| Rapeseed (Brassica napus.L) is one of the most important oil-producing crops in the world. Plant genetic transformation is a core research tool in plant biology and crop improvement. An efficient regeneration system is the foundation that can make sure good results to plant engineering technology. In this study, we intend to solve with the problem about the tissue culture and established a new tissue culture system from cotyledons and leaves of elite rapeseed cultivars; plant expression vector is directly related to transformation efficiency and genetic stability, also the safety to genetic modified plants, therefore, the construction of vector plays an important role in transformation.This study, we have amplified the homologous recombination fragments and constructed the vector to finish the rapessed chloroplast transformation.1We studied the differences of calli induction and defferentiation abilities for F4and T15cultivars, and found that there was a significant difference in defferentiation frequency between these two genotypes. F4was better for tissue culture than T15.2The construction for rapeseed tissue culture. We used cotyledons and leaves as explants to analysis the problems about rapeseed tissue culture.①MS+4mg/L6-BA+0.5mg/L NAA+6mg/L AgNO3was proper for defferentiation, with28.1%of defferentiation rate for this media;②The different seeding age leaf of F4for tissue culture showed great difference, this study showed the22-day leaf was the best explant for callus induction;③1/2MS+1mg/L IB A was suitable for rooting,100%was the rooting rate, the roots grew fast and flourishingly.3Construction of rapeseed chloroplast transformation vectors. The vectors contained different lengths of rbcL and accD region from the rapeseed chloroplast genome for integration via homologous recombination, and EPSPS, aadA were alien gene and marker gene respectively. They were formed in the same expression cassette as polycistron. The promoter and terminator were from tobacco. |
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