Transgenic Cowpea Expressing Synthetic BtCry1Ab Provides Enhanced Resistance to Maruca vitrata and Supports Sustainable Pod Borer Management

Transgenic Cowpea Expressing Synthetic BtCry1Ab Provides Enhanced Resistance to Maruca vitrata and Supports Sustainable Pod Borer Management | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Transgenic Cowpea Expressing Synthetic BtCry1Ab Provides Enhanced Resistance to Maruca vitrata and Supports Sustainable Pod Borer Management Muthuvel Jothi, Sanjeev Kumar, Devendra Kumar Maravi, Deepak Kumar, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8322435/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract Cowpea, a vital legume crop, suffers substantial yield losses due to insect pests, particularly the legume pod borer ( Maruca vitrata ). The narrow genetic base of cowpea and the absence of effective host resistance against M. vitrata have limited the success of conventional breeding-based pest management. Given the increasing incidence of insecticide resistance and environmental concerns associated with chemical control, there is an urgent need for host plant-based solutions that fit within integrated pest management (IPM) frameworks. To address this challenge, we engineered transgenic cowpea plants expressing a synthetic cry1Ab gene under the control of the CaMV35S promoter. High expression of Cry1Ab was detected in leaves and pods, the primary feeding sites of key lepidopteran pests. Bioassays with M. vitrata and Helicoverpa armigera larvae demonstrated strong resistance in the transgenic lines, evidenced by reduced pod damage, suppressed larval feeding, and high insect mortality compared to non-transgenic controls. These results confirm the effective expression and bioactivity of Cry1Ab in planta, highlighting its potential as a reliable pest control trait under pest pressure. Importantly, the transgenic plants showed no detectable metabolic changes by NMR profiling and displayed normal growth and development without yield penalties. These findings underline the role of Bt-cowpea as a sustainable, environmentally compatible, and economically viable approach to reducing pest burden and pesticide dependence in legume production systems. Overall, the synthetic cry1Ab-expressing cowpea lines represent a promising next-generation tool for durable and broad-spectrum protection against major lepidopteran pests, contributing to the long-term goals of sustainable pest management and agricultural resilience. Cowpea crop improvement Legume pod borer management Bacillus thuringiensis Insect resistance Sustainable pest management Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Key Messages Cowpea suffers severe yield losses due to pod borer with no durable resistance sources. No effective host plant resistance has been identified against pod borer. We designed a novel synthetic Btcry1Ab gene to achieve stronger insecticidal activity. Transgenic cowpea expressing Btcry1Ab showed high resistance to pod borer damage. This provides a sustainable strategy for legume pest management and yield improvement. Introduction Cowpea ( Vigna unguiculata L .) is an important grain legume that provides high-quality dietary protein to millions of people, particularly in Africa and Asia (Muthuvel et al. 2021). Its resilience to drought and poor soil conditions, coupled with its ability to improve soil fertility through nitrogen fixation, makes cowpea a cornerstone of sustainable, low-input agriculture. In addition to protein, cowpea seeds contain essential vitamins (e.g., thiamine folic acid, and niacin), iron, minerals, and dietary fiber (Kamara et al. 2010), thereby serving as a source of food and nutrition for both humans and livestock (Muthuvel et al. 2021). Cowpea is predominantly cultivated in India, Africa, the Middle East, and South America (FAOSTAT, 2023). However, its production in major consuming countries fall much short from meeting the domestic demand (Al-Hassan and Diao 2007). Among the factors limiting productivity, the Lepidopteran Pod Borer (LPB), Maruca vitrata , represents a key pest of economic significance in tropical legume ecosystems and a major constraint to integrated pest management (IPM) programs. Feeding internally on both vegetative and reproductive tissues, it causes yield losses of up to 72% in Asia and Africa (Abudulai et al. 2017; Kusi et al. 2019; Srinivasan et al. 2021). Cowpea is one of the major hosts for M. vitrata , with yield losses of up to 72% in Asia and sub-Saharan Africa. Due to its rapid growth, the pod borer exhibits strong adaptability in regions with high humidity, posing significant challenges for effective control. In field conditions, pod borer infestation begins at 25–30 days after the early vegetative stage inflicting maximum damage to flowering stage in cowpea. Young larvae form webs on the reproductive structures and bore into buds and pods to feed on developing seeds (Srinivasan et al. 2021). This pest’s concealed feeding behavior within floral structures limits the effectiveness of contact insecticides, making management particularly challenging under field conditions. Chemical insecticides remain the primary control method of M. vitrata infestation, yet their high cost, limited accessibility for resource-poor farmers, and negative impact on pollinators and other beneficial insects constrain their long-term utility. Moreover, recurrent insecticide use has contributed to the evolution of insecticide resistance, underscoring the need for alternative, ecologically sustainable control tactics within IPM frameworks. Host plant resistance offers the most sustainable option to manage LPB. However, breeding progress is limited because cultivated cowpea lacks effective resistance, genetic diversity, is narrow, and wild Vigna species with resistance are difficult to cross. This further restrict durable pest resistance development. Genetic transformation thus provides the most feasible strategy to overcome these constraints and develop durable resistance to M. vitrata (Muthuvel et al. 2021). Bacillus thuringiensis (Bt) Cry proteins are highly specific and effective against lepidopteran pests, with Cry1Ab showing activity against M. vitrata ( Acharjee and Higgins 2021). While Bt-cowpea lines have been developed and tested in Africa, their adaptability to Asian production systems remains uncertain due to differences in seasons, cultivation practices, and genetic backgrounds (Addae et al. 2020). In the context of pest science, region-specific evaluation of the Bt traits is critical for understanding pest population dynamics, resistance management, and environmental interactions. Recent studies, such as the expression of the Cry2Aa protein in Indian cowpea cultivar, have proven its region-specific effectiveness against M. vitrata (Kumar et al. 2021). However, variable expression levels, organ-specific differences, and late-season decline in Bt protein accumulation have led to incomplete protection (Bakshi et al. 2011; Jadhav et al. 2020). Such reduced effectiveness during commercial production may heighten the risk of resistant insect populations, which is an issue of major concern in applied pest science. To address this, enhancing gene expression by codon optimization offers a promising strategy to achieve stable, high-level expression and ensure reliable pest control. In this study, we developed transgenic cowpea expressing a synthetic Btcry1Ab gene driven by the CaMV35S promoter. The plants showed consistently high expression of Cry1Ab protein across vegetative and reproductive stages, resulting in durable resistance. Insect bioassays using leaves and immature pods revealed minimal feeding damage and high larval mortality of both M. vitrata and Helicoverpa armigera compared with non-transgenic controls. Our findings contribute to pest management science by demonstrating the potential of Bt-cowpea as a viable host plant resistance component within IPM systems. These results demonstrate that Btcry1Ab cowpea has strong potential for field-level deployment, providing an effective and sustainable pest management strategy that reduces reliance on synthetic insecticides while supporting stable legume production. Materials and methods Construction of binary vector and development of cowpea transgenics The Btcry1Ab construct was kindly provided by Prof. Illimar Altosaar, University of Ottawa, Canada (Shu et al. 2000). The coding sequences for the insecticidal protein Cry1Ab from B. thuringiensis were chemically synthesized with codon optimization tailored for plant expression systems (Cheng et al., 1998; Sardana et al., 1996). The full-length plant synthetic Btcry1Ab ORF (1.86 kb), with XhoI and KpnI fragment, was cloned into the intermediate vector pRT101 between a 35S promoter and nos terminator sequence. The PstI fragment of 35S::Btcry1Ab:nos-Ter was subsequently digested and sub-cloned into binary plant vector pCAMBIA2301. The plasmid construct was then mobilized into disarmed hyper-virulent Agrobacterium strain EHA105 and used for cowpea transformation. The T-DNA of binary plasmid pCAMBIA2301 includes neomycin phosphotransferase gene ( nptII ) and β-glucuronidase gene ( gus ) interrupted by catalase intron, both driven by the strong constitutive cauliflower mosaic virus (CaMV) 35S promoter. The cowpea plant transformation was carried out using cotyledonary node explants from an insect-sensitive cultivar cv. PUSA KOMAL (IARI New Delhi) as per our previous protocol (SI Fig. 1 ; Solleti et al. 2008; Bakshi et al. 2011; Kumar et al. 2017). The putative transgenic cowpea plants were established in soil: compost (1:1) and grown to maturity in greenhouse containment. The T 1, T 2 and T 3 seeds were harvested and grown in the soil inside a transgenic poly-house to raise their progeny lines. Molecular characterization of transgenic cowpea lines Genomic DNA was isolated from the young leaves of transgenic plants using the DNASure Plant Mini Kit (Nucleopore, Genetix, India). For screening of the cowpea transformants, PCR analyses were carried out using cry1Ab specific primers (Forward: 5’- TGTCCATCTGGTCCCTCTTC-3’ and Reverse: 5’-ATGGTGAAGCCGGTGAGTC-3’) to obtain a partial fragment of 628 bp product from the coding region of the cry1Ab gene. A 549 bp fragment of the nptII gene and 540 kb of gus gene was also amplified using gene specific primers ( nptII forward: 5’-GGTGGAGAGGCTATTCGGCTA-3’ and nptII reverse: 5’- GGTAGCCAACGCTATGTCCTGA-3’; gus forward: 5’-GGTGGGAAAGCGCGTTACAAG-3’; gus reverse: 5’-TGGATTCCGGCATAGTTAAA-3’). The amplified products were resolved by electrophoresis on 1% agarose gel and visualized by ethidium bromide staining (Malke 1990). Southern blotting was performed using the DIG labeling and detection kit according to the manufacturer’s instructions (Bio-Rad, Hercules, USA). The T 3 PCR positive transgenic and untransformed cowpea plants were chosen to extract genomic DNA (Maxiprep MN plant DNA maxi kit, Germany). 50–60 µg of total genomic DNA were digested using HindIII, fractionated in 0.8% agarose gel and transferred to a positively charged Zeta-probe membrane. The blot was hybridized with DIG labelled 1 kb PCR product, corresponding to the coding region of cry1Ab gene. The hybridized blot was processed for pre and post hybridization wash as per the manufacturer’s instructions. The detection of cry1Ab junction fragment was performed using the instructions of the DIG labeling and detection kit (Roche Diagnostics, Mannheim, Germany). Expression analysis of cry1Ab gene in transgenic lines Total RNA was isolated from the Southern positive plants using a NucleoSpin RNA Plant Kit (TAKARA, Clontech, Japan) and were subjected to cDNA synthesis using RevertAid™ H Minus first-strand cDNA synthesis kit (Fermentas, USA). Relative fold expression by real-time PCR (RT-PCR) was performed to quantitate the expression of cry1Ab gene using specific primers ( cry1Ab forward: 5’TGG TAC AAC ACT GGC TTG GA3’; cry1Ab reverse: 5’-ATGGGATTTGGGTGATTTGA-3’) using USB VeriQuest SYBR Green qPCR Master Mix (2X) (Affymetrix, USA) on a Rotor-Gene Q Real-Time PCR System (Quiagen, Germany). The VuUBQ1 gene (GenBank Accession No. FG859491) was used as an internal control. The experiment was repeated twice independently with three biological replicates each. The relative expression of cry1Ab in wild-type (WT) and transgenic cowpea lines was estimated by using the 2 −∆∆Ct method and student’s t-test was used to calculate significance. Analysis of Cry1Ab protein in transgenic lines The expression of the Cry1Ab protein was analyzed in T 3 transgenic lines generated from four independent transformation events by ELISA and Western blot hybridization. The detection of Cry1Ab protein in leaves and immature pods was assayed by ELISA using antibody coated plates as per the manufacturer’s instructions (Envirologix Quantiplate kit for Cry1Ab/Cry1Ac, Amar Immunodiagnostics, India). Total soluble proteins (TSP) from transgenic and non-transgenic cowpea plants using protein extraction buffer (50 mM Na 2 CO 3 , 100 mM NaCl, 0.05% TritonX-100, 0.05% Tween-20, 2 mM Phenylmethylsulfonyl fluoride and 1 µM leupeptin) and quantified using Bradford assay (Bradford 1976). The TSP were then added to the wells of antibody-coated plates. A secondary antibody Cry1Ab-Enzyme conjugate was then added to each well and the plates were kept for shaking at room temperature for 1 hr. After washing, a colored reaction was observed after adding the substrate in each well and absorbance was measured at 450 nm and comparative histogram was plotted. For Western blotting, 30 µg of the TSP was fractionated on 12% sodium dodecyl sulfate polyacrylamide gels with (SDS-PAGE) and blotted onto a PVDF membrane by wet transfer (GE Healthcare; Burnette 1981). The rabbit anti-Cry1Ac primary antibody (Amar immune diagnostics, India) in 1:500 dilution and goat anti rabbit conjugated with horseradish peroxidase (Promega) was used for detection. The blot was developed using substrate (SuperSignal West Dura, Thermo Scientific, USA) for 5 min and the reaction was stopped by washing the membrane with sterile distilled water. A single band of ~ 67 kDa corresponding to Cry1Ab protein was detected immunologically in all transgenic plants confirming the stable expression of Cry1Ab protein, whereas no such band was observed in the non-transgenic plants. Insect bioassays Maruca pod borer population were reared in an insect growth room at 27 ± 2°C, 16 h light photoperiod and 70 ± 10% relative humidity for optimal growth and mating for in vitro insect bioassays. Insect bioassays were conducted on T 3 transgenic cowpea lines of 45 days old, where fully expanded mid-canopy leaves and uniformly immature pods were sampled, and second-instar larvae were used to assess the efficacy of cry1Ab expression, as they are more robust and suitable for evaluating sub-lethal effects and realistic field damage (Addae et al 2020). Non-transformed plants served as the negative control. The leaves and immature pods of the non-transgenic and transgenic plants (confirmed by PCR, Southern, and Western blotting) were fed to the larvae separately to evaluate the insecticidal efficacy of the Cry1Ab protein. The ten second instar larvae were released on leaves and pods wrapped with cotton to sustain moisture in the bioassay dishes. Three replicates were carried out for each transgenic/non-transgenic line. In response to the feeding, the mass gain, life cycle and mortality of the larvae and the extent of damage onto the leaves and pods were recorded regularly up to four days after release of the larvae. Morpho-physiological analysis of transgenic cowpea lines The morpho-physiological analysis of the five cry1Ab lines were performed at greenhouse conditions. The level of Cry1Ab protein in the different progeny plants was determined by Western blot and were grouped into low, medium and high categories. The phenotypic parameters such as plant height, branch number and total number of seeds and pods formed per plant were observed to determine any off-target effects owing to the cry1Ab gene expression. To determine the effect of cry1Ab gene expression on seed mass, seed size, the average seed mass of 10 seeds collected from each line was also recorded. Metabolomic analysis using NMR Metabolite Extraction Samples were extracted according toKim et al. 2010 with minor modifications. Briefly, the frozen leaves were ground to fine powder using prechilled mortar and pestle and lyophilized overnight. The extraction was performed with approximately 25 mg of samples and 1 ml of aqueous methanol (80 % v/v) in micr-centrifuge tube. The samples were vortexed for 30 s and then the mixture was further extracted using ultrasonication water bath for 15 min (with 1 min sonication and 30 s pause) using UP200S, Ultrasonic Processor (Hielscher Ultrasonic GmbH, Germany). The extract was centrifuged at 10,000 rpm for 20 min at 4 0 C. The resulting supernatant was collected in fresh tube and the procedure repeated twice with the remaining pellets. The resulting supernatant from all extraction was combined and concentrated using SpeedVac (Eppendorf AG, Hamburg, Germany) for 3 h. Finally, the dried sample was dissolved in 600 µl of deuterated water (D 2 O) containing 0.05% (w/w) of 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid, sodium salt (TMSP) as an internal standard and transferred into a 5 mm NMR tubes (Sigma-Aldrich, Sant-Louis MO, USA) for further analysis. 