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Soybean

Summary

The University of Toledo, Monsanto, Pioneer Hi-Bred and University of Mississippi collectively have several patents granted in the United States, Canada, Australia and Europe which are directed to Agrobacterium transformation of soybean (Glycine max).

The University of Toledo has both an Australian and a United States patent which claim a non-tissue culture process to transform meristematic or mesocotyl cells of a soybean seed. An Agrobacterium strain of any species is used for the transformation and can contain either an armed (with tumor-causing genes) or a disarmed vector. A related European patent additionally claims a method for transforming beans (Phaseolus vulgaris).

In the United States patents granted to Monsanto, a cotyledon from a soybean seedling or an embryonic axis are used for transformation with Agrobacterium containing a disarmed vector. Additionally they claim the insertion of a gene encoding for neomycin phosphotransferase II (nptII) and a gene encoding EPSPS. The first gene confers resistance to kanamycin and neomycin among other antibiotics, and the later confers resistance to the herbicide glyphosate.

A  United States patent (US 6384301 B1) and published application (US 2002/0157139 A1) disclose a method for soybean transformation using a wounded embryonic axis isolated from germinating seed as target for Agrobacterium-mediated transformation. The explant is cultured in a medium containing a selection agent. The difference between the granted patent and the application is that in the patent application the wounding step is omitted, thereby providing an alternative protocol to obtain transgenic soybean plants.

In the European and Australian applications, Monsanto discloses a method to prepare a transformed germline of soybean starting with an embryonic axis of a soybean seed for transformation process.

The inventions disclosed by Pioneer Hi-Bred in patents granted in the United States (2), Australia (1) and Canada (1) use a hypocotyl or a cotyledonary node of a soybean seed transformed with any species of Agrobacterium. Detailed protocols for co-cultivation of a soybean explant with the bacterium are also claimed.

University of Mississippi owns a United States patent (US 5968830) on an Agrobacterium-mediated transformation method of a hypocotyl soybean explant, which also recites a method of regenerating soybean via organogenesis.

Soybean – Patents granted to The University of Toledo

Specific Patent Information

Patent Number Title, Independent Claims and Summary of Claims Assignee
US 5376543

  • Earliest priority – 21 December 1987
  • Filed – 7 December 1992
  • Granted – 27 December 1994
  • Expired – 29 January 2003
 Title – Agrobacterium mediated transformation of germinating plant seeds

Claim 1A non-tissue culture process for producing a transgenic soybean plant, which process comprises:

A) germinating a seed of a Glycine max plant for about 24 to 48 hours;
B) inoculating the meristematic or mesocotyl cells produced by the germinating seed of step (A), prior to differentiation of said cells, with an armed or disarmed Agrobacterium strain containing an Agrobacterium-derived vector, said vector containing a transferable gene; and
C) allowing the cells to differentiate into a mature plant.

Granted US 5376543 has expired due to non-payment of maintenance fees as reported by USPTO.

Production of a transgenic soybean plant through a non-tissue culture process. Meristematic or mesocotyl cells are inoculated with an armed or disarmed Agrobacterium vector having a gene of interest. The cells differentiate into a mature plant.

 University of Toledo
AU 648951 B2

  • Earliest priority – 21 December 1987
  • Filed – 27 April 1993
  • Granted – 5 May 1994
  • Expired – 18 July 2002
 Title – Agrobacterium mediated transformation of germinating plant seeds

Claim 1A non-tissue culture process for producing a transgenic plant, which process comprises:

A) germinating a seed of a Glycine max plant for about 24 to 48 hours;
B) inoculating the meristematic or mesocotyl cells produced by the germinating seed of step (A), prior to differentiation of said cells, with an armed or disarmed Agrobacterium strain containing an Agrobacterium-derived vector, said vector containing a transferable gene; and
C) allowing the cells to differentiate into a mature plant.

Granted AU 648951 recites the same process of producing a transgenic soybean plant in claim 1 as that of US 5376543 (see above).
EP 397687 B1

  • Earliest priority – 21 December 1987
  • Filed – 16 December 1988
  • Granted – 11 May 1994
  • Expected expiry – 16 December 2008
 Title – Agrobacterium mediated transformation of germinating plant seeds

Claim 1A non-tissue culture process for producing a transgenic plant, which comprises:

A) germinating a seed of a Phaseolus vulgaris or a Glycine max plant for 24 to 48 hours;
B) inoculating the meristematic or mesocotyl cells produced during germination, prior to differentiation of the seed, with a virulent or non-virulent Agrobacterium strain containing a transferable gene in an Agrobacterium -derived vector; and
C) allowing the cells to differentiate into a mature plant.

