建立……………………….......................................................75、台灣芭蕉幼雄花序衍生胚性細胞極化及非極化生長誘導...........................76一)、極化生長誘導轉換非極化生長……………….....................................76二)、pH緩衝劑對胞外pH變化及細胞極化生長發育之影響……………...76、平板培養與擬胚誘導………………………...................................................77一)、預處理對平板效力之探討……………….............................................77二)、培養基介質對體胚再生之影響……….................................................78三)、細胞團大小及濕度對平板之探討……….............................................78、 胚苗轉換………………………………….............................................79一)、PGR及培養介質對胚苗轉換之影響…………….................................79二)、BA及NAA對胚苗轉換之影響……………………..............................80三)、IBA及活性碳對臺灣芭蕉芽體發根之影響…………………………..80、 結語………………………………………………………………………….81考文獻.........................................................................................................................82錄、台灣芭蕉懸浮細胞及體胚發生建立之流程…………………………………...9