摘要
本研究以日龄1~3 d及7 d的小麦幼胚为受体，利用携带GUS基因的农杆菌LBA4404侵染，恢复培养至3~5叶期时，取第三叶进行GUS组织化学染色分析；并做两个优化对比处理：（1）抽真空处理：在侵染过程中，0.1 MPa抽真空2 min；（2）干燥处理：在共培养阶段，将幼胚转入铺有干燥无菌滤纸的平皿中。结果表明：GUS瞬时表达率随日龄增加而降低，以2~3 d龄的幼胚为最高；抽真空和干燥处理均能显著提高GUS瞬时表达率。最终，在优化处理条件下，获得77株转化苗中，40株第三叶GUS染色显示阳性，高达57.14%。进一步提取GUS阳性小麦叶片基因组DNA，进行PCR扩增，有6株转化苗扩增出目的条带，转化率为7.79%。初步说明以小麦幼胚的生长点作为转化受体是可行的。
Imm ature embryos of wheat were developed for 1~3 days and 7 days the immature embryos were transiently transformed with Agrobacterium tumefaciens LBA4404. The Agrobacterium tumefaciens LBA4404 that was used contained a gene reporter GUS, which was determined in the regenerated shoots of 3 to 5 leaves. Two optimal factors were compared which included：（1） Vacuuming at 0.1 MPa for 2 min;（2） Desiccation in the co-cu-lture phase, in which the immature embryos were dried and placed in the Petri dishes with filter papers. The results indicated that the GUS expression was affected by using aged immature wheat embryos at an maximum of 2~3 days;Thus vacuuming and desiccation could enhance the GUS expression more significantly. Finally, under the optimized condition 77 shoots were obtained and transient GUS frequency was about 57.14%. Detecting the target gene by PCR amplification, 6 plants were screened with the integrated tri101 gene, the frequency of transformation was 7.79%. This study showed that immature embryo of wheat could be feasible as transformation receptor.
出处
《分子植物育种》
CAS
CSCD
北大核心
2015年第4期781-786,共6页
Molecular Plant Breeding
基金
国家转基因生物新品种培育重大专项项目(2009ZX08002-002B)
郑州大学研究生自主创新项目(13L00303)共同资助
关键词
小麦
幼胚
生长点转化
抽真空
干燥
Wheat
Immature embryo
Apical meristem
Vacuuming
Desiccation