Granted EP 397687 is a national phase entry of WO 1989/05859.

Designated contracting States at the time of grant are: Austria (patent lapsed as reported by INPADOC), Belgium (patent lapsed as reported by INPADOC), Switzerland (patent lapsed as reported by INPADOC), Germany (patent lapsed as reported by INPADOC), France (patent lapsed as reported by INPADOC), United Kingdom (patent lapsed as reported by INPADOC), Italy, Liechtenstein, Luxembourg, Netherlands (patent lapsed as reported by INPADOC), Sweden (patent lapsed as reported by INPADOC).

The method for producing a transgenic soybean plant is the same as the one disclosed in the related United States and Australian patents (above mentioned), but transformation of beans (Phaseolus vulgaris) is also included in the claims (see section on beans).
Remarks
  1. National phase entry of WO 1989/05859 in Japan (JP H04/501201) is deemed to be withdrawn.
  2. Other national phase entries of WO 1989/05859 include Denmark (DK 126690) and Kuwait (KR 154872).

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

Soybean – Patents granted to and patent applications filed by Monsanto

Specific Patent Information

Patent Number Title, Independent Claims and Summary of Claims Assignee
US 5416011

  • Earliest priority – 22 July 1988
  • Filed – 23 November 1993
  • Granted – 16 May 1995
  • Expected expiry – 16 May 2012
 Title – Method for soybean transformation and regeneration

Claim 1A method for transforming soybeans which comprises:

A) preparing a cotyledon explant from a soybean seedling by:
(i) removing the hypocotyl region by cutting just below the cotyledonary node,
(ii) separating the two cotyledons at the cotyledonary node by tearing the cotyledons apart, and
(iii) removing the epicotyl from the cotyledon to which it remains attached, B) inserting a chimeric gene into the explant of part (A) by inoculation of at least the region adjacent to the axillary bud of the explant with a disarmed Agrobacterium tumefaciens vector containing said chimeric gene;
C) selecting transformed explant tissue, and
D) regenerating a differentiated transformed plant from the transformed explant tissue of part (C).
Claim 10A method for transforming soybeans which comprises:

A) preparing a cotyledon explant from a soybean seedling by:
(i) removing the hypocotyl region by cutting just below the cotyledonary node,
(ii) separating the two cotyledons at the cotyledonary node by tearing the cotyledons apart, and
(iii) removing the epicotyl from the cotyledon to which it is attached, and
(iv) wounding the explant by making at least one cut in the axillary bud region of the explant, B) inserting a chimeric gene into the explant of part (A) which gene encodes for neomycin phosphotransferase II by inoculation and co-cultivation of the explant with a disarmed Agrobacterium tumefaciens vector containing said chimeric gene;
C) selecting transformed explant tissue by growing the explant in the presence of kanamycin, and
D) regenerating a differentiated transformed plant from the transformed explant tissue of part (C).
Claim 12A method for transforming soybeans which comprises:

A) preparing a cotyledon explant from a soybean seedling by:
(i) removing the hypocotyl region by cutting just below the cotyledonary node,
(ii) separating the two cotyledons at the cotyledonary node, and
(iii) removing the epicotyl from the cotyledon to which it remains attached, B) inserting a chimeric gene into the explant of part (A) by inoculation of at least the region adjacent to the axillary bud of the explant with a disarmed Agrobacterium tumefaciens vector containing said chimeric gene;
C) selecting transformed explant tissue, and
D) regenerating a differentiated transformed plant from the transformed explant tissue of part (C).

Transformation of a cotyledon of a soybean seedling by inoculation with a disarmed A. tumefaciens vector having a chimeric gene. As part of the invention, the chimeric gene encodes for npt II.

