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.).

Alok Das,Jamal Ansari,Shallu Thakur,N P Singh,Subhojit Datta,P A Kumar,Alok Shukla,G K Sujayanand,Sushil K Chaturvedi

doi:10.3389/fpls.2017.01423

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.

Highlights

Chickpea (Cicer arietinum L.) is an important grain legume and holds third position in food legume production worldwide
The binary vector pBinAR originally derived from pBin19 (Hofgen and Willmitzer, 1990), harboring the chimeric Bt δ-endotoxin gene, cry1Aabc and neomycin phosphotransferase II as the plant selectable marker gene was used (Supplementary Figure S1)
A plant codon-optimized chimeric Bt insecticidal crystal proteins (ICPs) gene cry1Aabc was constructed with nucleotide sequences that encode three different domains I, II, and III from three different ICPs viz., cry1Aa, cry1Ab, and cry1Ac, respectively

Summary

Introduction

Chickpea (Cicer arietinum L.) is an important grain legume and holds third position in food legume production worldwide. It is an annual, self-pollinated, diploid (2n = 2x = 16) pulse species of Fabaceae family, with genome size of approximately 738 Mb (Varshney et al, 2013). India alone accounts for 70–75% of total global chickpea production and currently is the largest consumer. In India, chickpea is grown in an area of 8.5 m ha with average annual production of 8.83 m t (FAOSTAT, 2014). Two main types of chickpeas grown are smallseeded desi primarily consumed in the Middle East and Southeast Asia and larger-seeded kabuli is a valuable global commodity. Gram pod borer (Helicoverpa armigera Hubner) are major pest which attack chickpea starting from first fortnight after sowing, voraciously during flower and pod development stages resulting in yield loss of up to 20–30% annually

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