Establishment Of Regeneration System And Optimization Of Agrobacteriummediated Transformation Of Brachypodium Distachyon Mature Embryos

Brachypodium distachyon, a kind of temperate grass with small physical size, rapid life cycle, small genome, self-pollination, simple growth requirements, especially with more closely relationship to many cereal crops such as wheat, barley, oats, maize and many forage grasses and turf grasses, which can be used as a ideal model plant for cereal crops and grasses research. An efficient regeneration and transformation system of Brachypodium distachyon is crucial for the quality improvement and gene function identification of ceral crops. To establish the regeneration and genetic transformation system of Brachypodium distachyon, the mature embryos of Brachypodium distachyon BD21-3 were used as explants, and the callus induction, regeneration and the Agrobacterium infection were studied in this research. The main results were as follows:1. The callus induction, differentiation and its regeneration were studied using the mature embryos of Brachypodium distachyon BD21-3 as explants. The results showed that on the subculture media including 2.5mg·L-1 2, 4-D, 0.6mg·L-1 Cu SO4, 30g·L-1 sugar, 0.6mg·L-1 acid hydrolyzed casein and 7g·L-1 agar, the frequency of callus formation and embryonic callus formation were up to 93.83% and 62.28%, respectively. The maximum average regeneration frequency(39.72 ± 1.29%) was observed on the differential media containing 0.2mg·L-1 KT, and the regenerated plantlets were rooted more rapidly on the root media with 0.6mg·L-1 IAA.2. When using the stem and stem tip of Brachypodium distachyon as explants, the stem tip has the higher frequency of callus formation and regeneration than stem, which was 38.67% and 24.14%, respectively.3. Embryogenic calli derived from mature embryos were transformed by Agrobacterium GV3101 with a plasmid pCAMBIA3301 containing the GUS reporter gene. The GUS staining showed that the highest GUS activity was gained when the concentration of Agrobacterium was OD600 0.6 and infection incubation period was 5 min. This investigation laid a foundation for the establishment of genetic transformation system Brachypodium distachyon.