 Monsanto
US 5569834

  • Earliest priority – 22 July 1988
  • Filed – 14 February 1995
  • Granted – 29 October 1996
  • Expected expiry – 16 May 2012
 Title – Method for soybean transformation and regeneration

Claim 1A soybean plant comprising a chimeric gene and associated DNA resulting from an Agrobacterium tumefaciens -mediated transformation, said chimeric gene capable of conferring kanamycin resistance to said soybean plant, produced by the method which comprises:

A) preparing a cotyledon explant from a soybean seedling by:
(i) removing the hypocotyl region by cutting just below the cotyledonary node,
(ii) separating the two cotyledons at the cotyledonary node by tearing the cotyledons apart, and
(iii) removing the epicotyl from the cotyledon to which it remains attached, B) inserting a chimeric gene into the explant of part (A) by inoculation and co-cultivation of the explant with a disarmed Agrobacterium tumefaciens vector containing said chimeric gene;
C) selecting transformed explant tissue, and
D) regenerating a differentiated transformed plant from the transformed explant tissue of part (C).

United States granted patent US 5569834 is a divisional of now granted US 5416011 (see above).

Method to obtain a soybean plant resistant to kanamycin via A. tumefaciens transformation. The explant for transformation is a cotyledon isolated from a soybean seedling.
US 5824877

  • Earliest priority – 22 July 1988
  • Filed – 25 October 1996
  • Granted – 20 October 1998
  • Expected expiry –
  • 16 May 2012
 Title – Method for soybean transformation and regeneration

Claim 1A method for transforming soybean which comprises:

A) preparing a cotyledon explant from a soybean seedling by:
(i) removing the hypocotyl region by cutting just below the cotyledonary node,
(ii) separating the two cotyledons at the cotyledonary node by tearing the cotyledons apart, and
(iii) removing the epicotyl from the cotyledon to which it remains attached, B) inserting a chimeric gene into the explant of part (A) by inoculation and co-cultivation of the explant with a disarmed Agrobacterium tumefaciens vector containing said chimeric gene;
C) selecting transformed explant tissue, and
D) regenerating a differentiated transformed plant from the transformed explant tissue of part (C).
Claim 10A method for transforming soybean which comprises:

A) preparing a cotyledon explant from a soybean seedling by:
(i) removing the hypocotyl region by cutting just below the cotyledonary node,
(ii) separating the two cotyledons at the cotyledonary node by tearing the cotyledons apart, and
(iii) removing the epicotyl from the cotyledon to which it is attached, and
(iv) wounding the explant by making at least one cut in the axillary bud region of the explant, B) inserting a chimeric gene into the explant of part (A) which gene encodes for 5-enolpyruvylshikimatephosphate synthase by inoculation and co-cultivation of the explant with a disarmedAgrobacterium tumefaciens vector containing said chimeric gene;
C) selecting transformed explant tissue by growing the explant in the presence of glyphosate, and
D) regenerating a differentiated transformed plant from the transformed explant tissue of part (C).

United States granted patent US 5824877 is a divisional of now granted US 5569834 (see above).

As in the previous United States patents, a cotyledon explant is transformed by inoculation and co-cultivation with A. tumefaciens containing a gene encoding glyphosate resistance.
US 5959179

  • Earliest priority – 13 March 1996
  • Filed – 13 March 1996
  • Granted – 28 September 1999
  • Expected expiry – 13 March 2016
 Title – Method for transforming soybeans

Claim 1A method for transforming soybean, comprising:

A) preparing a cotyledon explant from a soybean seedling by:
(i) incubating said seedling at about 0ºC to about 10ºC for at least 24 hours;
(ii) removing the hypocotyl region by cutting in the region of from about 0.2 to about 1.5 cm below the cotyledonary node;
(iii) splitting and completely separating the remaining attached hypocotyl segment, also thereby separating the two cotyledons;
(iv) removing the epicotyl from the cotyledon to which it remains attached; and
(v) wounding the cotyledon in the region of the axillary bud; B) inserting DNA to be introduced into said explant of step (a) by inoculating at least the region adjacent to the axillary bud of the explant with a disarmed Agrobacterium tumefaciens vector containing said DNA;
C) selecting transformed explant tissue; and
D) regenerating a differentiated transformed plant from said transformed explant tissue of step (c).

