Journal: Plant physiology and biochemistry : PPB

Article Title: Combating aggressive weeds: Reinforcing herbicide resistance in pigeonpea (Cajanus cajan L.) through genome editing.

doi: 10.1016/j.plaphy.2025.109550

Figure Lengend Snippet: Fig. 1. Designing and development of the CRISPR-Cas9-based plant transformation vector for the incorporation of the desired mutations in the wild CcEPSPS gene where A. pCAMBIA1300-based NICTK-2_pCRISPR-Cas9 binary vector harboring the Cas9 expression cassette highlighted with NLS: nuclear localization signal, promoter sequence and restriction sites essential for cloning. B. The schematic representation depicting the standardized pipeline for the selection of the sgRNAs with high efficiency and no off-target activity and cloning of the selected sgRNAs in the NICTK-2_pCRISPR-Cas9 binary vector to develop complete CcEPSPS_NICTK- 2_pCRISPR-Cas9 plant transformation vector. C. Represents the sgRNA cassette where the effective AtU6-29 and AtU3b promoters drive the expression of the selected sequences (pink; highlighted PAM with red) of crRNA with scaffolds and terminator. D. Represents the sequence of the two selected sgRNAs confining the target region of the CcEPSPS gene. E. Represents the CcEPSPS donor repair template harboring the desired mutations in the target region ie., 182G > A (green), 183T > I (red), and 187P > S (blue) which will be utilized for the homology directed repair mechanism and development of mutated CcEPSPS. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The blocked blot was probed with 1:1000 dilution of Rabbit-raised anti-Cas9 primary antibody (specific to Cas9; procured from Rockland, USA) followed by incubation with 1:20000 dilution ratio of Goat anti-Rabbit HRP conjugated secondary antibody for 1H at RT.

Techniques: CRISPR, Transformation Assay, Plasmid Preparation, Expressing, Sequencing, Cloning, Selection, Activity Assay

Journal: Plant physiology and biochemistry : PPB

Article Title: Combating aggressive weeds: Reinforcing herbicide resistance in pigeonpea (Cajanus cajan L.) through genome editing.

doi: 10.1016/j.plaphy.2025.109550

Figure Lengend Snippet: Fig. 2. A. Representative images of stages in tissue culture of Pigeonpea (i) Seeds (seed coat removed) of pigeonpea on germination media (GM) (ii) Seed germination (iii) Excised embryonic axis (EA) explant on callus induction medium (CIM) (iv) Induction of callus on CIM (v) Initiation of multiple shooting from induced callus on the regeneration medium (REM) (vi) Regenerated shoots on the elongation media (EM) (vii) Rhizogenesis on rooting medium (RM) (viii) Rep resents the in vitro regenerated plants under hardening. B. The schematic representation of standardized protocol for biolistic transformation of the callus with 0.6 μm microcarrier (gold particle) coated with CcEPSPS_NICTK-2_pCRISPR-Cas9 plant transformation vector and donor template. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The blocked blot was probed with 1:1000 dilution of Rabbit-raised anti-Cas9 primary antibody (specific to Cas9; procured from Rockland, USA) followed by incubation with 1:20000 dilution ratio of Goat anti-Rabbit HRP conjugated secondary antibody for 1H at RT.

Techniques: In Vitro, Transformation Assay, Plasmid Preparation

Journal: Plant physiology and biochemistry : PPB

Article Title: Combating aggressive weeds: Reinforcing herbicide resistance in pigeonpea (Cajanus cajan L.) through genome editing.

