Micro RNA-induced gene silencing strategy for the delivery of siRNAs targeting Meloidogyne incognita in a model plant Nicotiana benthamiana.

BACKGROUND: Occurrence of multiple biotic stresses on crop plants result in drastic yield losses which may have severe impact on the food security. It is a challenge to design strategies for simultaneous management of these multiple stresses. Hence, establishment of innovative approaches that aid in their management is critical. Here, we have introgressed a micro RNA-induced gene silencing (MIGS) based combinatorial gene construct containing seven target gene sequences of cotton leaf curl disease (CLCuD), cotton leaf hopper (Amrasca biguttula biguttula), cotton whitefly (Bemisia tabaci) and root-knot nematode (Meloidogyne incognita). RESULTS: Stable transgenic lines of Nicotiana benthamiana were generated with the T-DNA harboring Arabidopsis miR173 target site fused to fragments of Sec23 and ecdysone receptor (EcR) genes of cotton leaf hopper and cotton whitefly. It also contained C2/replication associated protein (C2/Rep) and C4 (movement protein) along with ßC1 gene of betasatellite to target CLCuD, and two FMRFamide-like peptide (FLP) genes, Mi-flp14 and Mi-flp18 of M. incognita. These transgenic plants were assessed for the amenability of MIGS approach for pest control by efficacy evaluation against M. incognita. Results showed successful production of small interfering RNA (siRNA) through the tasiRNA (trans-acting siRNA) pathway in the transgenic plants corresponding to Mi-flp18 gene. Furthermore, we observed reduced Mi-flp14 and Mi-flp18 transcripts (up to 2.37 ± 0.12-fold) in females extracted from transgenic plants. The average number of galls, total endoparasites, egg masses and number of eggs per egg mass reduced were in the range 27-62%, 39-70%, 38-65% and 34-49%, respectively. More importantly, MIGS transgenic plants showed 80% reduction in the nematode multiplication factor (MF). CONCLUSION: This study demonstrates successful validation of the MIGS approach in the model plant, N. benthamiana for efficacy against M. incognita, as a prelude to translation to cotton. © 2021 Society of Chemical Industry.

Characterization and in planta validation of a CHI4 chitinase from Cajanus platycarpus (Benth.) Maesen for its efficacy against pod borer, Helicoverpa armigera (Hübner).

BACKGROUND: Pigeonpea, Cajanus cajan is one of the economically important legume food crops and a major source of dietary proteins. Management of pod borer, Helicoverpa armigera has been prominent among crop improvement programs. Lack of resistance sources in the cultivated germplasm and crossing incompatibility with pod borer-resistant wild relatives have prompted biotechnological interventions. Identification and exploitation of genes from pigeonpea wild relatives in host plant resistance towards the pod borer assumes pertinence. Dynamic transcriptome analysis of the wild relative vis a vis cultivated pigeonpea identified a CHI4 chitinase as one of the putative insect resistance genes. RESULTS: The study presents variations in important amino acids in CHI4 chitinases from C. cajan and its wild relative C. platycarpus. Comparative protein modeling and docking analysis of the two proteins demonstrated differences in substrate binding efficacy of the chitinase from C. platycarpus which resulted in a minimum binding energy of -8.7 kcal mol-1 . Furthermore, we successfully evaluated the insecticidal activity of the chitinase from C. platycarpus against H. armigera challenge through heterologous expression in tobacco. Molecular characterization of transgenic plants confirmed that their efficacy against H. armigera was a result of the integration of CHI4 from C. platycarpus. CONCLUSION: Docking analysis demonstrated effective substrate interaction as a possible reason for efficacy against pod borer in the chitinase from C. platycarpus. This was authenticated by successful overexpression and bioefficacy assessment against H. armigera in tobacco. The CHI4 gene from C. platycarpus can be useful in the mitigation of H. armegira in pigeonpea as well as in other crops. © 2021 Society of Chemical Industry.

Agrobacterium tumefaciens-mediated in planta transformation strategy for development of transgenics in cotton (Gossypium hirsutum L.) with GFP as a visual marker.

Cotton (Gossypium hirsutum L.), a mercantile crop plant, is grown worldwide for fiber and seed oil. As with other economically important crops, cotton is bogged down with many biotic and abiotic stress factors. Towards this, genetic engineering offers numerous protocols to engineer plants for better resilience. However, recalcitrance of cotton to plant tissue culture has been the major constraint for successful in vitro regeneration. Hence, alternate methods that evade tissue culture regeneration have been envisaged. Non tissue culture-based in planta transformation strategies are in vogue due to amenability and ease in the generation of transgenic plants. In the present study, we demonstrate the utility of an in planta transformation protocol and establishment of a stringent selection agent-based screening for the identification of transgenics. The genotype independent nature of the protocol was validated in cotton cv. Pusa 8-6 using GFP. Preliminary transformation efficiency of 28% was achieved with a screening efficiency of 20% in the presence of hygromycin. The proof of T-DNA integration by various molecular and expression analysis in T1 and T2 generations proved that this technique can be employed to generate transgenic cotton.

Genome Editing for Resistance to Insect Pests: An Emerging Tool for Crop Improvement.

