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Plumular meristem transformation system for chickpea: an efficient method to overcome recalcitrant tissue culture responses

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Abstract

Major biotic and abiotic stresses have led to the reduction of chickpea productivity, creating a strong barrier for its utilization as major food legume. Genetic transformation has revolutionized the chickpea improvement programs for biotic stress tolerance. Recent knowledge on genomic resources offers a range of approaches for genetic improvement of chickpea with a greater resolution, but relies on the transgenic establishment which is the major bottleneck in this process. Poor tissue culture responses along with insensitivity to in vitro rooting hindered the transgenic chickpea development through tissue culture based transformation methods. In the present report, Agrobacterium-mediated plumular meristem transformation was adapted in chickpea to bypass the recalcitrancy of regenerable tissue. Embryonic axis of immature seedlings of chickpea cultivar was decapitated followed by pricking of plumular meristem present in shoot apex and cotyledonary nodes. Pricking is followed by infection by Agrobacterium tumefaciens carrying binary vector pBI121. Three days co-cultivation was performed with infected explants in 6-benzylaminopurine and α-naphthaleneacetic acid supplemented modified Murashige and Skoog medium. Transient GUS expression was observed in co-cultivated explants and PCR based screening strategy allowed the establishment of primary transgenic events with a frequency of 60%. Kanamycin mediated stringent selection of T1 events helped to eliminate the chimeric plants and 44% of T1 progenies were confirmed through PCR. Further, Southern hybridization was performed to identify transgene integration in T1 events. Additionally, prominent GUS activity in T1 events confirmed the expression of transgene. Plumular transformation method, based on culture dependent Agrobacterium-infection and culture independent plant selection as well as establishment, was reported for the first time in chickpea. This transformation method will boost the recovery of novel genotypes with improved agronomic traits and mutant development for functional genomics in chickpea.

Key message

Agrobacterium-mediated transformation of plumular meristem in chickpea is established, following in vitro co-cultivation of explants and culture independent selection of transgenic plants, at higher frequency.

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Acknowledgements

The authors acknowledge the financial support of Indian Council of Agricultural Research (Project Code: NFBSFARA/PB2010/2010-11) and infrastructure support of University of Calcutta, St. Xavier’s College (Autonomous) and Bose Institute, Kolkata, India. S. Ganguly, S. Ghosh and A. Purohit thank West Bengal Higher Education Department - Swami Vivekananda Merit cum Means Scholarship (No. 52-Edn (B) l 5B-l s/2017), Department of Science and Technology, Govt. of India for INSPIRE fellowship (DST/INSPIRE Fellowship/2018/IF180158) and Council of Scientific and Industrial Research, India - CSIR direct SRF (File No. 08/548(0007)/2018 EMR-I), respectively, for providing fellowships.

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Authors and Affiliations

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

    Shreeparna Ganguly, Gourab Ghosh, Sanatan Ghosh & Arnab Purohit

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

    Rituparna Kundu Chaudhuri

  3. Division of Plant Biology, Bose Institute, P1/12 C.I.T. Scheme VII M, Kankurgachi, Kolkata, West Bengal, 700054, India

    Sampa Das

  4. Department of Genetics, University of Calcutta, 35, Ballygunge Circular Road, Kolkata, 700019, India

    Dipankar Chakraborti

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  1. Shreeparna Ganguly
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  4. Arnab Purohit
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  6. Sampa Das
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  7. Dipankar Chakraborti
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Contributions

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

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Correspondence to Dipankar Chakraborti.

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Communicated by Sergio J. Ochatt.

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Ganguly, S., Ghosh, G., Ghosh, S. et al. Plumular meristem transformation system for chickpea: an efficient method to overcome recalcitrant tissue culture responses. Plant Cell Tiss Organ Cult 142, 493–504 (2020). https://doi.org/10.1007/s11240-020-01873-8

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