Transformation Of Salt Tolerance Gene EhHOG Into Barley Immature Embryos And Callus Tissue By Agrobacterium Tumefaciens Infection

Barley (Hordeum vulgare L.), which is second only to wheat,rice and maize in graminaceous crop,Because of its high nutritional value and important applications, it is widely planted in the world, and one of the important model crops for molecular breeding. In recent years, genetic engineering approach has allowed breeders to produce a number of barley varieties improved in yield, quality, and response to environmental stress, which laid the groundwork for the development of a mature barley transgenic technology. With the deteriorating global environment, the increasing salinity in soil seriously impedes the normal growth and reproduction of barley. EhHog, a gene originated from the Dead Sea fungal and encoding a mitogen-activated protein kinase (MAPK), shows high tolerance to salinity. In the present study, we have constructed an efficient expression vector for a salt tolerance gene, HOG, which was introduced into barley using Agrobacterium-mediated transformation of immature embryos and callus from three varieties. The results were as follows:(1) Using immature embryos of barley varieties Golden promise, Huadamai4, and Huadamai7, we have studied the effect of sucrose and maltose on callus induction and differentiation from immature embryos. The results showed that maltose and sucrose can both be used as sugar sources in barley callus induction and differentiation from immature embryos, but maltose has a slightly higher callus-inducing and differentiating rate. Addition of lg/L acid casein hydrolyzate in the medium promoted barley callus induction.(2) Expression vector pCAMBIA1300was used as the basic skeleton, and addition of pUbi promoter and terminator tNos resulted in pCAMBIA13001. The gene HOG was PCR-amplified using specific primers containing restriction sites of BamHI and SacI, and cloned into pMD-18T vector. The HOG gene was then released by BamHI-SacI double digestion, and the recovered fragments was inserted into the corresponding sites in pCAMBIA13001by T4DNA ligase to obtain a recombinant plasmid pCAMBIA13001-B containing HOG gene. The recombinant plasmid was then introduced into the Agrobacterium AGL1using electroporation and used for barley genetic transformation.(3) The immature embryos and callus of three barley varieties were used as targets for Agrobacterium-mediated genetic transformation with the salt tolerance gene HOG. Preliminary PCR results showed that there are17positive transgenic plants from61resistant regenerated plantlets in the TO generation.