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Tissue Culture Independent Agrobacterium tumefaciens Mediated In Planta Transformation Method for Tropical Maize (Zea mays.L)

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Proceedings of the National Academy of Sciences, India Section B: Biological Sciences Aims and scope Submit manuscript

Abstract

Tropical maize is recalcitrant to tissue culture regeneration because of its poor response to in vitro regeneration after transformation. In this context, the present study has developed the tissue culture independent in planta transformation protocol for tropical maize by transferring plumular meristem cells of germinating seeds of tropical maize genotype through Agrobacterium tumefaciens infection. The protocol was developed by using Agrobacterium strain EHA105 containing vector pCAMBIA3301 carrying cry1Ab, gus and bar genes. The expression of transgene gus in T0 plants was confirmed by measuring the hydrolysis rate of the fluorescent substrate 4-methylumbelliferyl-beta-D-glucuronide (MUG) assay whereas the presence of cry1Ab gene was confirmed by polymerase chain reaction and T0 plants were allowed to grow in glass house into whole plant until maturity and were selfed to produce seeds of T1 generation. The presence of transgene and its segregation was studied in T1 generation through Southern and enzyme-linked immunosorbent assay confirming the presence of transgene and its expression respectively. The developed protocol is cost-effective, efficient and genotype independent without involvement of any tissue culture procedure and can generate a relatively large number of transgenic plants in approximately 75–90 days.

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Acknowledgments

The research work was funded by Indian Council of Agricultural Research (ICAR) through Network Project on Transgenics in Crops (NPTC 3031). The authors acknowledges Dr. P Anand Kumar, National Research Centre on Plant Biotechnology, New Delhi, India for providing the construct pCAMBIA3301 + cry1Ab. The authors also declare that they have no conflict of interest.

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Authors and Affiliations

  1. Directorate of Maize Research, ICAR, Pusa Campus, New Delhi, 110012, India

    Alok Abhishek, Chikkappa G. Karjagi, Pradyumn Kumar, Bhupender Kumar, Sain Dass & R. Sai Kumar

  2. SHIATS (Formerly Allahabad Agricultural Institute), Allahabad, India

    Pramod W. Ramteke

  3. Thermo Fisher Scientific (Life Science Group), Bengaluru, India

    Rita Kumari

Authors
  1. Alok Abhishek
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  2. Rita Kumari
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  4. Pradyumn Kumar
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  5. Bhupender Kumar
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  6. Sain Dass
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Corresponding author

Correspondence to Chikkappa G. Karjagi.

Electronic supplementary material

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40011_2014_454_MOESM1_ESM.pptx

Supplementary Fig. 1 Schematic diagram of plasmid construct and digestion pattern map of pCAMBIA3301 + cry1Ab. The T-DNA region of the transformation vector pCAMBIA3301 containing the uidA, bar and cry1Ab genes (total size: 15.837 kb). LB/RB– left/right T-DNA border sequences; P35S/35S A– CaMV35S promoter/terminator; bar– coding region of the phosphinothricin acetyltransferase gene; nosA– nopaline synthase terminator; gus gene coding region with intron sequence P - maize ubiquitin– promoter; cry1Ab - coding region

40011_2014_454_MOESM2_ESM.pptx

Supplementary Fig. 2 Qualitative expression profile of the cry1Ab protein by DAS-ELISA in Ten cry1Ab protein expressing T1 progeny leaves iT-2 and iT-3, transgenic lines. NC-negative control, PC- positive control

Supplementary material 3 (DOC 57 kb)

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Abhishek, A., Kumari, R., Karjagi, C.G. et al. Tissue Culture Independent Agrobacterium tumefaciens Mediated In Planta Transformation Method for Tropical Maize (Zea mays.L). Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. 86, 375–384 (2016). https://doi.org/10.1007/s40011-014-0454-0

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