Plants are challenged incessantly by several biotic and abiotic stresses during their entire growth period. As with other biotic stress factors, insect pests have also posed serious concerns related to yield losses due to which agricultural productivity is at stake. In plants, trait modification for crop improvement was initiated with breeding approaches followed by genetic engineering. However, stringent regulatory policies for risk assessment and lack of social acceptance for genetically modified crops worldwide have incited researchers toward alternate strategies. Genome engineering or genome editing has emerged as a new breeding technique with the ability to edit the genomes of plants, animals, microbes, and human beings. Several gene editing strategies are being executed with continuous emergence of variants. The scientific community has unraveled the utility of various editing tools from endonucleases to CRISPR/Cas in several aspects related to plant growth, development, and mitigation of stresses. The categorical focus on the development of tools and techniques including designing of binary vectors to facilitate ease in genome engineering are being pursued. Through this Review, we embark upon the conglomeration of various genome editing strategies that can be and are being used to design insect pest resistance in plants. Case studies and novel crop-based approaches that reiterate the successful use of these tools in insects as well as in plants are highlighted. Further, the Review also provides implications for the requirement of a specific regulatory framework and risk assessment of the edited crops. Genome editing toward insect pest management is here to stay, provided uncompromising efforts are made toward the identification of amiable target genes.

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

Efficient regeneration of explants devoid of intrinsic somaclonal variations is a cardinal step in plant tissue culture, thus, a vital component of transgenic technology. However, recalcitrance of economically important crops to tissue culture-based organogenesis ensues a setback in the use of transgenesis in the genetic engineering of crop plants. The present study developed an optimized, genotype-independent, nonconventional tissue culture-independent in planta strategy for the genetic transformation of flax/linseed. This apical meristem-targeted in planta transformation protocol will accelerate value addition in the dual purpose industrially important but recalcitrant fiber crop flax/linseed. The study delineated optimization of Agrobacterium tumefaciens-mediated transformation and stable T-DNA (pCambia2301:GUS:nptII) integration in flax. It established successful use of a stringent soilrite-based screening in the presence of 30 mg/L kanamycin for the identification of putative transformants. The amenability, authenticity, and reproducibility of soilrite-based kanamycin screening were further verified at the molecular level by GUS histochemical analysis of T0 seedlings, GUS and nptII gene-specific PCR, genomic Southern hybridization for stable integration of T-DNA, and expression analysis of transgenes by sqRT-PCR. This method resulted in a screening efficiency of 6.05% in the presence of kanamycin, indicating amenability of in planta flax transformation. The strategy can be a promising tool for the successful development of transgenics in flax.

Assessment of Pigeonpea (Cajanus cajan L.) transgenics expressing Bt ICPs, Cry2Aa and Cry1AcF under nethouse containment implicated an effective control against herbivory by Helicoverpa armigera (Hübner).

BACKGROUND: Pigeonpea is a source of quality proteins and the main constituent of a well-balanced diet for majority of Indian population. One of the major constraints in the production of pigeonpea is a polyphagous insect pest, Helicoverpa armigera. Non-availability of resistant sources in the germplasm and limitations in conventional breeding have been key factors for continued yield losses. Additionally, hazards of chemical fertilizers on the environment have prompted the scientific community to develop alternative strategies. Bacillus thuringiensis (Bt) insecticidal proteins (ICPs) have emerged as the most reliable source for the control of insect pests through transgenics. RESULTS: Transgenic pigeonpea plants harboring validated Bt ICPs, Cry2Aa and Cry1AcF were developed by a non-tissue culture based in planta transformation strategy and assessed for integration of Transfer-DNA (T-DNA) and efficacy against pod borer under in vitro conditions. For the first time this study demonstrates the successful evaluation of 19 transgenic pigeonpea events (11 with cry2Aa and 8 with cry1AcF) under soil and pot conditions in a nethouse containment. The stability in the performance was assessed stringently by deliberate H. armigera larval challenging. The trial identified ten promising events of both the genes that portrayed reduced damage to the herbivore. CONCLUSION: We present the first ever successful evaluation of pigeonpea transgenics with the ability to mitigate pod borer under nethouse conditions. The transgenics depicted molecular evidence for the stability of T-DNA integration, consistency in the expression of Cry proteins and resistance against H. armigera. These events can form a pool of useful transgenics to manage the devastating pod borer. © 2019 Society of Chemical Industry.

Molecular Interaction-Based Exploration of the Broad Spectrum Efficacy of a Bacillus thuringiensis Insecticidal Chimeric Protein, Cry1AcF.

Bacillus thuringiensis insecticidal proteins (Bt ICPs) are reliable and valuable options for pest management in crops. Protein engineering of Bt ICPs is a competitive alternative for resistance management in insects. The primary focus of the study was to reiterate the translational utility of a protein-engineered chimeric Cry toxin, Cry1AcF, for its broad spectrum insecticidal efficacy using molecular modeling and docking studies. In-depth bioinformatic analysis was undertaken for structure prediction of the Cry toxin as the ligand and aminopeptidase1 receptors (APN1) from Helicoverpa armigera (HaAPN1) and Spodoptera litura (SlAPN1) as receptors, followed by interaction studies using protein-protein docking tools. The study revealed feasible interactions between the toxin and the two receptors through H-bonding and hydrophobic interactions. Further, molecular dynamics simulations substantiated the stability of the interactions, proving the broad spectrum efficacy of Cry1AcF in controlling H. armigera and S. litura. These findings justify the utility of protein-engineered toxins in pest management.