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.

Keywords: GM crops; GUS; apical meristem; in planta transformation; nptII; transgenic flax/linseed.

FIGURE 1

(A) Schematic representation of the pCAMBIA2301 vector used for the development of transgenic flax, (B) restriction analysis of pCAMBIA2301, and (C) details of the restriction profile of pCAMBIA2301.

FIGURE 2

Overview of apical meristem targeted in planta transformation strategy for the development of transgenics in flax. (A) Immersed seeds for germination, (B) removal of seed coat of immersed seeds to facilitate Agrobacterium infection, (C) apical meristematic region of seedling was punctured with an insulin syringe, (D) co-cultivation of embryos in AB minimal medium, (E) post-infection embryos and soilrite planting, (F) recovery of plants on soilrite, and (G) establishment of recovered plants in the greenhouse.

FIGURE 3

GUS expression in primary transformants. (A) GUS expression in the shoot apical region of (i,ii) primary transformants, and (iii) wild type, (B) sections of the apical meristematic region showing GUS expression within cells in the primary transformants (i), and sections of wild type (ii) showing absence of GUS expression.

FIGURE 4

(A) Recovery of primary transformants in the greenhouse, (i) transfer of healthy plants to pots, (ii,iii) transgenic plant showing normal growth, flowering, and bud set, (iv) transgenic plant with mature capsules, and (B) an overview of seed pool collected from 10 transgenic and wild type plants.

FIGURE 5

Screening of putative transformants under kanamycin selection. (A) Transfer of seedlings of independent T₀ plants after kanamycin treatment, (B) cup tray screening for antibiotic resistance, (i) overview of cup tray with planted seeds, (ii) initial signs of kanamycin induced necrosis after 4^th day of treatment, (C) variation in root and shoot lengths of plants after 10^th day of kanamycin treatment, (i) treated wild type, (ii) untreated wild type, (iii,iv) transgenic plants with phenotype at par with untreated wild type, (D) an overview of (i) treated wild type plants exhibiting severe necrosis, (ii) untreated wild type, (iii) putative transgenics, and (E) an overview of the variable response in T₁ seedlings to kanamycin treatment exhibiting resistance and susceptibility to kanamycin.

FIGURE 6

Variations in root and shoot lengths of wild type (untreated and treated) and T₁ generation transgenic plants in response to 30 mg/L kanamycin.

FIGURE 7

PCR analysis of T₁ transgenic plants and wild type plants showing amplification of 750 bp nptII gene (A) and 1 kb GUS gene (B) [Lane M: 1 kb marker (Thermo scientific), Lane B: water blank (all PCR components except template DNA), Lane WT: wild type plant, Lanes 1-26: putative T₁ transgenic plants obtained after kanamycin screening, Lane P: binary vector pCambia2301 as positive control], (C) genomic Southern analysis of transgenic plants probed with DIG-labeled 750 bp nptII gene fragment, Lane L: Lambda HindIII DNA digest, Lane WT: wild type, Lanes 1–4: transgenic plants (P4-2, P5-1, P6-3, P9-1, respectively), Lane P: linearized plasmid of pCambia 2301, (D) sqRT-PCR analysis for the assessment of transcript accumulation of (i) nptII, (ii) GUS genes, and (iii) total RNA as loading control. [Lane B: water blank (all PCR components except template DNA), Lane WT: wild type plant, and transgenic plants P4-2, P5-1, P6-3, P7-1, P9-1, Lane P: binary vector pCambia2301 as positive control].