农杆菌介导的玉米原位转化方法改良(英文)

A Modified Method of Agrobacterium-Mediated in Planta Transformation of Maize

原位转化是一种简便的植物转基因方法，在拟南芥中已经应用较为成熟，在玉米上的应用并不多见。本文在玉米开花期间，将含有目标载体pCAMBIA1301的农杆菌菌株LBA4404的细胞悬浮液直接涂抹到事先授粉约8小时后并去除掉苞叶和花丝的幼穗上。转化后种子的幼苗，经过gus活性染色和潮霉素基因（hpt）的PCR扩增，证实有部分的种子T—DNA整合进入了基因组。在潮霉素筛选后，有2．6％抗性苗存活。其中57．7％抗性植株表现出GUS阳性，相当于全部检测幼苗的1．5％。这一结果通过潮霉素基因的PCR扩增，得到进一步的证实。

In planta transformation is a simple and convenient method of plant gene transformation. In Arabidopsis thaliana, it has been successfully used. To date, however, little application is found in maize. In this thesis, cell suspension of Agrobacterium tumefaciens line LBA4404 （ pCAMBIA1301 ）, carrying activated vir genes, was applied onto the young ears with bracts and silks removed, which had been previously pollinated for approximate 8 h with the pollen of the same cuhivar during flowering in field. Partially, integration of T-DNA into maize genome was confirmed by hystochemical staining （the gus reporter gene） and PCR （the hpt reporter gene） of the seedling tissues, obtained from the trans-formed seeds. Analysis of the gus gene expression showed the presence of about 57.7 % of GUS-positive plants out of the total number of hygromycin-resistant seedlings examined, or 1.5 % of the total number of seedlings. This result was further confirmed by amplification of the hpt gene.