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High-frequency generation of transgenic tobacco plants after modified leaf disk cocultivation withAgrobacterium tumefaciens

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Abstract

A modified protocol for theAgrobacterium tumefaciens-mediated transformation of tobacco (Nicotina tabacum L.) leaf disks was developed for greater recovery of transgenic plants. Modifications include transformation ofAgrobacterium by a freeze-thaw procedure, initial cocultivation of leaf disks andAgrobacterium under vacuum, subsequent growth with nurse cells for one week, rooting of shoots in medium lacking carbenicillin, longer, growth in rooting medium, and a shortened “hardening” step. By this procedure, an average of 1.3 kanamycin-resistant calli were obtained per leaf disk, and 38% of, the callus cultures used were regenerated to produce 133 independently transformed tobacco plants.

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Abbreviations

2,4-D:

2,4-diphenoxyacetic acid

GUS:

β-glucuronidase

MS:

Murashige-Skoog

nptII :

gene coding for neomycin phosphotransferase

tml :

tumor morphology large

uidA :

gene coding for GUS. Additional abbreviations are listed under Materials and Methods

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Authors and Affiliations

  1. Department of Plant Pathology and Crop Physiology, Louisiana State University, 302 Life Sciences Building, 70803, Baton Rouge, LA, USA

    Mark D. Burow, Caryl A. Chlan, Partha Sen, Andreana Lisca & Norimoto Murai

Authors
  1. Mark D. Burow
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  2. Caryl A. Chlan
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  3. Partha Sen
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  4. Andreana Lisca
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  5. Norimoto Murai
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Burow, M.D., Chlan, C.A., Sen, P. et al. High-frequency generation of transgenic tobacco plants after modified leaf disk cocultivation withAgrobacterium tumefaciens . Plant Mol Biol Rep 8, 124–139 (1990). https://doi.org/10.1007/BF02669766

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  • DOI: https://doi.org/10.1007/BF02669766

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