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

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.

Amenability of an Agrobacterium tumefaciens-mediated shoot apical meristem-targeted in planta transformation strategy in Mango (Mangifera indica L.)

“…Detection of visible scorable markers like GUS and GFP provide an early signal of transformability and/ or successful transformation of infected tissues. 38 Therefore, in this study, GFP expression and GUS histochemical analysis were performed in their specific transformants to identify primary transformants.…”

“… 31 Among several in planta transformation techniques, apical meristem mediated transformation targets T-DNA to the growing shoot apical meristematic regions in vitro and allows the development of plants ex vitro . The methodology has been unequivocally proved in different crops like field bean, 32 groundnut, 33 capsicum, 34 chili, 35 pigeon pea, 36 , 37 flax 38 and cotton. 39 , 40 …”

Generation of High-Value Genomic Resource in Rice: A “Subgenomic Library” of Low-Light Tolerant Rice Cultivar Swarnaprabha

“…Many advances have been made in the field of genomics [ 52 , 53 , 54 ] and transgenics [ 55 , 56 ] to impart biotic/abiotic tolerance [ 57 , 58 ] or engineer a trait in crops [ 59 ], but the genomic resources for low-light tolerance in plants are meagre. Therefore, a subgenomic library yielding a large number of clones that harbor photoreceptors or other agronomically important (intrinsic yield genes) traits is required.…”

GWAS supported by computer vision identifies large numbers of candidate regulators of in planta regeneration in Populus trichocarpa

“…However, the ability to impart new traits by these methods is limited to crop species with genotypes that can reliably undergo transformation and regeneration (TR), typically via in vitro tissue culture methods. Various in planta approaches show promise to enable efficient TR without labor-intensive tissue culturing or the materials and sterile conditions it requires, but nonetheless require plant transformation and organ regeneration ( Maher et al 2020 ; Kesiraju et al 2021 ; Bomzan et al 2022 ; Lian et al 2022 ). TR requires developmental responses to a series of hormone treatments and amenability to gene insertion, and the capacities for both vary greatly between and within species ( Altpeter et al 2016 ).…”