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Efficient regeneration of explants devoid of intrinsic somaclonal variations is a cardinal step in plant tissue culture, thus, a vital component of transgenic technology. However, recalcitrance of economically important crops to tissue culture-based organogenesis ensues a setback in the use of transgenesis in the genetic engineering of crop plants. The present study developed an optimized, genotype-independent, nonconventional tissue culture-independent  in planta  strategy for the genetic transformation of flax/linseed. This apical meristem-targeted  in planta  transformation protocol will accelerate value addition in the dual purpose industrially important but recalcitrant fiber crop flax/linseed. The study delineated optimization of  Agrobacterium tumefaciens- mediated transformation and stable T-DNA (pCambia2301: GUS : nptII ) integration in flax. It established successful use of a stringent soilrite-based screening in the presence of 30 mg/L kanamycin for the identification of putative transformants. The amenability, authenticity, and reproducibility of soilrite-based kanamycin screening were further verified at the molecular level by GUS histochemical analysis of T 0  seedlings,  GUS  and  nptII  gene-specific PCR, genomic Southern hybridization for stable integration of T-DNA, and expression analysis of transgenes by sqRT-PCR. This method resulted in a screening efficiency of 6.05% in the presence of kanamycin, indicating amenability of  in planta  flax transformation. The strategy can be a promising tool for the successful development of transgenics in flax.

frontiers in plant science

An Apical Meristem-Targeted in planta Transformation Method for the Development of Transgenics in Flax (Linum usitatissimum): Optimization and Validation