1 H-NMR Spectroscopy and spectral data processing The NMR spectra for all samples were acquired using Bruker Avance III 600 MHz spectrometer with a DCH CryoProbe (BrukerBioSpin GmbH, Rheinstetten, Germany), operating at 600 MHz and 298 K temperature, with 90 o pulse width of 12 kHz. One-dimensional 1 H-NMR spectra with water pre-saturation were acquired using a standard Bruker pulse-acquire sequence during a relaxation delay of 4 s and a mixing time of 100 ms, 32 scans were collected into 64 k data points over a spectral width of 4801.54 Hz and pulse width of 9.46 µs, with an acquisition time of 6.82 s. Water suppression was obtained by pre-saturation sequence present in the routine experiments. The obtained 1 H-NMR spectrum was manually corrected for phase and baseline using Mestrenova, and the chemical shift was referenced to TMS at 0.000 ppm, to assess the signal quality and to determine the relative proportion of metabolites. The spectral regions of 0.5–10.0 were bucketed into bins (0.04 ppm) using Mestrenova Software (Version 6.0.2–5475; Mestrelab Research, S.L. Spain). Spectral regions 4.84 containing residual signals from water was excluded to remove the residual water by using Mestrenova software. The resulting data was subjected to multivariate analysis (MVDA). Multivariate Statistical Analysis for Metabolomic The dataset was imported into the MetaboAnalyst 5.0 ( https://www.metaboanalyst.ca/ ), and then, the MVDA was performed by PCA and PLS-DA. All the variables were mean centered and log-transformed for multivariate analysis. Furthermore, OPLS-DA was applied to each sample for pairwise comparison between transgenic samples and the control wild type sample using MetaboAnalyst 5.0. The data were statistically analyzed with one-way analysis of variance (ANOVA), and the significant differences were determined using Tukey’s test to evaluate the significant differences between the transgenic samples and the control wild type sample. Differences with p < 0.05 were considered significant. Metabolite identification Metabolite identification was carried out based on the databases such as Biological Magnetic Resonance Data Bank (BMRB; http://www.bmrb.wisc.edu/ ) and Human Metabolome Database (HMDB; https://hmdb.ca/ ) and published literature (Maravi et al. 2022). To determine the possible differences in the related metabolic pathways between control and transgenic samples, pathway analysis was carried out using MetaboAnlyst 5.0 and their contributions and biological interpretations were discussed based on the Kyoto Encyclopedia of Genes and Genome (KEGG) database ( https://www.genome.jp/kegg/pathway.html ). Statistical analysis Statistical analyses were performed using GraphPad Prism, version 8.0 (GraphPad software, USA). The assumptions of normality and homogeneity of variances were evaluated using the Shapiro-Wilk test and Brown-Forsythe and Welch test, respectively. Data met the criteria for parametric analysis, and therefore replicated datasets (n = 3 biological replicates) from each experiment were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. The experiment was repeated twice, and a representative image is shown. Results Development of transgenic cowpea lines with stable cry1Ab integration Transgenic cowpea plants were produced using Agrobacterium -mediated transformation using cotyledonary node explants from the commercial cowpea variety PUSA KOMAL (Kumar et al. 2017). The explants produced an average of 5.4 elongated shoots per explant, with a 92 ± 2% response rate and a maximum transformation efficiency of 3.92% after three selection cycles on kanamycin-supplemented medium (Kumar et al. 2017). PCR analysis was used to confirm the presence of the nptII , gus , and cry1Ab transgenes in the putative T 1 plants, using gene-specific primers (Fig. 1 A; SI Table 1 ). Out of 65 plants that survived on the kanamycin selection, 43 tested positives for amplification, yielding fragments of 549 bp, 540 bp, and 628 bp for the nptII , gus , and cry1Ab genes, respectively. No amplification products were detected in non-transformed control plants (Fig. 1 B-D). Based on high transgene expression and PCR results, five independent transgenic lines were selected for further analysis to study the segregation patterns of the integrated cry1Ab , gus , and nptII genes in subsequent generations. For each of these five lines, we analyzed a minimum of 10–12 plants in the T 2 generation, and at least 8–10 homozygous plants per line in T 3 generation. Segregation ratios for cry1Ab , gus , and nptII were recorded in T 1 and T 2 generation and were consistent with Mendelian inheritance patterns for a single locus insertion. In T 3 generation, we selected one homozygous plant per independent line (confirmed by PCR and expression analysis) for insect bioassay and phenotypic evaluation. The segregation ratios of transgenic lines adhered to a Mendelian inheritance pattern (3:1; P < 0.05), indicating that the cry1Ab gene was inherited as a single dominant locus (data not shown). To verify the expression of cry1Ab , relative fold expression by real-time PCR (RT-PCR) was conducted on PCR-positive plants. In the T 3 lines 17, 30, and 38, expression levels of cry1Ab were found to be 3- to 10-fold higher in leaves and 6- to 13-fold higher in immature pods relative to the Vuubiquitin gene. No amplification was observed in control non-transformed plants (Fig. 1 E and F). Expression levels varied significantly between leaves and pods across the transgenic lines. Notably, lines #12 and #5 exhibited the highest cry1Ab expression in leaves and immature pods, respectively, while line #38 showed the lowest expression in both tissues among the transgenic lines (Fig. 1 E and F). The stable integration and copy number of the cry1Ab gene were further assessed in the T 3 lines by Southern blot analysis. HindIII-digested genomic DNA hybridized with a cry1Ab probe displayed hybridization bands larger than 4.2 Kbp, confirming the successful integration of the cry1Ab gene into the cowpea genome. Southern blotting also confirmed that three out of five lines contained a single copy of the cry1Ab gene, indicating a single T-DNA insertion event. The lines #17 and #30 showed two copies of cry1Ab gene. No hybridization signal was observed in the non-transformed control plants (Fig. 1 G). Two (line #17 and #30) transgenic lines showing similar Southern hybridization patterns were independently generated. The similarity likely reflects preferential integration or conserved restriction site environments. Transgenic cowpea lines express higher Cry1Ab protein The qualitative and quantitative measurement of the Cry1Ab protein expression was analyzed by Western blotting and ELISA, respectively. The total soluble protein from the leaves and immature pods of transgenics and non-transformed control plant was used to test Cry1Ab protein. The Cry1Ab protein that are hybridized with the polyclonal Cry1Ab antibodies was detected as a signal band of molecular weight ~ 67 kDa in all tested lines (Fig. 2 A and B). The intensity of the Cry1Ab protein in all the transgenic lines, except #38, was higher in leaves. In immature pods, lines #17 and #38 were showed low Cry1Ab intensity than the rest of the transgenic lines. Faint non-specific bands were detected, likely due to antibody cross-reactivity, but do not interfere with interpretation of Cry1Ab expression. These results on expression of Cry1Ab toxin protein demonstrate stable and consistent expression in most of the transgenic lines. No band was detected in the case of the untransformed control (non-transgenic) plant. The concentration of Cry1Ab protein in leaves and immature pods was quantified in five independent transgenic lines using ELISA (Fig. 2 C and D). Since the cry1Ab gene was driven by CaMV35S constitutive promoter, the Cry1Ab protein was expressed in both tissues. We observed that the Cry1Ab protein was highly expressed across the three generations in transgenic cowpea lines. The protein expression ranged from 2.8 to 10.8 mg/g in leaf tissues and 5.0 to 10.7 mg/g in immature pods in T 1 transgenics (SI Fig. 2 A and B). The T 2 transgenics recorded a maximum level of Cry1Ab protein expression, 2.9 to 10.1 mg/g leaf tissue and 5.6 to 10.1 mg/g young pods (SI Fig. 2 C and D). In T 3 generation, the Cry1Ab protein expression observed to be 5.5 to 7.5 mg/g in leaf tissue and 5.5 to 8 mg/g in immature pods (Fig. 2 C and D). The accumulation of Cry1Ab proteins was higher in the reproductive tissues (young pods) compared to vegetative tissues (leaf) across all the three generations in tested events. Among the five transgenic lines tested, cry1Ab expression level in lines #12, #17 and #30 were found higher, in both vegetative as well as reproductive tissues at T 3 generation. The concentration of Cry1Ab proteins in transgenic lines were stabilized in subsequent generation. The T 3 transgenic lines of the respective T 1 parents, showed 5.7% higher expression (on an average) of Cry1Ab protein in leaves and pods of all transgenic lines. Insect bioassay of transgenic cowpea leaves and immature pods showed higher mortality of M. vitrata and H. armigera The entomocidal activity of cowpea transgenic plants, expressing moderate to high levels of BtCry1Ab, was evaluated through insect feeding bioassays. The mortality rate for M. vitrata larvae ranged from 33–60%, while H. armigera larvae exhibited a mortality range of 66–96% when fed on leaves of T 3 transgenic plants (Fig. 3 ). Similarly, in the detached pod assay, mortality rates ranged from 60–90% for M. vitrata and 46–63% for H. armigera in the T 3 transgenic lines (Fig. 3 ). Notably, transgenic lines #30 and #38 showed the highest mortality for both M. vitrata and H. armigera larvae in leaf and pod bioassays. Significant differences in mean larval mortality were observed between the transgenic lines and the control. Additionally, surviving larvae on the transgenic plants exhibited retarded growth compared to those on non-transgenic controls (data not shown). For M. vitrata larvae, Cry1Ab protein concentrations greater than 6 mg g − 1 fresh mass in the pods caused over 70% mortality. However, at similar Cry1Ab protein concentrations, H. armigera larvae exhibited relatively lower mortality (over 53%). In contrast, H. armigera larvae showed higher mortality (> 83%) than M. vitrata in transgenic leaves expressing > 6 mg g − 1 fresh mass Cry1Ab toxin (Fig. 2 C and D; Fig. 3 ). Although larval mortality was positively correlated with Cry1Ab protein levels, the reason for the differences in mortality between M. vitrata and H. armigera in leaves and pods remains unclear. NMR Metabolite identification The 1 H NMR spectrum obtained at 600 MHz for the transgenic and non-transgenic cowpea pods (SI Fig. 3 ). The NMR results revealed the variation 40 major metabolites non-transgenic cowpea pods. These 40 metabolites include amino acids, organic acids, sugars, and other aromatic compounds (SI Table 2). 1 H NMR performed on immature pods of transgenic and non-transgenic cowpea, the 600MHz spectra exhibited in three main regions (SI Fig. 3 ). The first region (0.5-3.0 ppm) contained signals of amino acids and some organic acids were identified. The second region (3.0-5.8 ppm) comprised signals of carbohydrates. Sucrose (5.41 ppm), fructose (4.11 ppm), beta glucose (4.64 ppm), alpha glucose (5.23 ppm) were the most abundant carbohydrates in this region. In the third region (5.8–9.5 ppm), were relatively weak signals which are corresponds to the aromatic groups from phenolic compounds and aromatic amino acids. The most abundant signals in the aromatic region, phenylalanine (7.43 ppm), tryptophan (7.71 ppm), fumarate (6.53 ppm) and formic acid (8.54 ppm) were identified (SI Fig. 3 ). Multivariate statistical analysis for metabolomic Identification of metabolite variation of transgenic and non-transgenic leaves and pods was carried out by principle component analysis (PCA). In our current study, the first three components explained 78.6% of whole data: PC1, 41; PC2, 19.8; PC3, 17.8%, respectively (Fig. 4 ). The score plots clearly showed that samples from the transgenics and non-transgenics were well separated (Fig. 4 A). However, lines #12 (TR2), #17 (TR3), #30 (TR4), and #38 (TR5) samples showed noticeable overlaps indicating that these samples not be efficiently separated. The control group was negatively influenced by PC1 and positively influenced by PC2, whereas, line #5 (TR1) group was negatively influenced by both PC1 and PC2. The lines #12 (TR2), #17 (TR3), #30 (TR4), and #38 (TR5) groups were negatively influenced by PC1. The loading plots indicated that the discriminative metabolites responsible for the variable separations. Metabolites such as isocitrate and isoleucine are represented in PC1. PC2 was mainly characterized by proline, fructose and malate (Fig. 4 B). The VIP analysis revealed that the top 12 metabolites (out of 30) were identified as discriminating metabolites (VIP ≥ 1) between transgenic and non-transgenic tissues (P < 0.05). Significant higher contents of glucose-6-phosphate, glucose, betaine, mannose, citrate, N-carbomylaspartate, galactose, trans-4-hydroxy proline, glutamate, valine, methionine, and proline were upregulated by transgenic cowpea pods (SI Fig. 4 ). To effectively compare the differences caused by cry1Ab expression, pairwise comparisons were performed using the supervised OPLS-DA approach. The random permutation test (100 times) on the OPLS-DA model confirmed the difference between control and transgenic cowpea (Fig. 5 A-E). The S-plot also confirmed the higher relative content of methionine, proline, galactose, threonine, fructose, valine, and glucose-6-phosphate in transgenic pods compared to control (Fig. 5 F-J). Metabolic Pathway Analysis The Web tool, MetaboAnalyst 5.0, was used to perform the biological pathway analysis. Metabolic pathway analysis (MetPA) suggested that thirteen pathways might be altered by the cry1Ab expression in cowpea based on the statistically significant value (p 0.1. This includes, (1) starch and sucrose metabolism, (2) alanine, aspartate and glutamate metabolism, (3) galactose metabolism, (4) valine, leucine and isoleucine biosynthesis, (5) cysteine and methionine metabolism, (6) glyoxylate and dicarboxylate metabolism, (7) arginine and proline metabolism, (8) amino sugar and nucleotide sugar metabolism, (9) glycine, serine and threonine metabolism, (10) arginine biosynthesis, and (11) citrate cycle (TCA cycle), (12) carbon fixation by Calvin cycle, (13) glutathione metabolism. The relationship between several metabolites and relevant metabolic pathways and the primary metabolites process is depicted in (Fig. 6 and Table 1 ). Table 1 The metabolite pathway majorly altered in transgenic cowpea pods expressing cry1Ab . Total number of pathways and hits along with their negative log values and false discovery rate (FDR), and their impact values are mentioned. Pathway name Total Hits -log ( p ) FDR Impact 1 Starch and sucrose metabolism 22 7 6.6988 1.84E-05 0.72922 2 Alanine, aspartate and glutamate metabolism 22 6 5.3342 0.000213 0.57914 3 Galactose metabolism 27 6 4.7706 0.00052 0.36582 4 Valine, leucine and isoleucine biosynthesis 22 4 2.9363 0.026631 0 5 Cysteine and methionine metabolism 46 5 2.5288 0.05156 0.1793 6 Glyoxylate and dicarboxylate metabolism 29 4 2.4733 0.05156 0.27842 7 Arginine and proline metabolism 32 4 2.3139 0.055568 0.14435 8 Amino sugar and nucleotide sugar metabolism 52 5 2.293 0.055568 0.11106 9 Glycine, serine and threonine metabolism 33 4 2.2647 0.055568 0.16478 10 Arginine biosynthesis 18 3 2.1757 0.061389 0.23148 11 Citrate cycle (TCA cycle) 20 3 2.0438 0.075611 0.19387 12 Carbon fixation by Calvin cycle 21 3 1.9835 0.079632 0.05923 13 Glutathione metabolism 26 3 1.7256 0.13312 0.05801 Agronomic performance and segregational analysis: The agronomic traits of T 3 transgenic plants expressing cry1Ab and their non-transgenic counterparts were assessed to determine if the incorporation of the Bt gene resulted in any phenotypic changes (Fig. 7 ). Morphologically, there