Regeneration Of Barley And Wheat Leaf-base Segments And Their Transformation

Barley and wheat are the main cereal crops in the world.It is important to breed excellent cultivars though genetic engineering.Now immature embryos and immature embryos-derived callus are used as explants in the transformation of barley and wheat. However,the transformation of immature embryo is limited of genotype.So it is necessary to establish an easy transformation without limit of period and genotype.In this study we have elementary established regeneration and transformation systems of leaf-base segments using Agrobacterium tumefaciens.The bar gene was the selection marker in the transformation.The plants resistant to PPT were obtained through identification by PCR and Southern blot analysis.The main results are followings:（1） The transgenic plants were both obtained for two barley cultivars though the transformation of calli derived from leaf-base segments by Agrobacterium tumefaciens.The E 32380 obtained 6 plants and the Epi 2 3 plants after PCR identification.One of them was sterility and the other were fertility normal.It was demonstrated that the bar gene had been intergraded into the T₁ generation seedlings and expressed in transgenic plants by PCR analysis and swearing herbicide on the leaf.The Southern blots of the transformants here investigated reveal simple integration site with only one copy of the transgene.（2） We designed three disposals including control experiment,sonication-assisted Agrobacterium-mediated transformation and negative pressure to increase the transformation frequency.The transgenic frequency was increased compared with the frequency of control experiment.However,when we used the technology,called sonication -assisted Agrobacterium-mediated transformation（SAAT）,involves subjecting the plant tissue to ultrasound in the presence of Agrobacterium,the frequency was decreased compared with the control experiment.Maybe the calli were damaged badly.（3） The regeneration system of wheat leaf base and their transformation system have been established preliminar.The bar gene was the selection marker in the transformation and in order to insert Rs-D2 gene into the wheat genome.At last,a transgenic plant was obtained according to the PCR and Southern blot.（4） After the barley plants planted in the soil,swearing 200 mg/L PPT on the leaf of the 97 plants and analysising the resistent result.8 plants of transgenic were resistent,but one of them were not and some resistent were no-transgenic.Maybe the gene inserted into the genome was not expression.So,it is a method to decrease the no-transgenic plants.