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南苜蓿组织和原生质体培养及转化试验

TISSUE AND PROTOPLAST CULTURE AND TRANSFORMATION OF MEDICAGO HISPIDA

  • 摘要: 主要探讨南苜蓿子叶和下胚轴外植体、子叶原生质体培养中的器官发生及遗传转化。南苜蓿子叶和下胚轴外植体培养在附加IAA 0.5—1mg/L和细胞分裂素(BA或ZT)0.5—2mg/L的MS培养基上,在有光照的条件下诱导形成不定芽,进而再生成完整植株。子叶原生质体培养在附加2.4-D0.5mg/L和KT 0.2mg/L的B5液体培养基中,细胞分裂频率可达30—41%,原生质体来源的愈伤组织在MSB培养基(MS无机盐+B5维生素)上分化出芽。采用子叶外植体(或原生质体)-农杆菌共培养法获得转化植株(或转化组织)。GUS活性检测和胭脂碱分析表明,外源基因在转化细胞内得到稳定表达。

     

    Abstract: In the present report, we studied the organogenesis in culture in vitro of hypocotyl and cotyledon explants and cotyledon protolasts of Medicago hispida and also studied transformation of the cotyledon explants and protoplasts of M. hispida mediated by Agrobacterium tumefaciens. When the explants were cultured on MS medium supplemented with IAA 0.5—1mg/L and cytokinin (BA or ZT) 0.5—2mg/L, the shoots differentiated from the explant-derived calli in condition of light (25℃; 12h/day) at 30—40 days of culture and further developed to form intact plantlets after they were subcultured on the same medium composition. The purified protoplasts isolated from cotyledons of M. hispida were cultured in B5 liquid medium containing 2,4-D 0.5mg/L and KT 0.2mg/L. It was observed that the division frequency of the cells was about 30—41%. The shoots were induced from protoplast-derived calli after they were transferred and subcultured onto MSB medium. Using the method of co-cultivation, cotyledon explants and protoplast-derived cells of M. hispida were transformed by Agrobacterium tumefaciens strains PGV 2260 (pBI 121) or C58Cl (pBZ 6111), and transformants were abtained. Enzyme activity of GUS and NOS assay demonstrated that foreign genes expressed in the transformed plants and calli.

     

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