Establishing Of Efficient Regeneration System For Barley Mature Embryos And Improving Amylose By Gene Transfer

Barley has great development potential as feed crop and main raw material for beer production in China. The study of improving barley strains had fastened on barley resistibility and quality, rare reports of extracting amylose from barley appeared. Amylose was important industrial raw material used wildly over in 30 fields. We put forward the study of utilizing maize starch branch enzyme sbel , sbe2b genes to transform barley and analysing starch structure in transformed barley. The aim of our research was to discover whether SBE transformed in barley genome regulate starch structure and to illustrate the possibility breeding new barley varieties by maize SBE.Barley mature embryo was good trial material for biotechnology study and fit for innovating barley variety resource due to free of time and quantity restrictions.Yet barley mature embryo could hardly produce embryonic callus and most cultivars were difficult to regenerate, which hindered barley gene engineering. So it was significant to build an efficient regeneration system for barley mature embryo by studying factors that influence barley mature embryo culture and plant regeneration. Ten barley elite cultivars were studied in this research to determine the influence of different culture conditions on the embryo-derived callus induction and green plant regeneration. We built an efficient regeneration system for barley mature embryo and screened out 5 elite cultivars 87-3175, 87-0053, 97-4010, 97-6004 and 208813-509 with better performance in callus induction and plant regeneration for genetic transformation.On the base of barley regeneration system, we transformed 5 barley cultivars with expression vectors carrying sbe1, sbe2b gene and bar marker gene by particlebombardment.The results were as follow, the resistant calli rates of 5 barley cultivars were10.32%-17.13%, green plantlets rate were 0-14.29%. Finally, 28 regenerated independent transformants, including 11 independent transformants of 87-3175, 9 independent transformants of 87-0053, 3 independent transformants of 97-4010 and 5 independent transformants of 208813-509, were transplanted to field. Only 4 independent transformants of 87-3175, 5 independent transformants of 87-0053 and 3 independent transformants of 208813-509 were transplanted successfully. PCR analysis of 10 regenerated plants showed that 7 plants produced the same size of 0.5kb specific DNAband as that produced from the vector p35Scpbar by PCR, 2 plants had the same size of 2.4kb specific DNA band as that produced from vector prEBE2 by PCR, 3 plants had the same size of 2.5kb specific DNA bands as that from vector prEBEl by PCR. The sequences of the PCR bands were identified with that of genes bar, sbe2b and sbel respectively. It indicated that genes bar, sbe2b and sbel had been integrated into barley genome.