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Tissue Culture- and Selection-Independent Agrobacterium tumefaciens-Mediated Transformation of a Recalcitrant Grain Legume, Cowpea (Vigna unguiculata L. Walp)

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Abstract

A simple and generally fast Agrobacterium-mediated transformation system with no tissue culture and selection steps has been developed for the first time in a recalcitrant food legume, cowpea. The approach involves wounding of 1-day-old germinated seeds with a needle or sonication either alone or in combination of vacuum infiltration with A. tumefaciens EH105 (pCAMBIA2301) carrying a β-glucuronidase (GUS) gene (uidA) and a neomycin phosphotransferase (nptII) gene for stable transformation. Sonicated and vacuum infiltrated seedlings showed the highest transient GUS activity in 90% of the explants. The sprouted co-cultured seeds directly established in soil and without selection were allowed to develop into plants which on maturity produced T0 seeds. The presence of the alien genes, nptII and uidA in T0 plants and their integration into the genome of T1 plants were confirmed by polymerase chain reaction (PCR) and Southern blot analyses, respectively. The transgenes were inherited in the subsequent T2 generation in a Mendelian fashion and their expression was confirmed by semi-quantitative PCR. The transformation frequency of 1.90% was obtained with sonication followed by vacuum infiltration with Agrobacterium. This approach provides favorable circumstances for the rapid meristem transformation and likely makes translational research ease in an important recalcitrant food legume, cowpea.

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References

  1. Gonçalves, A., Goufo, P., Barros, A., Domínguez-Perles, R., Trindade, H., Rosa, E. A., & Rodrigues, M. (2016). Cowpea (Vigna unguiculata L. Walp.), a renewed multipurpose crop for a more sustainable agrifood system: Nutritional advantages and constraints. Journal of the Science of Food and Agriculture, 96, 2941–2951.

    Article  Google Scholar 

  2. Togola, A., Boukar, O., Belko, N., Chamarthi, S. K., Fatokun, C., Tamo, M., & Oigiangbe, N. (2017). Host plant resistance to insect pests of cowpea (Vigna unguiculata L. Walp.): Achievements and future prospects. Euphytica, 213, 239. https://doi.org/10.1007/s10681-017-2030-1

    Article  Google Scholar 

  3. Sindhu, M., Kumar, A., Yadav, H., Chaudhary, D., Jaiwal, R., & Jaiwal, P. K. (2019). Current advances and future directions in genetic enhancement of a climate resilient food legume crop, cowpea (Vigna unguiculata L. Walp.). Plant Cell, Tissue and Organ Culture, 139, 429–453. https://doi.org/10.1007/s11240-019-01695-3

    Article  Google Scholar 

  4. Hiei, Y., Ohta, S., Komari, T., & Kumashiro, T. (1994). Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. The Plant Journal, 6, 271–282. https://doi.org/10.1046/j.1365-313X.1994.6020271.x

    Article  CAS  PubMed  Google Scholar 

  5. Popelka, J. C. S., Gollasch, S., Moore, A., Molvig, L., & Higgins, T. J. V. (2006). Genetic transformation of cowpea (Vigna unguiculata) and stable transmission of the transgenes to progeny. Plant Cell Reports, 25, 304–312.

    Article  CAS  Google Scholar 

  6. Chaudhury, D., Madanpotra, S., Jaiwal, R., Saini, R., Kumar, P. A., & Jaiwal, P. K. (2007). Agrobacterium tumefaciens-mediated high frequency genetic transformation of an Indian cowpea (Vigna unguiculata L. Walp.) cultivar and transmission of transgenes into progeny. Plant Science, 172, 692–700.

    Article  CAS  Google Scholar 

  7. Bakshi, S., Sadhukhan, A., Mishra, S., & Sahoo, L. (2011). Improved Agrobacterium-mediated transformation of cowpea via sonication and vacuum infiltration. Plant Cell Reports, 30, 2281–2292.

    Article  CAS  Google Scholar 

  8. Chhikara, S., Chaudhary, D., Yadav, M., Sainger, M., & Jaiwal, P. K. (2011). A non-tissue culture approach for developing transgenic Brassica juncea L. plants with Agrobacterium tumefaciens. In Vitro Cellular & Developmental Biology-Plant, 48, 7–14.

    Article  Google Scholar 

  9. Clough, S. J., & Bent, A. F. (1998). Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal, 16, 735–743. https://doi.org/10.1046/j.1365-313x.1998.00343.x

    Article  CAS  PubMed  Google Scholar 

  10. Bent, A. F. (2000). Arabidopsis in planta transformation: Uses, mechanisms and prospects for transformation of other species. Plant Physiology, 124, 1540–1547. https://doi.org/10.1104/PP.124.4.1540

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Feldmann, K. A., & Marks, M. D. (1987). Agrobacterium-mediated transformation of germinating seeds of Arabidopsis thaliana: A non-tissue culture approach. Molecular Genetics and Genomics, 208, 1–9.

