Floral Transformation

Floral Transformation – patent granted to Paradigm Genetics Inc.

The invention disclosed in this patent comprises an alternative protocol to adjust the cell density of the Agrobacteria used in the transformation step. This method should be useful in high throughput transformation protocols.  According to the INPADOC patent family information, this patent is granted only in the United States.

Specific patent information

Patent Number Title, Independent Claims and Summary of Claims Assignee
US 6353155 B1

  • Earliest priority – 30 June 2000
  • Filed – 30 June 2000
  • Granted – 5 March 2002
  • Expected expiry – 30 June 2020
Title – Method for transforming plants

Claim 1
A method for preparing a transgenic plant or seed comprising:a) growing a suspension of Agrobacterium cells until the optical density of the suspension is about 2 to about 2.4 at a wavelength of 600 nanometers, wherein said Agrobacterium cells contain at least one plasmid having a DNA sequence of interest flanked by T-DNA borders;
b) diluting said Agrobacterium cells of said suspension with an aqueous medium so that the optical density of the suspension is reduced to about 0.6 to about 1.5, wherein said diluting is not followed by pelleting via centrifugation with subsequent resuspension; and
c) treating the flower of said plant with said diluted suspension so that the Agrobacterium cells in said diluted suspension can transform said plant with the DNA-sequence of interest;
d) optionally, cultivating said treated plant to produce seed; and
e) optionally, growing plants from said seed and selecting for transgenic plants having said DNA sequence of interest.
Claim 10
A method for preparing a transgenic plant or seed comprising:a) growing a suspension of Agrobacterium cells until growth of Agrobacterium cells in the suspension is substantially completed, wherein said Agrobacterium cells contain at least one plasmid having a DNA sequence of interest flanked by T-DNA borders;
b) diluting said suspension with an aqueous medium to reduce the concentration of Agrobacterium cells and any other components in the growth medium and allow the Agrobacterium cells to infect the plant without harming it, wherein said diluting is not followed by pelleting via centrifugation with subsequent resuspension;
c) treating the flower of said plant with said diluted suspension so that the Agrobacterium cells in said diluted suspension can transform said plant with the DNA-sequence of interest;
d) optionally, cultivating said treated plant to produce seed; and
e) optionally, growing plants from said seed and selecting for transgenic plants having said DNA sequence of interest.
Claim 17
A method for preparing a transgenic plant or seed comprising:a) growing a suspension of Agrobacterium cells until growth of Agrobacterium cells in the suspension is substantially completed, wherein said Agrobacterium cells contains at least one plasmid having a DNA sequence of interest flanked by T-DNA borders;
b) diluting said Agrobacterium cells in said suspension with about 2 to about 10 volumes aqueous medium per volume of suspension, wherein said diluting is not followed by pelleting via centrifugation with subsequent resuspension;
c) treating the flower of said plant with said diluted suspension so that the Agrobacterium cells in said diluted suspension can transform said plant with the DNA-sequence of interest;
d) optionally, cultivating said treated plant to produce seed; and
e) optionally, growing plants from said seed and selecting for transgenic plants having said DNA sequence of interest.

United States patent US 6353155 claims

  • a method to transform floral tissue in vivo by treatment with an Agrobacterium cell suspension adjusted to a certain density by dilution with an aqueous solution.
  • The claims exclude pelleting by centrifugation after the dilution step, but not by other means.
  • Claim 10 states that Agrobacterium should be allowed to infect a plant without harming it.  The patent does not define what “harm” means.

The invention comprises an alternative protocol to adjust the cell density of the Agrobacteria used in the transformation step. According to the inventors, centrifugation and resuspension are avoided by direct dilution of the bacteria in an aqueous solution, thereby allowing sequential treatment of many more plants. The method is well-suited for the transformation of Arabidopsis thaliana (Brassicaceae), a model plant where such a high throughput transformation protocol might be required. No other plants are used in the only example provided as part of the patent specification.

Paradigm Genetics Inc.

Note: Patent information on this page was last updated on 23 February 2006.