小麦茎生长点转化研究初报

doi:10.3321/j.issn:1000-7091.2005.01.004

摘要/Abstract

摘要： 为了躲避转基因技术对组织培养的过分依赖,本研究以小麦为材料尝试了一种对生长点直接进行转化的方法,并初步证明了这一方法的可行性。具体做法是:将种子萌芽,然后剥去胚芽鞘暴露出生长点,再用玻璃纤维制作的小刷子将生长点刺伤,最后用带有外源基因的农杆菌进行侵染处理。用携带hph-GUS基因的LBA4404农杆菌,侵染处理1-11日龄幼苗(生长点),共培养7 d后检测新生芽中GUS的瞬时表达情况;侵染1-2日龄幼苗(生长点),取长成植株的2-3朵小花进行GUS稳定表达检测。又用携带BADH和npt Ⅱ基因的AGL1农杆菌菌株侵染1日龄的幼苗(生长点),在含100 mg/L卡那霉素的蛭石中进行选择。结果表明,GUS的瞬时表达率随被处理幼苗的日龄增加而降低,以2日龄幼苗为最高(10. 7%)。用花序检测GUS的稳定表达,被侵染受体为1日龄幼苗时的表达率高于2日龄的幼苗。蔗糖的存在并不提高GUS基因在花序中表达的频率。不过花序表现为GUS阳性的植株后代, 经PCR检测后并没有证实GUS基因存在。用AGL1菌株进行转化,获得了13. 6%的抗卡那霉素的绿色植株,但只有3株呈现PCR阳性,且只有1株结了实。

关键词: 小麦, In planta, 生长点转化, GUS, npt Ⅱ-BADH

Abstract: Realizing the importance of in planta transformation for wheat to obviate the inconvenience of callus based transformation through Agrobacterium and particle bombardment an attempt was made for direct transformation of apical meristem. The study provides a positive clue for success of in planta transformation of wheat apical meristem. Coleoptiles of growing seedlings were cut and exposed meristems were wounded with fiberglass brush and inoculated with Agrobacterium.Apical meristems of 1一11 days old seedlings were inocufated with Agrobacterium tumefaciens LBA4404 containing hph-GUS genes in its plasmid and transient GUS expression was determined in regenerated shoots one week after co-cultivation. When apical meristems from 1一2 days old seedlings were inoculated then 2 to 3 florets from inflorescences were tested for stable GUS expression. One-day-old seedlings were inoculated with Agrobacterium tumefaciens AGL1 harboring nptII and BADH genes in its plasmid and regenerated seedlings were selected on 100 mg/L kanamycin in vermiculite. Results indicate that transient GUS frequency decreased with increasing age of the seedlings with maximum(10.7%)in two days old seedlings. Numbers of GUS positive inflorescences were more when apical meristems from one-day-old seedlings were inoculated compared with 2 days old seedlings. Presence of glucose did not enhance the frequency of GUS expression in inflorescences. PCR test did not confirm the presence of GUS transgene in the progeny of plants with GUS positive inflorescences. Sixty-three kanamycin resistant green plants(13.6%)were obtained but only three plants were found PCR positive. Only one plant produced seeds.

Key words: Wheat, In planta, Apical meristem, Transformation, GUS, nptII-BADH

中图分类号:

S513

RAZZAQ Abdul, 张艳敏, 杨帆, 游颜杰, 赵和, 马峙英, 王海波. 小麦茎生长点转化研究初报[J]. 华北农学报, 2005, 20(1): 17-22. doi: 10.3321/j.issn:1000-7091.2005.01.004.

RAZZAQ Abdul, ZHANG Yan-min, YANG Fan, YOU Yan-jie, ZHAO He, MA Zhi-ying, WANG Hai-bo. In planta Transformation of Wheat Apical Meristem:A Preliminary Study[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2005, 20(1): 17-22. doi: 10.3321/j.issn:1000-7091.2005.01.004.

http://www.hbnxb.net/CN/Y2005/V20/I1/17

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