were no observable differences between the transgenic and non-transgenic plants. Statistical analysis revealed no significant differences (P < 0.05) in key agronomic traits, including plant height, seed number per plant, seed length, and ten seed mass of transgenics were not different from non-transgenic plants (Fig. 7 ). Line #30 showed lower number of branches compared to other transgenics and non-transgenic plants. However, the pod number per plant was observed to be higher in line #30 and #38 compared to other plants (Fig. 7 ). Furthermore, transgenic plants exhibited no differences in growth or fertility compared to non-transgenic plants. Discussion Cowpeas are cost-effective protein-rich grain legumes critical for nutritional security and sustainable agriculture in developing countries. The legume pod borer ( M. vitrata ) is the most devastating insect pest of cowpea. Till date, no source of complete-resistance has been identified in the gene pool of cultivated cowpea. Given the heavy reliance on synthetic insecticides for pod borer control and the consequent risk of pest resurgence, environmental contamination, and resistance evolution, developing insect-resistant cowpea varieties remains a top priority in pest management programs. Amelioration of insect damage can add to cowpea production, reduce insecticide use and help in sustainable production (Muthuvel et al. 2021). Sources of resistance to the insect pest have not been identified in cowpea germplasm, which limited the approach of conventional breeding to develop resistant varieties (Horn and Shimelis 2020; Smith 2021). Genetic engineering of crops using genes encoding insecticidal crystal proteins or δ-endotoxins from B thuringiensis (Bt) has been shown to confer resistance to a variety of insects and pests in various crops (Huang 2021; Tilgam et al. 2021). Among others, Cry1Ab has been demonstrated to be highly toxic against early instars of M. vitrata (Srinivasan et al. 2021). The incorporation of Bt genes into legumes, therefore, aligns with sustainable pest management strategies and the goals of IPM frameworks that emphasize host plant resistance as a first line of defense. Previous field studies have evidenced that cry1Ab provides effective protection for cowpea against M. vitrata . Artificial infestation of M. vitrata larvae, along with an integrated pest management strategy, resulted in nearly complete protection in transgenic cowpea lines expressing cry1Ab protein compared to non-transgenic cowpea lines (Nboyine et al. 2024). These findings suggest that cry1Ab could be a promising candidate for integrating insect resistant cowpea breeding pipelines. We have developed transgenic cowpea lines expressing a codon-optimized cry1Ab gene from B. thuringiensis and verified their efficacy against M. vitrata as well as H. armigera , both major lepidopteran pests of legumes. The inclusion of both pests in bioassays underscores the relevance of this study to pest science, as H. armigera is a notorious polyphagous pest known for rapid resistance development and cross-infestation across several legume crops. Cowpea transformation protocol has been standardized and used to generate many transgenic lines to confer various biotic and abiotic stress tolerance in our lab (Kumar et al. 2017, 2022). Following the same protocol, we have generated 65 putative transgenic cowpea lines. The transmission of the transgene/inheritance was confirmed by PCR analysis at each generation and molecular analyses was calculated to verify Mendelian segregation. Interestingly, a few transgenic events were found to be positive for the selectable marker nptII but negative for both cry1Ab and gus gene. This may be due to partial T-DNA integration or genomic rearrangements during T-DNA integration or chimerism in regenerated plants could result in such incomplete or tissue-specific transgene presence. Another possible explanation includes false positives during PCR screening, particularly if low-copy or truncated insertions occurred. These factors highlight the complexity of T-DNA integration and underscore the need for thorough molecular validation of transgenic events. We have observed few homozygous lines in T 2 generation, based on PCR analysis. However, in most of the cases we have obtained mixed populations of homo and hemizygous lines T 2 generation. The five homozygous PCR confirmed progenies with Mendelian segregation were used for characterization of the transgenic lines. Southern blotting revealed single (three lines) and double (two lines) copy of the cry1Ab gene in the HindIII-digested genomic DNA of the selected transgenic cowpea lines when probed with a sequence specific to cry1Ab , indicating its stable integration in the cowpea genome. The presence of the expected hybridization signals with genomic DNA fragments (> 4.2 kb) in the majority of the transformed plants showed that the probed genes cry1Ab remained intact upon integration into the cowpea genome (Fig. 1 G). It is preferable to have low-copy (one to three) insertions of the transgene into the plant genome using A. tumefaciens , as they tend to remain stable over several generations. The transgenic cowpea plants have shown an independent pattern of transgene expression due to the complex and random integration of foreign genes in the host genome following Agrobacterium -mediated transformation. Therefore, the inheritance of foreign genes in transgenic plants may exhibit complex patterns for both single and multiple genes. Stable expression across generations is particularly critical for pest management programs aimed at reducing pest population pressure over multiple cropping cycles. Given the poor expression levels of native Bt insecticidal proteins in higher eukaryotes (Jadhav et al. 2020; Li et al. 2022), it's critical to ensure optimal expression of insecticidal proteins for effective control of targeted insects (Liu et al. 2020). The optimal expression of Bt genes in plants is controlled by various factors including codon preference, AT content, mRNA destabilization sequences and putative polyadenylation signals (Watts et al. 2021). Codon optimization has been previously employed in various crops, such as cotton (Zafar et al. 2022; Siddiqui et al. 2023), soybean (Fang et al. 2024), chickpea (Singh et al. 2022), and cowpea (Kumar et al. 2021), to enhance expression levels. In our current study, we adopted a similar approach to maximize cry1Ab expression in transgenic cowpea. This involved increasing the GC content of the coding sequence and removing polyadenylation and mRNA destabilization sequences without altering the amino acid sequence. Additionally, codon usage was optimized to enhance translation in plants by incorporating plant-preferred codons. Relative fold expression by real-time PCR analysis of cry1Ab transcripts in transgenic cowpea plants, driven by the constitutive CaMV35S promoter, revealed higher expression levels of cry1Ab transcripts in both leaves and immature pods (Fig. 1 E and F). In our transgenic lines, cry1Ab expression in leaves and immature pods reached 3 to 10-fold and 6 to 13-fold expression relative to ubiquitin, respectively. Notably, the expression of cry1Ab in these tissues surpasses that reported in previous studies on cowpea (Addae et al. 2020; Majumder et al. 2020; Eckerstorfer et al. 2022). Given the critical importance of achieving optimal expression of cry genes in field crops for effective pest control strategies, our transgenic lines emerge as potential candidates for integrating existing Cry lines into comprehensive field studies. Furthermore, our investigation revealed variations in cry1Ab expression across independent transgenic cowpea lines (Fig. 1 E and F). These variations likely arise from factors such as positional effects, transgene copy numbers, and the choice of promoter for transgene expression. Notably, similar fluctuations in cry gene expression have been documented in numerous other crops engineered to confer resistance against lepidopteran insects (Siddiqui et al. 2023; Fang et al. 2024; Singh et al. 2022; Kumar et al. 2021). This underscores the importance of meticulous optimization and rigorous evaluation protocols in transgenic crop development. The results from PCR, Southern blotting, and RT-PCR analyses of transgenic cowpea plants decisively confirmed the stable integration without any rearrangements of the cry1Ab gene in transgenic plants and also in subsequent generations. The effectiveness of selected cowpea lines expressing the Cry1Ab protein was assessed against MPB larvae by feeding them leaves and pods. Western blot analysis, followed by ELISA, confirmed stable cry gene expression. Among the T 3 progenies, line #30 exhibited the highest Cry1Ab expression (7.6 µg/g in leaves and 8 µg/g in pods), while line #5 showed the lowest expression (7.6 µg/g in leaves and 8 µg/g in pods). The expression-based selection process successfully identified the high-expressing events in this research. Notably, the pods accumulated 6.6% more toxin on average than the leaves in the corresponding transgenic lines. Previous studies have documented the variability in Bt -endotoxin expression among transgenic plants driven by CaMV35S promoters. This variability has been attributed to factors such as the position effect of gene integration, surrounding flanking sequences, chromatin context, increased DNA methylation with plant age, and physiological changes affecting the stability of foreign proteins within plant tissues (To et al. 2021; Rurek and Smolibowski 2024). In contrast, our analysis of transgenic cowpea lines demonstrated that the expression of the Cry1Ab protein, as verified through Western blot and ELISA assays, remained robust and consistent in both leaves and immature pods across three successive generations. This stability highlights the potential for reliable expression of Bt -endotoxins in cowpea, minimizing the concerns raised in earlier observations. The Bt -toxin expression levels observed in this study surpass those reported for other grain legumes (Acharjee and Higgins 2021; Singh et al. 2023) and represent the highest recorded Cry protein levels in cowpea (Addae et al. 2020; Kumar et al. 2021). This higher expression may be attributed to the use of a codon-optimized cry1Ab gene, specifically engineered to enhance mRNA stability, eliminate polyadenylation sites and splicing sequences, and optimize ATG consensus flanking nucleotides for efficient translation initiation. Additionally, the transgene's integration into a transcriptionally active region of the host genome likely contributed to this robust expression (Watts et al. 2021). Codon optimization has been extensively employed in Bt -cry genes to enhance insect resistance across various plant species. High-level expression of codon-optimized cry1Ac , cry1Ab , and cry1C genes has been previously demonstrated in transgenic cotton, tomato, and tobacco, respectively (Zafar et al. 2022; Siddiqui et al. 2023; Fernandes et al. 2023; Wang et al. 2024). Furthermore, increased expression of Cry2Aa in cowpea has been shown to improve resistance against lepidopteran pests (Singh et al. 2018; Kumar et al. 2021). The expression levels of a transgene are significantly influenced by the choice of promoter driving its transcription and translation into functional protein. Historically, the CaMV35S promoter has been widely used to achieve constitutive expression of cry1Ab in transgenic cowpea, and the first pod-borer-resistant cowpea released for commercial use in Nigeria utilized the CaMV35S promoter for gene regulation ( https://aaccnet.confex.com/aaccnet/2020/meetingapp.cgi/Paper/5721 ). However, recent studies have shown that the green tissue-specific RuBisCO small subunit (rbcS) promoter can drive Cry1Ac expression to levels 1.5 times higher than those achieved with CaMV35S and Ubi promoters in chickpea (Boruah et al. 2023; Hazarika et al. 2021). These findings suggest that future research should explore the use of the rbcS promoter to achieve enhanced endotoxin protection through higher transgene expression. Our study adds to the expanding body of research focused on the effectiveness of transgenic plants expressing insecticidal proteins in controlling major lepidopteran pests. Through in vitro insect feeding bioassays using larvae of M. vitrata and H. armigera , we observed a marked increase in insect-induced damage in non-transgenic control compared to transgenics. This was directly linked to the presence of Cry1Ab toxins in the leaves and immature pods of the transgenic plants. Notably, all five transgenic lines exhibited resistance to insect damage, with some variation in effectiveness, likely due to differences in cry1Ab gene expression levels. Our results are consistent with previous studies on the role of transgenic plants in pest management. For instance, cowpea lines expressing the Cry2Aa protein showed substantial protection against Maruca pod borer larvae, with mortality rates exceeding 90% (Kumar et al. 2021). Similarly, chickpea plants co-expressing cry1Ab and cry1Ac genes exhibited enhanced toxicity, providing broader protection against pests such as H. armigera (Koul et al. 2022). Some Bt -transgenic chickpea lines even achieved 100% mortality, underscoring the potential of Bt -transgenics as an effective pest management tool (Hazarika et al. 2021). Such line-dependent efficacy data are vital for pest management programs, as they aid in selecting elite events for resistance durability testing and resistance management studies under variable field conditions. Moreover, our cry1Ab cowpea transgenics demonstrated enhanced insecticidal efficacy not only against M. vitrata but also against H. armigera . Interestingly, although Cry1Ab protein levels were lower in the leaves compared to pods of transgenic cowpea lines, higher H. armigera mortality was observed in leaf-feeding assays. This apparent contradiction can be attributed to several factors. Firstly, H. armigera larvae show a feeding preference for softer leaf tissues during early instars, leading to greater ingestion of Cry1Ab despite lower expression levels in leaves (Zalucki et al. 1994; Sharma et al., 2005). Secondly, Cry1Ab may be more evenly distributed or bioavailable in leaf tissues, whereas physical structures in pods may hinder effective ingestion. Additionally, plant-derived secondary metabolites such as flavones, commonly found in leaves, may interact synergistically with Cry toxins to enhance their toxicity (Wang et al., 2021). Furthermore, recent studies suggest that Cry1Ab’s toxicity depends on binding to specific receptors in the insect midgut, including prohibitin and cadherin-like proteins, whose expression or accessibility could vary depending on the tissue consumed (Sena da Silva et al., 2021). Therefore, the observed mortality is likely influenced not just by absolute Cry1Ab levels, but also by tissue-specific feeding dynamics, toxin accessibility, plant metabolite interactions, and receptor-mediated mechanisms. The observed reduction in larval mortality in transgenic cowpea compared to larvae fed on non-transgenic control plants confirms the effective expression of the toxin protein in these transgenic lines. These findings provide a strong foundation for future crop protection strategies, suggesting that integrating Cry1Ab proteins into cowpea could be a promising approach to controlling M. vitrata and H. armigera infestations, thereby minimizing crop damage and enhancing agricultural sustainability. Metabolomics has emerged as a powerful tool for providing deep insights into crop biology. The information obtained from metabolomic analyses can be effectively utilized in assessing phenotypic changes, identifying biomarkers, and tracking gene expression, while also enhancing the interpretation of other genomic data (Makhumbila et al. 2022). NMR metabolomics, in particular, is highly effective in studying plant defenses, both constitutive and induced, against biotic stressors(Mascellani Bergo et al. 2024). Our study revealed that the majority of metabolites in transgenic cowpea were upregulated, including amino acids, sugars, and other key metabolites (Fig. 6 ). Notably, carbohydrates such as sucrose and fructose were significantly more abundant in transgenics compared to non-transgenic cowpea. These results align with those of (Chang et al. 2012), who reported elevated levels of sucrose, mannitol, and glutamic acid in transgenic rice expressing cry1Ac and sck genes. Metabolic pathway analysis further highlighted the upregulation of the amino acid metabolism pathway, TCA cycle, and carbohydrate metabolism pathway (Fig. 6 ; Table 1 ). Our findings indicate that while no new metabolites were detected, there was a significant alteration in the abundance of existing ones. Similar outcomes were observed in maize transgenics overexpressing cry proteins (Liu et al. 2021; Liu et al. 2023). The use of both targeted and untargeted NMR metabolomics was crucial in evaluating the metabolite profile variations in cowpea leaves and pods resulting from transgene expression. From a pest science perspective, such analyses are crucial for biosafety assessments, ensuring that transgenic plants maintain normal physiology while exhibiting enhanced pest resistance, thereby minimizing unintended ecological risks. Conclusion In conclusion, our study demonstrated that the synthetic cry1Ab gene is a valuable strategy for enhancing MPB resistance. This is the first report of a transgenic cowpea variety using a synthetic cry1Ab gene, which also conferred resistance to H. armigera . This represents a major advancement toward developing genetically engineered host plant resistance that complements existing IPM strategies and reduce reliance on broad-spectrum insecticides. Incorporating such Bt cowpea lines into IPM frameworks could help suppress pest populations, delay the evolution of insect resistance through gene pyramiding, and reduce the frequency of insecticide sprays. The cry1Ab gene is a strong candidate for co-expression with other cry genes in developing Bt cowpea resistant to multiple insects, including MPB and H. armigera . Such combinations could delay resistance evolution, provide broad-spectrum protection, increase yields, boost the income of farmers, and reduce pesticide use. Our findings demonstrate that the transgenic cowpea lines not only show high insect mortality and stable Cry1Ab expression but also maintain normal metabolic function, ensuring biosafety and agronomic performance. Taken together, this work positions Bt cowpea as a valuable component in the next generation of pest management technologies- offering an effective, sustainable, and scientific approach to mitigating lepidopteran damage in grain legumes. Declarations Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Competing Interests The authors have no relevant financial or non-financial interest to disclose. Author Contribution Statement LS conceived and designed research, contributed new reagents/analytical tools and corrected the manuscript. MJ designed, conducted experiments and analyzed data and wrote the manuscript. DKM, SK, DK assisted MJ in some experiments. IA provided the synthetic cry1Ab construct. 