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Maize

Summary

The University of Toledo (US), Pioneer Hi-Bred (US), and Stine Biotechnology (US) have been granted United States patents directed toAgrobacterium-mediated transformation of maize. The main difference among them is the type of maize tissue used as target for transformation:

  • The University of Toledo claims a maize seedling wounded in a specific area for inoculation of the bacterium.
  • Pioneer Hi Bred claims an immature embryo of maize and a medium formula for cultivation of a transformed maize embryo
  • Stine Biotechnology also employs immature embryos as target tissue for transformation with Agrobacterium in their patent. Claims in their applications disclose a method to improve the production of so-called Type II callus (a preferred target tissue for the transformation of corn) by including a monosaccharide like glucose in addition to the usual sucrose in the medium.

Maize – Specific Patent Information – part 1

Patent Number Title, Independent Claims and Summary of Claims Assignee
US 5177010

  • Earliest priority – 30 June 1986
  • Filed – 5 September 1990
  • Granted – 5 Jan 1993
  • Expected expiry – 4 September 2010
 Title – Process for Transforming Corn and the Products Thereof

Claim 1A method of producing transformed corn, said method comprising:

A) making a wound in a corn seedling with newly emerging radicle and stem, said wound being made in an area of the seedling containing rapidly dividing cells, wherein said area extends from the base of the scutellar node to slightly beyond the coleoptile node; and
B) inoculating the wound with vir+ A. tumefaciens.

Transformation of a corn seedling by inoculating vir+ A. tumefaciens in a wounded area located between the scutellar node to slightly beyond the coleoptile node. This area presents a high rate of cell division.

University of Toledo
US 2002/0002711 A1

  • Earliest priority – 27 June 1994
  • Filed – 10 June 1998
  • Abandoned – 28 May 2002
 Title – Process for Transforming Germinae and the Products Thereof

Claim 11. A transformed Gramineae.
Claim 7
A fertile transgenic Zea mays plant comprising stably incorporated exogenous DNA.
Claim 10
A transformed corn plant having a transformed gene, the plant produced by an Agrobacterium-mediated transformation or direct gene transfer transformation.
Claim 11
A transformed corn plant produced by an Agrobacterium or direct cell transfer whereby the plant or other differentiated organs or tissues provide an expression of exogenous DNA in the corn product.
Claim 12
A transformed corn plant produced by a regenerated protoplast or single cell cultures.
Claim 13A transformed corn plant produced by a method equivalent to an Agrobacterium tumefaciens-mediated transformation.

Transformed corn plant having a transformed gene produced by: Agrobacterium -mediated transformation or an equivalent method, direct cell transfer, regenerated protoplasts and single cell cultures. Fertile transgenic Zea mays having an exogenous gene and transformed Gramineae are also part of the filed claims.

This patent application has been abandoned (failure to respond to a USPTO action) according to the USPTO.

Goldman and Graves
Remarks A related patent has been granted in Japan (JP3234534, which is a divisional of now granted JP 2693443 claiming Agrobacterium-mediated transformation of Gramineae).
US 5981840

  • Earliest priority – 24 January 1997
  • Filed – 24 January 1997
  • Granted – 9 Nov 1999
  • Expected expiry – 23 January 2017
 Title – Method for Agrobacterium-Mediated Transformation

Claim 1A method for transforming maize using Agrobacterium comprising the steps of:

A) contacting at least one immature embryo from a maize plant with Agrobacterium capable of transferring at least one gene to the embryo;
B) co-cultivating the embryo with Agrobacterium;
C) culturing the embryo in a medium comprising N6 salts, an antibiotic at concentrations capable of inhibiting the growth of Agrobacterium, and a selective agent to select for embryos expressing the gene; and
D) regenerating plants expressing the gene.
Claim 4A method for transforming maize using Agrobacterium comprising the steps of:

A) contacting at least one immature embryo from a maize plant with Agrobacterium capable of transferring at least one gene to the embryo in a medium comprising N6 salts;
B) co-cultivating the embryo with Agrobacterium in a medium comprising N6 salts;
C) culturing the embryo in a medium comprising N6 salts, an antibiotic at concentrations capable of inhibiting the growth of Agrobacterium, and a selective agent to select for embryos expressing the gene; and
D) regenerating plants expressing the gene in a medium comprising MS salts.
Claim 7A method for transforming maize using Agrobacterium comprising the steps of:

