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Inhibition of enzymatic browning of Agrobacterium-transformed tissue

Patent Applications filed by the University of Minnesota

The present applications describe methods for inhibiting enzymatic browning of plant tissue, cells or parts of a plant in response to wounding. The disclosure describes agents that inhibit the activity or production of enzymes associated with browning such as polyphenol oxidase (PPO) and peroxidase (POD), chelators of metals required for enzymatic activity, and sulphydryl-containing agents that also inhibit PPO activity.

Specific Patent Information

Patent Number Title, Independent Claims and Summary of Claims Assignee
US 2004/187177 AA

  • Earliest priority – 15 December 1999
  • Filed – 30 March 2004
  • Application pending
 Title – Method to enhance Agrobacterium -mediated transformation of plants

Claim 1
A method for transforming plant explant tissue, comprising:a) contacting a cotyledon explant from a plant seedling infected with an Agrobacterium containing DNA to be introduced into the explant with an agent that inhibits enzymatic browning of a wounded plant, plant tissue or plant cell so as to yield transformed explant tissue; and

b) identifying transformed explant tissue.
Claim 22
A method to identify an agent that enhances Agrobacterium-mediated transformation of a plant cell, plant tissue or plant part, comprising:a) contacting the plant cell, plant tissue or plant part with Agrobacterium containing DNA to be introduced into the plant cell, plant tissue or plant part and the agent so as to yield a transformed plant cell, plant tissue or plant, wherein the agent is not a phenolic compound; and

b) detecting or determining whether the agent enhances Agrobacterium-mediated transformation of the plant cell, plant tissue or plant part relative to Agrobacterium-mediated transformation of a plant cell, plant tissue or plant part in the absence of the agent.
Claim 37
A method for the stable transformation of plant tissue or cells, comprising:a) contacting plant tissue or cells with an Agrobacterium containing DNA and an agent selected from the group consisting of a sulfhydryl-containing agent, an iron chelator, a copper chelator, an inhibitor of plant polyphenol oxidase and an inhibitor of plant peroxidase; and

b) identifying stably transformed plant tissue or cells.
Claim 44
A plant medium comprising: an amount of an agent effective to inhibit the enzymatic browning of a plant organ, tissue or cell, wherein the agent is selected from the group consisting of a sulfhydryl-containing agent, an iron chelator, a copper chelator, an inhibitor of polyphenol oxidase and an inhibitor of peroxidase.
Claim 57
A method for the stable transformation of monocot plant tissue or cells, comprising:a) contacting monocot plant tissue or cells with an Agrobacterium containing a recombinant DNA and one or more agents selected from the group consisting of a sulfhydryl-containing agent, methionine, an iron chelator, a copper chelator, an inhibitor of plant polyphenol oxidase and an inhibitor of plant peroxidases, which one or more agents are present in solid media in an amount effective to enhance the stable transformation of the monocot plant tissue or cells relative to corresponding monocot plant tissue or cells contacted with Agrobacterium in the absence of the one or more agents; and

b) identifying stably transformed plant tissue or cells.
Claim 62
A method for the stable transformation of plant tissue or cells, comprising:a) contacting plant tissue or cells with an Agrobacterium containing a recombinant DNA and one or more agents selected from the group consisting of a sulfhydryl-containing agent, methionine, an iron chelator, a copper chelator, an inhibitor of plant polyphenol oxidase and an inhibitor of plant peroxidases, which one or more agents are present in solid media in an amount effective to enhance the stable transformation of the tissue or cells relative to corresponding tissue or cells contacted with Agrobacterium in the absence of the one or more agents; and

b) identifying stably transformed plant tissue or cells.

This application is a continuation of now granted US 6759573 (see below).

The claims are not limited to the transformation any particular plant type.