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Supplementary Files SI.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 08 Jan, 2026 Reviews received at journal 08 Jan, 2026 Reviews received at journal 03 Jan, 2026 Reviewers agreed at journal 19 Dec, 2025 Reviewers agreed at journal 18 Dec, 2025 Reviewers agreed at journal 12 Dec, 2025 Reviewers invited by journal 12 Dec, 2025 Editor assigned by journal 11 Dec, 2025 Submission checks completed at journal 10 Dec, 2025 First submitted to journal 09 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8322435","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":559411258,"identity":"2f51e81c-e205-4c9f-bb2f-9008ed882fac","order_by":0,"name":"Muthuvel Jothi","email":"","orcid":"","institution":"Osaka Metropolitan University","correspondingAuthor":false,"prefix":"","firstName":"Muthuvel","middleName":"","lastName":"Jothi","suffix":""},{"id":559411259,"identity":"65ceaf1b-b3d0-449e-b8da-dd92f61c5d0e","order_by":1,"name":"Sanjeev Kumar","email":"","orcid":"","institution":"International Centre for Genetic Engineering and 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1","display":"","copyAsset":false,"role":"figure","size":400227,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDevelopment and molecular characterization of transgenic \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ecry1Ab\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e lines\u003c/strong\u003e. \u003cstrong\u003e(A)\u003c/strong\u003e T-DNA region of the \u003cem\u003ecry1Ab\u003c/em\u003e construct of the binary vector pCAMBIA2301-35Spro::Cry1Ab. \u003cstrong\u003e(B-D)\u003c/strong\u003e PCR detection of \u003cem\u003ecry1Ab\u003c/em\u003e, \u003cem\u003enptII\u003c/em\u003e, and \u003cem\u003egus\u003c/em\u003e using gene specific primers. Lanes, M: 1 kb DNA marker; P: plasmid positive control; NT: non-transgenic cowpea; B: blank as a negative control; Line#5-38: transgenic \u003cem\u003ecry1Ab\u003c/em\u003e lines. \u003cstrong\u003e(E-F)\u003c/strong\u003e RT-PCR based relative fold expression analysis of \u003cem\u003ecry1Ab\u003c/em\u003e in three independent T\u003csub\u003e3\u003c/sub\u003e transgenic cowpea lines and non-transgenic (NT) in leaves and pods. VuUbiquitin is used as a housekeeping gene. Bars represent mean values ± standard deviation (SD; error bar) from n=3 biological replicates. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparison test in GraphPad Prism 8.0 Bars shares at least one common letter (a, b) are not significantly different from each other (P\u0026gt; 0.05), while bars labelled with different letters indicate statistically significant differences (P\u0026lt; 0.05) \u003cstrong\u003e(G)\u003c/strong\u003e Southern blot of HindIII digested genomic DNA of T\u003csub\u003e3\u003c/sub\u003e transgenic lines (Line #5-38), non-transgenic (NT), and plasmid (P) hybridized with \u003cem\u003ecry1Ab\u003c/em\u003e probe.\u003c/p\u003e","description":"","filename":"Picture1.png","url":"https://assets-eu.researchsquare.com/files/rs-8322435/v1/a83b824b3bc00b9707c9b8b1.png"},{"id":98416973,"identity":"ca382186-7b1f-4d1d-aeb7-1e90e18b4d59","added_by":"auto","created_at":"2025-12-17 14:59:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":294678,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression and quantification of Cry1Ab protein in transgenic cowpea. \u003c/strong\u003eWestern blot analysis of \u003cem\u003ecry1Ab\u003c/em\u003e gene in T\u003csub\u003e3\u003c/sub\u003e transgenic lines (Line#5-38) and non-transgenic (NT) leaves \u003cstrong\u003e(A)\u003c/strong\u003e and pods \u003cstrong\u003e(B)\u003c/strong\u003e. A single band of ~67 kDa corresponding to Cry1Ab was shown by a black arrow. Purified Cry1Ab protein was used as positive control (P) and proteins from non-transgenic plants were used as a negative (NT). \u003cstrong\u003e(C-D)\u003c/strong\u003e Quantitative estimation of Cry1Ab protein in T\u003csub\u003e3\u003c/sub\u003e transgenic lines and non-transgenic (NT) using Cry1Ab-enzyme conjugate. Average quantity of total Cry1Ab protein of the transgenics is represented as mg g\u003csup\u003e−1\u003c/sup\u003e fresh mass ± standard deviation. Bars represent mean values ± standard deviation (SD; error bar) from n=3 biological replicates. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparison test in GraphPad Prism 8.0. Bars shares at least one common letter (a, b and c) are not significantly different from each other (P\u0026gt; 0.05), while bars labelled with different letters indicate statistically significant differences (P\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Picture2.png","url":"https://assets-eu.researchsquare.com/files/rs-8322435/v1/b060588f5a97378b2b1fb297.png"},{"id":98441316,"identity":"133fe985-52e0-488c-bb31-4a14ea6e188a","added_by":"auto","created_at":"2025-12-17 17:05:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":298493,"visible":true,"origin":"","legend":"\u003cp\u003eEfficacy of transgenic cowpea lines against \u003cem\u003eMaruca vitrata\u003c/em\u003e and \u003cem\u003eHelicoverpa armigera\u003c/em\u003elarvae by detached leaf and pod bioassay from transgenics (Line #5-38) and non-transgenics (NT). Mean percentage values ± standard error (SE) from n=3 biological replicates are provided. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparison test in GraphPad Prism 8.0. Values shares at least one common letter (a, b, c, and d) are not significantly different from each other (P\u0026gt; 0.05), while values labelled with different letters indicate statistically significant differences (P\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Picture3.png","url":"https://assets-eu.researchsquare.com/files/rs-8322435/v1/b37820125371a5a2b28e2a5a.png"},{"id":98441430,"identity":"1b11b96b-6ebd-4eb6-af20-ed1c073c2066","added_by":"auto","created_at":"2025-12-17 17:05:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":212849,"visible":true,"origin":"","legend":"\u003cp\u003eThe principal component analysis (PCA) score plot \u003cstrong\u003e(A)\u003c/strong\u003e and loading plot \u003cstrong\u003e(B)\u003c/strong\u003e illustrate the \u003csup\u003e1\u003c/sup\u003eH NMR spectra of transgenic (line #5:TR1, line #12:TR2, line #17:TR3, line #30:TR4, and line #38:TR5) and non-transgenic (WT) cowpea pods. Distinct colors are used to represent different lines. The T\u003csub\u003e3\u003c/sub\u003e transgenic lines were analyzed using three biological replicates per line.\u003c/p\u003e","description":"","filename":"Picture4.png","url":"https://assets-eu.researchsquare.com/files/rs-8322435/v1/2e4e368eca37bd65e058eafb.png"},{"id":98416978,"identity":"0d90e945-f671-48b4-91cb-aa474e81ca6d","added_by":"auto","created_at":"2025-12-17 14:59:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":253007,"visible":true,"origin":"","legend":"\u003cp\u003eStatistical analysis of transgenic (line #5:TR1, line #12:TR2, line #17:TR3, line #30:TR4, and line #38:TR5) and non-transgenic (WT) samples. OPLS-DA score plot \u003cstrong\u003e(A-E)\u003c/strong\u003e and corresponding loadings \u003cem\u003eS\u003c/em\u003e-plot \u003cstrong\u003e(F-J)\u003c/strong\u003e for discrimination of transgenic and non-transgenic cowpea pods. The T\u003csub\u003e3\u003c/sub\u003e transgenic lines were analyzed using three biological replicates per line (n=3).\u003c/p\u003e","description":"","filename":"Picture5.png","url":"https://assets-eu.researchsquare.com/files/rs-8322435/v1/92f1dc0631e17590af905ad4.png"},{"id":98416976,"identity":"10887c4f-4173-473d-9603-0f568c6d5088","added_by":"auto","created_at":"2025-12-17 14:59:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":80377,"visible":true,"origin":"","legend":"\u003cp\u003eMetabolic pathways analysis from pods of transgenic cowpea lines expressing cry1Ab. Analysis carried out using MetaboAnalyst 5.0. Differences were considered statistically significant with P values \u0026lt; 0.05 and an impact factor threshold \u0026gt; 0. Altered metabolic pathways of \u003cem\u003ecry1Ab\u003c/em\u003e cowpea samples was observed. (1) Starch and sucrose metabolism, (2) Alanine, aspartate and glutamate metabolism, (3) Galactose metabolism, (4) Valine, leucine and isoleucine biosynthesis, (5) Cysteine and methionine metabolism, (6) Glyoxylate and dicarboxylate metabolism, (7) Arginine and proline metabolism, (8) Amino sugar and nucleotide sugar metabolism, (9) Glycine, serine and threonine metabolism, (10) Arginine biosynthesis, and (11) Citrate cycle (TCA cycle), (12) Carbon fixation by Calvin cycle, (13) Glutathione metabolism.\u003c/p\u003e","description":"","filename":"Picture6.png","url":"https://assets-eu.researchsquare.com/files/rs-8322435/v1/f5acd013f96161edf6dfd5d3.png"},{"id":98440904,"identity":"d6c302da-64c8-4b6f-bd6d-fc48793484ae","added_by":"auto","created_at":"2025-12-17 17:04:35","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":544712,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of agronomical performance.\u003c/strong\u003eTransgenic and non-transgenic cowpea lines were evaluated under field condition (poly-house). The physiological parameters including plant height \u003cstrong\u003e(A)\u003c/strong\u003e, branch number \u003cstrong\u003e(B)\u003c/strong\u003e, pod number/plant \u003cstrong\u003e(C)\u003c/strong\u003e, seed number/plant \u003cstrong\u003e(D)\u003c/strong\u003e, ten seed mass \u003cstrong\u003e(E)\u003c/strong\u003e, and ten seed length \u003cstrong\u003e(F)\u003c/strong\u003e were shown. Bars represent mean values ± standard deviation (SD) from n=3 biological replicates. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparison test in GraphPad Prism 8.0. Bars shares at least one common letter (a, b) are not significantly different from each other (P\u0026gt; 0.05), while bars labelled with different letters indicate statistically significant differences (P\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Picture7.png","url":"https://assets-eu.researchsquare.com/files/rs-8322435/v1/3eb6710ad5160ecc2d7cadb8.png"},{"id":98622492,"identity":"6f517b86-c37e-47f7-be0f-df4abd60b23d","added_by":"auto","created_at":"2025-12-19 16:56:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3270688,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8322435/v1/4382b77e-394e-4371-96f2-7ed679871eed.pdf"},{"id":98441737,"identity":"432f9ffc-35f1-4c58-92e2-6daa04fd5a32","added_by":"auto","created_at":"2025-12-17 17:05:45","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2008045,"visible":true,"origin":"","legend":"","description":"","filename":"SI.docx","url":"https://assets-eu.researchsquare.com/files/rs-8322435/v1/19da36658480bb3f642ddb24.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Transgenic Cowpea Expressing Synthetic BtCry1Ab Provides Enhanced Resistance to Maruca vitrata and Supports Sustainable Pod Borer Management","fulltext":[{"header":"Key Messages","content":"\u003cul\u003e\n \u003cli\u003eCowpea suffers severe yield losses due to pod borer with no durable resistance sources.\u003c/li\u003e\n \u003cli\u003eNo effective host plant resistance has been identified against pod borer.\u003c/li\u003e\n \u003cli\u003eWe designed a novel synthetic \u003cem\u003eBtcry1Ab\u003c/em\u003e gene to achieve stronger insecticidal activity.\u003c/li\u003e\n \u003cli\u003eTransgenic cowpea expressing \u003cem\u003eBtcry1Ab\u003c/em\u003e showed high resistance to pod borer damage.\u003c/li\u003e\n \u003cli\u003eThis provides a sustainable strategy for legume pest management and yield improvement.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eCowpea (\u003cem\u003eVigna unguiculata L\u003c/em\u003e.) is an important grain legume that provides high-quality dietary protein to millions of people, particularly in Africa and Asia (Muthuvel et al. 2021). Its resilience to drought and poor soil conditions, coupled with its ability to improve soil fertility through nitrogen fixation, makes cowpea a cornerstone of sustainable, low-input agriculture. In addition to protein, cowpea seeds contain essential vitamins (e.g., thiamine folic acid, and niacin), iron, minerals, and dietary fiber (Kamara et al. 2010), thereby serving as a source of food and nutrition for both humans and livestock (Muthuvel et al. 2021). Cowpea is predominantly cultivated in India, Africa, the Middle East, and South America (FAOSTAT, 2023). However, its production in major consuming countries fall much short from meeting the domestic demand (Al-Hassan and Diao 2007).\u003c/p\u003e \u003cp\u003eAmong the factors limiting productivity, the Lepidopteran Pod Borer (LPB), \u003cem\u003eMaruca vitrata\u003c/em\u003e, represents a key pest of economic significance in tropical legume ecosystems and a major constraint to integrated pest management (IPM) programs. Feeding internally on both vegetative and reproductive tissues, it causes yield losses of up to 72% in Asia and Africa (Abudulai et al. 2017; Kusi et al. 2019; Srinivasan et al. 2021). Cowpea is one of the major hosts for \u003cem\u003eM. vitrata\u003c/em\u003e, with yield losses of up to 72% in Asia and sub-Saharan Africa. Due to its rapid growth, the pod borer exhibits strong adaptability in regions with high humidity, posing significant challenges for effective control. In field conditions, pod borer infestation begins at 25\u0026ndash;30 days after the early vegetative stage inflicting maximum damage to flowering stage in cowpea. Young larvae form webs on the reproductive structures and bore into buds and pods to feed on developing seeds (Srinivasan et al. 2021). This pest\u0026rsquo;s concealed feeding behavior within floral structures limits the effectiveness of contact insecticides, making management particularly challenging under field conditions. Chemical insecticides remain the primary control method of \u003cem\u003eM. vitrata\u003c/em\u003e infestation, yet their high cost, limited accessibility for resource-poor farmers, and negative impact on pollinators and other beneficial insects constrain their long-term utility. Moreover, recurrent insecticide use has contributed to the evolution of insecticide resistance, underscoring the need for alternative, ecologically sustainable control tactics within IPM frameworks.