    Article  CAS  Google Scholar 

  12. Kapildev, G., Chinnathambi, A., Sivanandhan, G., Rajesh, M., Vasudevan, V., Mayavan, S., Arun, M., Jeyaraj, M., Alharbi, S. A., Selvaraj, N., & Ganapathi, A. (2016). High-efficient Agrobacterium-mediated in planta transformation in black gram (Vigna mungo (L.) Hepper). Acta Physiologia Plantarum, 38, 205. https://doi.org/10.1007/s11738-016-2215-6

    Article  CAS  Google Scholar 

  13. Karthik, S., Pavan, G., Sathish, S., Siva, R., Kumar, P. S., & Manickavasagam, M. (2018). Genotype-independent and enhanced in planta Agrobacterium tumefaciens-mediated genetic transformation of peanut [Arachis hypogaea (L.)]. 3 Biotechnology, 8, 202. https://doi.org/10.1007/s13205-018-1231-1

    Article  Google Scholar 

  14. Karmakar, S., Kutubuddin, A. M., Gayen, D., Karmakar, A., Das, K., Sarkar, S. N., Datta, K., & Datta, S. K. (2019). Development of a rapid and highly efficient Agrobacterium-mediated transformation system for pigeon pea [Cajanus cajan (L.) Millsp]. GM Crops & Food, 10, 115–138. https://doi.org/10.1080/21645698.2019.1625653

    Article  Google Scholar 

  15. Kesiraju, K., Mishra, P., Bajpai, A., Sharma, M., Rao, U., & Sreevathsa, R. (2020). Agrobacterium tumefaciens-mediated in planta transformation strategy for development of transgenics in cotton (Gossypium hirsutum L.) with GFP as a visual marker. Physiology and Molecular Biology of Plants, 26, 2319–2327. https://doi.org/10.1007/s12298-020-00887-y

    Article  CAS  PubMed  Google Scholar 

  16. Sainger, M., Jaiwal, A., Sainger, P. A., Chaudhary, D., Jaiwal, R., & Jaiwal, P. K. (2017). Advances in genetic improvement of Camelina sativa for biofuel and industrial bioproducts. Renewable and Sustainable Energy Reviews, 68, 623–637.

    Article  CAS  Google Scholar 

  17. Trieu, A. T., Burleigh, S. H., Kardailsky, I. V., Maldonado-Mendoza, I. E., Versaw, W. K., Blaylock, L. A., Shin, H., Chiou, T. J., Katagi, H., Dewbre, G. R., Weigel, D., & Harrison, M. J. (2000). Transformation of Medicago truncatula via infiltration of seedlings or flowering plants with Agrobacterium. The Plant Journal, 22, 531–541.

    Article  CAS  Google Scholar 

  18. Curtis, I. S., & Nam, H. G. (2001). Transgenic radish (Raphanus sativus L. longipinnatus Bailey) by floral-dip method-plant development and surfactant are important in optimizing transformation efficiency. Transgenic Research, 10, 363–371.

    Article  CAS  Google Scholar 

  19. Yasmeen, A., Mirza, B., Inayatullah, S., Safdar, N., Jamil, M., Ali, S., & Choudhry, M. F. (2009). In planta transformation of tomato. Plant Molecular Biology Reports, 27, 20–28.

    Article  CAS  Google Scholar 

  20. Bastaki, N. K., & Cullis, C. A. (2014). Floral-dip transformation of flax (Linum usitatissimum) to generate transgenic progenies with a high transformation rate. Journal of Visualized Experiments, 94, 52189. https://doi.org/10.3791/52189

    Article  CAS  Google Scholar 

  21. Saha, P., & Blumwald, E. (2016). Spike-dip transformation of Setaria viridis. The Plant Journal, 86, 89–101. https://doi.org/10.1111/tpj.13148

    Article  CAS  PubMed  Google Scholar 

  22. Jefferson, R. A. (1987). Assaying chimearic genes in plants: The GUS gene fusion system. Plant Molecular Biology, 204, 387–405.

    Article  Google Scholar 

  23. Rogers, S. O., & Bendich, A. J. (1989). Extraction of DNA from plant tissues. In S. B. Gelvin, R. A. Schilperoort, & D. P. S. Verma (Eds.), Plant molecular biology manual (pp. 73–83). Springer. https://doi.org/10.1007/978-94-009-0951-9_6

    Chapter  Google Scholar 

  24. Sambrook, K. J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning: A laboratory manual (2nd ed.). Cold Spring Harbor Laboratory Press.

    Google Scholar 

  25. Saifi, S. S., Passricha, N., Tuteja, R., Kharb, P., & Tuteja, N., et al. (2020). In planta transformation: A smart way of crop improvement. In N. Tuteja, et al. (Eds.), Advancement in crop improvement techniques (pp. 351–362). Elsevier Publ.

    Chapter  Google Scholar 

  26. Trick, H., & Finer, J. J. (1997). SAAT: Sonication-assisted Agrobacterium-mediated transformation. Transgenic Research, 6, 329–336.

