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Development of pod borer-resistant transgenic chickpea using a pod-specific and a constitutive promoter-driven fused cry1Ab/Ac gene

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Abstract

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We studied pod-specific msg promoter from soybean and developed different transgenic lines of chickpea expressing fused cry1Ab/Ac constitutively and pod specifically for resistance against the destructive pest Helicoverpa armigera.

Abstract

Crystal (Cry) proteins derived from the soil bacterium Bacillus thuringiensis (Bt) play an important role in controlling infestation of Helicoverpa armigera, which has been considered a serious problem in chickpea productivity. This study was undertaken to overcome the problem by introducing fused cry1Ab/Ac insecticidal gene under the control of pod-specific soybean msg promoter as well as rice actin1 promoter into chickpea var. DCP 92-3 by Agrobacterium-mediated transformation. Transgenic chickpea lines were characterized by real-time PCR, ELISA and insect bioassay. Expression of fused cry gene under constitutive and pod-specific promoter results in increase of 77- and 110-fold, respectively, compared to non-transgenic control plants. Levels of Cry toxins produced under the control of actin1 and soybean msg promoter were also estimated by ELISA in the leaves and pods, respectively. The higher expression of fused cry gene caused a lethal effect in larvae. The results of insect bioassay study revealed significant reduction in the survival rate of H. armigera reared on transgenic chickpea twigs as well as on pods. Pod-specific promoter-driven fused cry gene provides better and significant management strategy of pest control of chickpea without phenotypic cost.

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Abbreviations

Bt :

Bacillus thuringiensis

Cry:

Crystal protein

TSP:

Total soluble protein

ELISA:

Enzyme-linked immunosorbent assay

WT:

Wild type

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Acknowledgments

The financial support from the Department of Biotechnology (DBT), Govt. of India, in the form of DBT Programme Support [Sanction no. BT/COE/01/06/05] is thankfully acknowledged. The fellowship from the Council of Scientific and Industrial Research (CSIR), Govt. of India [Sanction no. -09/028(0749)/2009-EMR-1] to K. Ali Molla is highly acknowledged. We thankfully acknowledge Yunliu Fan for providing the Bt construct. The authors thank the Department of Biochemistry, University of Calcutta, for the ELISA analysis. The authors are also grateful to Prof. Kailash Chandra Bansal, Director, National Bureau of Plant Genetic Resources, India, and Dr. V. V. Ramamurthy and Dr. S. Subramanian, Division of Entomology, Indian Agricultural Research Institute, New Delhi, for arrangement of insect larvae of H. armigera for the bioassay.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Department of Botany, University of Calcutta, 35, Ballygunge Circular Road, Kolkata, 700019, West Bengal, India

    Moumita Ganguly, Kutubuddin Ali Molla, Subhasis Karmakar, Karabi Datta & Swapan Kumar Datta

  2. Division of Crop Sciences, Indian Council of Agricultural Research (ICAR), Krishi Bhavan, Dr. Rajendra Prasad Road, New Delhi, 110114, India

    Swapan Kumar Datta

Authors
  1. Moumita Ganguly
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  2. Kutubuddin Ali Molla
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  3. Subhasis Karmakar
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  4. Karabi Datta
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  5. Swapan Kumar Datta
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Corresponding author

Correspondence to Karabi Datta.

Additional information

Communicated by Emmanuel S. Guiderdoni.

Electronic supplementary material

Below is the link to the electronic supplementary material.

122_2014_2397_MOESM1_ESM.pptx

Figure S1 Stable integration of fused cry1Ab/Ac in transgenic chickpea plants with both actin-Bt and msg-Bt. Genomic DNA was digested with HindIII/XbaI releasing 1849 bp fragment and hybridized with 800 bp PCR product of cry1Ab/Ac. NC-Negative control. (PPTX 200 kb)

122_2014_2397_MOESM2_ESM.pptx

Figure S2 Histochemical GUS staining (blue) of different parts of chickpea plants transformed with the msg-gus and actin-gus construct (A) flower from msg-gus transformed plants exhibited blue staining (B) non transformed control flower (C) Flower from actin-gus transformed plants exhibited blue staining (D) outer covering layer of pod transformed with msg-gus (E) young pod from msg-gus transformed plant showed blue stain. Each bar represents 2 mm. (PPTX 315 kb)

122_2014_2397_MOESM3_ESM.ppt

Figure S3 Dip stick assay showing clear bands for CRY protein expression in transgenic chickpea, while wild type did not show the positive band. Figure (PPT 802 kb)

122_2014_2397_MOESM4_ESM.pptx

Figure S4 Physiomorphological nature of insect used in the bioassay after 96 h. (A) Dead, shrinked and decomposing insects fed on the transgenic plants with actin-Bt. (B) Live, healthy, developing insect fed on WT non-transformed control plants. (PPTX 221 kb)

Supplementary material 5 (DOCX 12 kb)

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Ganguly, M., Molla, K.A., Karmakar, S. et al. Development of pod borer-resistant transgenic chickpea using a pod-specific and a constitutive promoter-driven fused cry1Ab/Ac gene. Theor Appl Genet 127, 2555–2565 (2014). https://doi.org/10.1007/s00122-014-2397-5

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