A) contacting at least one immature embryo from a maize plant with Agrobacterium capable of transferring at least one gene to the embryo in a medium comprising N6 or MS salts;
B) co-cultivating the embryo with Agrobacterium in a medium comprising MS salts;
C) culturing the embryo in a medium comprising N6 salts, an antibiotic at concentrations capable of inhibiting the growth of Agrobacterium, and a selective agent to select for embryos expressing the gene; and
D) regenerating plants expressing the gene in a medium comprising MS salts.
Claim 10A method for optimizing the production of transgenic maize plants of a first genotype using Agrobacterium-mediated transformation comprising the steps of:

A) isolating immature embryos from maize;
B) separating the embryos into treatment groups;
C) incubating each treatment group separately in a medium comprising N6 or MS salts and in a suspension of Agrobacterium at concentrations ranging from about 1×108 cfu/ml to about 1×1010 cfu/ml;
D) co-cultivating the embryos with Agrobacterium on a solid medium;
E) culturing the embryos in a medium comprising N6 salts, an antibiotic at concentrations capable of inhibiting the growth of Agrobacterium, and a selective agent to select for embryos transformed by Agrobacterium;
F) identifying the treatment group with the highest transformation frequency; and
G) using the concentration of Agrobacterium generating the highest transformation frequency to transform other embryos from the first genotype.
Claim 13A method for transforming maize using Agrobacterium comprising the steps of:

A) contacting at least one immature embryo from a maize plant with Agrobacterium capable of transferring at least one gene to the embryo;
B) co-cultivating the embryo with Agrobacterium;
C) culturing the embryo in a medium containing salts other than MS salts, an antibiotic at concentrations capable of inhibiting the growth of Agrobacterium, and a selective agent to select for embryos expressing the gene; and
D) regenerating plants expressing the gene.

Transformation of an immature embryo of maize by contact and co-cultivation with Agrobacterium having a gene of interest. The media used for culturing the embryo contains N6 or MS salts, an antibiotic against Agrobacterium, and a selective agent for embryos expressing the gene. Regeneration of plants expressing the gene is also recited in the claims.

Pioneer Hi-Bred
AU 727849 B2

  • Earliest priority – 24 January 1997
  • Filed – 23 January 1998
  • Granted – 4 January 2001
  • Expected expiry – 22 January 2018
 Title – Methods for Agrobacterium-mediated transformation

Claim 1A method for transforming maize using Agrobacterium comprising the steps of:

A) contacting at least one immature embryo from a maize plant with Agrobacterium capable of transferring at least one gene to the embryo;
B) co-cultivating the embryo with Agrobacterium;
C) culturing the embryo in a medium comprising N6 salts, an antibiotic at concentrations capable of inhibiting the growth of Agrobacterium, and a selective agent to select for embryos expressing the gene; and
D) regenerating plants expressing the gene.

Claim 1 states the same method of maize transformation as that of US 5981840.
EP 971578 A1

  • Earliest priority – 24 January 1997
  • Filed – 23 January 1998
  • Application deemed to be withdrawn – 25 June 2003
 Title– Methods for Agrobacterium-mediated Transformation

Claim 1A method for transforming maize using Agrobacterium comprising the steps of:

A) contacting tissue from a maize plant with Agrobacterium capable of transferring at least one gene to the tissue in a non-LS salt medium;
B) co-cultivating the tissue with Agrobacterium in a non-LS salt medium;
C) culturing the tissue in a non-LS salt medium and a selective agent to select for tissue expressing the gene; and
D) regenerating, in a non-LS salt medium, plants expressing the gene.

The transformation method disclosed in these applications is very similar to the method claimed in the United States patent US 5981840. The difference consists in the use of a co-cultivation medium free of LS salts.
Remarks
  1. A divisional patent of now granted US 5981840 has been granted in the US (US 6822144), which claims a stably transformed maize plant (limited to PHN46 or PHJ90 inbred plants) using Agrobacterium to transform an immature embryo.
  2. National phase entry of WO 1998/32326 in Canada (CA 2278618 AA) is pending.

Note: Patent information on this page was last updated on 7 February 2006.

Maize – Specific Patent Information – part 2

Patent Number Title, Independent Claims and Summary of Claims Assignee
US 6420630 B1

  • Earliest priority – 1 December 1998
  • Filed – 1 December 1998
  • Granted – 16 July 2002
  • Expected expiry – 30 November 2018
 Title – Methods for tissue culturing and transforming elite inbreds of Zea mays L.

Claim 1A method for transforming a line of corn using Agrobacterium comprising the steps of:
(a) initiating co-cultivation of an immature embryo from said line with Agrobacterium capable of transferring at least one gene to tissue of said line to produce an infected embryo;
(b) applying heat shock treatment during said co-cultivation;
(c) culturing the infected embryo to initiate callus on a medium comprising an antibiotic;
(d) culturing the resulting callus tissue on a medium comprising a selective agent;
(e) selecting transformed callus tissue comprising growing Type II callus; and
(f) regenerating transgenic plants from said Type II callus.