\u003c/p\u003e \u003cp\u003eHost plant resistance offers the most sustainable option to manage LPB. However, breeding progress is limited because cultivated cowpea lacks effective resistance, genetic diversity, is narrow, and wild Vigna species with resistance are difficult to cross. This further restrict durable pest resistance development. Genetic transformation thus provides the most feasible strategy to overcome these constraints and develop durable resistance to \u003cem\u003eM. vitrata\u003c/em\u003e (Muthuvel et al. 2021).\u003c/p\u003e \u003cp\u003e \u003cem\u003eBacillus thuringiensis (Bt)\u003c/em\u003e Cry proteins are highly specific and effective against lepidopteran pests, with Cry1Ab showing activity against \u003cem\u003eM. vitrata (\u003c/em\u003eAcharjee and Higgins 2021). While Bt-cowpea lines have been developed and tested in Africa, their adaptability to Asian production systems remains uncertain due to differences in seasons, cultivation practices, and genetic backgrounds (Addae et al. 2020). In the context of pest science, region-specific evaluation of the Bt traits is critical for understanding pest population dynamics, resistance management, and environmental interactions. Recent studies, such as the expression of the Cry2Aa protein in Indian cowpea cultivar, have proven its region-specific effectiveness against \u003cem\u003eM. vitrata\u003c/em\u003e (Kumar et al. 2021). However, variable expression levels, organ-specific differences, and late-season decline in Bt protein accumulation have led to incomplete protection (Bakshi et al. 2011; Jadhav et al. 2020). Such reduced effectiveness during commercial production may heighten the risk of resistant insect populations, which is an issue of major concern in applied pest science. To address this, enhancing gene expression by codon optimization offers a promising strategy to achieve stable, high-level expression and ensure reliable pest control.\u003c/p\u003e \u003cp\u003eIn this study, we developed transgenic cowpea expressing a synthetic \u003cem\u003eBtcry1Ab\u003c/em\u003e gene driven by the CaMV35S promoter. The plants showed consistently high expression of Cry1Ab protein across vegetative and reproductive stages, resulting in durable resistance. Insect bioassays using leaves and immature pods revealed minimal feeding damage and high larval mortality of both \u003cem\u003eM. vitrata\u003c/em\u003e and \u003cem\u003eHelicoverpa armigera\u003c/em\u003e compared with non-transgenic controls. Our findings contribute to pest management science by demonstrating the potential of Bt-cowpea as a viable host plant resistance component within IPM systems. These results demonstrate that \u003cem\u003eBtcry1Ab\u003c/em\u003e cowpea has strong potential for field-level deployment, providing an effective and sustainable pest management strategy that reduces reliance on synthetic insecticides while supporting stable legume production.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of binary vector and development of cowpea transgenics\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eBtcry1Ab\u003c/em\u003e construct was kindly provided by Prof. Illimar Altosaar, University of Ottawa, Canada (Shu et al. 2000). The coding sequences for the insecticidal protein Cry1Ab from \u003cem\u003eB. thuringiensis\u003c/em\u003e were chemically synthesized with codon optimization tailored for plant expression systems (Cheng et al., 1998; Sardana et al., 1996). The full-length plant synthetic \u003cem\u003eBtcry1Ab\u003c/em\u003e ORF (1.86 kb), with XhoI and KpnI fragment, was cloned into the intermediate vector pRT101 between a 35S promoter and nos terminator sequence. The PstI fragment of \u003cem\u003e35S::Btcry1Ab:nos-Ter\u003c/em\u003e was subsequently digested and sub-cloned into binary plant vector pCAMBIA2301. The plasmid construct was then mobilized into disarmed hyper-virulent \u003cem\u003eAgrobacterium\u003c/em\u003e strain EHA105 and used for cowpea transformation. The T-DNA of binary plasmid pCAMBIA2301 includes neomycin phosphotransferase gene (\u003cem\u003enptII\u003c/em\u003e) and β-glucuronidase gene (\u003cem\u003egus\u003c/em\u003e) interrupted by catalase intron, both driven by the strong constitutive cauliflower mosaic virus (CaMV) 35S promoter. The cowpea plant transformation was carried out using cotyledonary node explants from an insect-sensitive cultivar cv. \u003cem\u003ePUSA KOMAL\u003c/em\u003e (IARI New Delhi) as per our previous protocol (SI Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Solleti et al. 2008; Bakshi et al. 2011; Kumar et al. 2017). The putative transgenic cowpea plants were established in soil: compost (1:1) and grown to maturity in greenhouse containment. The T\u003csub\u003e1,\u003c/sub\u003e T\u003csub\u003e2\u003c/sub\u003e and T\u003csub\u003e3\u003c/sub\u003e seeds were harvested and grown in the soil inside a transgenic poly-house to raise their progeny lines.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMolecular characterization of transgenic cowpea lines\u003c/h3\u003e\n\u003cp\u003eGenomic DNA was isolated from the young leaves of transgenic plants using the DNASure Plant Mini Kit (Nucleopore, Genetix, India). For screening of the cowpea transformants, PCR analyses were carried out using \u003cem\u003ecry1Ab\u003c/em\u003e specific primers (Forward: 5\u0026rsquo;- TGTCCATCTGGTCCCTCTTC-3\u0026rsquo; and Reverse: 5\u0026rsquo;-ATGGTGAAGCCGGTGAGTC-3\u0026rsquo;) to obtain a partial fragment of 628 bp product from the coding region of the \u003cem\u003ecry1Ab\u003c/em\u003e gene. A 549 bp fragment of the \u003cem\u003enptII\u003c/em\u003e gene and 540 kb of \u003cem\u003egus\u003c/em\u003e gene was also amplified using gene specific primers (\u003cem\u003enptII\u003c/em\u003e forward: 5\u0026rsquo;-GGTGGAGAGGCTATTCGGCTA-3\u0026rsquo; and \u003cem\u003enptII\u003c/em\u003e reverse: 5\u0026rsquo;- GGTAGCCAACGCTATGTCCTGA-3\u0026rsquo;; \u003cem\u003egus\u003c/em\u003e forward: 5\u0026rsquo;-GGTGGGAAAGCGCGTTACAAG-3\u0026rsquo;; \u003cem\u003egus\u003c/em\u003e reverse: 5\u0026rsquo;-TGGATTCCGGCATAGTTAAA-3\u0026rsquo;). The amplified products were resolved by electrophoresis on 1% agarose gel and visualized by ethidium bromide staining (Malke 1990).\u003c/p\u003e \u003cp\u003eSouthern blotting was performed using the DIG labeling and detection kit according to the manufacturer\u0026rsquo;s instructions (Bio-Rad, Hercules, USA). The T\u003csub\u003e3\u003c/sub\u003e PCR positive transgenic and untransformed cowpea plants were chosen to extract genomic DNA (Maxiprep MN plant DNA maxi kit, Germany). 50\u0026ndash;60 \u0026micro;g of total genomic DNA were digested using HindIII, fractionated in 0.8% agarose gel and transferred to a positively charged Zeta-probe membrane. The blot was hybridized with DIG labelled 1 kb PCR product, corresponding to the coding region of \u003cem\u003ecry1Ab\u003c/em\u003e gene. The hybridized blot was processed for pre and post hybridization wash as per the manufacturer\u0026rsquo;s instructions. The detection of \u003cem\u003ecry1Ab\u003c/em\u003e junction fragment was performed using the instructions of the DIG labeling and detection kit (Roche Diagnostics, Mannheim, Germany).\u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression analysis of\u003c/b\u003e \u003cb\u003ecry1Ab\u003c/b\u003e \u003cb\u003egene in transgenic lines\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTotal RNA was isolated from the Southern positive plants using a NucleoSpin RNA Plant Kit (TAKARA, Clontech, Japan) and were subjected to cDNA synthesis using RevertAid\u0026trade; H Minus first-strand cDNA synthesis kit (Fermentas, USA).\u003c/p\u003e \u003cp\u003eRelative fold expression by real-time PCR (RT-PCR) was performed to quantitate the expression of \u003cem\u003ecry1Ab\u003c/em\u003e gene using specific primers (\u003cem\u003ecry1Ab\u003c/em\u003e forward: 5\u0026rsquo;TGG TAC AAC ACT GGC TTG GA3\u0026rsquo;; \u003cem\u003ecry1Ab\u003c/em\u003e reverse: 5\u0026rsquo;-ATGGGATTTGGGTGATTTGA-3\u0026rsquo;) using USB VeriQuest SYBR Green qPCR Master Mix (2X) (Affymetrix, USA) on a Rotor-Gene Q Real-Time PCR System (Quiagen, Germany). The \u003cem\u003eVuUBQ1\u003c/em\u003e gene (GenBank Accession No. FG859491) was used as an internal control. The experiment was repeated twice independently with three biological replicates each. The relative expression of \u003cem\u003ecry1Ab\u003c/em\u003e in wild-type (WT) and transgenic cowpea lines was estimated by using the 2\u003csup\u003e\u0026minus;∆∆Ct\u003c/sup\u003e method and student\u0026rsquo;s t-test was used to calculate significance.\u003c/p\u003e\n\u003ch3\u003eAnalysis of Cry1Ab protein in transgenic lines\u003c/h3\u003e\n\u003cp\u003eThe expression of the Cry1Ab protein was analyzed in T\u003csub\u003e3\u003c/sub\u003e transgenic lines generated from four independent transformation events by ELISA and Western blot hybridization. The detection of Cry1Ab protein in leaves and immature pods was assayed by ELISA using antibody coated plates as per the manufacturer\u0026rsquo;s instructions (Envirologix Quantiplate kit for Cry1Ab/Cry1Ac, Amar Immunodiagnostics, India). Total soluble proteins (TSP) from transgenic and non-transgenic cowpea plants using protein extraction buffer (50 mM Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e, 100 mM NaCl, 0.05% TritonX-100, 0.05% Tween-20, 2 mM Phenylmethylsulfonyl fluoride and 1 \u0026micro;M leupeptin) and quantified using Bradford assay (Bradford 1976). The TSP were then added to the wells of antibody-coated plates. A secondary antibody Cry1Ab-Enzyme conjugate was then added to each well and the plates were kept for shaking at room temperature for 1 hr. After washing, a colored reaction was observed after adding the substrate in each well and absorbance was measured at 450 nm and comparative histogram was plotted.\u003c/p\u003e \u003cp\u003eFor Western blotting, 30 \u0026micro;g of the TSP was fractionated on 12% sodium dodecyl sulfate polyacrylamide gels with (SDS-PAGE) and blotted onto a PVDF membrane by wet transfer (GE Healthcare; Burnette 1981). The rabbit anti-Cry1Ac primary antibody (Amar immune diagnostics, India) in 1:500 dilution and goat anti rabbit conjugated with horseradish peroxidase (Promega) was used for detection. The blot was developed using substrate (SuperSignal West Dura, Thermo Scientific, USA) for 5 min and the reaction was stopped by washing the membrane with sterile distilled water. A single band of ~\u0026thinsp;67 kDa corresponding to Cry1Ab protein was detected immunologically in all transgenic plants confirming the stable expression of Cry1Ab protein, whereas no such band was observed in the non-transgenic plants.\u003c/p\u003e\n\u003ch3\u003eInsect bioassays\u003c/h3\u003e\n\u003cp\u003eMaruca pod borer population were reared in an insect growth room at 27\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 16 h light photoperiod and 70\u0026thinsp;\u0026plusmn;\u0026thinsp;10% relative humidity for optimal growth and mating for in vitro insect bioassays. Insect bioassays were conducted on T\u003csub\u003e3\u003c/sub\u003e transgenic cowpea lines of 45 days old, where fully expanded mid-canopy leaves and uniformly immature pods were sampled, and second-instar larvae were used to assess the efficacy of cry1Ab expression, as they are more robust and suitable for evaluating sub-lethal effects and realistic field damage (Addae et al 2020). Non-transformed plants served as the negative control. The leaves and immature pods of the non-transgenic and transgenic plants (confirmed by PCR, Southern, and Western blotting) were fed to the larvae separately to evaluate the insecticidal efficacy of the Cry1Ab protein. The ten second instar larvae were released on leaves and pods wrapped with cotton to sustain moisture in the bioassay dishes. Three replicates were carried out for each transgenic/non-transgenic line. In response to the feeding, the mass gain, life cycle and mortality of the larvae and the extent of damage onto the leaves and pods were recorded regularly up to four days after release of the larvae.\u003c/p\u003e\n\u003ch3\u003eMorpho-physiological analysis of transgenic cowpea lines\u003c/h3\u003e\n\u003cp\u003eThe morpho-physiological analysis of the five \u003cem\u003ecry1Ab\u003c/em\u003e lines were performed at greenhouse conditions. The level of Cry1Ab protein in the different progeny plants was determined by Western blot and were grouped into low, medium and high categories. The phenotypic parameters such as plant height, branch number and total number of seeds and pods formed per plant were observed to determine any off-target effects owing to the \u003cem\u003ecry1Ab\u003c/em\u003e gene expression. To determine the effect of \u003cem\u003ecry1Ab\u003c/em\u003e gene expression on seed mass, seed size, the average seed mass of 10 seeds collected from each line was also recorded.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMetabolomic analysis using NMR\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eMetabolite Extraction\u003c/h2\u003e \u003cp\u003eSamples were extracted according toKim et al. 2010 with minor modifications. Briefly, the frozen leaves were ground to fine powder using prechilled mortar and pestle and lyophilized overnight. The extraction was performed with approximately 25 mg of samples and 1 ml of aqueous methanol (80 % v/v) in micr-centrifuge tube. The samples were vortexed for 30 s and then the mixture was further extracted using ultrasonication water bath for 15 min (with 1 min sonication and 30 s pause) using UP200S, Ultrasonic Processor (Hielscher Ultrasonic GmbH, Germany). The extract was centrifuged at 10,000 rpm for 20 min at 4\u003csup\u003e0\u003c/sup\u003e C. The resulting supernatant was collected in fresh tube and the procedure repeated twice with the remaining pellets. The resulting supernatant from all extraction was combined and concentrated using SpeedVac (Eppendorf AG, Hamburg, Germany) for 3 h. Finally, the dried sample was dissolved in 600 \u0026micro;l of deuterated water (D\u003csub\u003e2\u003c/sub\u003eO) containing 0.05% (w/w) of 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid, sodium salt (TMSP) as an internal standard and transferred into a 5 mm NMR tubes (Sigma-Aldrich, Sant-Louis MO, USA) for further analysis.\u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cb\u003e1\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eH-NMR Spectroscopy and spectral data processing\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe NMR spectra for all samples were acquired using Bruker Avance III 600 MHz spectrometer with a DCH CryoProbe (BrukerBioSpin GmbH, Rheinstetten, Germany), operating at 600 MHz and 298 K temperature, with 90\u003csup\u003eo\u003c/sup\u003e pulse width of 12 kHz. One-dimensional \u003csup\u003e1\u003c/sup\u003eH-NMR spectra with water pre-saturation were acquired using a standard Bruker pulse-acquire sequence during a relaxation delay of 4 s and a mixing time of 100 ms, 32 scans were collected into 64 k data points over a spectral width of 4801.54 Hz and pulse width of 9.46 \u0026micro;s, with an acquisition time of 6.82 s. Water suppression was obtained by pre-saturation sequence present in the routine experiments. The obtained \u003csup\u003e1\u003c/sup\u003eH-NMR spectrum was manually corrected for phase and baseline using Mestrenova, and the chemical shift was referenced to TMS at 0.000 ppm, to assess the signal quality and to determine the relative proportion of metabolites. The spectral regions of 0.5\u0026ndash;10.0 were bucketed into bins (0.04 ppm) using Mestrenova Software (Version 6.0.2\u0026ndash;5475; Mestrelab Research, S.L. Spain). Spectral regions 4.84 containing residual signals from water was excluded to remove the residual water by using Mestrenova software. The resulting data was subjected to multivariate analysis (MVDA).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eMultivariate Statistical Analysis for Metabolomic\u003c/h3\u003e\n\u003cp\u003eThe dataset was imported into the MetaboAnalyst 5.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.metaboanalyst.ca/\u003c/span\u003e\u003cspan address=\"https://www.metaboanalyst.ca/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and then, the MVDA was performed by PCA and PLS-DA. All the variables were mean centered and log-transformed for multivariate analysis. Furthermore, OPLS-DA was applied to each sample for pairwise comparison between transgenic samples and the control wild type sample using MetaboAnalyst 5.0. The data were statistically analyzed with one-way analysis of variance (ANOVA), and the significant differences were determined using Tukey\u0026rsquo;s test to evaluate the significant differences between the transgenic samples and the control wild type sample. Differences with \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significant.