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Development of transgenic pigeonpea using high throughput plumular meristem transformation method

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Abstract

Tissue culture based poor regeneration along with restricted rooting responses are considered to be major hindrances for in vitro transgenic pigeonpea development. Present study was designed to establish a novel method of Agrobacterium tumefaciens mediated plumular meristem transformation in pigeonpea for improvement of transgenic development frequency. Three days old decapitated seedlings of pigeonpea cultivar ICPL 87119 were pricked at plumular meristem region under in vitro conditions. After infecting with Agrobacterium binary vector pBI121, the explants were co-cultivated in 6-benzylaminopurine and α-naphthaleneacetic acid supplemented modified- Murashige and Skoog medium. Transformed seedling with well-developed tap root system were established in soil. GUS activity as well as PCR based confirmation of transgene presence was demonstrated in transgenic events. Transformation frequency of 72% was achieved for the first time in pigeonpea. Further, kanamycin mediated stringent selection was used for the screening of T1 seeds. Established T1 progenies were analysed by PCR and Southern blot, to confirm transgene integration and copy number, respectively. This is the first report of transgenic pigeonpea development, where the combination of culture based Agrobacterium-infection and culture independent plant establishment, coupled with PCR based selection method was found to be most preferable for faster and frequent establishment of transgenic plants. This method will contribute to large scale transgenic pigeonpea development for its improvement and satisfy the requirement of routine transformation experiments for T-DNA insertion mutagenesis.

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Acknowledgements

The authors thank Indian Council of Agricultural Research (Project code: NFBSFARA/PB2010/2010-11) for financial assistance, WBDBT (Project No. 335/WBBDC/IP-2/2013) for infrastructural grant and St. Xavier’s College (Autonomous), Kolkata for providing infrastructure.

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Author notes

  1. Shreeparna Ganguly and Gourab Ghosh have contributed equally to this work.

Authors and Affiliations

  1. Post Graduate Department of Biotechnology, St. Xavier’s College (Autonomous), 30, Mother Teresa Sarani, Kolkata, West Bengal, 700016, India

    Shreeparna Ganguly, Gourab Ghosh, Arnab Purohit & Dipankar Chakraborti

  2. Department of Botany, Krishnagar Govt. College, Krishnagar, West Bengal, 741101, India

    Rituparna Kundu Chaudhuri

Authors
  1. Shreeparna Ganguly
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  2. Gourab Ghosh
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  3. Arnab Purohit
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  4. Rituparna Kundu Chaudhuri
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  5. Dipankar Chakraborti
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Contributions

SG, GG and DC conceived and designed the experiments. SG, GG and AP conducted all the experiments. SG and DC drafted the manuscript. RKC and DC were responsible for data analysis, manuscript editing, and supervision of the work. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Dipankar Chakraborti.

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Conflict of interest

The work is presented in the manuscript with the consent of all authors. The authors declare that they have no conflict of interest.

Additional information

Communicated by Sergio J. Ochatt.

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Supplementary Fig. 1

Establishment of T1 pigeonpea plants after three weeks of kanamycin mediated selection. Pot 1, Untransformed pigeonpea plants obtained without kanamycin treatment. Pot 2, Untransformed pigeonpea plants obtained after treating the seeds with 100 mg l-1 kanamycin. Pot 3, T1 transgenic plants obtained after treating the seeds with 100 mg l-1 kanamycin. Bar represents 1 cm. (TIF 9757 KB)

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Ganguly, S., Ghosh, G., Purohit, A. et al. Development of transgenic pigeonpea using high throughput plumular meristem transformation method. Plant Cell Tiss Organ Cult 135, 73–83 (2018). https://doi.org/10.1007/s11240-018-1444-3

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  • DOI: https://doi.org/10.1007/s11240-018-1444-3

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