农杆菌介导转化油菜中几种影响玻璃化频率的因素

在用农杆菌介导法转化甘蓝型油菜的组织培养过程中,常会伴生着试管苗玻璃化现象的发生,此现象的发生会降低转基因的频率.本试验结果表明,影响玻璃化发生的因素较多,包括激素体积质量分数、蔗糖体积质量分数、琼脂体积质量分数和接种密度等.其中,激素6-BA和ZT体积质量分数对诱芽率和玻璃化率都有显著影响,随着体积质量分数的升高,玻璃化率升高,以6-BA体积质量分数为1mg/L,ZT体积质量分数为2mg/L时结果较为理想.蔗糖体积质量分数和琼脂体积质量分数对玻璃化率也有显著影响,当二者体积质量分数升高,玻璃化率降低,同时诱芽率也降低.综合分析,当蔗糖体积质量分数为50g/L,琼脂体积质量分数为10g/L时最适宜.接种密度对玻璃化的发生有显著影响,密度越高,玻璃化倾向越严重,而密度对诱芽率影响不明显.试验结果分析发现,接种密度为20～30个/皿最适宜.

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To this day,Agrobacterium-mediated transformation is the most widely used method in plant gene engineering,its mechanism is best understood and its effects are most satisfactory.In tissue culture for transformation of Brassica napus mediated by A.tumefaciens,vitrification of the regenerated plantlets often occurs,which normally reduces the frequency of gene transformation.The experiment reported in this paper showed that many factors affected vitrification,including phytohormone concentration,sucrose concentration,agar concentration and inoculum density.The concentration of 6-BA and ZT had significant influence on budding rate and vitrification rate.Vitrification rate increased with their concentration.6-BA 1 mg/L plus ZT 2 mg/L gave quite satisfactory results.Sucrose concentration and agar concentrations also had significant influence on vitrification rate.Vitrification and budding rate decreased with increasing sucrose and agar concentration.Sucrose 50 g/L plus agar 10 g/L was advisable.Meanwhile,inoculum density had significant influence on the occurrence of vitrification.Vitrification tended to be more serious with higher inoculum density,but budding rate was little influenced by it.A density of 20-30 inocula per dish was appropriate.