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Agrobacterium-mediated transformation in chickpea (Cicer arietinum L.) with an insecticidal protein gene: optimisation of different factors

Physiology and Molecular Biology of Plants volume 16, pages 273–284 (2010)Cite this article

Abstract

Agrobacterium-mediated transformation in chickpea was developed using strain LBA4404 carrying nptII, uidA and cryIAc genes and transformants selected on Murashige and Skoog’s basal medium supplemented with benzyladenine, kinetin and kanamycin. Integration of transgenes was demonstrated using polymerase chain reaction and Southern blot hybridization of T0 plants. The expression of CryIAc delta endotoxin and GUS enzyme was shown by enzyme linked immunosorbent assay and histochemical assay respectively. The transgenic plants (T0) showed more tolerance to infection by Helicoverpa armigera compared to control plants. Various factors such as explant source, cultivar type, different preculture treatment period of explants, co-cultivation period, acetosyringone supplementation, Agrobacterium harboring different plasmids, vacuum infiltration and sonication treatment were tested to study the influence on transformation frequency. The results indicated that use of epicotyl as explant, cultivar ICCC37, Agrobacterium harboring plasmid pHS102 as vector, preculture of explant for 48 h, co-cultivation period of 2 days at 25°C and vacuum infiltration for 15 min produced the best transformation results. Sonication treatment of explants with Agrobacteria for 80 s was found to increase the frequency of transformation.

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Abbreviations

BA:

Benzyladenine

ELISA:

Enzyme Linked Immunosorbent Assay

IBA:

Indole butyric acid

Kan:

Kanamycin

Kn:

Kinetin

MS:

Murashige and Skoog’s medium

PCR:

Polymerase chain reaction

SR:

Shoot regeneration medium

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Acknowledgements

The authors are thankful to Dr. S.F. D’Souza and Dr. S.K. Apte for their encouragement, ICRISAT, AP and MPKV, Rahuri for providing the seed material. S. Indurker is grateful to UGC for the fellowship.

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Affiliations

  1. Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai, 400 085, India

    Shivani Indurker & Susan Eapen

  2. Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai, 400 085, India

    Hari S. Misra

Authors
  1. Shivani Indurker
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  2. Hari S. Misra
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  3. Susan Eapen
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Correspondence to Susan Eapen.

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Indurker, S., Misra, H.S. & Eapen, S. Agrobacterium-mediated transformation in chickpea (Cicer arietinum L.) with an insecticidal protein gene: optimisation of different factors. Physiol Mol Biol Plants 16, 273–284 (2010). https://doi.org/10.1007/s12298-010-0030-x

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  • DOI: https://doi.org/10.1007/s12298-010-0030-x

Keywords

  • Agrobacterium tumefaciens
  • Chickpea
  • Cicer arietinum
  • cryIAc gene
  • Genetic transformation