Transformation Of Maize Immature Embryos With Trehalose Synthase Gene

Drought is the major limiting factor in the production of maize, and maize is much easier suffering from drought than other gramineous plants by the physiology of itself. The drought tolerance which is controlled by microeffect polygene are not the same in different gene type. It is bottlenecks in maize improvement. Therefore, transgenic operation is an useful technology to overcome reproductive isolation among species and utilize drought tolerance exotic genes.In recent years, a large number of drought tolerance genes are transferred into maize. But the transgenic plants can not fit the damand of maize production, because the anti-drought ability of the gene is not enough and it is not consistant with the metabolization of maize itself (Song et al., 2000; Wang et al., 2002; Song et al., 2005 ) . It is very important to make use of hight anti-drought ability of the gene which is more consistant with maize.Trehalos-6-phosphate synthase (TPS) is one of them for trait of adversity tolerance. Trehalose has been shown to stabilise proteins and membranes under stress conditions, especially during desiccation. By replacing water through hydrogen bonding to polar residues, trehalose prevents the denaturation of proteins and the fusion of membranes (Crowe J H et al., 1992) . So it's very significant to transfer the related gene which has the strong ability of drought tolerance into maize.Abundant study on the genetic transformation of maize take embryonic callus induced from immature embryo as recipient. There were only few materials can highly and stable induce embryonic callus due to genetype. The research of mazie gene transform were restrict in long-playing cultureing, cockamamie manipulateing, low seedling rate.It is necessary to advance gene trasform rate by other recipient.Therefore, maize immature embryos were taken as recipient in this study ,and transformed by Agrobacterium tumefaciens EHA105 with TPS1 gene from saccharomyces cerevisiae.The resulta as following:1) The transform process is much shorter for takeing immature embryos as recipient. The immature embryos were concultured with Agrobacterium tumefaciens which contained exotic gene. The process of induceing immature embryos into embyronic calli and selected with antibiotic were cut down,bring immature embryos into seedling directly.That make study much shorter and easier than taking embyronic calli as recipient.2) Optimize the system which takes immature embryos as recipient. Immature embryos is sensitive to agrobacterium tumefaciens,and that make highly brown ratio and fail transform.This study optimize the system by selecting length of immature embryos and concentration of Cys, the results indicated that the 1.5-2.0 mm of immature embryos were better than other length, and adding Cys 200mg/L in coculture can reduce brown ratio (29.3%) effectively, almost half of antitheses (53.7%).3) Result of specific PCR test showed the exotic gene had been transformed in maize. Taking 5 plants as 1 mix-sample to extract DNA, after transplants grow to 5 leaves period .Then test DNA by specific PCR and repeat test if there was positive. Finally one plant was detected to be positive. It is more efficiently and accurately to test mix-sample.