Expression of a Chimeric Gene Encoding Insecticidal Crystal Protein Cry1Aabc of Bacillus thuringiensis in Chickpea (Cicer arietinum L.) Confers Resistance to Gram Pod Borer (Helicoverpa armigera Hubner.)

Abstract

Domain swapping and generation of chimeric insecticidal crystal protein is an emerging area of insect pest management. The lepidopteran insect pest, gram pod borer (Helicoverpa armigera H.) wreaks havoc to chickpea crop affecting production. Lepidopteran insects were reported to be controlled by Bt (cryI) genes. We designed a plant codon optimized chimeric Bt gene (cry1Aabc) using three domains from three different cry1A genes (domains I, II, and III from cry1Aa, cry1Ab, and cry1Ac, respectively) and expressed it under the control of a constitutive promoter in chickpea (cv. DCP92-3) to assess its effect on gram pod borer. A total of six transgenic chickpea shoots were established by grafting into mature fertile plants. The in vitro regenerated (organogenetic) shoots were selected based on antibiotic kanamycin monosulfate (100 mg/L) with transformation efficiency of 0.076%. Three transgenic events were extensively studied based on gene expression pattern and insect mortality across generations. Protein expression in pod walls, immature seeds and leaves (pre- and post-flowering) were estimated and expression in pre-flowering stage was found higher than that of post-flowering. Analysis for the stable integration, expression and insect mortality (detached leaf and whole plant bioassay) led to identification of efficacious transgenic chickpea lines. The chimeric cry1Aabc expressed in chickpea is effective against gram pod borer and generated events can be utilized in transgenic breeding program.

Applications of Protein Engineering and Directed Evolution in Plant Research

“…However, it is important to subsequently test promising protein variants in a plant system. A recent example of this was performed by Das et al (2017). The authors found that feeding chickpea (Cicer arietinum) leaves expressing the Cry1Aabc protein, which had been generated by chimeragenesis, to larvae of the gram pod borer (Helicoverpa armigera Hubner) led to significantly increased mortality when compared to control leaves not expressing the protein.…”

A modified pod specific promoter for high level heterologous expression of genes in legumes

“…Alternatively the constitutive promoter deployed in a particular crop species may either completely fail or demonstrate low levels of expression in the desired tissue/ organ leading to ineffective levels of intended phenotype. For instance, deployment of CaMV35S promoter for expression of Cry protein in chickpea revealed low expression levels in post-flowering stage (116 DAS) compared to that of pre-flowering stage (88 DAS) (Das et al, 2017). To this end, usage of tissue specific or inducible promoters that function in post-flowering stages and more specifically during pod development can overcome such a bottleneck.…”

Symbiotic nitrogen fixation and endophytic bacterial community structure in Bt-transgenic chickpea (Cicer arietinum L)

“…Conventional breeding approaches for insect resistance have limited success in chickpea due to lack of sufficient resistance source and incompatibility with many of the wild relatives 5 . Success of Bt-cotton in India led extensive research on developing transgenic crops especially, insect resistance Bt-chickpea [6][7][8][9][10][11][12] . Site of insertion of transgene in plant genome is critical as it could alter plant metabolism and toxicity of Bt-protein [13][14][15][16] .…”

“…Site of insertion of transgene in plant genome is critical as it could alter plant metabolism and toxicity of Bt-protein [13][14][15][16] . Similarly, insertion of the transgene in active coding region of genome might be responsible for hemizygous state of some Bt-chickpea events with distorted segregation ratio 12 . Altered plant metabolism of genetically modified (GM) plants along with constitutive expression of chimeric Bt-transgenes and neomycin phospho-transferase II (nptII) can also influence the diversity, and richness of endophytic bacterial community that are often described as an important modulating agent of plants' fitness to adapt against environmental stresses.…”