Establishment Of Marker-free Transformation System And Transferring Of HMW-GS Genes In Wheat

HMW is the important component of storage protein in wheat grains,and determines the dough elasticity and processing quality.A wheat somatic variation line AS208 developed from tissue culture of Lunxuan987 missed 1Bx20 and 1By20 subunits at Glu-B1 locus,which leads its decreased bread quality comparing the parent of Lunxuan987.However,AS208 have potential application to identify the function of other HMW-GSs and breed ideal weak-gluten wheat varieties.The purpose of this study is to establish the transformation system of AS208 and some Chinese commercial wheat cultivars including Jimai22 and AK58 as well as the technique of obtaining marker-free transgenic wheat plants mediated by Agrobacterium firstly.Then several HMW-GS genes will be transferred into AS208 by the developed approach.The main results achieved in this study are as follows:1.By using wheat immature embryos as explants,transformation system of some important materials such as AS208,Kenong199,Xinchun9,CB037 and Fielder were set up by Agrobacterium-mediated.Moreover,15 commercial Chinese hexaploid wheat varieties were successfully transformed via this transformation method,with efficiency of up to 27.7%.The transgenic plants were confirmed by Quickstix strips,indicating that positive wheat plants was obtained by a rate more than 90%.2.By optimizing tissue culture conditions and modifying medium composition,efficient regeneration system for wheat mature embryos was generated.Transgenic wheat plants were successfully achieved from the mature embryos of Verry and CB037 via Agrobacterium-mediated transformation.Further study is conducting for efficient transformation efficiency of this tissue.3.By Agrobacterium-mediated transformed system,an expression vector with two independent T-DNA regions carrying GUS and bar genes was transferred into wheat varieties of Kenong199,Xinchun9,CB037 and Fielder,and transgenic T₀ plants were confirmed by Quickstix strips,histochemical staining,PCR and Southern blot analysis.Results indicated that the average co-integration frequency of the GUS and the bar genes located on the two independent T-DNA regions was 49.0%in T₀ plants.Marker-free transgenic wheat plants were obtained by a rate of 14.5%in T₁generation through detecting the GUS and the bar genes by the molecular techniques.Based on the analysis by Southern blot,it was deduced that the ratio and size of the two T-DNA regions might affect integration copy numbers of the selection marker and the target genes in T₀ transgenic wheat plants,further affecting the separation ratio of the two genes in T₁ generation.4.According to the testing results by Quickstix strips and PCR analysis,the bar gene silencing was detected in 33.5%in T₁ positive plants,but the GUS gene was never found to be silenced in T₁ plants.Bisulphite genomic sequencing suggested that DNA methylation in the 35S promoter of the bar gene regulatory region might be the main reason for bar gene silencing in the transgenic plants.5.Employing Agrobacterium-mediated wheat immature embryos transformation system,with the two T-DNA vectors carrying the HMW-GS genes including 1By8,1Bx20,1By20 and 1Sx2.3,respectively,were transferred into AS208.In total,sixty-nine transgenic plants were confirmed by the use of Quickstix strips.All of the positive transgenic plants were tested by a specific molecular marker to Glu-1B locus,and four transgenic plants were proved to have 1By8,eight transgenic plants to have1Bx20,five transgenic plants to have 1By20,four transgenic plants to have 1Sx2.3.Further,only the1Sx2.3 gene was found to express in the transgenic plants other than the other three transferred HMW-GS genes revealed by SDS-PAGE.