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Genetic transformation and regeneration of Sesbania drummondii using cotyledonary nodes

  • Genetic Transformation and Hybridization
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Abstract

Sesbania drummondii (Rydb.) Cory is a source for phytopharmaceuticals. It also hyperaccumulates several toxic heavy metals. Development of an efficient gene transfer method is an absolute requirement for the genetic improvement of this plant with more desirable traits due to limitations in conventional breeding methods. A simple protocol was developed for Agrobacterium-mediated stable genetic transformation of Sesbania. Agrobacterium tumefaciens strain EHA 101 containing the vector pCAMBIA 1305.1 having hptII and GUS plus genes was used for the gene transfer experiments. Evaluation of various parameters was carried out to assess the transformation frequency by GUS expression analysis. High transformation frequency was achieved by using 7-day-old precultured cotyledonary node (CN) explants. Further, the presence of acetosyringone (150 μM), infection of explants for 30–45 min and 3 days of cocultivation proved to be critical factors for greatly improving the transformation efficiency. Stable transformation of S. drummondii was achieved, and putative transgenic shoots were obtained on medium supplemented with hygromycin (25 mg l−1). GUS histochemical analysis of the putative transgenic tissues further confirmed the transformation event. Genomic Southern blot analysis was performed to verify the presence of transgenes and their stable integration. A transformation frequency of 4% was achieved for CN explants using this protocol.

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Abbreviations

BA:

6-Benzylaminopurine

CN:

Cotyledonary node

GUS:

β-d-glucuronidase

hptII:

Hygromycin phosphotransferase

IAA:

Indole acetic acid

IBA:

Indole butyric acid

LB:

Luria broth

NAA:

α-Naphthaleneacetic acid

MS:

Murashige and Skoog’s medium

PCR:

Polymerase chain reaction

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Acknowledgments

Authors would like to acknowledge Satish Cheepala for technical assistance and Dr. Linda Gonzales for critical review of the manuscript. Financial support for this work was provided by the Applied Research and Technology Program and the Faculty Scholarship Award from the Graduate studies of Western Kentucky University.

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Authors and Affiliations

  1. Department of Biology, Western Kentucky University, 1906 College Heights Blvd. # 11080, Bowling Green, KY, 42101-1080, USA

    Priya Padmanabhan & Shivendra V. Sahi

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  1. Priya Padmanabhan
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  2. Shivendra V. Sahi
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Correspondence to Shivendra V. Sahi.

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Communicated by P. Ozias-Akins.

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Padmanabhan, P., Sahi, S.V. Genetic transformation and regeneration of Sesbania drummondii using cotyledonary nodes. Plant Cell Rep 28, 31–40 (2009). https://doi.org/10.1007/s00299-008-0618-6

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  • DOI: https://doi.org/10.1007/s00299-008-0618-6

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