A method for transforming a soybean cotyledon by inoculating into the axillary bud region a disarmed A. tumefaciens vector containing DNA of interest.
WO 1998/00557 A2

  • Earliest priority – 28 June 1996
  • Filed – 28 May 1997
  • OPI – 8 January 1998
 Title – Methods of optimizing substrate pools and biosynthesis of poly-‘beta’-hydroxybutyrate-co-poly-‘beta’-hydroxyvalerate in bacteria and plants

Claim 37
A method for transforming soybean, comprising:
(a) preparing a cotyledon explant from a soybean seedling by:
(i) incubating said seedling at about 0˚C to about 10˚C for at least 24 hours;
(ii) removing the hypocotyl region by cutting in the region of from about 0.2 to about 1.5 cm below the cotyledonary node;
(ii) splitting and completely separating the remaining attached hypocotyl segment, also thereby separating the two cotyledons;
(iii) removing the epicotyl from the cotyledon to which it remains attached; and
(iv) wounding the cotyledon in the region of said axillary bud;
(b) inserting DNA to be introduced into said explant of step (a) by inoculating at least the region adjacent to the axillary bud of the explant with a disarmed Agrobacterium tumefaciens vector containing said DNA;
(c) selecting transformed explant tissue; and
(d) regenerating a differentiated transformed plant from said transformed explant tissue of step (c).

PCT application WO 1998/00557 recites genes and methods for optimizing levels of substrates employed in the biosynthesis of copolymers of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) in plants and bacteria using recombinant DNA technology. Claim 37 is one of the independent claims that recite a method to transform soybean using a disarmed A. tumefaciens containing DNA that is to be introduced.
US 6384301

  • Earliest priority – 14 January 1999
  • Filed – 20 December 2001
  • Granted – 24 October 2002
  • Expected expiry – 20 December 2021
 Title – Soybean Agrobacterium transformation method

Claim 1
A method of making germline-transformed soybean plants using Agrobacterium mediation, the method comprising:(a) initiating the germination of a soybean seed;
(b) isolating the embryonic axis including the embryonic meristem from the soybean seed to prepare an explant;
(c) wounding the explant;
(d) exposing the explant to a disarmed Agrobacterium vector comprising a heterologous genetic construct comprising a selectable marker gene wherein the heterologous genetic construct is transferred into at least one cell in the explant;
(e) culturing the explant in the presence of a selection agent in a manner allowing identification of soybean cells of the explant to which the heterologous genetic construct has been transferred;
(f) inducing formation of one or more shoots from the explant, the shoot comprising germline transformed cells;
(g) cultivating the shoot into a whole fertile mature soybean plant.

A method for soybean transformation using a wounded embryonic axis isolated from germinating seed as a target for Agrobacterium-mediated transformation. The explant is cultured in a medium containing a selection agent.
US 7002058

  • Earliest priority – 14 January 1999
  • Filed – 20 December 2001
  • Granted – 21 February 2006
  • Expected expiry – 20 December 2021
 Title – Soybean transformation method

Claim 1
A method of making germline-transformed soybean plants using Agrobacterium mediation, the method comprising:(a) initiating the germination of a soybean seed;
(b) isolating the embryonic axis including the embryonic meristem from the soybean seed to prepare an explant;
(c) exposing the explant to a disarmed Agrobacterium vector comprising a heterologous genetic construct comprising a selectable marker gene wherein the heterologous genetic construct is transferred into at least one cell in the explant;
(d) culturing the explant in the presence of a selection agent in a manner allowing identification of soybean cells of the explant to which the heterologous genetic construct has been transferred;
(e) inducing formation of one or more shoots from the explant, the shoot comprising germline transformed cells;
(f) cultivating the shoot into a whole fertile mature soybean plant.
Claim 11
A method of transforming a soybean cell using Agrobacterium comprising:(a) initiating the germination of a soybean seed;
(b) isolating the embryonic axis including the embryonic meristem from the soybean seed to prepare an explant;
(c) exposing the explant to a disarmed Agrobacterium vector comprising a heterologous genetic construct comprising a selectable marker gene wherein the heterologous genetic construct is transferred into at least one cell in the explant;
(d) culturing the explant in the presence of a selection agent in a manner allowing identification of soybean cells of the explant to which the heterologous genetic construct has been transferred.

United States granted patent US 7002058 is a continuation of now granted US 6384301.

