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Tissue specific expression of potent insecticidal, Allium sativum leaf agglutinin (ASAL) in important pulse crop, chickpea (Cicer arietinum L.) to resist the phloem feeding Aphis craccivora

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Abstract

The phloem sap-sucking hemipteran insect, Aphis craccivora, commonly known as cowpea aphid, cause major yield loss of important food legume crop chickpea. Among different plant lectins Allium sativum leaf agglutinin (ASAL), a mannose binding lectin was found to be potent antifeedant for sap sucking insect A. craccivora. Present study describes expression of ASAL in chickpea through Agrobacterium-mediated transformation of “single cotyledon with half embryo” explant. ASAL was expressed under the control of CaMV35S promoter for constitutive expression and phloem specific rolC promoter for specifically targeting the toxin at feeding site, using pCAMBIA2301 vector containing plant selection marker nptII. Southern blot analysis demonstrated the integration and copy number of chimeric ASAL gene in chickpea and its inheritance in T1 and T2 progeny plants. Expression of ASAL in T0 and T1 plants was confirmed through northern and western blot analysis. The segregation pattern of ASAL transgene was observed in T1 progenies, which followed the 3:1 Mendelian ratio. Enzyme linked immunosorbant assay (ELISA) determined the level of ASAL expression in different transgenic lines in the range of 0.08–0.38% of total soluble protein. The phloem tissue specific expression of ASAL gene driven by rolC promoter has been monitored by immunolocalization analysis of mature stem sections. Survival and fecundity of A. craccivora decreased to 11–26% and 22–42%, respectively when in planta bioassay conducted on T1 plants compared to untransformed control plant which showed 85% survival. Thus, through unique approach of phloem specific expression of novel insecticidal lectin (ASAL), aphid resistance has been successfully achieved in chickpea.

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Acknowledgements

We are grateful to Bose institute for providing infrastructure and other facilities to undergo the study. The authors are thankful to Swiss Agency for Development and Cooperation, Government of Switzerland and the Department of Biotechnology, Government of India under the Indo-Swiss Collaboration in Biotechnology for financial contributions. The help of Dr. K. K. Sharma, ICRISAT, Dr. B. K. Sarmah, AAU, Prof. Thomas Hohn and Prof. Barbara Hohn, Basel, Switzerland are sincerely acknowledged. Technical help from Mr. Arup Dey is being acknowledged.

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  1. Dipankar Chakraborti

    Present address: Umeå Plant Science Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences (SLU), 901 83, Umeå, Sweden

  2. Anindya Sarkar

    Present address: Department of Molecular Genetics, Section of Virology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, 44195, USA

Authors and Affiliations

  1. Plant Molecular and Cellular Genetics, Bose Institute, P1/12 C.I.T. Scheme VII (M), Kolkata, 700054, India

    Dipankar Chakraborti, Anindya Sarkar, Hossain Ali Mondal & Sampa Das

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  1. Dipankar Chakraborti
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Correspondence to Sampa Das.

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Supplementary material 1 (DOC 30 kb)

11248_2009_9242_MOESM2_ESM.jpeg

Artificial diet bioassay of (a) ASAL, (b) DEA, (c) CEA and (d) ATL against A. craccivora. Graphs showing mean survival per replicates, and each bar represents mean ± SE (JPEG 651 kb)

11248_2009_9242_MOESM3_ESM.jpeg

Schematic representation of the T-DNA region of binary vector constructs (a) pCAMBIA35SASAL and (b) pCAMBIArolCASAL showing the restriction sites. ASAL, gusA and neomycin phosphotransferase (nptII) coding genes are shown within T-DNA. LB, left border of T-DNA; RB, right border of T-DNA; CaMV35S Pr., cauliflower mosaic virus 35S promoter; rolC Pr., Agrobacterium rhizogenes rolC gene promoter; CaMV35SpolyA, cauliflower mosaic virus 35S terminator; nos polyA, nopaline synthase polyA terminator (JPEG 448 kb)

11248_2009_9242_MOESM4_ESM.jpeg

PCR analyses for segregation of ASAL gene in randomly chosen T1 progenies. (a) Lane 1, GenerulerTM (MBI Fermentus) marker; lane 2, pCAMBIA35S ASAL plasmid as positive control; lane 3, untransformed DNA as negative control; lanes 4–17, DNA samples of fourteen randomly selected T1 progenies of 35SASAL line cp212. Lanes 8, 10 and 13 did not show any amplification. (b) Lane 1, GenerulerTM (MBI Fermentus) marker; lane 2, pCAMBIArolCASAL plasmid as positive control; lane 3, untransformed DNA as negative control; lanes 4–18, DNA samples of fifteen randomly selected T1 progenies of rolCASAL line cp101. Lanes 10, 14, 17 and 18 did not show any amplification (JPEG 1769 kb)

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Chakraborti, D., Sarkar, A., Mondal, H.A. et al. Tissue specific expression of potent insecticidal, Allium sativum leaf agglutinin (ASAL) in important pulse crop, chickpea (Cicer arietinum L.) to resist the phloem feeding Aphis craccivora . Transgenic Res 18, 529–544 (2009). https://doi.org/10.1007/s11248-009-9242-7

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