| Brachypodium distachyon, a monocot grass plant and widely distributed in temperate areas, has been regarded as an new potential model plant species and studied extensively in recent years because of its smaller genome, less chromosomes, shorter plant height, rapid life cycle, undemanding growth requirements, and different ploidies similar to Triti-cum including diploid, tetraploid, and hexaploid. 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 and transgene expression when used to transform Brachypodium distachyon. The main content just as follows:1. Optimization experiment of high frequency regeneration system of Brachypodium distachyonIn this paper, we studied the effect of different2,4-D concentration on embryogenic calli induction and effect of different age of callus on differentiation in, BD21, BD21-3and BDR018Brachypodium distachyon accessions. The results indicated that, when the mature embryos were cultured on MS2.5induction medium supplemented with0.6mg/L CUSO4, the embryogenic callus induction efficiency were86.2%,85.0%and72.3%and they were higher than others. The regeneration efficiency was98.1%,99.3%and83.3%when the ages of calli were7weeks.2. Establishment of genetic transformation system of Brachypodium distachyonEmbryonic calluses of BD21, BD21-3Brachypodium distachyon accessions were used as target materials of transformation. They are bombarded with plasmid pDM803, J34and J53. The results indicated that, the average efficiency of transformation was7.2%and13.5%bombarded with plasmid J34, while the lowest efficiency of transformation was bombarded with plasmid pDM803. Transformation efficiency was strongly influenced by0.01%Silwet L-77, vacuum infiltration and acetosyringone concentrations. The highest transformation efficiency of13.8%was achieved. 3. Detection of the resistant plantsPlasmid pDM803and pDM805contained reporter gene (gus) and selective marker gene (bar). The results indicated that,80.3%in leaves of the transgenic lines derived from Agrobacterium-mediated transformation showed different blue colours, while only51.3%of the transgenic lines produced by particle bombardment showed different blue colours. It means the stable expression of the gus gene. PCR was carried out to verify bar expression in transgenic plants. A357bp DNA 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 T1lines; the results showed that the segregation ratio of transgene coincident with Mendelian’s segregation law. |