    Article  CAS  Google Scholar 

  27. Santarém, E. R., Trick, H. N., Essing, J. S., & Finer, J. J. (1998). Sonication assisted Agrobacterium-mediated transformation of soybean immature cotyledons: Optimization of transient expression. Plant Cell Reports, 17, 752–759.

    Article  Google Scholar 

  28. Tang, W., Sederoff, R., & Whetten, R. (2001). Regeneration of transgenic loblolly pine (Pinus taeda L.) from zygotic embryos transformed with Agrobacterium tumefaciens. Planta, 213, 981–989.

    Article  CAS  Google Scholar 

  29. Liu, Z., Park, B. J., Kanno, A., & Kameya, T. (2005). The novel use of a combination of sonication and vacuum infiltration in Agrobacterium-mediated transformation of kidney bean (Phaseolus vulgaris L.) with lea gene. Molecular Breeding, 16, 189–197.

    Article  CAS  Google Scholar 

  30. Subramanyam, K., Rajesh, M., Jaganath, B., Vasuki, A., Theboral, J., Elayaraja, D., Karthik, S., Manickavasagam, M., & Ganapathi, A. (2013). Assessment of factors influencing the Agrobacterium-mediated in planta seed transformation of brinjal (Solanum melongena L.). Applied Biochemistry and Biotechnology, 171, 450–468. https://doi.org/10.1007/s12010-013-0359-z

    Article  CAS  PubMed  Google Scholar 

  31. Chen, Y., Lange, A., Vaghchhipawala, Z., Ye, X., & Saltarikos, A. (2020). Direct germline transformation of cotton meristem explants with no selection. Frontiers in Plant Science, 11, 575283. https://doi.org/10.3389/fpls.2020.575283

    Article  PubMed  PubMed Central  Google Scholar 

  32. Solleti, S. K., Bakshi, S., & Sahoo, L. (2008). Additional virulence genes in conjunction with efficient selection scheme and compatible culture regime enhance recovery of stable transgenic plants in cowpea via Agrobacterium tumefaciens-mediated transformation. Journal of Biotechnology, 135, 97–104. https://doi.org/10.1016/j.jbiotec.2008.02.008

    Article  CAS  PubMed  Google Scholar 

  33. Bett, B., Gollasch, S., Moore, A., Harding, R., & Higgins, T. J. V. (2019). An improved transformation system for cowpea (Vigna unguiculata L. Walp.) via sonication and a kanamycin–geneticin selection regime. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2019.00219

    Article  PubMed  PubMed Central  Google Scholar 

  34. Ilori, C. O., & Pellegrineschi, A. (2011). Transgene expression in cowpea (Vigna unguiculata (L.) Walp.) through Agrobacterium transformation of pollen in flower buds. African Journal of Biotechnology, 10, 11821–11828.

    CAS  Google Scholar 

  35. Bakshi, S., Saha, B., Roy, N. K., Mishra, S., Panda, S. K., & Sahoo, L. (2012). Successful recovery of transgenic cowpea (Vigna unguiculata) using the 6-phosphomannose isomerase gene as the selectable marker. Plant Cell Reports, 31, 1093–1103.

    Article  CAS  Google Scholar 

  36. Che, P., Chang, S., Simon, M. K., Zhang, Z., Shaharyar, A., Ourada, J., O’Neill, D., Torres-Mendoza, M., Guo, Y., Marasigan, K. M., Vielle-Calzada, J. P., Ozias-Akins, P., Albertsen, M. C., & Jones, T. J. (2021). Developing a rapid and highly efficient cowpea regeneration, transformation and genome editing system using embryonic axis explants. The Plant Journal. https://doi.org/10.1111/tpj.15202

    Article  PubMed  PubMed Central  Google Scholar 

  37. Okeyo-Ikawa, R., Amugune, N. O., Njoroge, N. C., Asami, P., & Holton, T. (2016). In planta seed transformation of Kenyan cowpeas (Vigna unguiculata) with P5CS gene via Agrobacterium tumefaciens. Journal of Agricultural Biotechnology and Sustainable Development, 8, 32–45.

    Article  CAS  Google Scholar 

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Acknowledgements

The authors express their gratitude to UGC, New Delhi for the award of BSR Faculty Fellowship to PKJ and for the funding of a major research project (F. No-42-189/2013 (SR) to DC. AK is grateful to MDU, Rohtak for the award of University Research Fellowship.

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Authors and Affiliations

  1. Centre for Biotechnology, M. D. University, Rohtak, 124001, India

    Anil Kumar, Manish Sainger, Darshna Chaudhary & Pawan K. Jaiwal

  2. Department of Zoology, M. D. University, Rohtak, 124001, India

    Ranjana Jaiwal

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  1. Anil Kumar
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Kumar, A., Sainger, M., Jaiwal, R. et al. Tissue Culture- and Selection-Independent Agrobacterium tumefaciens-Mediated Transformation of a Recalcitrant Grain Legume, Cowpea (Vigna unguiculata L. Walp). Mol Biotechnol 63, 710–718 (2021). https://doi.org/10.1007/s12033-021-00333-8

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