Establishment Of Regeneration System For Barley And Transferring Dwarf Gene Bnrga-ds Into Barley

As the fourth widest cultivated cereal crop after wheat, rice and corn in the world, barley has played significance role in modern agriculture with high nutrition value. Barley is also widely used as genetic research model. In recent years, researchers have long been challenged by developing a suitable transgenic technique in barley. But many technical problems are still needed to be resolved, such as different genotypes has different response to media.Regeneration systems of barley for mature embryos, immature embryos and shoot segments of the seedlings of different barley genotypes were established, which provide the basis for genetic transformation. To abtain lodging-resistant, and dwarf barley variety, the dwarf gene Bnrga-ds from rapeseed was transferred into barley immature embryos using Agrobacterium tumefatiens method. The target gene in the barley genomes of TO transformants was verified using PCR amplification. The following results were obtained:(1) Six barley elite varieties (Chuannong4060, Zaofengyihao, Huadamai 4, Huadamai 5, Huadamai 6, Huadamai 7) were used to determine the effect of different culture conditions on callus induction from the mature embryo and plant regeneration. An efficient regeneration system for barley mature embryo was established. The study revealed that Dicamba and 2,4-D showed no significant difference in inducing callus formation. We found that Huadamai 4, Huadamai 5, and Huadamai 6 have better performance in callus induction and plant regeneration. The calli induction rate of Huadamai 4 reached to 97.45%, green point rate was 78.26%. As many green points failed to form a plantlet, the green plantlets rate was only 23.91%.(2) Immature embryos of eight barley varieties were cultured in vitro to screen better genotypes for transformation. Golden Promise and Harrington were used as controls. As a result, all the genotypes showed good performance except Zaofengyihao. However, once the calli were subcultured more than two months, the ability of shoot regeneration gradually reduced.(3) Using the shoot segments of barley seedlings of two varieties Huadamai 7 as explant, we have examined the effect of different media on inducing the multiple shoot clumps. The multiple shoot clumps regeneration system of barley has been established with the improved MS medium which contain 1.0mg/l TDZ and 1mg/16-BA as induction medium. When cultured on the improved MS medium containing 2.0mg/l 2,4-D and 2.5mg/l 6-BA, the shoot segments induced calli, which then formed regeneration shoots. It was difficult to produce root in the medium with no hormone. Strong roots were induced on the medium containing 0.5mg/l IBA. The establishment of the multiple shoot clumps regeneration system of barley provides a foundation for barley transformation.(4) Using the optimized system, the rapeseed dwarf gene Bnrga-ds was transferred into the immature embryos from Huadamai 4, Huadamai 5, Huadamai 6, Huadamai 7. PCR assay detected 23 positive transgenic plants from 117 resistant plantlets in the TO generation.(5) The rapeseed dwarf gene Bnrga-ds was transferred into the immature embryos calli from Huadamai 6. The effect of Agrobactrium concentration on genetic transformation was investigated. When infection time is 10 minutes and time Co-cultivation is three days,0.6-0.7 was the most optimal Agrobactrium concentration.