菊花花蕾培殖體利用農桿菌轉殖花色基因

Abstract

菊花‘Red Windmill’、‘Pink Flame’、‘Linker’、‘Margenta Linker’葉片培殖體培養在含hygromycin之培養基時，當hygromycin濃度達25 mg/L時，死亡率達到100％；而濃度為20 mg/L時，致死率仍達到87％以上；濃度在10 mg/L時，除‘Margenta Linker’培殖體致死率為66％外，其他品種致死率均達到80％以上。 不同花蕾培殖體型式轉殖花色基因後再生情況，發現以完整總花托再生芽體情況最佳，其次分別為去除小花未去總苞之花蕾上半部組織與總花托之上半部。含有總苞組織，轉殖花色基因後培殖體褐化率、農桿菌復發程度均為最高。以花瓣尖端著色但未超過1㎝的花蕾作為培殖體，存活培殖體數量最多。 菊花器官培殖體經農桿菌轉殖花色基因後，發現以花蕾培殖體存活率較高，農桿菌復發率較低，再生芽體數量較多。培殖體使用莖段或小花梗經農桿菌轉殖花色基因後，均無法再生芽體。‘Red Windmill’、‘Pink Flame’、‘Linker’、‘Margenta Linker’轉殖花色基因後，每片培殖體再生芽體數量分別為0.76、0.76、0.33、1，遠高於葉片及葉柄培殖體。 花蕾培殖體轉殖花色基因再生之植株，經抗生素hygromycin篩選後總共獲得149株植株，從當中選拔出34株花色變異植株。其中包含了含有hpt基因之轉殖植株6株，hpt & ANS基因轉殖株2株，及hpt & F3’5’H基因轉殖株1株。葉片轉殖花色基因再生之植株試驗，經抗生素hygromycin篩選後獲得20株植株，其中包含hpt & F3’5’H基因轉殖株7株。 選拔後34株花色變異植株，其中轉入hpt & ANS基因的‘Margenta Linker’轉殖株2株無法正常開花。而轉入hpt & F3’5’H基因的‘Biaritz’轉殖株1株，經花青素分析後，並未發現飛燕草素存在。使用葉片獲得hpt & F3’5’H基因的‘Linker’轉殖株7株，同樣無飛燕草素。

The lethal rate of leaf explants in Chrysanthemum ‘Red windmill’, ‘Pink Flame’, ‘Linker’ and ‘Margenta Linker’ was up to 100% when they were cultured in medium containing hygromycin at 25 mg/L. When hygromycin in medium decreased to 20 mg/L, the leathal rate was still over 87%. As well as they were cultured on 15 mg/L hygromycin medium, the lethal rate was over 80%, except ‘Margenta Linker’ explants (66%). Floral explants with involucre were easier browning and relapsing than those without involucre. And the whole receptacle explants regenerated more shoots than parts of receptacle explants regenerated. In addition, flower bud with 1㎝ ray florets had higher survival rate. After transforming pigment gene by Agrobacterium tumefacien-mediated, flower bud explants were more survival and less relapsing, and regenerated more shoots. Each receptacle explant of chrysanthemum of Red windmill’, ‘Pink Flame’, ‘Linker’ and ‘Margenta Linker’ regenerated 0.76, 0.76, 0.33, and 1, respectively. The regeneration efficiency of receptacle was higher than leaf and petiole explant. Through hygromycin selection, there were 149 plants regenerated from floral explants. There were 34 plants with different types of flower, containing 6 plants with hpt gene, 2 plants with hpt and ANS genes and 1 plant with hpt and F3’5’H genes. There were also 20 plants from leaf explants, containing 7 plants with hpt and F3’5’H. In the 8 plants with hpt and F3’5’H genes, there were no delphinidin detected. And the 2 plants with hpt and ANS genes from ‘Margenta Linker’ didn’t develop flower normally.