A method of transforming maize by co-cultivating Agrobacterium carrying a gene of interest with immature embryos. Heat shock is applied to enhance DNA integration.

Stine Biotechnology
US 6919494 B2

  • Earliest priority – 1 December 1998
  • Filed – 30 July 2001
  • Granted – 19 July 2005
  • Expected expiry – 29 July 2021
 Title – Methods for tissue culturing and transforming elite inbreds of Zea mays L.

Claim 1A method for producing a corn plant comprising the steps of:

(a) co-cultivating an immature embryo from said tissue at a temperature of about 18˚C. to 20˚C. with Agrobacterium capable of transferring at least one DNA sequence of interest to said tissue to produce an infected embryo;

(b) culturing the infected embryo on a medium comprising an antibiotic to produce a resulting tissue;

(c) culturing said resulting tissue on a medium comprising a selective agent and an antibiotic;

(d) selecting transformed tissue having Type II callus; and

(e) regenerating transgenic plants from said Type II callus.
Claim 3A method for transforming a line of corn comprising the steps of:

(a) co-cultivating an immature embryo from said line with Agrobacterium capable of transferring at least one DNA sequence of interest to said tissue of said line to produce an infected embryo;

(b) culturing the infected embryo to initiate callus on a medium comprising an antibiotic and a compound selected from the group consisting of glucose, maltose, lactose, sorbitol and mannitol, wherein the concentration of said compound is from 5 g/L to 30 g/L;

(c) culturing the resulting callus tissue on a medium comprising a selective agent and an antibiotic;

(d) selecting transformed callus tissue comprising growing Type II callus; and

(e) regenerating transgenic plants from said growing Type II callus.
Claim 4A method for producing a transformed corn plant using Agrobacterium comprising the steps of:

(a) initiating co-cultivation of an immature embryo from said tissue with Agrobacterium capable of transferring at least one DNA sequence of interest to said tissue to produce an infected embryo;

(b) applying heat shock treatment during said co-cultivation;

(c) culturing the infected embryo to initiate callus on a medium comprising an antibiotic and glucose;

(d) culturing the resulting callus tissue on a medium comprising a selective agent and an antibiotic;

(e) selecting transformed callus tissue having Type II callus; and

(f) regenerating transgenic plants from said Type II callus.

Granted patent of application US 2002/0104131 (see below).

Continuation of US Patent Application 09/203,679 (now US Patent 6420630 B1)
US 2002/0104131 A1

  • Earliest priority – 1 December 1998
  • Filed – 30 July 2001
  • Granted as US 6919494 (see above)
 Title – Methods for tissue culturing and transforming elite inbreds of Zea mays L.

Claim 1 (not granted in US 6919494)A method for stimulating a high frequency production of Type II callus from immature embryos of elite corn inbreds which comprises culturing said embryos on a solid medium comprising sucrose and a monosaccharide sugar, wherein the concentration of said monosaccharide sugar is between about 5 g/L and about 30 g/L.
Claim 4
A method for transforming elite lines of corn using Agrobacterium comprising the steps of:(a) co-cultivating an immature embryo from said elite line with Agrobacterium capable of transferring at least one gene to tissue of said elite line on a solid medium to produce an infected embryo;(b) culturing the infected embryo on a solid medium comprising an antibiotic;

(c) culturing the resulting tissue on a solid medium comprising a selective agent to select for transformed tissue;

(d) selecting transformed tissue with growing Type II callus capable of forming water tower embryo structures; and

(e) regenerating plants from said embryo structures.
Claim 22
A method for transforming elite lines of corn using Agrobacterium comprising the steps of:(a) co-cultivating an immature embryo from said elite line with Agrobacterium capable of transferring at least one gene to tissue of said elite line on a solid medium to produce an infected embryo;(b) culturing the infected embryo on a solid medium comprising an antibiotic and a monosaccharide sugar in an amount of from 5 g/L to 30g/L;

(c) culturing the resulting tissue on a solid medium comprising an antibiotic and a selective agent;

(d) culturing the resulting tissue on a solid medium comprising a selective agent to select for transformed tissue;

(e) selecting transformed tissue with growing Type II callus capable of forming water tower embryo structures; and

(f) regenerating plants from said embryo structures.
Claim 32
A method for transforming elite lines of corn using Agrobacterium comprising the steps of:(a) co-cultivating at a temperature of 19[deg.] C. an immature embryo from said elite line with Agrobacterium capable of transferring at least one gene to tissue of said elite line on a solid medium to produce an infected embryo, said Agrobacterium is selected from Agrobacterium one to two days after rescue from frozen glycerol stocks;(b) culturing the infected embryo on a solid medium comprising an antibiotic at a concentration of 15 mg/L to 75 mg/L and a monosaccharide sugar selected from the group consisting of glucose, maltose, lactose, sorbitol and mannitol in an amount of from 5 g/L to 30 g/L;

(c) culturing the resulting tissue on a solid medium comprising an antibiotic and a selective agent;

(d) culturing the resulting tissue on a solid medium comprising a selective agent to select for transformed tissue;

(e) selecting transformed tissue with growing Type II callus capable of forming water tower embryo structures; and

(f) regenerating plants from said embryo structures.

