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Agrobacterium-Mediated Genetic Transformation of Pogostemon cablin (Blanco) Benth. Using Leaf Explants: Bactericidal Effect of Leaf Extracts and Counteracting Strategies

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Abstract

An optimized protocol for Agrobacterium tumefaciens-mediated transformation of patchouli using leaf disk explants is reported. In vitro antibacterial activity of leaf extracts of the plants revealed Agrobacterium sensitivity to the extracts. Fluorometric assay of bacterial cell viability indicated dose-dependent cytotoxic activity of callus extract against Agrobacterium cells. Addition of 0.1% Tween 20 and 2 g/l L-glutamine to Agrobacterium infection medium counteracted the bactericidal effect and significantly increased the T-DNA delivery to explants. A short preculture of explants for 2 days followed by infection with Agrobacterium in medium containing 150 μM of acetosyringone were found essential for efficient T-DNA delivery. Cocultivation for 3 days at 22 °C in conjunction with other optimized factors resulted in maximum T-DNA delivery. The Agrobacterium-mediated transformation of leaf disk explants were found significantly related to physiological age of the explants, age and origin of the of the donor plant. Leaf explants from second node of the 3-month-old in vivo plants showed highest transformation efficiency (94.3%) revealed by transient GUS expression assay. Plants selected on medium containing 20 mg/l kanamycin showed stable GUS expression in leaves and stem. The elongated shoots readily developed roots on kanamycin-free rooting medium and on transfer to soil, plants were successfully established. Polymerase chain reaction (PCR) and reverse-transcriptase PCR analysis in putative plants confirmed their transgenic nature. The established transformation method should provide new opportunities for the genetic improvement of patchouli for desirable trait.

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Acknowledgments

We thank Prof. K. Veluthambi, MKU, Madurai, India for Agrobacterium strain and the Center for Application of Molecular Biology to International Agriculture (CAMBIA), Australia for pCAMBIA2301. The research was partially supported by a program support grant from the Department of Biotechnology (DBT), Government of India. AP and DPS are grateful to DBT and SB to the Council of Scientific and Industrial Research (CSIR) for Senior Research Fellowship.

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  1. Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, 781039, Assam, India

    Anamika Paul, Souvika Bakshi, Debee Prasad Sahoo & Lingaraj Sahoo

  2. Department of Biotechnology, Gauhati University, Guwahati, 781014, Assam, India

    Anamika Paul & Mohan Chandra Kalita

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Paul, A., Bakshi, S., Sahoo, D.P. et al. Agrobacterium-Mediated Genetic Transformation of Pogostemon cablin (Blanco) Benth. Using Leaf Explants: Bactericidal Effect of Leaf Extracts and Counteracting Strategies. Appl Biochem Biotechnol 166, 1871–1895 (2012). https://doi.org/10.1007/s12010-012-9612-0

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