Comparative Analysis Of Transgenic Brachypodium Distachyon Plants Obtained By Agrobacterium-mediated Transformation

The use of a model species is a very important tool to genetically analyses plants. Recently, in the ongoing process of developing Brachypodium distachyon as a model plant for temperate cereals and forage grasses. The prominent desirable features B. distachyon boasts a simple genome, small size and a relatively rapid lifecycle. Following the deep research, people have clearly realized that, it is possible to be a grass model. We have developed a high-throughput Agrobacterium-mediated transformation system for a diploid accession. The main content just as follows:1. Optimization experiment of high frequency regeneration system of brachypodium distachyon.In this paper, the effect of different size of immature embryo on embryogenic calli induction and regeneration of B. distachyon was studied. The results indicated that, when the size of immature embryos between 0.3mm and 1mm, the embryogenic callus induction was 85%,85%,74% for the DB21,BD21-3,BDR018respectively. And BDR018 is the highest compared with the others on regeneration efficiency.2. Establishment of genetic transformation system of BDR018.Embryogenic callus, derived from immature embryos of the accession BDR018, were transformed with Agrobacterium tumefaciens strain one T-DNA plasmid PDM805. The bar gene was used as a selectable marker and selection during tissue culture was performed using 5mg/L bialaphos in both callus induction and regeneration media, and subcultured every 2 or 3 weeks. On average 21.1% of 5～8-week-old calli co-inoculated with Agrobacterium regenerated stable transgenic plants. in addition, the effect of different liquid medium on transformation efficiency of bar gene. MG/L was the beast, and stable transformation frequencies of up to 42.9%, which to our knowledge is the highest transformation efficiency reported in the same age, were observed.3. Detection of the resistant plants.The results showed that all the plants are positive in leaf test, and 91.4% in leaves of the transgenic lines derived from Agrobacterium-mediated transformation showed different blue colors, It means the stable expression of the bar gene.RT-PCR was carried out to verify bar expression in transgenic plants. A 400 bp cDNA fragment of bar was specifically amplified from all the transgenic lines, but not from the non-transformed control plants. That means the stable expression of bar gene and showed the resistance of herbicide. Sprayed basta to 8 T1 lines, the results showed that the segregation ratio of transgene coincident with Mendelian's segregation law. It is mean that bar gene was still in the transgenic BDR018.