doi: 10.1016/j.plaphy.2025.109550

Figure Lengend Snippet: Fig. 3. Molecular characterization of putative positive T0 and T1 edited plants. A. Illustrate the amplification with Cas9 specific primer in putative positive T0 plants showing Cas9 integration. B. Amplification with EPSPS target site overlapping primer set1 of the Cas9 positive T0 plants showing desired mutation. C. Illustrate the amplification with EPSPS target site overlapping primer set2 of the Cas9 positive T0 plants showing desired mutation. D. Amplification of a portion spanning the target site of the EPSPS gene from putative positive T0 plants utilized for sequencing. E. Illustrate the amplification with Cas9 specific primers of putative positive T1 plants showing Cas9 integration. F. Amplification of a portion spanning the target site of the EPSPS gene from positive T1 plants. Where, M- 1 kb DNA ladder, NTC- no template control, WT-wild type, PC- positive control, and WC- water control. G. Representative Southern blot analysis of established T1 transformants showing the single copy number of Cas9 gene in screened transgenic pigeonpea plants. These observations were obtained by the digestion of genomic DNA from eight T1 plants using restriction enzyme EcoRV and Cutsmart 2 buffer obtained from NEB. Lane M 100 ng DNA molecular weight marker III, Digoxigenin labeled, lane S1-8 20 μg genomic DNA, EcoRV, lane WT 20 μg genomic DNA of untransformed plant, EcoRV, lane PC 5 μg positive control plasmid DNA. H. Representative Western blot analysis of established T1 transformants showing Cas9 protein expression in T1 edited pigeonpea plants. These observations were obtained by the total protein extraction from eight edited plants using Cas9 specific primary antibody. Where in S1-8 lane, 40–45 μg/ml of plant extracts were loaded and for positive control (PC) 1ug/ul Cas9 protein was loaded. The marker used in the lane M was of size 10–180 KDa.

Article Snippet: The blocked blot was probed with 1:1000 dilution of Rabbit-raised anti-Cas9 primary antibody (specific to Cas9; procured from Rockland, USA) followed by incubation with 1:20000 dilution ratio of Goat anti-Rabbit HRP conjugated secondary antibody for 1H at RT.

Techniques: Amplification, Mutagenesis, Sequencing, Control, Positive Control, Southern Blot, Transgenic Assay, Molecular Weight, Marker, Labeling, Plasmid Preparation, Western Blot, Expressing, Protein Extraction

Journal: Plant physiology and biochemistry : PPB

Article Title: Combating aggressive weeds: Reinforcing herbicide resistance in pigeonpea (Cajanus cajan L.) through genome editing.

doi: 10.1016/j.plaphy.2025.109550

Figure Lengend Snippet: Fig. 5. The molecular characterization of T2 edited plants. A. Illustrate the amplification with Cas9 specific primers of T2 edited plants showing Cas9 integration (T2.1

Article Snippet: The blocked blot was probed with 1:1000 dilution of Rabbit-raised anti-Cas9 primary antibody (specific to Cas9; procured from Rockland, USA) followed by incubation with 1:20000 dilution ratio of Goat anti-Rabbit HRP conjugated secondary antibody for 1H at RT.

Techniques: Amplification

Journal: Plant physiology and biochemistry : PPB

Article Title: Combating aggressive weeds: Reinforcing herbicide resistance in pigeonpea (Cajanus cajan L.) through genome editing.

doi: 10.1016/j.plaphy.2025.109550

Figure Lengend Snippet: Fig. 6. Physiological analysis of WT, TC and edited T2 plants under glyphosate treatment. A. Seed germination analysis: seeds of WT and T2 generation edited seeds were germinated on germination medium supplemented with gradually increasing concentration of glyphosate (0–50 mM) in a glass jar under controlled tissue culture conditions. Additionally, the effect of 4 mM of glyphosate on the growth of edited (T2 generation) and WT seeds displayed that all the edited seeds germinated while WT seeds did not germinate. Data was recorded after two week of incubation. B. Glyphosate spray analysis: 42-days-old T2 plants and C. 28-days-old T2 plants were sprayed with 6 ml/L commercial glyphosate (Roundup: 41.0% w/v; Monsanto Inc., Montreal, QC, Canada) under controlled greenhouse conditions (RH = 85%; Temp. = 28±2 ◦C). The data was recorded after one week. D. Post glyphosate treatment the edited plants were allowed to grow till maturity and the recovered Cas9- free T2 plants displayed optimum growth. E. Weed competition assay: the WT and edited plants seeds were germinated along with associated weeds on a vermiculite tray and they were sprayed with 6 ml/L Roundup solution and after one week only edited plants survived.

Article Snippet: The blocked blot was probed with 1:1000 dilution of Rabbit-raised anti-Cas9 primary antibody (specific to Cas9; procured from Rockland, USA) followed by incubation with 1:20000 dilution ratio of Goat anti-Rabbit HRP conjugated secondary antibody for 1H at RT.

Techniques: Concentration Assay, Incubation, Competitive Binding Assay