Biotechnology

The possibility of out-segregating marker genes (during sexual crossing) to produce transgenic rice plant using Agrobacterium was investigated and demonstrated to be feasible using a novel pGreen/pSoup dual binary system. We cloned into one T-DNA (pSoup plasmid) expression units containing the aph1V and gfp genes and into a second T-DNA (pGreen) the expression units containing the bar and the gus genes. Both plasmids were introduced into Agrobacterium strain AGL1 which was used to transform calli derived from matured embryo of Nipponbarre rice seeds. Transformed calli were selected using Hygromycin and/or Phosphinotricin (PPT) and hundreds of independently transformed fertile plants were regenerated. Transformation efficiency with Agrobacterium strain AGL1, when both plasmids were selected for, was found to range from 23-44%. Co-transformation efficiency was 100% when both plasmids were selected for with Hygromycin and L- Phosphinotricin (PTT) and 71% when only pSoup was selected using Hygromycin selection. Fertility in all cases was more than 85%. Phenotypic and genotypic analyses were perfomed on progenies using 64 T₁ seeds from 68 independent T₀ plants (50 plants selected on both Hygromycin and PPT and 9 plants selected on Hygromycin alone and 9 plants selected on PPT alone). Plants were phenotypically observed for green fluorescence (GFP) and GUS coloration using UV microscopy and GUS histochemical assay, respectively. Four phenotypic categories were obtained: (1) GFP+ and GUS+ progenies that contained and expressed both T-DNAs from the pGreen and pSoup plasmids; (2) GFP+ and GUS- progenies containing and expressing the T-DNA from pSoup plasmid and not expressing or not containing the T-DNA from the pGreen plasmid; (3) GFP- and GUS+ progenies containing and expressing T-DNA from pGreen plasmid and not expressing or not containing T-DNA from pSoup plasmid; (4) GFP- and GUS- not containing or not expressing the T-DNAs in plasmids pGgreen and pSoup. Polymerase chain reaction (PCR) and Dot blot analyses were used to determine the presence of the four transgenes (bar, gusA, gfp and aph1V) in all progenies exhibiting no phenotype. Both the PCR and dot blot data showed that unlinked T-DNA integration could be obtained as well as linked integration. Of the 50 T_o plants (PPT & Hygromycin selection) evaluated at the structural level, 56% showed unlinked T-DNAs. The observed genotypic segregation analysis data were subjected to statistical analysis and compared to the theoretical Mendelian values. Our results indicated that Agrobacterium- mediated “clean gene” technology using pGreen / pSoup dual binary vector can produce marker free-transgenic rice plants. Most of the observed and analysed integration patterns of the T-DNAs in different combination (up to 4 loci and 30 different permutations) falls within two and three loci

Development of Agro-bacterium-mediated clean gene (marker free) technology for rice transformation using a novel dual binary plasmid pgreen / psoup

A.S. Afolabi, J. Snape & P. Vain