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Establishment of the regeneration and Agrobacterium-mediated transformation system of genus Haworthia
利用花梗誘導出'三仙壽' (Haworthia 'Sansenjyu') 之癒傷組織，因增生速率較快適合作為試驗材料。利用不同濃度之BA與NAA組合誘導癒傷組織進行增生或分化，實驗發現以600 lux之光度培養下，培養基添加2 mgL-1 BA可獲得較多的癒傷組織增生倍數為33-44倍；以5500 lux之光度與不添加或只添加1 mgL-1 BA較能誘導癒傷組織芽分化高達38-81%，但培植體於5500 lux下較會發生褐化為12-21%。三仙壽植株以MS培養基添加0.4 mgL-1 NAA與0.4 mgL-1 IBA培養四週後可誘導出最多17條根，且NAA會使得植株分化出粗大的根系。將組培苗分別以帶根或不帶根之型式出瓶，發現兩者生長速率差異不大，三仙壽無需帶根出瓶。
試驗三仙壽癒傷組織對篩選抗生素hygromycin與cefotaxime之耐受性，結果為三仙壽癒傷組織適合以MS培養基培養於600 lux下 5 mgL-1 hygromycin進行篩選。以三仙壽癒傷組織作為農桿菌感染材料，感染農桿菌LBA4404攜帶pCAMBIA1305.1質體，菌液以OD600為0.2的濃度，感染三仙壽之癒傷組織10分鐘，共培養4天後以無菌水添加250 mgL-1 cefotaxime清洗，感染後30天可觀察到表現。癒傷組織感染後恢復八週進行篩選，篩選濃度為5 mgL-1 hygromycin和250 mgL-1 cefotaxime，篩選4週後可獲得轉殖株，轉殖植株呈現嵌合體。|
Haworthia is a genus of succulent plants. Some species with leaf epidermal windows have been well accepted by people. Hybrid breeding were utilized for new cultivars creation, but it was not easy and took a long time. The microprogation can propagation large amount of consistency plant. Used the transformation ways to create new varieties and shorten the breeding time, then take the microprogation way to accelerated growth. Regeneration and Agrobacterium mediated transformation system of Haworthia succulents were established in this study. The Haworthia 'Sansenjyu' calli were induced from flower stems and been used to be experimental materials. The calli were propagated to 33-44 times by using MS medium containing 2 mg/L BA under 600 lux. To differentiation more shoot, calli cultured in MS medium containing 0 or 1 mg/L BA under 5500 lux with the best efficiency by 38-81%. However, the explants growth under 5500 lux may become browning by 12-21%. The plants were placed on the medium with 0.4 mg/L NAA and 0.4 mg/L IBA to induce maximum number by 17 roots. The plantlets with or without root from tissue culture were acclimatized to ex vivo. The growth rate of two plants were not much different. H. 'Sansenjyu' is not necessary to root in vito. The hygromycin selection concentrations by calli cultivated in MS selection medium by 5 mgL-1 hygromycin under 600 lux. Agrobacterium LBA4404 strain carrying pCAMBIA1305.1 vetor containing GUS gene were dilute to OD600 of 0.2 and add to calli for 10 minutes. After co-culture for 4 days and remove Agrobacterium with 250 mg/L cefotaxime, GUS expression were observed 30 days later. The calli after infected were grown for 8 weeks and then transfer to selection medium containing 5 mg/L hygromycin and 250 mg/L cefotaxime. Chimeric transgenic plants were obtained after 4 weeks selection.
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