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花蓮亞蔬五號番茄農桿菌基因轉殖系統之建立
Establishment of the System for Agrobacterium-Mediated Transformation of Tomato 'Hualien ADRVC No. 5'
摘要
為了建立花蓮亞蔬五號番茄之基因轉殖系統，以8－10天苗齡之子葉及下胚軸切段做為培植體，結果顯示以BA5、S1-1及MSG1培養基再生芽體較多，培養基添加Timentin 150mg/L之芽體再生率與不添加處理沒有顯著差異。以農桿菌LBA4404菌系內含PBI121之質體作為轉殖之載體，以感染菌液濃度O. D. 600為0.6－0.8，與培植體浸泡30分鐘，共培養時間為2天，共同培養基之AS濃度以200－400μM較佳。另以再生培養基B5（內含BA 5 mg/L）、S1-1（內含kinetin 2mg/L，NAA 0.02mg/L）及MSG1（內含zeatin 1mg/L）配合不同抗生素篩選方法誘導植株再生，每培植體平均再生芽數為1.5－3.4個，以MSG1培養基之篩選後再生率較高，先以低濃度抗生素篩選再以高濃度抗生素篩選可獲得GUS基因全株表達之植株，並已經由PCR檢測及南方雜交檢測確定之。目前正利用此技術轉殖Bt基因試驗。
並列摘要
The aim of this research is to establish the plant gene transformation system for tomato cultivar 'Hualien AVRDC No. 5'. The 8-10 days old cotyledons and hypocotyls of tomato were used as explants. The better regeneration media were BA5, S1-1, and MSG1. Regeneration rate was not siginificant different between regeneration media with and without Timetin 150mg/l. The plasmids and the Agrobacterium strain was binary vector PBI121 and LBA4404. The transformation efficiency would be better if the O.D.600 of bacteria infection medium was 0.6-0.8, infection time was 30 min., and the cocultured period was 2 days. The regeneration media B5 (added with BA 5 mg/L), SI-1(added with kinetin 2mg/L，NAA 0.02mg/L) and MSG1(added with zeatin 1mg/L) were used as selection media combined with different selection methods. There were 1.5-3.4 regenerated shoots per explant after selection. The transformation efficiency was highest when explants were selected on the MSG1 medium. It could be obtained the transformants expressed the GUS gene in the whole plants under the selection methods that treated with low concentration antibiotic first and high concentration antibiotic latter. This technique was proved f by histochemical GUS assay, PCR and Southern blot assay. It is expected that insect resistance genes will be transferred into tomato plants by employing this technique.