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMetabolite identification\u003c/h2\u003e \u003cp\u003eMetabolite identification was carried out based on the databases such as Biological Magnetic Resonance Data Bank (BMRB; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.bmrb.wisc.edu/\u003c/span\u003e\u003cspan address=\"http://www.bmrb.wisc.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Human Metabolome Database (HMDB; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://hmdb.ca/\u003c/span\u003e\u003cspan address=\"https://hmdb.ca/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and published literature (Maravi et al. 2022). To determine the possible differences in the related metabolic pathways between control and transgenic samples, pathway analysis was carried out using MetaboAnlyst 5.0 and their contributions and biological interpretations were discussed based on the Kyoto Encyclopedia of Genes and Genome (KEGG) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genome.jp/kegg/pathway.html\u003c/span\u003e\u003cspan address=\"https://www.genome.jp/kegg/pathway.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using GraphPad Prism, version 8.0 (GraphPad software, USA). The assumptions of normality and homogeneity of variances were evaluated using the Shapiro-Wilk test and Brown-Forsythe and Welch test, respectively. Data met the criteria for parametric analysis, and therefore replicated datasets (n\u0026thinsp;=\u0026thinsp;3 biological replicates) from each experiment were analyzed using one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s multiple comparison test. The experiment was repeated twice, and a representative image is shown.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eDevelopment of transgenic cowpea lines with stable\u003c/strong\u003e \u003cstrong\u003ecry1Ab\u003c/strong\u003e \u003cstrong\u003eintegration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTransgenic cowpea plants were produced using \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation using cotyledonary node explants from the commercial cowpea variety PUSA KOMAL (Kumar et al. 2017). The explants produced an average of 5.4 elongated shoots per explant, with a 92\u0026thinsp;\u0026plusmn;\u0026thinsp;2% response rate and a maximum transformation efficiency of 3.92% after three selection cycles on kanamycin-supplemented medium (Kumar et al. 2017).\u003c/p\u003e\n\u003cp\u003ePCR analysis was used to confirm the presence of the \u003cem\u003enptII\u003c/em\u003e, \u003cem\u003egus\u003c/em\u003e, and \u003cem\u003ecry1Ab\u003c/em\u003e transgenes in the putative T\u003csub\u003e1\u003c/sub\u003e plants, using gene-specific primers (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA; SI Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Out of 65 plants that survived on the kanamycin selection, 43 tested positives for amplification, yielding fragments of 549 bp, 540 bp, and 628 bp for the \u003cem\u003enptII\u003c/em\u003e, \u003cem\u003egus\u003c/em\u003e, and \u003cem\u003ecry1Ab\u003c/em\u003e genes, respectively. No amplification products were detected in non-transformed control plants (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB-D). Based on high transgene expression and PCR results, five independent transgenic lines were selected for further analysis to study the segregation patterns of the integrated \u003cem\u003ecry1Ab\u003c/em\u003e, \u003cem\u003egus\u003c/em\u003e, and \u003cem\u003enptII\u003c/em\u003e genes in subsequent generations. For each of these five lines, we analyzed a minimum of 10\u0026ndash;12 plants in the T\u003csub\u003e2\u003c/sub\u003e generation, and at least 8\u0026ndash;10 homozygous plants per line in T\u003csub\u003e3\u003c/sub\u003e generation. Segregation ratios for \u003cem\u003ecry1Ab\u003c/em\u003e, \u003cem\u003egus\u003c/em\u003e, and \u003cem\u003enptII\u003c/em\u003e were recorded in T\u003csub\u003e1\u003c/sub\u003e and T\u003csub\u003e2\u003c/sub\u003e generation and were consistent with Mendelian inheritance patterns for a single locus insertion. In T\u003csub\u003e3\u003c/sub\u003e generation, we selected one homozygous plant per independent line (confirmed by PCR and expression analysis) for insect bioassay and phenotypic evaluation. The segregation ratios of transgenic lines adhered to a Mendelian inheritance pattern (3:1; P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating that the \u003cem\u003ecry1Ab\u003c/em\u003e gene was inherited as a single dominant locus (data not shown).\u003c/p\u003e\n\u003cp\u003eTo verify the expression of \u003cem\u003ecry1Ab\u003c/em\u003e, relative fold expression by real-time PCR (RT-PCR) was conducted on PCR-positive plants. In the T\u003csub\u003e3\u003c/sub\u003e lines 17, 30, and 38, expression levels of \u003cem\u003ecry1Ab\u003c/em\u003e were found to be 3- to 10-fold higher in leaves and 6- to 13-fold higher in immature pods relative to the \u003cem\u003eVuubiquitin\u003c/em\u003e gene. No amplification was observed in control non-transformed plants (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE and F). Expression levels varied significantly between leaves and pods across the transgenic lines. Notably, lines #12 and #5 exhibited the highest \u003cem\u003ecry1Ab\u003c/em\u003e expression in leaves and immature pods, respectively, while line #38 showed the lowest expression in both tissues among the transgenic lines (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE and F).\u003c/p\u003e\n\u003cp\u003eThe stable integration and copy number of the \u003cem\u003ecry1Ab\u003c/em\u003e gene were further assessed in the T\u003csub\u003e3\u003c/sub\u003e lines by Southern blot analysis. HindIII-digested genomic DNA hybridized with a \u003cem\u003ecry1Ab\u003c/em\u003e probe displayed hybridization bands larger than 4.2 Kbp, confirming the successful integration of the \u003cem\u003ecry1Ab\u003c/em\u003e gene into the cowpea genome. Southern blotting also confirmed that three out of five lines contained a single copy of the \u003cem\u003ecry1Ab\u003c/em\u003e gene, indicating a single T-DNA insertion event. The lines #17 and #30 showed two copies of \u003cem\u003ecry1Ab\u003c/em\u003e gene. No hybridization signal was observed in the non-transformed control plants (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eG). Two (line #17 and #30) transgenic lines showing similar Southern hybridization patterns were independently generated. The similarity likely reflects preferential integration or conserved restriction site environments.\u003c/p\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eTransgenic cowpea lines express higher Cry1Ab protein\u003c/h2\u003e\n \u003cp\u003eThe qualitative and quantitative measurement of the Cry1Ab protein expression was analyzed by Western blotting and ELISA, respectively. The total soluble protein from the leaves and immature pods of transgenics and non-transformed control plant was used to test Cry1Ab protein. The Cry1Ab protein that are hybridized with the polyclonal Cry1Ab antibodies was detected as a signal band of molecular weight\u0026thinsp;~\u0026thinsp;67 kDa in all tested lines (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA and B). The intensity of the Cry1Ab protein in all the transgenic lines, except #38, was higher in leaves. In immature pods, lines #17 and #38 were showed low Cry1Ab intensity than the rest of the transgenic lines. Faint non-specific bands were detected, likely due to antibody cross-reactivity, but do not interfere with interpretation of Cry1Ab expression. These results on expression of Cry1Ab toxin protein demonstrate stable and consistent expression in most of the transgenic lines. No band was detected in the case of the untransformed control (non-transgenic) plant.\u003c/p\u003e\n \u003cp\u003eThe concentration of Cry1Ab protein in leaves and immature pods was quantified in five independent transgenic lines using ELISA (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC and D). Since the \u003cem\u003ecry1Ab\u003c/em\u003e gene was driven by CaMV35S constitutive promoter, the Cry1Ab protein was expressed in both tissues. We observed that the Cry1Ab protein was highly expressed across the three generations in transgenic cowpea lines. The protein expression ranged from 2.8 to 10.8 mg/g in leaf tissues and 5.0 to 10.7 mg/g in immature pods in T\u003csub\u003e1\u003c/sub\u003e transgenics (SI Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA and B). The T\u003csub\u003e2\u003c/sub\u003e transgenics recorded a maximum level of Cry1Ab protein expression, 2.9 to 10.1 mg/g leaf tissue and 5.6 to 10.1 mg/g young pods (SI Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC and D). In T\u003csub\u003e3\u003c/sub\u003e generation, the Cry1Ab protein expression observed to be 5.5 to 7.5 mg/g in leaf tissue and 5.5 to 8 mg/g in immature pods (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC and D). The accumulation of Cry1Ab proteins was higher in the reproductive tissues (young pods) compared to vegetative tissues (leaf) across all the three generations in tested events. Among the five transgenic lines tested, \u003cem\u003ecry1Ab\u003c/em\u003e expression level in lines #12, #17 and #30 were found higher, in both vegetative as well as reproductive tissues at T\u003csub\u003e3\u003c/sub\u003e generation. The concentration of Cry1Ab proteins in transgenic lines were stabilized in subsequent generation. The T\u003csub\u003e3\u003c/sub\u003e transgenic lines of the respective T\u003csub\u003e1\u003c/sub\u003e parents, showed 5.7% higher expression (on an average) of Cry1Ab protein in leaves and pods of all transgenic lines.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eInsect bioassay of transgenic cowpea leaves and immature pods showed higher mortality of\u003c/strong\u003e \u003cstrong\u003eM. vitrata\u003c/strong\u003e \u003cstrong\u003eand\u003c/strong\u003e \u003cstrong\u003eH. armigera\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe entomocidal activity of cowpea transgenic plants, expressing moderate to high levels of BtCry1Ab, was evaluated through insect feeding bioassays. The mortality rate for \u003cem\u003eM. vitrata\u003c/em\u003e larvae ranged from 33\u0026ndash;60%, while \u003cem\u003eH. armigera\u003c/em\u003e larvae exhibited a mortality range of 66\u0026ndash;96% when fed on leaves of T\u003csub\u003e3\u003c/sub\u003e transgenic plants (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Similarly, in the detached pod assay, mortality rates ranged from 60\u0026ndash;90% for \u003cem\u003eM. vitrata\u003c/em\u003e and 46\u0026ndash;63% for \u003cem\u003eH. armigera\u003c/em\u003e in the T\u003csub\u003e3\u003c/sub\u003e transgenic lines (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Notably, transgenic lines #30 and #38 showed the highest mortality for both \u003cem\u003eM. vitrata\u003c/em\u003e and \u003cem\u003eH. armigera\u003c/em\u003e larvae in leaf and pod bioassays. Significant differences in mean larval mortality were observed between the transgenic lines and the control. Additionally, surviving larvae on the transgenic plants exhibited retarded growth compared to those on non-transgenic controls (data not shown). For \u003cem\u003eM. vitrata\u003c/em\u003e larvae, Cry1Ab protein concentrations greater than 6 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e fresh mass in the pods caused over 70% mortality. However, at similar Cry1Ab protein concentrations, \u003cem\u003eH. armigera\u003c/em\u003e larvae exhibited relatively lower mortality (over 53%). In contrast, \u003cem\u003eH. armigera\u003c/em\u003e larvae showed higher mortality (\u0026gt;\u0026thinsp;83%) than \u003cem\u003eM. vitrata\u003c/em\u003e in transgenic leaves expressing\u0026thinsp;\u0026gt;\u0026thinsp;6 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e fresh mass Cry1Ab toxin (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC and D; Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Although larval mortality was positively correlated with Cry1Ab protein levels, the reason for the differences in mortality between \u003cem\u003eM. vitrata\u003c/em\u003e and \u003cem\u003eH. armigera\u003c/em\u003e in leaves and pods remains unclear.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eNMR\u003c/h2\u003e\n \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\n \u003ch2\u003eMetabolite identification\u003c/h2\u003e\n \u003cp\u003eThe \u003csup\u003e1\u003c/sup\u003eH NMR spectrum obtained at 600 MHz for the transgenic and non-transgenic cowpea pods (SI Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The NMR results revealed the variation 40 major metabolites non-transgenic cowpea pods. These 40 metabolites include amino acids, organic acids, sugars, and other aromatic compounds (SI Table 2). \u003csup\u003e1\u003c/sup\u003eH NMR performed on immature pods of transgenic and non-transgenic cowpea, the 600MHz spectra exhibited in three main regions (SI Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The first region (0.5-3.0 ppm) contained signals of amino acids and some organic acids were identified. The second region (3.0-5.8 ppm) comprised signals of carbohydrates. Sucrose (5.41 ppm), fructose (4.11 ppm), beta glucose (4.64 ppm), alpha glucose (5.23 ppm) were the most abundant carbohydrates in this region. In the third region (5.8\u0026ndash;9.5 ppm), were relatively weak signals which are corresponds to the aromatic groups from phenolic compounds and aromatic amino acids. The most abundant signals in the aromatic region, phenylalanine (7.43 ppm), tryptophan (7.71 ppm), fumarate (6.53 ppm) and formic acid (8.54 ppm) were identified (SI Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eMultivariate statistical analysis for metabolomic\u003c/h2\u003e\n \u003cp\u003eIdentification of metabolite variation of transgenic and non-transgenic leaves and pods was carried out by principle component analysis (PCA). In our current study, the first three components explained 78.6% of whole data: PC1, 41; PC2, 19.8; PC3, 17.8%, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). The score plots clearly showed that samples from the transgenics and non-transgenics were well separated (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). However, lines #12 (TR2), #17 (TR3), #30 (TR4), and #38 (TR5) samples showed noticeable overlaps indicating that these samples not be efficiently separated. The control group was negatively influenced by PC1 and positively influenced by PC2, whereas, line #5 (TR1) group was negatively influenced by both PC1 and PC2. The lines #12 (TR2), #17 (TR3), #30 (TR4), and #38 (TR5) groups were negatively influenced by PC1. The loading plots indicated that the discriminative metabolites responsible for the variable separations. Metabolites such as isocitrate and isoleucine are represented in PC1. PC2 was mainly characterized by proline, fructose and malate (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\n \u003cp\u003eThe VIP analysis revealed that the top 12 metabolites (out of 30) were identified as discriminating metabolites (VIP\u0026thinsp;\u0026ge;\u0026thinsp;1) between transgenic and non-transgenic tissues (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Significant higher contents of glucose-6-phosphate, glucose, betaine, mannose, citrate, N-carbomylaspartate, galactose, trans-4-hydroxy proline, glutamate, valine, methionine, and proline were upregulated by transgenic cowpea pods (SI Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eTo effectively compare the differences caused by \u003cem\u003ecry1Ab\u003c/em\u003e expression, pairwise comparisons were performed using the supervised OPLS-DA approach. The random permutation test (100 times) on the OPLS-DA model confirmed the difference between control and transgenic cowpea (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA-E). The S-plot also confirmed the higher relative content of methionine, proline, galactose, threonine, fructose, valine, and glucose-6-phosphate in transgenic pods compared to control (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF-J).