Construction of a set of small and useful binary Ti plasmid vectors for plant transformation

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1.By appropriately combining replication protein repA [3 types : high-copy type, wild-type, and low-copy type (LC)] and staA or par fragments conferring plasmid stability upon Agrobacteria, very small plasmid backbones enabling replication in both E.coli and Agrobacterium were constructed. Then, Pnos::Hyg^R marker and P35S::gusA reporter cassettes were introduced into the shuttle vectors to generate five mini-binary vectors. The novel binary vectors were very small (8.6〜9.65 kb) and carry 4 multicloning sites between Hyg^R and gusA genes. In addition, I got three Gateway-type binary vectors by substituting gusA with the Gateway cassette.2.The abovementioned five mini-binary vectors, after introducing into Agrobacterium, were transformed into rice based on tissue culture and into Arabidopsis by using floral-dip method (without tissue culture). The transformation frequencies were high enough to obtain many transgenic plants from both rice and Arabidopsis. Southern blot analysis indicated that relatively low-copy numbers (one to three) of T-DNAs were integrated into transgenic rice plants, especially when transformed with pSTARH301G carrying repA-LC + staA.3.Rice acetolactate synthase (ALS) gene bearing two different point mutations in the coding sequence (CDS), which confers herbicide tolerance upon rice plants, was further changed to delete a single HindIII site in the CDS. This fragment (mALS) was subcloned along with 1.37 or 0.56 kb promoter regions. The two mALS cassettes were individually introduced into the two mini-binary vectors, pSTARH301G and pPARH5O1G, to generate four different vectors harboring the long or short mALS herbicide-tolerant marker gene. All the four vectors were introduced into rice via Agrobacterium, and gave good transformation frequencies, indicating even mALS with short promoter could work excellently in rice transformation.