利用花粉管導入法建立甘藍基因轉殖系統之研究

Study on Genetic Transformation of Cabbage via Pollen Tube Pathway

以甘藍栽培品種初秋為接受親，利用花粉管基因導入法轉殖含GUS報導基因及NPTH篩選基因之pRT99gus質體，所得之第一代種子播種150粒成活137株；分別以噴施kanamycin篩選及進行南方氏雜交分析，結果顯示34株（24.82%）對kanamycin具抗性，29株（21.16%）具有南方氏雜交條帶，同時具有抗kanamycin又具雜交條帶者有13株（9.49%）。將具有南方氏雜交條帶之T1植株自花授粉採收種子，分析其中一個T1之後代T2，具有南氏雜交條帶之比例為3/4，顯示外來基因可以遺傳至T2世代。故由分子層次證實外來基因可經由花粉管導入法進入植物基因組，且遺傳至子代。

The purpose of this study is to establish an efficient, stable and practical gene transfer system for cabbage (Brassica oleracea L. var. capitata L.). The plasmid pRT99gus containing the reporter gene GUS encoding β- glucuronidase and selectable marker NPTII gene was introduced by injection into placenta tissue of recipient. The cultivated cabbage KYcross variety was used as recipient plant. By spraying kanamycin (100mg/l) on cabbage plant, 34 out of 137 T1 plants showed resistant to kanamycin. Based on DNA blot hybridization analysis, 29 out of 137 showed positive signals. Among them, 13 plants showed both resistant to kanamycin and hybridization signal. After selfing, the progeny of the T1 plants was further assessed for the transmission of integrative transformation. The proportion of the progeny (T2) showing positive hybridization signals was 3/4 for one of the Tl plants. This result suggests that exogenous DNA can be integrated into cabbage genome and inherited to the next generation. Thus, gene transfer through pollen tube pathway was proved to be feasible on the molecular level.