AUTHOR=Kesiraju Karthik , Tyagi Shaily , Mukherjee Soumyadeep , Rai Rhitu , Singh Nagendra K. , Sreevathsa Rohini , Dash Prasanta K. 

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

JOURNAL=Frontiers in Plant Science

VOLUME=11

YEAR=2021

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2020.562056

DOI=10.3389/fpls.2020.562056

ISSN=1664-462X

ABSTRACT=<p>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 <italic>in planta</italic> strategy for the genetic transformation of flax/linseed. This apical meristem-targeted <italic>in planta</italic> transformation protocol will accelerate value addition in the dual purpose industrially important but recalcitrant fiber crop flax/linseed. The study delineated optimization of <italic>Agrobacterium tumefaciens-</italic>mediated transformation and stable T-DNA (pCambia2301:<italic>GUS</italic>:<italic>nptII</italic>) 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<sub>0</sub> seedlings, <italic>GUS</italic> and <italic>nptII</italic> 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 <italic>in planta</italic> flax transformation. The strategy can be a promising tool for the successful development of transgenics in flax.</p>