Positive selection based on hormone signal transduction

Nippon Paper Industries Co. Ltd holds patents regarding vectors and methods of plant transformation using an auxin biosynthesis gene and/or cytokinin biosynthesis gene as positive selectable markers.

Technology overview

The patent family represented by US 6767735 and EP 1033409 discloses the use of plant hormone signal transduction genes as positive selection markers in a plant transformation vector, which can be excised after selection in order to generate selection marker-free transgenic plants.  This method eliminates selection of false-positive plants that have not acquired the gene but show a morphological abnormality phenotype due to the influence of hormone produced by the true transformants close by.

The Japanese patent application JP 2001275667 claims the invention of use of a cytokinin-related gene in plant transformation.  Addition of the cytokinin-related gene enhances transformation efficiency, as well as being useful as a positive selection marker gene (that can later be excised using transposons or a site-specific recombination system, or controlled by use of an inducible promoter upstream of the cytokinin-related gene).  The application states that the reason for enhanced transformation efficiency is unknown, but relates the effect to cytokinins causing enhanced cell division of the plant.

Another patent family, represented by the United States patent application  US 2004/163143, discloses a method of plant transformation using a vector that contains a gene coding for an enzyme that synthesises auxin from an auxin precursor (auxin synthesis gene).  By adding the auxin precursor to the selection medium, transformants will synthesise auxin and produce adventitious shoots.  The auxin synthesis gene can later be excised using a transposon or site-specific recombination system after selection to regenerate physiologically normal transformants with only the desired gene.  Although not claimed in this published application, the applicant suggests the parallel introduction of a cytokinin synthesis gene to control the auxin/cytokinin ratio in the plant.  The applicant states that the ‘appropriate’ auxin/cytokinin ratio in transformants (as opposed to non-transformants) will promote shoot generation.

Specific patent information

Patent/Application Number Title, Independent Claims and Summary Assignee
US 6767735

  • Earliest priority – 06 Jan 1999 (JP)
  • Filed – 05 Jan 2000
  • Granted – 27 Jul 2004
  • Expected expiry – 05 Jan 2020
Title – Vector for introducing a gene into a plant using a selectable marker

Claim 1 
A vector for introducing a gene into a plant, which comprises:a desired gene,

a cytokinin signal transduction gene which comprises the CKI1 gene from Arabidopsis thaliana and a cytokinin synthesis gene together as selectable marker genes, and

a removable DNA element,

wherein the selectable marker genes are positioned such that they behave integrally with the removable DNA element, and wherein the desired gene is positioned such that it does not behave integrally with the removable DNA element.

Nippon PaperIndustries Co. Ltd.

EP 1033409

  • Earliest priority – 06 Jan 1999 (JP)
  • Filed – 05 Jan 2000
  • Granted – Pending
  • Expected expiry – N/A
Title – Vector for introducing a gene into a plant using a selectable marker

Claim 1
A vector for introducing a gene into a plant, which comprisesa desired gene, and
a plant hormone signal transduction gene as a selectable marker gene.

The claim is drawn to a vector comprises a desired gene and a plant hormone signal transduction gene for selection.  A method for producing a transgenic plant cell is also claimed in the independent claim 10, which comprises the transformation of plant cells with the vector and the selection of transformed cells in the presence of plant hormones.

Remarks

Related patent applications were also filed in Canada (CA 2292798) and Japan (JP 2001218583).
JP 2001275667

  • Earliest priority – 31 March 2000
  • Filed – 31 March 2000
  • Granted – Pending
  • Expected expiry – N/A
Title – Method of enhancing gene-transferring efficiency for monocotyledon

Claim 1

A method of increasing transformation efficiency of a monocotyledonous plant upon obtaining transformed monocotyledonous plant tissue or body, characterised by introducing into said monocotyledonous plant cell
a desired gene, and
a cytokinin-related gene.

Remarks

There is no family information for this Japanese patent application according to INPADOC.
US 2004/163143

  • Earliest priority – 26 Jul 2002
  • Filed -26 Jul 2002
  • Granted – Pending
  • Expected expiry – N/A
Title – Method for efficiently producing transgenic plant using auxin precursor

Claim 1
A method for producing a transgenic plant, which comprises:(A) introducing a vector into a plant cell, wherein the vector is a vector for gene introduction into a plant and comprises: a desired gene, and a selectable marker gene comprising a gene encoding an enzyme which synthesizes auxin from an auxin precursor;
(B) culturing the plant cell into which the genes are introduced by the vector, in the presence of an auxin precursor and/or an analogue thereof to thereby prepare a redifferentiated tissue, and detecting and selecting the redifferentiated tissues; and
(C) culturing the redifferentiated tissue selected in (B) to redifferentiate a plant individual.
Claim 5
A vector for gene introduction into a plant, which comprises:a desired gene, and

a selectable marker gene comprising

an indoleacetamide hydrolase, iaaH, gene and

an isopentenyl transferase, ipt, gene

and being free of an tryptophan monooxygenase, iaaM, gene.

Definitions from the specification:

“Auxin” is a plant hormone and is known to accelerate elongation, proliferation and division of cells.

“Auxin precursor and/or an analogue” is a substance which is converted into auxin or a substance having a physiological activity similar to that of auxin (hereinafter also referred to as “auxin-like substance” as a whole) by the expression of the auxin precursor-auxin synthesis gene introduced as a selectable marker gene.

Remarks

Related patent applications were also filed in Japan (JP 2004057066), Australia (AU 2003221268 A1), Europe (EP 1384785 A1), Canada (CA 2436046 A1 ) and China (CN 1473936 A).