An Agrobacterium-mediated transformation method of soybean, where the embryonic axis does not require wounding before it is exposed to a disarmed Agrobacterium in comparison to claim 1 of US 6384301 (see above), where wounding of the embryogenic axis is included in the transformation step.
US 2006/0059589 A1

  • Earliest priority – 14 January 1999
  • Filed – 4 November 2005
  • Application pending
 Title – Soybean transformation method

Claim 11
A method of producing a transformed soybean cell comprising:(a) initiating the germination of a soybean seed;
(b) substantially removing at least one cotyledon from the germinating seed;
(c) exposing at least a portion of the meristematic region in the germinating seed;
(d) exposing one or more cells in the meristematic region to a disarmed Agrobacterium vector comprising a heterologous genetic construct, wherein said genetic construct comprises a selectable marker gene, and said genetic construct is transferred into at least one cell in the meristematic region;
(e) culturing the cells of the meristematic region in the presence of a selection agent to identify a transformed soybean cell that contains the genetic construct.
Claim 25
A method of producing a fertile transgenic soybean plant comprising:(a) initiating the germination of a soybean seed;
(b) substantially removing at least one cotyledon from the germinating seed;
(c) exposing at least a portion of the meristematic region in the germinating seed;
(d) exposing one or more cells in the meristematic region to a disarmed Agrobacterium vector comprising a heterologous genetic construct, wherein said genetic construct comprises a selectable marker gene, and said genetic construct is transferred into at least one cell in the meristematic region;
(e) culturing the cells of the meristematic region in the presence of a selection agent in a manner that identifies a transformed soybean cell that contains the genetic construct;
(f) inducing formation of one or more shoots from the meristematic region, wherein said shoot comprises at least one transformed cell, to produce a transgenic soybean shoot;
(g) cultivating the transgenic soybean shoot into a fertile transgenic soybean plant.

United States patent application US 2006/0059589 is a divisional of now granted US 7002058 (see above).

Claims 1-10 in this application have been cancelled according to the publication.

This application recites an Agrobacterium-mediated transformation method of soybean, where cells in the meristematic tissue are exposed to a disarmed Agrobacterium.
WO 2000/42207 A2

  • Earliest priority – 4 November 1999
  • Filed – 12 January 2000
  • OPI – 20 July 2000
 Title – Soybean transformation method

Claim 1
A method of preparing a germline-transformed soybean plant using Agrobacterium mediation, the method comprising:
(a)   initiating the germination of a soybean seed;
(b)  isolating the embryonic axis from the soybean seed to prepare an explant;
(c)   wounding the explant;
(d)   exposing the explant to a disarmed Agrobacterium vector comprising a heterologous genetic construct including a selectable marker gene under conditions in which the heterologous genetic construct is transferred into at least one cell in the explant;
(e)   culturing the explant in the presence of a selection agent in a manner capable of identifying soybean cells of the explant to which the heterologous genetic construct has been transferred;
(f)   inducing formation of one or more shoots from the explant, the shoot comprising germline transformed cells;
(g)   cultivating the shoot into a whole fertile mature soybean plant.

PCT application WO 2000/42207 recites an Agrobacterium-mediated transformation method of soybean, where a wounded embryonic axis is exposed to a disarmed Agrobacterium.
Remarks
  1. National phase entry of WO 1998/00557 in Australia (AU 31443/97) has lapsed on 25 March 1999.
  2. National phase entry of WO 1998/00557 in Canada (CA 2259251) is pending.
  3. National phase entry of WO 2000/42207 in Australia (AU 2000/28488) has lapsed on 18 August 2005.
  4. National phase entry of WO 2000/42207 in Canada (CA 2359868) has lapsed on 12 January 2004.
  5. National phase entry of WO 2000/42207 in Europe (EP 1141346) is deemed to be withdrawn on 3 March 2004.
  6. National phase entry of WO 2000/42207 in Japan (JP 2002534129) is pending.

Note: Patent information on this page was last updated on 24 March 2006.

Soybean – Patent applications filed by Monsanto

Actual pending claims

EP 1 141 346 A1* & AU 28488/00
Claim 1A method of preparing a germline-transformed soybean plant using Agrobacterium mediation, the method comprising: A) initiating the germination of a soybean seed;
B) isolating the embryonic axis from the soybean seed to prepare an explant;
C) wounding the explant;
D) exposing the explant to a disarmed Agrobacterium vector comprising a heterologous genetic construct including a selectable marker gene under conditions in which the heterologous genetic construct is transferred into at least one cell in the explant;
E) culturing the explant in the presence of a selection agent in a manner capable of identifying soybean cells of the explant to which the heterologous genetic construct has been transferred;
F) inducing formation of one or more shoots from the explant, the shoot comprising germline transformed cells;
G) cultivating the shoot into a whole fertile mature soybean plant.

