Patents granted to Japan Tobacco

A method for transforming a monocotyledon callus, comprising contacting a cultured tissue of a monocotyledon during dedifferentiation wherein said dedifferentiation is obtained by culturing an explant on a dedifferentiation-inducing medium for not less than 7 days or a dedifferentiated cultured tissue of a monocotyledon, with a bacterium belonging to the genus Agrobacterium containing a desired gene.

A method for transforming a monocotyledon with a desired gene, comprising:

A) contacting a cultured tissue of said monocotyledon during dedifferentiation thereof, or a dedifferentiated cultured tissue of said monocotyledon, with a suspension of Agrobacterium tumefaciens having a cell population of 10⁶ to 10¹¹ cells/ml for 3-10 minutes, and then
B) culturing said cultured tissue of said monocotyledon during dedifferentiation thereof, or said dedifferentiated cultured tissue of said monocotyledon, on a solid medium for several days together with said Agrobacterium tumefaciens, or
C) adding said Agrobacterium tumefaciens to culture medium in which said cultured tissue of said monocotyledon during dedifferentiation thereof or said dedifferentiated cultured tissue of said monocotyledon is cultured, and continuously culturing said cultured tissue of said monocotyledon during dedifferentiation or said dedifferentiated cultured tissue of said monocotyledon together with said Agrobacterium tumefaciens,

wherein said dedifferentiated cultured tissue of said monocotyledon is selected from the group consisting of a tissue cultured during the process of callus formation which is cultured for not less than 7 days after an explant is placed on a dedifferentiation-inducing medium and a callus, and
wherein said Agrobacterium tumefaciens contains plasmid pTOK162, and said desired gene is present between border sequences of the T region of said plasmid pTOK162, or wherein said desired gene is present in another plasmid contained in said Agrobacterium tumefaciens.

A method for transforming a monocot plant comprising:

i) culturing an explant of said monocot plant, or a tissue isolated from said explant, for one to six days in a medium comprising at least one auxin to obtain a cultured tissue;
ii) co-culturing the cultured tissue from step i) with an Agrobacterium bacterium comprising a polynucleotide of interest;
iii) selecting cultured tissue into which the polynucleotide of interest has been introduced; and
iv) culturing the selected tissue on a regeneration medium to obtain a transformed monocot plant.

A method for transforming a tissue of a monocot plant comprising:

i) culturing an explant of an immature tissue of a monocot plant for one to six days on a medium comprising at least one auxin and that induces dedifferentiation of the cells of the explanted tissue to obtain a dedifferentiating or dedifferentiated cultured immature tissue;
ii) contacting the dedifferentiating or dedifferentiated cultured immature tissue with cells of Agrobacterium bacteria that comprise a vector comprising at least one virulence gene of a Ti plasmid, a left T-DNA border, a right T-DNA border and a polynucleotide of interest located between the left T-DNA border and the right T-DNA border;
thereby obtaining a transformed plant tissue.

A method for obtaining a transformed monocot plant comprising

i) culturing an explant of an immature tissue of a monocot plant for one to six days on a medium comprising at least one auxin and that induces dedifferentiation of the cells of the explanted tissue to obtain a dedifferentiating or dedifferentiated cultured immature tissue;
ii) contacting the dedifferentiating or dedifferentiated cultured immature tissue with cells of Agrobacterium bacteria that comprise a vector comprising at least one virulence gene of a Ti plasmid, a left T-DNA border, a right T-DNA border and a polynucleotide of interest located between the left T-DNA border and the right T-DNA border; thereby obtaining a transformed plant tissue; and
iii) culturing the transformed plant tissue on at least one regeneration medium, thereby obtaining a transformed monocot plant.

A method for transforming a monocotyledon, comprising contacting a cultured tissue of said monocotyledon during dedifferentiation thereof obtained by culturing an explant on a dedifferentiation-inducing medium for less than 7 days with a bacterium belonging to the genus Agrobacterium containing a super binary vector having the virulence region of Ti plasmid pTiBo542 contained in Agrobacterium tumefaciens A281, left and right border sequences of T-DNA of a Ti plasmid or an Ri plasmid of a bacterium belonging to the genus Agrobacterium, and a desired gene located between said left and right border sequences.

A method for transforming a monocotyledon, comprising contacting a cultured tissue of said monocotyledon during dedifferentiation thereof obtained by culturing an explant derived from an immature tissue on a dedifferentiation-inducing medium for less than 7 days with a bacterium belonging to the genus Agrobacterium containing a desired gene and containing a vector having the virulence region of Ti plasmid contained in Agrobacterium tumefaciens.

A method for transforming a monocotyledon comprising transforming a cultured tissue during dedifferentiation process or a dedifferentiated cultured tissue of said monocotyledon with a bacteria belonging to genus Agrobacterium containing a desired gene.