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An efficient plantlet regeneration system in Indian flax var JRF2 developed through in vitro tissue culture using hypocotyl explants with successful rooting, acclimatization, hardening and rearing of the rooted plantlets ex vitro up to maturity with viable seed setting. Clonal fidelity test using RAPD and ISSR markers involving genomic DNA of in vitro culture derived plantlets showed no variation in the amplicon profiles in comparison to the control plant. Developed plants were apparently looking normal morphologically similar with no ectopic expression of any abnormal characters prospecting ample scope of in vitro culture in undertaking diverse cell technological researches including transgenic development. To harness these benefits gleaned from this work, efforts were made to develop an optimized protocol for Agrobacterium-mediated genetic transformation involving LBA4404 (pCAMBIA1305.2) through both floral-dipping of buds and by transforming in vitro developed embryogenic calli. Transgenic status of the transformants was confirmed at transient level through GUS histochemical assay and PCR. Thus the developed protocol offers immense scope for its use in different biotechnological researches especially in transgenic development for genetic enhancement and value addition in Indian flax.