Patents granted to Monsanto

• contacting plant cells, which are susceptible to genetic transformation by Agrobacterium cells, with Agrobacterium tumefaciens cells containing a co-integrated Ti plasmid comprising a disarmed T-DNA region which comprises in sequence:

(i) a left Agrobacterium T-DNA border sequence,
(ii) a chimeric selectable marker gene which functions in plant cells comprising: (a) a promoter which functions in plant cells;
(b) a structural coding sequence encoding a neomycin phosphotransferase; and
(c) a 3í non-translated region encoding a polyadenylation signal, and (iii) a right Agrobacterium T-DNA border sequence.

(i) culturing a microorganism containing

• a first plasmid containing:

(a) at least one first gene that is expressed in plant cells and
(b) at least one second gene which serves as a marker in a selected microorganism, and

• a Ti second plasmid, under conditions which allow the first plasmid to recombine with the Ti second plasmid in the microorganism, thereby creating a third plasmid having at least one T-DNA border on each side of the first gene, said gene being located in said first plasmid between a region of homology from the T-region of the Ti plasmid and a T-DNA border;

(ii) selecting those microorganisms containing the third plasmid;
(iii) inserting the third plasmid, or a portion thereof, into plant cells; and
(iv) culturing the plant cells under conditions which allow a segment of DNA from the third plasmid to be inserted into the genome of the plant cells.

• inserting into the plant cells a co-integrated plasmid, or a portion thereof, which was formed by a single crossover event between a first plasmid and a Ti second plasmid, wherein the co-integrated plasmid contains a region comprising:

(i) a first T-DNA border;
(ii) a gene which expressed in plant cells; and
(iii) a second T-DNA border which is complementary to the first T-DNA border,
wherein the region does not contain any sequences which would render the plant cells incapable of being regenerated into morphologically normal plants.

A) a chimeric plasmid comprising a gene which functions in plants to express an encoded polypeptide, said plasmid comprising in sequence:

(i) a region of DNA which is homologous to T-DNA located near the left T-DNA border of a tumor-inducing plasmid of Agrobacterium and which is capable of causing in vivo recombination of the chimeric plasmid with said tumor-inducing plasmid of Agrobacterium by a single crossover event;
(ii) a gene comprising a promoter which functions in plant cells, a structural coding sequence and a 3í non-translated region encoding a polyadenylation signal, said promoter and polyadenylation signal being operably linked to said structural coding sequence; and
(iii) an Agrobacterium plasmid T-DNA right border sequence which enables the transfer and incorporation of T-DNA into the genome of a plant cell;
said chimeric plasmid containing no plant tumorigenic genes between and including the region (i), the gene (ii) and the border sequence (iii), and

B) a Ti plasmid capable of transferring the T-DNA region into the genome of a plant cell,

such that the co-integrated plasmid comprises a T-DNA region having the following elements in sequence:
1) a left T-DNA border sequence;
2) the gene (ii) of the chimeric plasmid;
3) at least one right T-DNA border sequence;
wherein said T-DNA border sequences enable the transfer of the T-DNA region into the genome of a plant cell, and wherein, between and including the left T-DNA border sequence, the gene (ii) and the right T-DNA border sequence or (if there is more than one right T-DNA border sequence) between the gene (ii) and the right T-DNA border sequence which is nearest to the left T-DNA border sequence, there are no genes which would render a transformed plant cell tumorous or incapable of regeneration into a morphologically normal plant, and there is no duplication of DNA sequences, which duplication could, through homologous recombination, result in loss of the gene (ii).