农杆菌介导的葡萄柚转化再生体系建立研究

【目的】建立邓肯葡萄柚上胚轴遗传转化再生体系,为葡萄柚基因转化研究奠定基础。【方法】以邓肯葡萄柚上胚轴为外植体,研究上胚轴不定芽和生根诱导、遗传转化的影响因子。【结果】在添加TDZ0.50～1.00mg/L的培养基上,上胚轴不定芽诱导率为72.7%～81.6%;无菌枝条的最适生根条件为1/2MT添加ABT6号1.0mg/L,生根率可达81.3%。不同抗菌素中,以替门汀杀菌效果较好,且对外植体出芽无明显抑制作用。转化芽选择初期,5～7d的暗培养有利于卡那霉素抗性芽的诱导发生。PCR检测结果显示,7株抗性植株中有6株呈阳性。【结论】建立了邓肯葡萄柚上胚轴植株再生繁殖体系,为其基因转化奠定了基础。

Establishment regeneration system for Citrus. paradisi Macf. cv Duncan by Agrobacterium-mediated transformation

Objective ]Transformation regeneration system of epicotyls derived from Citrus paradisi Macf. cv Duncan seedling were established in order to provide useful reference for its gene transformation. Method]Epicotyls of seedling were used as explants to investigate the influence factors on induction and differentiation of adventitious buds, rooting of plantlets, and genetic transformation. Result]The induction rate of adventitious buds derived from epicotyles were ranged from 72.7% to 81.6% in media with 0.50-1.00 mg/L TDZ. Comparing three types of growth regulators, the higher rooting rate （81.3%） were observed in 1/2MT medium with 1.0 mg/L of ABT 6 rooting powder. Among different antibiotics, ti- mentin showed better sterilizing effects than cefotaxime sodium and ceftriaxone sodium on agrobacterium-mediated trans~ formation plantlets culture. Timentin had no inhibition on buds induction of explants. During initial transformation stage of buds, 5-7 days of darkness culture was beneficial to induce Kan anti-buds. Among 7 transgenic plantlets, 6 plantlets showed positive in PCR detection. Conclusion]Regeneration system of Citrus paradisi Macf. cv Duncan ecopityls was es- tablished and provided base on its genetic transformation.