Agrobacterium-mediated genetic transformation of chickpea for insect resistance

Introduction

How long can chickpea production bear the losses inflicted by Helicoverpa armigera? Each year, the gram pod borer destroys 20-40% of chickpea harvests across South Asia, and conventional insecticide sprays are neither cost-effective for small-holders nor environmentally acceptable [1] . Chickpea ranks as the world's third most important food legume, with India accounting for roughly 70% of global output [2] . Yet decades of germplasm screening have failed to identify durable, high-level resistance within the genus Cicer [3] . Bt crystal proteins, especially Cry1Ac, have been deployed in cotton and maize against lepidopteran pests with well-documented success [4] . But chickpea transformation has lagged behind. Recalcitrance to in vitro regeneration and Agrobacterium infection remains a persistent obstacle [5] . Particle bombardment often produces complex, multi-copy insertions prone to silencing [6] , whereas Agrobacterium-mediated methods deliver low-copy, stable integrations [7] . Earlier groups reported chickpea transformation via Agrobacterium, but efficiencies stayed below 5% in most cases [8] . Factors affecting outcome include explant type, strain, infection duration, and co-cultivation conditions [9] . This research compared five explant types for Agrobacterium-mediated delivery of cry1Ac into chickpea cultivar JG-62 and evaluated transgene integration, expression, and bioactivity against H. armigera. The work was done at the Department of Crop Improvement, Saurashtra College of Agricultural Sciences, Junagadh, Gujarat, between June 2021 and March 2023 [10] .

Study area description

All laboratory and greenhouse work was conducted at the Plant Biotechnology Laboratory, Department of Crop Improvement, Saurashtra College of Agricultural Sciences, Junagadh (21.52°N, 70.47°E), Gujarat. Junagadh falls in agro-climatic zone XIII with semi-arid climate receiving 800-1000 mm annual rainfall. The greenhouse maintained 28 ± 2°C day and 22 ± 2°C night temperatures, 55-70% RH. The tissue culture facility had a laminar flow bench, growth chamber at 25 ± 1°C with 50 µmol m⁻² s⁻¹ fluorescent lighting, and an autoclave. Insect bioassays were carried out in a separate insect-proof chamber at 27 ± 1°C, 65 ± 5% RH, with a 12:12 h light:dark cycle.

Material and Methods Material

Seeds of chickpea cultivar JG-62 were obtained from the Pulse Research Station, Junagadh Agricultural University, and surface-sterilised with 0.1% mercuric chloride for 4 minutes. Five explant types were prepared: cotyledonary nodes from 5-day-old seedlings, embryo axes from imbibed seeds, half-seeds with intact embryo axes, shoot apices from 7-day-old seedlings, and epicotyl segments from 10-day-old seedlings. Agrobacterium strain EHA105 harbouring pCAMBIA2301 with synthetic cry1Ac under CaMV 35S and nptII under NOS promoter was grown in YEM broth with kanamycin (50 mg/L) and rifampicin (25 mg/L) at 28°C to OD600 of 0.6-0.8. Acetosyringone (100 µM) was added 1 hour before infection. Media were based on MS salts [11] , pH 5.8, solidified with 0.8% agar. Comprehensive interpretation PCR screening of 94 kanamycin-resistant plants detected cry1Ac in 79 individuals (83.6% confirmation rate). Southern blot revealed one to three hybridising bands per line. Single-copy lines JG-T1 and JG-T9 showed the highest Cry1Ac expression (0.34 and 0.29 µg/g FW) and highest 96h mortality (78.3% and 76.1%). Line JG-T12, with two copies but only 0.09 µg/g, had the lowest transgenic-line mortality at 53.7%, possibly from partial silencing.

Experimental design

Discussion

The 14.7% transformation efficiency with cotyledonary nodes is among the highest reported for chickpea. Mehrotra and colleagues obtained about 3.1% with embryo axes of a kabuli type, while Ganguly and co-workers reached 8.6% with a half-seed method [8,9] . The superiority of cotyledonary nodes likely reflects their higher density of competent cells at the axillary meristem, which are receptive to Agrobacterium attachment and T-DNA transfer [10] . The inverse relationship between copy number and expression in line JG-T12 fits the well-known pattern of repeat-induced gene silencing [6] . Single-copy integrants generally express transgenes more stably, which has practical value for breeding programmes needing consistent expression across generations.

Larval mortality at 96 hours in the best line (78.3%) falls within the range seen in Bt cotton at comparable protein levels [4] . However, detached-leaf bioassays don't fully replicate field conditions. Whole-plant cage assays and confined field trials will be needed before these lines can advance toward regulatory evaluation. The ELISAmeasured Cry1Ac levels (0.09-0.34 µg/g FW) are modest compared to commercial Bt cotton events, which often reach 1-3 µg/g. Switching to a stronger or tissue-specific promoter, such as the chickpea pod-wall promoter described by Singh et al. [12] , or stacking cry1Ac with cry2Ab, may improve performance.

Conclusion

This research confirmed that Agrobacterium-mediated transformation of chickpea with synthetic cry1Ac is feasible at efficiencies sufficient to generate a usable pool of transgenic events. Cotyledonary nodes from cultivar JG-62 gave 14.7% efficiency, significantly higher than the four other explant types. PCR and Southern blot verified stable integration of one to three copies in T0 plants, and ELISA detected Cry1Ac at 0.09-0.34 µg/g fresh weight. The best transgenic line caused 78.3% neonate H. armigera mortality within 96 hours on detached leaves. Single-copy lines expressed higher and more consistent protein levels than multi-copy lines. Going forward, T1 and T2 progeny analyses, whole-plant cage trials, and confined field experiments are needed to assess whether this protection holds under natural pest pressure. Combining cry1Ac with cry2Ab could widen the toxin spectrum and delay resistance evolution in H. armigera.

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