优良大麦品种花30幼胚遗传转化体系的优化

Optimization of Genetic Transformation System for Immature Embryos of Improved Barley(Hordeum vulgare L.)Variety Hua 30

以大麦花培基因型花30的幼胚为外植体,设置不同的培养基类型、不同激素配比及碳源,研究其对幼胚愈伤组织诱导及绿苗分化的影响,以此建立和优化一个适于优良大麦品种遗传转化的高效组织培养体系。结果表明:在N6、MS和B5的组合改良培养基下,以蔗糖为碳源,附加2mg/L 2,4-D、1mg/L ABA时,有最高的愈伤组织诱导率,且愈伤质量最好。Cu2+的添加具有抑制幼胚直接发芽成苗和改善愈伤组织质量的双重功效。添加2mg/L 6-BA对愈伤组织的分化效果比较理想。为了提高农杆菌介导转化大麦外源基因的瞬时表达率和优化遗传转化体系,利用花30幼胚产生的愈伤组织为受体材料,通过检测GUS基因的瞬时表达情况,研究了农杆菌介导的大麦遗传转化中菌液的浓度、侵染时间以及共培养天数对遗传转化的影响,结果表明:当菌液浓度OD600=0.5的条件下,侵染15min,共培养2d表现出最佳的GUS瞬时表达率。

In order to establish a highly efficient plant regeneration system suitable for gene transformation,the immature embryos of Hua 30,which is a improved barley variety derived from anther culture,was used as explants to study the effects of different medium,hormone combination and carbon sources on callus induction and plant regeneration.The results indicated that the combination medium of modified N6,MS and B5 ＋ 2mg/L 2,4-D ＋ 1mg/L ABA ＋ 30g/L sucrose was optimal for callus induction.The addition of Cu2＋ had effects on inhibiting immature embryos germination and improving callus quality on callus induction.The highest regeneration frequency could be obtained by adding 2mg/L 6-BA in differentiation medium.Meanwhile,some factors that affect transient expression including bacterial concentrations,infection time and co-culture period were investigated by detecting transient expression of GUS gene to increase the frequency of transient expression of exogenes mediated by Agrobacterium tumefaciens and to optimize the transformation conditions in barley variety Hua 30.The results showed that the higher transient expression frequency of GUS gene could be resulted from Agrobacterium tumefaciens bacterial concentration of OD600=0.5,15min infection,2d co-cultivation.