* The PCT application WO 0042207 A2 was converted into the present European application.

Soybean – Patents granted to and patent applications filed by Pioneer Hi-Bred

Specific Patent Information

Patent Number Title, Independent Claims and Summary of Claims Assignee
US 5563055

  • Earliest priority – 27 July 1992
  • Filed – 28 March 1994
  • Granted – 8 October 1996
  • Expired – 11 November 2004
 Title – Method of Agrobacterium-mediated transformation of cultured soybean cells

Claim 1
A method for transforming soybean cells, comprising the steps of(A) providing a complete plant medium that supports rapid division of plant cells, said medium comprising
(i) a virulence-inducing amount of a signal molecule,
(ii) a growth promoting amount of an auxin, and
(iii) Agrobacterium tumefaciens bacteria in log growth phase, such that said bacteria are present in said medium in a concentration of about 108 viable cells per ml, wherein said bacteria contain a chimeric gene and said medium is buffered at a pH below 6.0;
(B) introducing into a first portion of said medium a plurality of germinated soybean seeds, from each of which seeds seed coat and radicle have been removed, and separating the cotyledons of each seed so as to expose the cotyledonary node of each seed, whereby a plurality of explants is produced; then
(C) macerating said cotyledonary node, without cutting entirely through each of said explants to the abaxial side thereof, and thereafter maintaining said explants in said first portion, at room temperature, for at least about 30 minutes; then
(D) transferring said explants to a second portion of said medium in solidified form, such that said explants are embedded in said medium, adaxial side up and level with the surface of said medium, and culturing said explants for about 3 days at about 22° C.;
(E) treating said explants in counterselection medium;
(F) cultivating said explants in agarose-solidified selection medium, wherein said explants are embedded adaxial side down in said selection medium, whereby transformed cells in said explants are favored; and then
(G) selecting transformed cells from said explants.

Transformation of cotyledonary nodes of soybean seeds in a medium containing A. tumefaciens, an auxin, and a signal molecule that induces virulence. Transformed tissue is subsequently selected.

Pioneer Hi-Bred
AU 670316 B2

  • Earliest priority – 27 July 1992
  • Filed – 26 July 1993
  • Granted – 11 July 1996
  • Expired – 24 February 2005
 Title – An improved method of Agrobacterium-mediated transformation of cultured soybean cells

Claim 1A genotype-independent method for producing a transgenic soybean plant which comprises:

A) co-cultivating an explant derived from a hypocotyl or cultured cotyledonary nodes of a germinated soybean seed with cells of anAgrobacterium species containing a chimeric gene at a concentration of 108 to 3 x 108 cells/ml and in the presence of a signal compound selected from the group consisting of acetosyringone, alfa-hydroxyacetosyringone, acetovanillone, syringaldehyde, syringic acid, sinapinic acid and mixtures thereof;
B) maintaining a temperature for co-cultivation of from 18 to 28°C; and
C) inducing virulence of the Agrobacterium by decreasing the pH of the plant culture media below pH 6.0.

Transformation of a hypocotyl or a cotyledonary node of a soybean seed by co-cultivation with Agrobacterium in a medium having specific signal compounds that induce Agrobacterium virulence, in order to regenerate transgenic soybean plants.

A divisional patent of now granted AU 670316 has also been granted (AU 691423 B2, expired 24 February 2005), however the claims do not recite an Agrobacterium-mediated transformation method of soybean, but a method to regenerate soybean plants from cotyledonary nodes.
CA 2140910 C

  • Earliest priority – 27 July 1992
  • Filed – 26 July 1993
  • Granted – 23 March 1999
  • Lapsed – 26 July 2005
 Title – An improved method of Agrobacterium-mediated transformation of cultured soybean cells

Claim 1A genotype-independent method for producing a transgenic soybean plant which comprises:

A) co-cultivating an explant derived from a hypocotyl or cultured cotyledonary node of a germinated soybean seed with cells of anAgrobacterium species containing a chimeric gene at a concentration of 108 to 3 x 108 cells/ml and in the presence of a signal compound selected from the group consisting of acetosyringone, alpha-hydroxyacetosyringone, acetovanillone, syringaldehyde, syringic acid, sinapinic acid and mixtures thereof;
B) maintaining a temperature for co-cultivation of from about 22 to about 25°C;
C) inducing virulence of the Agrobacterium by decreasing the pH of the plant culture media below pH 6.0; and
D) regenerating a transgenic soybean plant.