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eMetabolic Pathway Analysis\u003c/h2\u003e\n \u003cp\u003eThe Web tool, MetaboAnalyst 5.0, was used to perform the biological pathway analysis. Metabolic pathway analysis (MetPA) suggested that thirteen pathways might be altered by the \u003cem\u003ecry1Ab\u003c/em\u003e expression in cowpea based on the statistically significant value (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and impact factor\u0026thinsp;\u0026gt;\u0026thinsp;0.1. This includes, (1) starch and sucrose metabolism, (2) alanine, aspartate and glutamate metabolism, (3) galactose metabolism, (4) valine, leucine and isoleucine biosynthesis, (5) cysteine and methionine metabolism, (6) glyoxylate and dicarboxylate metabolism, (7) arginine and proline metabolism, (8) amino sugar and nucleotide sugar metabolism, (9) glycine, serine and threonine metabolism, (10) arginine biosynthesis, and (11) citrate cycle (TCA cycle), (12) carbon fixation by Calvin cycle, (13) glutathione metabolism. The relationship between several metabolites and relevant metabolic pathways and the primary metabolites process is depicted in (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e and Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe metabolite pathway majorly altered in transgenic cowpea pods expressing \u003cem\u003ecry1Ab\u003c/em\u003e. Total number of pathways and hits along with their negative log values and false discovery rate (FDR), and their impact values are mentioned.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePathway name\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHits\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e-log (\u003cem\u003ep\u003c/em\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFDR\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eImpact\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStarch and sucrose metabolism\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.6988\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.84E-05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.72922\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlanine, aspartate and glutamate metabolism\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.3342\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.000213\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.57914\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGalactose metabolism\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.7706\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00052\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.36582\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eValine, leucine and isoleucine biosynthesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.9363\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.026631\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCysteine and methionine metabolism\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.5288\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.05156\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.1793\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlyoxylate and dicarboxylate metabolism\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.4733\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.05156\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.27842\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eArginine and proline metabolism\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.3139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.055568\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.14435\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAmino sugar and nucleotide sugar metabolism\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.293\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.055568\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.11106\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlycine, serine and threonine metabolism\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.2647\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.055568\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.16478\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eArginine biosynthesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.1757\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.061389\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.23148\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCitrate cycle (TCA cycle)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.0438\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.075611\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.19387\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCarbon fixation by Calvin cycle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.9835\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.079632\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.05923\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlutathione metabolism\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.7256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.13312\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.05801\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eAgronomic performance and segregational analysis:\u003c/h2\u003e\n \u003cp\u003eThe agronomic traits of T\u003csub\u003e3\u003c/sub\u003e transgenic plants expressing \u003cem\u003ecry1Ab\u003c/em\u003e and their non-transgenic counterparts were assessed to determine if the incorporation of the Bt gene resulted in any phenotypic changes (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). Morphologically, there were no observable differences between the transgenic and non-transgenic plants. Statistical analysis revealed no significant differences (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in key agronomic traits, including plant height, seed number per plant, seed length, and ten seed mass of transgenics were not different from non-transgenic plants (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). Line #30 showed lower number of branches compared to other transgenics and non-transgenic plants. However, the pod number per plant was observed to be higher in line #30 and #38 compared to other plants (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). Furthermore, transgenic plants exhibited no differences in growth or fertility compared to non-transgenic plants.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCowpeas are cost-effective protein-rich grain legumes critical for nutritional security and sustainable agriculture in developing countries. The legume pod borer (\u003cem\u003eM. vitrata\u003c/em\u003e) is the most devastating insect pest of cowpea. Till date, no source of complete-resistance has been identified in the gene pool of cultivated cowpea. Given the heavy reliance on synthetic insecticides for pod borer control and the consequent risk of pest resurgence, environmental contamination, and resistance evolution, developing insect-resistant cowpea varieties remains a top priority in pest management programs. Amelioration of insect damage can add to cowpea production, reduce insecticide use and help in sustainable production (Muthuvel et al. 2021). Sources of resistance to the insect pest have not been identified in cowpea germplasm, which limited the approach of conventional breeding to develop resistant varieties (Horn and Shimelis 2020; Smith 2021). Genetic engineering of crops using genes encoding insecticidal crystal proteins or δ-endotoxins from \u003cem\u003eB thuringiensis\u003c/em\u003e (Bt) has been shown to confer resistance to a variety of insects and pests in various crops (Huang 2021; Tilgam et al. 2021). Among others, Cry1Ab has been demonstrated to be highly toxic against early instars of \u003cem\u003eM. vitrata\u003c/em\u003e (Srinivasan et al. 2021). The incorporation of Bt genes into legumes, therefore, aligns with sustainable pest management strategies and the goals of IPM frameworks that emphasize host plant resistance as a first line of defense. Previous field studies have evidenced that cry1Ab provides effective protection for cowpea against \u003cem\u003eM. vitrata\u003c/em\u003e. Artificial infestation of \u003cem\u003eM. vitrata\u003c/em\u003e larvae, along with an integrated pest management strategy, resulted in nearly complete protection in transgenic cowpea lines expressing \u003cem\u003ecry1Ab\u003c/em\u003e protein compared to non-transgenic cowpea lines (Nboyine et al. 2024). These findings suggest that \u003cem\u003ecry1Ab\u003c/em\u003e could be a promising candidate for integrating insect resistant cowpea breeding pipelines.\u003c/p\u003e \u003cp\u003eWe have developed transgenic cowpea lines expressing a codon-optimized \u003cem\u003ecry1Ab\u003c/em\u003e gene from \u003cem\u003eB. thuringiensis\u003c/em\u003e and verified their efficacy against \u003cem\u003eM. vitrata\u003c/em\u003e as well as \u003cem\u003eH. armigera\u003c/em\u003e, both major lepidopteran pests of legumes. The inclusion of both pests in bioassays underscores the relevance of this study to pest science, as H. armigera is a notorious polyphagous pest known for rapid resistance development and cross-infestation across several legume crops. Cowpea transformation protocol has been standardized and used to generate many transgenic lines to confer various biotic and abiotic stress tolerance in our lab (Kumar et al. 2017, 2022). Following the same protocol, we have generated 65 putative transgenic cowpea lines. The transmission of the transgene/inheritance was confirmed by PCR analysis at each generation and molecular analyses was calculated to verify Mendelian segregation. Interestingly, a few transgenic events were found to be positive for the selectable marker \u003cem\u003enptII\u003c/em\u003e but negative for both \u003cem\u003ecry1Ab\u003c/em\u003e and \u003cem\u003egus\u003c/em\u003e gene. This may be due to partial T-DNA integration or genomic rearrangements during T-DNA integration or chimerism in regenerated plants could result in such incomplete or tissue-specific transgene presence. Another possible explanation includes false positives during PCR screening, particularly if low-copy or truncated insertions occurred. These factors highlight the complexity of T-DNA integration and underscore the need for thorough molecular validation of transgenic events. We have observed few homozygous lines in T\u003csub\u003e2\u003c/sub\u003e generation, based on PCR analysis. However, in most of the cases we have obtained mixed populations of homo and hemizygous lines T\u003csub\u003e2\u003c/sub\u003e generation. The five homozygous PCR confirmed progenies with Mendelian segregation were used for characterization of the transgenic lines.\u003c/p\u003e \u003cp\u003eSouthern blotting revealed single (three lines) and double (two lines) copy of the \u003cem\u003ecry1Ab\u003c/em\u003e gene in the HindIII-digested genomic DNA of the selected transgenic cowpea lines when probed with a sequence specific to \u003cem\u003ecry1Ab\u003c/em\u003e, indicating its stable integration in the cowpea genome. The presence of the expected hybridization signals with genomic DNA fragments (\u0026gt;\u0026thinsp;4.2 kb) in the majority of the transformed plants showed that the probed genes \u003cem\u003ecry1Ab\u003c/em\u003e remained intact upon integration into the cowpea genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). It is preferable to have low-copy (one to three) insertions of the transgene into the plant genome using \u003cem\u003eA. tumefaciens\u003c/em\u003e, as they tend to remain stable over several generations. The transgenic cowpea plants have shown an independent pattern of transgene expression due to the complex and random integration of foreign genes in the host genome following \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation. Therefore, the inheritance of foreign genes in transgenic plants may exhibit complex patterns for both single and multiple genes. Stable expression across generations is particularly critical for pest management programs aimed at reducing pest population pressure over multiple cropping cycles.\u003c/p\u003e \u003cp\u003eGiven the poor expression levels of native \u003cem\u003eBt\u003c/em\u003e insecticidal proteins in higher eukaryotes (Jadhav et al. 2020; Li et al. 2022), it's critical to ensure optimal expression of insecticidal proteins for effective control of targeted insects (Liu et al. 2020). The optimal expression of \u003cem\u003eBt\u003c/em\u003e genes in plants is controlled by various factors including codon preference, AT content, mRNA destabilization sequences and putative polyadenylation signals (Watts et al. 2021). Codon optimization has been previously employed in various crops, such as cotton (Zafar et al. 2022; Siddiqui et al. 2023), soybean (Fang et al. 2024), chickpea (Singh et al. 2022), and cowpea (Kumar et al. 2021), to enhance expression levels. In our current study, we adopted a similar approach to maximize \u003cem\u003ecry1Ab\u003c/em\u003e expression in transgenic cowpea. This involved increasing the GC content of the coding sequence and removing polyadenylation and mRNA destabilization sequences without altering the amino acid sequence. Additionally, codon usage was optimized to enhance translation in plants by incorporating plant-preferred codons. Relative fold expression by real-time PCR analysis of \u003cem\u003ecry1Ab\u003c/em\u003e transcripts in transgenic cowpea plants, driven by the constitutive CaMV35S promoter, revealed higher expression levels of \u003cem\u003ecry1Ab\u003c/em\u003e transcripts in both leaves and immature pods (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and F). In our transgenic lines, \u003cem\u003ecry1Ab\u003c/em\u003e expression in leaves and immature pods reached 3 to 10-fold and 6 to 13-fold expression relative to ubiquitin, respectively. Notably, the expression of \u003cem\u003ecry1Ab\u003c/em\u003e in these tissues surpasses that reported in previous studies on cowpea (Addae et al. 2020; Majumder et al. 2020; Eckerstorfer et al. 2022). Given the critical importance of achieving optimal expression of cry genes in field crops for effective pest control strategies, our transgenic lines emerge as potential candidates for integrating existing Cry lines into comprehensive field studies. Furthermore, our investigation revealed variations in \u003cem\u003ecry1Ab\u003c/em\u003e expression across independent transgenic cowpea lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and F). These variations likely arise from factors such as positional effects, transgene copy numbers, and the choice of promoter for transgene expression. Notably, similar fluctuations in cry gene expression have been documented in numerous other crops engineered to confer resistance against lepidopteran insects (Siddiqui et al. 2023; Fang et al. 2024; Singh et al. 2022; Kumar et al. 2021). This underscores the importance of meticulous optimization and rigorous evaluation protocols in transgenic crop development. The results from PCR, Southern blotting, and RT-PCR analyses of transgenic cowpea plants decisively confirmed the stable integration without any rearrangements of the \u003cem\u003ecry1Ab\u003c/em\u003e gene in transgenic plants and also in subsequent generations.\u003c/p\u003e \u003cp\u003eThe effectiveness of selected cowpea lines expressing the Cry1Ab protein was assessed against MPB larvae by feeding them leaves and pods. Western blot analysis, followed by ELISA, confirmed stable cry gene expression. Among the T\u003csub\u003e3\u003c/sub\u003e progenies, line #30 exhibited the highest Cry1Ab expression (7.6 \u0026micro;g/g in leaves and 8 \u0026micro;g/g in pods), while line #5 showed the lowest expression (7.6 \u0026micro;g/g in leaves and 8 \u0026micro;g/g in pods). The expression-based selection process successfully identified the high-expressing events in this research. Notably, the pods accumulated 6.6% more toxin on average than the leaves in the corresponding transgenic lines. Previous studies have documented the variability in \u003cem\u003eBt\u003c/em\u003e-endotoxin expression among transgenic plants driven by CaMV35S promoters. This variability has been attributed to factors such as the position effect of gene integration, surrounding flanking sequences, chromatin context, increased DNA methylation with plant age, and physiological changes affecting the stability of foreign proteins within plant tissues (To et al. 2021; Rurek and Smolibowski 2024). In contrast, our analysis of transgenic cowpea lines demonstrated that the expression of the Cry1Ab protein, as verified through Western blot and ELISA assays, remained robust and consistent in both leaves and immature pods across three successive generations. This stability highlights the potential for reliable expression of \u003cem\u003eBt\u003c/em\u003e-endotoxins in cowpea, minimizing the concerns raised in earlier observations.