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Transfer and Targeted Overexpression of γ-Tocopherol Methyltransferase (γ-TMT) Gene Using Seed-Specific Promoter Improves Tocopherol Composition in Indian Soybean Cultivars

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Abstract

Soybean oil contains high levels of tocopherols which are an important source of vitamin E in human diet. The conversion of γ- to α-tocopherol catalyzed by γ-tocopherol methyltransferase (γ-TMT) is found to be the rate limiting factor in soybean which influences the tocopherol composition. Using Agrobacterium-mediated transformation, we overexpressed the γ-TMT gene of Perilla frutescens under the control of the seed-specific promoter vicillin in cultivar Pusa 16. Transgene integration and expression was confirmed in five independently transformed GUS positive soybean plants by polymerase chain reaction (PCR), Southern hybridization, and reverse transcriptase-PCR (RT-PCR). High-performance liquid chromatography (HPLC) analysis showed that overexpression of Pf-γ-TMT resulted in efficient conversion of γ-tocopherol to α-tocopherol and concomitant increase in seed α-tocopherol content in RT-PCR positive plants. The protocol was successfully applied to three more cultivars PK 416, Gujarat soybean 1, and VL soya 1 in which seeds of transformed plants showed elevated level of α-tocopherol than wild-type seeds.

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Acknowledgments

The authors are thankful to the Department of Biotechnology (DBT) of Ministry of Science and Technology, New Delhi, Government of India, for the financial support (BT/PR9622/AGR/02/464/2007). The corresponding author is thankful to University Grants Commission (UGC), Govt. of. India, for providing fellowship under UGC–BSR scheme. Subramanyam and Sivanandhan are thankful to the Council of Scientific and Industrial Research (CSIR), Govt. of India for the award of Senior Research Fellowship (SRF) to carry out his doctoral work. J. Theboral is thankful to University Grants Commission (UGC), New Delhi, India for providing Moulana Azad Fellowship to carry out her doctoral work.

Conflict of Interest

The authors declare that they have no competing interests.

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Authors and Affiliations

  1. Department of Biotechnology & Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli, 620024, Tamil Nadu, India

    Muthukrishnan Arun, Kondeti Subramanyam, Jeevaraj Theboral, Ganeshan Sivanandhan, Manoharan Rajesh, Gnanajothi Kapil Dev, Balusamy Jaganath, Markandan Manickavasagam & Andy Ganapathi

  2. Department of Genetic Engineering, School of Bioengineering, SRM University, Kattankulathur, Kancheepuram District, 603203, Tamil Nadu, India

    Muthukrishnan Arun

  3. Plant Biotechnology Division, Department of Biotechnology, Bharathiar University, Coimbatore, 641046, Tamil Nadu, India

    Shanmugam Girija

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  1. Muthukrishnan Arun
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Correspondence to Andy Ganapathi.

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Arun, M., Subramanyam, K., Theboral, J. et al. Transfer and Targeted Overexpression of γ-Tocopherol Methyltransferase (γ-TMT) Gene Using Seed-Specific Promoter Improves Tocopherol Composition in Indian Soybean Cultivars. Appl Biochem Biotechnol 172, 1763–1776 (2014). https://doi.org/10.1007/s12010-013-0645-9

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