Claims are the same as for the Australian patent AU 670316.
WO 1994/02620 A2

  • Earliest priority – 27 July 1992
  • Filed – 26 July 1993
  • OPI – 3 February 1994
 Title – An improved method of Agrobacterium-mediated transformation of cultured soybean cells

Claim 1
A genotype-independent method for producing a transgenic soybean plant which comprises:a. cocultivating an explant derived from a hypocotyl or cultured cotyledonary nodes of a germinated soybean seed with Agrobacterium species containing a chimeric gene in the presence of a signal compound selected from the group consisting of acetosyringone, a-hydroxyacetosyringone, acetovanillone, syringaldehyde, syringic acid, and sinapinic acid and mixtures thereof;
b. maintaining a temperature for cocultivation of from 18 to 28°C; and
c. inducing virulence of the Agrobacterium by decreasing the pH of the plant culture media below pH 6.0.

PCT application WO 1994/02620 recites a method to produce a transgenic soybean plant.
Remarks
  1. National phase entry of WO 1994/02620 in Europe (EP 652965) is deemed to be withdrawn on 12 march 2003.
  2. National phase entry of WO 1994/02620 in Japan (JP 2952041) has been granted on 20 September 1999, which has lapsed on 9 July 2004. A divisional of now granted JP 2952041 (JP H10/014425) has been rejected by the JPO, the notice of which was sent to the applicant on 20 April 2000.
  3. Other national phase entries of WO 1994/02620 include Argentina (AR 247920), Brazil (BR 9306802), Hungary (HU 70467).

Note: Patent information on this page was last updated on 24 March 2006.

Soybean – Patent assigned to University of Mississipppi

Specific Patent Information

Patent Number Title, Independent Claims and Summary of Claims Assignee
US 5968830

  • Earliest priority – 28 March 1997
  • Filed – 28 March 1997
  • Granted – 19 October 1999
  • Expected expiry – 28 March 2017
 Title – Soybean transformation and regeneration methods

Claim 1A method of regenerating soybeans (G. max) via organogenesis, comprising:

A) obtaining hypocotyl explants from germinated seedlings of soybean plants whose regeneration is desired,
B) maintaining said hypocotyl explants on a shoot induction medium comprising a cytokinin until shoots form at the acropetal end of said hypocotyl explant,
C) excising shoots from said hypocotyl explant and maintaining said shoots on a shoot elongation medium until said shoots are competent on a rooting medium, and maintaining said shoots on a rooting medium until rooted plantlets are formed, and
D) transplanting said plantlets to soil.
Claim 7A method for transforming soybean plants to express exogenous DNA, comprising:

A) obtaining a hypocotyl explant from germinated seedlings of a soybean plant,
B) maintaining said explant on a shoot induction medium comprising a cytokinin for 16-32 hours,
C) bombarding said explant, with the acropetal end facing up, with microparticles of an inert metal coated with exogenous DNA comprising a plasmid which comprises an expression gene, said expression gene encoding the expression of a protein exogenous to said soybean plant,
D) maintaining said bombarded hypocotyl explant on a medium selective for growth of transformed tissues, followed by maintenance on a shoot elongation medium and preparing plantlets from shoots so obtained.
Claim 10A method of transforming soybeans, comprising:

A) obtaining a hypocotyl soybean explant from germinated seedlings, and
B) maintaining on its side or upright on a shoot induction medium comprising a cytokinin with a basipetal end thereof submerged in said medium,
C) pre-culturing and/or co-culturing an Agrobacterium strain with a virulence enhancing substance, and
D) adding said pre- or co-cultured bacteria to the acropetal end of said upright hypocotyl explant or by complete submersion,
E) co-incubating said hypocotyl explant for a time sufficient to permit transfection of said hypocotyl explant by said bacteria,
F) disinfecting said hypocotyl explant, maintaining said hypocotyl explant on a shoot induction medium and assaying said explant for expression of introduced foreign DNA, and regenerating hypocotyl explants positive for expression of said DNA.

Transformation of a hypocotyl soybean explant by cultivation with Agrobacterium in a medium having a virulence-inducing signal and cytokinin. A method for regeneration of soybeans (G. max) via organogenesis is also disclosed. Additionally, the explant can be transformed by microbombardment.

 University of Mississippi

Note: Patent information on this page was last updated on 27 March 2006.