Abstract
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 T0 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.
Highlights
Unpredictable climatic vagaries, burgeoning global population, and brimming buffer stocks are major concerns that threaten the sustainability of agriculture across all agro-ecological regions of the world
Innumerable strategies that target T-DNA to discrete parts of plants and avoid tissue culture-induced somaclonal variations have emerged to aid genetic engineering of crop plants (Kaur and Sugani, 2019). One such methodology is an apical meristem-targeted in planta transformation protocol that targets T-DNA to the growing shoot apical meristem in vitro and allows the development of plants ex vitro (Singh et al, 2018)
We present successful optimization of the apical meristem-targeted in planta transformation
Summary
Unpredictable climatic vagaries, burgeoning global population, and brimming buffer stocks are major concerns that threaten the sustainability of agriculture across all agro-ecological regions of the world. Innumerable strategies that target T-DNA to discrete parts of plants and avoid tissue culture-induced somaclonal variations have emerged to aid genetic engineering of crop plants (Kaur and Sugani, 2019). One such methodology is an apical meristem-targeted in planta transformation protocol that targets T-DNA to the growing shoot apical meristem in vitro and allows the development of plants ex vitro (Singh et al, 2018). Our successful demonstration of a non-tissue culture-based methodology targeting the apical meristem to develop genetically modified flax can be a bona fide approach to be incorporated in varietal improvement programs in flax through genetic modification
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