The Research About Genetic Transformation Efficiency Of BAR Gene In Tall Fescue (Festuca Arundinacea Schreb.) Plants Obtained By Agrobacterium-mediated Transformation And Particle Bombardment

Tall fescue (Festuca arundinacea Schreb.)is an important perennial cool-season grass species which is widely planted for turf and forage purposes in temperate regions throughout the world. For a long time, biotechnology has been widely applied to improve its characteristics. Agrobacterium-mediated transformation and particle bombardment are the two most widely used methods for genetically modifying grasses. Here we compare these two systems for transformation efficiency, transgene copy number and transgene expression when used to transform tall fescue. The main content just as follows:1. Optimization experiment of high frequency regeneration system of tall fescue.In this paper, the effect of different 6-BA concentration on embryogenic calli induction and regeneration of tall fescue was studied. The results indicated that, when the embryos were cultured on MS5 induction medium supplemented with 0.2mg/L 6-BA, the embryogenic callus induction and regeneration efficiency were 3.3% and 82%, respectively. And they are the highest compared with the others.2. Establishment of genetic transformation system of tall fescue.The bar gene was used as a selectable marker and selection during tissue culture was performed using 2mg/l bialaphos in both callus induction and regeneration media.And subcultured every 2 or 3 weeks.3.Detection of the resistant plants.The results showed that average transformation efficiency across the four callus lines used in the experiments was 10.5% for Agrobacterium-mediated transformation and 11.5% for particle bombardment. And 53% in leaves of the transgenic lines derived from Agrobacterium-mediated transformation showed different blue colours, while only 20% of the transgenic lines produced by particle bombardment showed that.It means the stable expression of the uidA gene.Sprayed Basta to all the transgennic lines,and after 7 days we found that all the control plants died but nothing change for transgennic lines. In addition, 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.A Southern blot analysis was performed, a low transgene copy number was observed in most transgenic plants obtained by either method. Similar transgene integration patterns and co-integration frequencies of bar and uidA were observed in both gene transfer systems. It showed that of the transgenic lines transformed through Agrobacterium transformation 63% contained the expected 3.7-kb fragment of the bar gene, in comparison, 50% of the lines obtained by particle bombardment showed the presence of the expected 3.7-kb bar fragment. Plants showed only the expected fragment without any indication of T-DNA rearrangement are 32% and 20%,respectively. Hybridization with the uidA probe revealed that 47% of the lines obtained from Agrobacterium transformation and 30% of the bombarded lines contained no more than two bands of the expected size of 5 kb or greater.This indicates that for the efficient production of transgenic tall fescue plants with stable and predictable transgene expression, the Agrobacterium-mediated method offers considerable advantages over particle bombardment.