花粉介导的植物转基因方法不需要组织培养过程, 且操作简便易行。为明确该方法的细胞学基础, 以离体玉米(Zea mays)郑单958花粉为材料, 在超声波作用下将含有绿色荧光蛋白(GFP)基因的质粒与花粉共处理, 对处理过的花粉进行体外培养及人工授粉, 并利用荧光显微镜对GFP基因在玉米花粉粒、花粉管及胚中的表达进行示踪观察。结果表明: 处理组和对照组均有部分花粉粒呈强烈的绿色荧光, 因此通过观察花粉粒荧光来确定GFP基因是否表达不可靠; 处理组花粉管较对照呈现强烈的绿色荧光; GFP基因在玉米胚中表达可以作为鉴定转化体的证据。该实验首次利用荧光观察为花粉介导植物转基因方法提供了可视的细胞学证据。
Pollen-mediated gene transformation does not require tissue culture and is easy to apply. In the present study, we investigate the cytological basis for the transformation approach. Fresh pollen was taken from maize plants (Zea mays cv ‘Zhengdan 958’) and treated under ultrasonication mixed with plasmid DNA harboring green fluorescence protein (GFP) gene. Treated pollen was artificially pollinated onto silks of maize ears. The expression of GFP gene in pollen grains, pollen tubes and embryo were observed under a fluorescence microscope. Pollen grains had a strong green autoflorescent background and so were not suitable for observing the expression of exotic GFP gene. Treated pollen tubes produced strong green florescence as compared with the control. The expression of GFP gene in embryo could be used to confirm the expression of exotic genes. Our study provides cytological evidence for pollen-mediated transformation.
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