A method in which formation of Type II callus (a preferred form of target tissue for the transformation with Agrobacterium) from immature embryos is enhanced by adding sucrose and a monosaccharide to the medium.

Continuation of US Patent Application 09/203,679 (now US Patent US 6420630 B1)
US 2002/0104132 A1

  • Earliest priority – 1 December 1998
  • Filed – 30 July 2001
  • Abandoned – 19 September 2005
 Title – Methods for tissue culturing and transforming elite inbreds of Zea mays L.

Claim 1A method for stimulating a high frequency production of Type II callus from immature embryos of elite corn inbreds which comprises

  • culturing said embryos on a solid medium comprising sucrose and a monosaccharide sugar, wherein the concentration of said monosaccharide sugar is between about 5 g/L and about 30 g/L.
Claim 4A method for transforming elite lines of corn using Agrobacterium comprising the steps of:

(a) co-cultivating an immature embryo from said elite line with Agrobacterium capable of transferring at least one gene to tissue of said elite line on a solid medium to produce an infected embryo;

(b) culturing the infected embryo on a solid medium comprising an antibiotic;

(c) culturing the resulting tissue on a solid medium comprising a selective agent to select for transformed tissue;

(d) selecting transformed tissue with growing Type II callus capable of forming water tower embryo structures; and

(e) regenerating plants from said embryo structures.
Claim 22A method for transforming elite lines of corn using Agrobacterium comprising the steps of:

(a) co-cultivating an immature embryo from said elite line with Agrobacterium capable of transferring at least one gene to tissue of said elite line on a solid medium to produce an infected embryo;

(b) culturing the infected embryo on a solid medium comprising an antibiotic and a monosaccharide sugar in an amount of from 5 g/L to 30g/L;

(c) culturing the resulting tissue on a solid medium comprising an antibiotic and a selective agent;

(d) culturing the resul ting tissue on a solid medium comprising a selective agent to select for transformed tissue;

(e) selecting transformed tissue with growing Type II callus capable of forming water tower embryo structures; and

(f) regenerating plants from said embryo structures.
Claim 32A method for transforming elite lines of corn using Agrobacterium comprising the steps of:

(a) co-cultivating at a temperature of 19[deg.] C. an immature embryo from said elite line with Agrobacterium capable of transferring at least one gene to tissue of said elite line on a solid medium to produce an infected embryo, said Agrobacterium is selected from Agrobacterium one to two days after rescue from frozen glycerol stocks.;

(b) culturing the infected embryo on a solid medium comprising an antibiotic at a concentration of 15 mg/L to 75 mg/L and a monosaccharide sugar selected from the group consisting of glucose, maltose, lactose, sorbitol and mannitol in an amount of from 5 g/L to 30 g/L;

(c) culturing the resulting tissue on a solid medium comprising an antibiotic and a selective agent;

(d) culturing the resulting tissue on a solid medium comprising a selective agent to select for transformed tissue;

(e) selecting transformed tissue with growing Type II callus capable of forming water tower embryo structures; and

(f) regenerating plants from said embryo structures.

A method as in the application above but specifically designed for elite corn inbred lines.

Divisional application from US Patent Application 09/203,679 (now US Patent US 6420630 B1), this application has been abandoned due to failure to respond to a USPTO action according to the USPTO.
US 2005/0278802 A1

  • Earliest priority – 1 December 1998
  • Filed – 18 July 2005
  • Application pending
 Title – Methods for tissue culturing and transforming elite inbreds of Zea mays L.

Claim 35A method for producing a corn plant comprising the steps of:

(a) culturing a corn embryo on a medium comprising a compound selected from the group consisting of glucose, maltose, lactose, sorbitol, and mannitol, wherein said compound is in an amount of from about 5 g/L to 30 g/L, to produce a type II callus; and

(b) regenerating a plant.

This is a divisional application of now granted US 6420630.

All of the independent claims preceeding claim 35 has been cancelled.

Note: Patent information on this page was last updated on 7 February 2006.