玉米基因转化的离体子房注射及其转基因植株的鉴定

Microinjection of Genes into in Vitro Ovaries of Maize and Identification of Transformed Plants

用微量注射法对玉米 (ZeamaysL .)进行基因转化已获得成功 ,但这种方法是在田间进行的 ,污染较大 ,操作时劳动强度大 ,转化率低 ,而且受气温影响 ,气温下降则种子不能成熟。采用离体注射培养法 ,取下授粉 2 4h后的玉米雌穗 ,剥去苞叶 ,在超净工作台进行微量注射 ,然后切成小块放在培养瓶中 ,在光照培养箱内培养 ,3～ 4周可直接获得种子。这种方法克服了非离体培养的缺点 ,提高了转化效率。种子萌发后进行植株抗性筛选和分子检测 ,得到了具抗性的、PCR和Southern杂交结果均为阳性的转基因植株。

Transformation of gene into maize (Zea mays L.) ovaries by microinjection has been successfully achieved. However, this method was laborious and was carried out in the field which led to serious contamination of the ovaries, low ratio of transformation and was significantly influenced by lower atmospheric temperature causing seed immaturity. The method of in vitro injection was used in which the maize ears were removed from the plant after they were pollinated for 24 h, then, the ear coats were stripped off to expose the ovaries in which several LL of transformed gene solution were injected in clean bench area using glass microneedle. After all the ovaries were injected, the ear was cut into several segments, which then were put into a glass bottle containing culture medium and incubated in a growth chamber with fluorescent lamps. Seeds could be directly obtained from the segments in three to four weeks after culture. This method overcomes the shortcomings of the in vivo seed culture and increases the transformation efficiency. Transformed plants after the seed germination were screened by antibiotic selection and identified at molecular levels, indicating that transgenic plants with positive antibiotic resistance, and confirmed by PCR, Southern blotting were obtained.