Regents of the University of Minnesota
US 6759573

  • Earliest priority – 15 December 1999
  • Filed – 15 December 2000
  • Granted – 6 July 2004
  • Expected expiry – 15 July 2021 (213 day-extension from 14 December 2020)
 Title – Method to enhance Agrobacterium -mediated transformation of plants

Claim 1
A method for stable transformation of leguminous plant tissue or cells, comprising:a) contacting leguminous plant tissue or cells with an Agrobacterium comprising a recombinant DNA and one or more sulfhydryl-containing agents which one or more agents are present in solid media in an amount effective to enhance the stable transformation of the leguminous plant tissue or cells relative to corresponding plant tissue or cells contacted with the Agrobacterium in the absence of the one or more agents, wherein the plant tissue or cells are embryogenic somatic cells, immature embryo, meristem, or a cotyledon explant, and wherein the stable transformation is enhanced by at least 0.5%; and
b) identifying stably transformed plant tissue or cells.

Granted patent US 6759573 recites a method of Agrobacterium-mediated transformation that is limited to those of leguminous plant tissue or cells.

The claims require that the sulfhydryl-containing agents are in a solid media.  Thus, if the agents are added in solution such a method might not be covered by the claim.

The patent was first published as US 2001/34888 A1.
WO 2001/44459 A2

  • Earliest priority – 15 December 1999
  • Filed – 15 December 2000
  • Publication date – 21 June 2001
  • Expected expiry- N/A
 Title – Method to enhance Agrobacterium -mediated transformation of plants

Claim 1

A method for transforming plant explant tissue, comprising:

a) contacting a cotyledon explant from a plant seedling infected with an Agrobacterium containing DNA to be introduced into the explant with an agent that inhibits enzymatic browning of a wounded plant, plant tissue or plant cell so as to yield transformed explant tissue; and
b) identifying transformed explant tissue.
Claim 22
A method to identify an agent that enhances Agrobacterium-mediated transformation of a plant cell, plant tissue or plant part, comprising:a) contacting the plant cell, plant tissue or plant part with Agrobacterium containing DNA to be introduced into the plant cell, plant tissue or plant part and the agent so as to yield a transformed plant cell, plant tissue or plant, wherein the agent is not a phenolic compound; and
b) detecting or determining whether the agent enhances Agrobacterium-mediated transformation of the plant cell, plant tissue or plant part relative to Agrobacterium-mediated transformation of a plant cell, plant tissue or plant part in the absence of the agent.
Claim 37
A method for the stable transformation of plant tissue or cells, comprising:a) contacting plant tissue or cells with an Agrobacterium containing DNA and an agent selected from the group consisting of a sulfhydryl-containing agent, an iron chelator, a copper chelator, an inhibitor of plant polyphenol oxidase and an inhibitor of plant peroxidase; and
b) identifying stably transformed plant tissue or cells.
Claim 44
A plant medium comprising: an amount of an agent effective to inhibit the enzymatic browning of a plant organ, tissue or cell, wherein the agent is selected from the group consisting of a sulfhydryl-containing agent, an iron chelator, a copper chelator, an inhibitor of polyphenol oxidase and an inhibitor of peroxidase.

The claims as filed of the PCT application recite:

  • a method for inhibiting enzymatic browning in cotyledon explant to be infected with Agrobacterium by introducing an agent that inhibits browning
  • a method to identify an agent that enhances Agrobacterium-mediated transformation of a plant cell, tissue or plant part  so long as the agent is not a phenolic compound
  • methods where agents that inhibit browning are either put into contact with the explant to be transformed or present in the plant medium. The agents are metal chelators, sulphydryl-containing agents or other enzyme inhibitors.
  • The degree of enhancement required to fall within the scope of the claims is unclear (only that it’s more transformation than observed without the agent) and the disclosure provides a number of different assays to judge enhancement by.
Remarks
  1. National phase entry of WO 2001/44459 in Australia (AU 200122672) has lapsed on 15 August 2002.
  2. National phase entry of WO 2001/44459 in Canada (CA 2394367) and EP (EP 1240341) are pending.
  3. Other national phase entries of WO 2001/44459 include: Brazil (BR 200016367)

Note: Patent information on this page was last updated on 6 March 2006.