The present invention provides development of an improved protocol to screen the marker free transgenic rice plants during tissue culture if the phenotypic characteristics of the gene of interest are reported earlier. The protocol is validated by taking Pea DNA helicase 45 (PDH45) gene as example. The invention provides construction of a marker free binary vector and its successful application to produce marker-free transgenic rice plants. The invention also provides a screening method for selecting transgenic rice plants during tissue culture. To generate selectable marker-free transgenic rice plants, a reporter gene free binary vector pCAMBIA 1300 is used by cloning the gene of interest in place of hygromycin. Using this pCAMBIA 1300 binary vector with the PDH45 gene, marker-free T1 transgenic. rice plants expressing PDH are produced by Agrobacterium-mediated stable transformation using 200 mM NaC1 as a screening agent, during tissue culture. This salt screening protocol can be easily adopted and used to efficiently generate and screened marker-free transgenic rice plants which may improve the strategy. against. stress tolerance and public acceptance of GM rice.