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eBt\u003c/em\u003e-toxin expression levels observed in this study surpass those reported for other grain legumes (Acharjee and Higgins 2021; Singh et al. 2023) and represent the highest recorded Cry protein levels in cowpea (Addae et al. 2020; Kumar et al. 2021). This higher expression may be attributed to the use of a codon-optimized \u003cem\u003ecry1Ab\u003c/em\u003e gene, specifically engineered to enhance mRNA stability, eliminate polyadenylation sites and splicing sequences, and optimize ATG consensus flanking nucleotides for efficient translation initiation. Additionally, the transgene's integration into a transcriptionally active region of the host genome likely contributed to this robust expression (Watts et al. 2021). Codon optimization has been extensively employed in \u003cem\u003eBt\u003c/em\u003e-cry genes to enhance insect resistance across various plant species. High-level expression of codon-optimized \u003cem\u003ecry1Ac\u003c/em\u003e, \u003cem\u003ecry1Ab\u003c/em\u003e, and \u003cem\u003ecry1C\u003c/em\u003e genes has been previously demonstrated in transgenic cotton, tomato, and tobacco, respectively (Zafar et al. 2022; Siddiqui et al. 2023; Fernandes et al. 2023; Wang et al. 2024). Furthermore, increased expression of Cry2Aa in cowpea has been shown to improve resistance against lepidopteran pests (Singh et al. 2018; Kumar et al. 2021).\u003c/p\u003e \u003cp\u003eThe expression levels of a transgene are significantly influenced by the choice of promoter driving its transcription and translation into functional protein. Historically, the CaMV35S promoter has been widely used to achieve constitutive expression of \u003cem\u003ecry1Ab\u003c/em\u003e in transgenic cowpea, and the first pod-borer-resistant cowpea released for commercial use in Nigeria utilized the CaMV35S promoter for gene regulation (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://aaccnet.confex.com/aaccnet/2020/meetingapp.cgi/Paper/5721\u003c/span\u003e\u003cspan address=\"https://aaccnet.confex.com/aaccnet/2020/meetingapp.cgi/Paper/5721\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). However, recent studies have shown that the green tissue-specific RuBisCO small subunit (rbcS) promoter can drive Cry1Ac expression to levels 1.5 times higher than those achieved with CaMV35S and Ubi promoters in chickpea (Boruah et al. 2023; Hazarika et al. 2021). These findings suggest that future research should explore the use of the rbcS promoter to achieve enhanced endotoxin protection through higher transgene expression.\u003c/p\u003e \u003cp\u003eOur study adds to the expanding body of research focused on the effectiveness of transgenic plants expressing insecticidal proteins in controlling major lepidopteran pests. Through in vitro insect feeding bioassays using larvae of \u003cem\u003eM. vitrata\u003c/em\u003e and \u003cem\u003eH. armigera\u003c/em\u003e, we observed a marked increase in insect-induced damage in non-transgenic control compared to transgenics. This was directly linked to the presence of Cry1Ab toxins in the leaves and immature pods of the transgenic plants. Notably, all five transgenic lines exhibited resistance to insect damage, with some variation in effectiveness, likely due to differences in \u003cem\u003ecry1Ab\u003c/em\u003e gene expression levels. Our results are consistent with previous studies on the role of transgenic plants in pest management. For instance, cowpea lines expressing the Cry2Aa protein showed substantial protection against Maruca pod borer larvae, with mortality rates exceeding 90% (Kumar et al. 2021). Similarly, chickpea plants co-expressing \u003cem\u003ecry1Ab\u003c/em\u003e and \u003cem\u003ecry1Ac\u003c/em\u003e genes exhibited enhanced toxicity, providing broader protection against pests such as \u003cem\u003eH. armigera\u003c/em\u003e (Koul et al. 2022). Some \u003cem\u003eBt\u003c/em\u003e-transgenic chickpea lines even achieved 100% mortality, underscoring the potential of \u003cem\u003eBt\u003c/em\u003e-transgenics as an effective pest management tool (Hazarika et al. 2021). Such line-dependent efficacy data are vital for pest management programs, as they aid in selecting elite events for resistance durability testing and resistance management studies under variable field conditions.\u003c/p\u003e \u003cp\u003eMoreover, our \u003cem\u003ecry1Ab\u003c/em\u003e cowpea transgenics demonstrated enhanced insecticidal efficacy not only against \u003cem\u003eM. vitrata\u003c/em\u003e but also against \u003cem\u003eH. armigera\u003c/em\u003e. Interestingly, although Cry1Ab protein levels were lower in the leaves compared to pods of transgenic cowpea lines, higher \u003cem\u003eH. armigera\u003c/em\u003e mortality was observed in leaf-feeding assays. This apparent contradiction can be attributed to several factors. Firstly, \u003cem\u003eH. armigera\u003c/em\u003e larvae show a feeding preference for softer leaf tissues during early instars, leading to greater ingestion of Cry1Ab despite lower expression levels in leaves (Zalucki et al. 1994; Sharma et al., 2005). Secondly, Cry1Ab may be more evenly distributed or bioavailable in leaf tissues, whereas physical structures in pods may hinder effective ingestion. Additionally, plant-derived secondary metabolites such as flavones, commonly found in leaves, may interact synergistically with Cry toxins to enhance their toxicity (Wang et al., 2021). Furthermore, recent studies suggest that Cry1Ab\u0026rsquo;s toxicity depends on binding to specific receptors in the insect midgut, including prohibitin and cadherin-like proteins, whose expression or accessibility could vary depending on the tissue consumed (Sena da Silva et al., 2021). Therefore, the observed mortality is likely influenced not just by absolute Cry1Ab levels, but also by tissue-specific feeding dynamics, toxin accessibility, plant metabolite interactions, and receptor-mediated mechanisms. The observed reduction in larval mortality in transgenic cowpea compared to larvae fed on non-transgenic control plants confirms the effective expression of the toxin protein in these transgenic lines. These findings provide a strong foundation for future crop protection strategies, suggesting that integrating Cry1Ab proteins into cowpea could be a promising approach to controlling \u003cem\u003eM. vitrata\u003c/em\u003e and \u003cem\u003eH. armigera\u003c/em\u003e infestations, thereby minimizing crop damage and enhancing agricultural sustainability.\u003c/p\u003e \u003cp\u003eMetabolomics has emerged as a powerful tool for providing deep insights into crop biology. The information obtained from metabolomic analyses can be effectively utilized in assessing phenotypic changes, identifying biomarkers, and tracking gene expression, while also enhancing the interpretation of other genomic data (Makhumbila et al. 2022). NMR metabolomics, in particular, is highly effective in studying plant defenses, both constitutive and induced, against biotic stressors(Mascellani Bergo et al. 2024). Our study revealed that the majority of metabolites in transgenic cowpea were upregulated, including amino acids, sugars, and other key metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Notably, carbohydrates such as sucrose and fructose were significantly more abundant in transgenics compared to non-transgenic cowpea. These results align with those of (Chang et al. 2012), who reported elevated levels of sucrose, mannitol, and glutamic acid in transgenic rice expressing \u003cem\u003ecry1Ac\u003c/em\u003e and \u003cem\u003esck\u003c/em\u003e genes. Metabolic pathway analysis further highlighted the upregulation of the amino acid metabolism pathway, TCA cycle, and carbohydrate metabolism pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Our findings indicate that while no new metabolites were detected, there was a significant alteration in the abundance of existing ones. Similar outcomes were observed in maize transgenics overexpressing cry proteins (Liu et al. 2021; Liu et al. 2023). The use of both targeted and untargeted NMR metabolomics was crucial in evaluating the metabolite profile variations in cowpea leaves and pods resulting from transgene expression. From a pest science perspective, such analyses are crucial for biosafety assessments, ensuring that transgenic plants maintain normal physiology while exhibiting enhanced pest resistance, thereby minimizing unintended ecological risks.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our study demonstrated that the synthetic \u003cem\u003ecry1Ab\u003c/em\u003e gene is a valuable strategy for enhancing MPB resistance. This is the first report of a transgenic cowpea variety using a synthetic \u003cem\u003ecry1Ab\u003c/em\u003e gene, which also conferred resistance to \u003cem\u003eH. armigera\u003c/em\u003e. This represents a major advancement toward developing genetically engineered host plant resistance that complements existing IPM strategies and reduce reliance on broad-spectrum insecticides. Incorporating such Bt cowpea lines into IPM frameworks could help suppress pest populations, delay the evolution of insect resistance through gene pyramiding, and reduce the frequency of insecticide sprays. The \u003cem\u003ecry1Ab\u003c/em\u003e gene is a strong candidate for co-expression with other cry genes in developing \u003cem\u003eBt\u003c/em\u003e cowpea resistant to multiple insects, including MPB and \u003cem\u003eH. armigera\u003c/em\u003e. Such combinations could delay resistance evolution, provide broad-spectrum protection, increase yields, boost the income of farmers, and reduce pesticide use. Our findings demonstrate that the transgenic cowpea lines not only show high insect mortality and stable Cry1Ab expression but also maintain normal metabolic function, ensuring biosafety and agronomic performance. Taken together, this work positions Bt cowpea as a valuable component in the next generation of pest management technologies- offering an effective, sustainable, and scientific approach to mitigating lepidopteran damage in grain legumes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interest to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLS conceived and designed research, contributed new reagents/analytical tools and corrected the manuscript. MJ designed, conducted experiments and analyzed data and wrote the manuscript. DKM, SK, DK assisted MJ in some experiments. IA provided the synthetic \u003cem\u003ecry1Ab\u003c/em\u003e construct. VK conducted insect bioassay. All authors read and approved the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbudulai M, Kusi F, Seini SS, et al (2017) Effects of planting date, cultivar and insecticide spray application for the management of insect pests of cowpea in northern Ghana. Crop Protection 100:168\u0026ndash;176. https://doi.org/10.1016/J.CROPRO.2017.07.005Acharjee S, Higgins TJV (2021) Genetic Engineering of Grain Legumes: Their Potential for Sustainable Agriculture and Food and Nutritional Security. In: Saxena, K.B., Saxena, R.K., Varshney, R.K. (eds) Genetic Enhancement in Major Food Legumes. 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Inductive and synergistic interactions between plant allelochemical flavone and Bt toxin Cry1Ac in Helicoverpa armigera. Insect Science, 28(6):1756-1765. https://doi.org/10.1111/1744-7917.12897\u003c/li\u003e\n\u003cli\u003eWang Y, Wang, M, Zhang Y, et al (2024) Resistance to both aphids and nematodes in tobacco plants expressing a \u003cem\u003eBacillus thuringiensis\u003c/em\u003e crystal protein. Pest Management Science 80(7):3098-3106. https://doi.org/10.1002/ps.8013\u003c/li\u003e\n\u003cli\u003eWatts A, Sankaranarayanan S, Watts A, et al (2021) Optimizing protein expression in heterologous system: Strategies and tools. Meta Gene 29:100899. https://doi.org/10.1016/j.mgene.2021.100899\u003c/li\u003e\n\u003cli\u003eZafar M.M, Mustafa G, Shoukat F, et al. (2022) Heterologous expression of \u003cem\u003ecry3Bb1\u003c/em\u003e and \u003cem\u003ecry3\u003c/em\u003e genes for enhanced resistance against insect pests in cotton. Scientific Reports 12:10878. https://doi.org/10.1038/s41598-022-13295-x\u003c/li\u003e\n\u003cli\u003eZalucki M. P, Murray D. A. H, Gregg P. C et al (1994). Ecology of Helicoverpa-Armigera (Hubner) and Heliothis-Punctigera (Wallengren) in the inland of Australia-larval sampling and host-plant relationships during winter and spring. Australian Journal of Zoology, 42(3):329-346. https://doi.org/10.1071/ZO9940329\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"phytoparasitica","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pypa","sideBox":"Learn more about [Phytoparasitica](http://link.springer.com/journal/12597)","snPcode":"12600","submissionUrl":"https://submission.nature.com/new-submission/12600/3","title":"Phytoparasitica","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Cowpea crop improvement, Legume pod borer management, Bacillus thuringiensis, Insect resistance, Sustainable pest management","lastPublishedDoi":"10.21203/rs.3.rs-8322435/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8322435/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCowpea, a vital legume crop, suffers substantial yield losses due to insect pests, particularly the legume pod borer (\u003cem\u003eMaruca vitrata\u003c/em\u003e). The narrow genetic base of cowpea and the absence of effective host resistance against \u003cem\u003eM. vitrata\u003c/em\u003e have limited the success of conventional breeding-based pest management. Given the increasing incidence of insecticide resistance and environmental concerns associated with chemical control, there is an urgent need for host plant-based solutions that fit within integrated pest management (IPM) frameworks. To address this challenge, we engineered transgenic cowpea plants expressing a synthetic \u003cem\u003ecry1Ab\u003c/em\u003e gene under the control of the CaMV35S promoter. High expression of Cry1Ab was detected in leaves and pods, the primary feeding sites of key lepidopteran pests. Bioassays with \u003cem\u003eM. vitrata\u003c/em\u003e and \u003cem\u003eHelicoverpa armigera\u003c/em\u003e larvae demonstrated strong resistance in the transgenic lines, evidenced by reduced pod damage, suppressed larval feeding, and high insect mortality compared to non-transgenic controls. These results confirm the effective expression and bioactivity of Cry1Ab in planta, highlighting its potential as a reliable pest control trait under pest pressure. Importantly, the transgenic plants showed no detectable metabolic changes by NMR profiling and displayed normal growth and development without yield penalties. These findings underline the role of Bt-cowpea as a sustainable, environmentally compatible, and economically viable approach to reducing pest burden and pesticide dependence in legume production systems. Overall, the synthetic cry1Ab-expressing cowpea lines represent a promising next-generation tool for durable and broad-spectrum protection against major lepidopteran pests, contributing to the long-term goals of sustainable pest management and agricultural resilience.\u003c/p\u003e","manuscriptTitle":"Transgenic Cowpea Expressing Synthetic BtCry1Ab Provides Enhanced Resistance to Maruca vitrata and Supports Sustainable Pod Borer Management","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-17 14:59:31","doi":"10.21203/rs.3.rs-8322435/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-08T17:22:18+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-08T09:26:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-04T01:30:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"187767349290743942154969618792242313000","date":"2025-12-19T06:26:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"50467214342920634846920593633860646437","date":"2025-12-19T03:03:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"26255890429305437903558137193143088151","date":"2025-12-12T09:12:08+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-12T07:00:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-11T18:29:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-11T04:22:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Phytoparasitica","date":"2025-12-10T03:03:13+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"phytoparasitica","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pypa","sideBox":"Learn more about [Phytoparasitica](http://link.springer.com/journal/12597)","snPcode":"12600","submissionUrl":"https://submission.nature.com/new-submission/12600/3","title":"Phytoparasitica","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"fa20b0b3-99b3-48e5-9b2c-182782f61f38","owner":[],"postedDate":"December 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-01-08T17:38:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-17 14:59:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8322435","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8322435","identity":"rs-8322435","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}