Precise breeding
AssigneeJ.R. Simplot Company
InventorsCaius Rommens, Jingsong Ye, Jaime Menendez-Humara, Hua Yan, Kathy Swords
Life of this patent
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Abstract
The present invention relates to a method for identifying and isolating native plant nucleic acid sequences that may function as T-DNAs or T-DNA border-like sequences, effecting the transfer of one polynucleotide into another polynucleotide. The present invention also provides a modified tuber, such as a genetically modified mature tuber, that comprises at least one trait that is not exhibited by a non-modified tuber of the same species.
Claims43
A method for isolating a plant polynucleotide that comprises a T-DNA border-like sequence, comprising
- (i) fragmenting a plant genome;
- (ii) ligating a polynucleotide of known sequence to a plant DNA fragment to produce a ligated DNA;
- (iii) producing a PCR product from the ligated DNA that comprises (a) a sequence that is homologous to a part of a T-DNA border sequence, (b) a DNA sequence from the plant genome, and (c) a sequence from the polynucleotide of known sequence, wherein the sequences of (a) and (b) are linked;
- (iv) sequencing the PCR product;
- (v) designing at least one PCR primer based on the DNA sequence from the plant genome;
- (vi) using the PCR primer of (v) in an inverse PCR of plant genomic DNA to identify a sequence from the plant genomic DNA that is a T-DNA border-like sequence.
A method for producing a modified plant that does not contain a T-DNA comprising (1) transforming a plant cell with (i) an Agrobacterium-transformation vector that comprises a desired polynucleotide within a P-DNA, and (ii) an Agrobacterium-transformation vector that comprises a selectable marker within a T-DNA;
- (2) obtaining from said transformed plant cell a transformed plant that comprises at least one copy of said P-DNA and at least one copy of said T-DNA in its genome;
- (3) self-fertilizing or cross-fertilizing the transformed plant to produce progeny plants that segregate for the T-DNA and P-DNA;
- and (4) screening the progeny plants to identify a modified plant that does not comprise said T-DNA, but does comprise said P-DNA.
A modified tuber, comprising a level of acrylamide that is at least about 99%, 98%, 97%. 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% lower than the level of acrylamide normally associated with a wild-type tuber of the same species as the species of the modified tuber.
A modified tuber comprising a level of amylose that is at least about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% greater than the level of amylose of a wild-type tuber of the same species as said modified tuber.
A modified, mature tuber comprising a level of amylose that is about 1 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times greater than the level of amylose of a wild-type tuber of the same species.
A modified tuber comprising a level of cold-induced glucose that is at least about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% lower than the level of glucose in a wild-type tuber of the same species as said modified tuber.
A modified, mature tuber comprising a 5-fold reduction in acrylamide levels compared to the level of acrylamide in a wild-type tuber of the same species.
A modified tuber comprising a level of phosphate that is at least about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% lower than the level of phosphate in a wild-type tuber of the same species as said modified tuber.
A modified tuber comprising a level of polyphenol oxidase activity that is at least about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% lower than the level of polyphenol oxidase activity associated with a wild-type tuber of the same species as the species of the modified tuber.
A modified tuber comprising at least one cell that overexpresses an inactive polyphenol oxidase gene, wherein the level of polyphenol oxidase activity in the modified tuber is reduced by 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% in comparison to the level of polyphenol oxidase activity in a wild-type tuber of the same species as the modified tuber.
A method for producing a transgenic plant that does not contain a T-DNA, comprising
- co-transforming a plant tissue with a P-DNA vector and a T-DNA vector;
- growing plantlets from the transformed plant tissue by incubating the plant tissue on media that contains a substance that destroys cells which do not contain the P-DNA vector or the T-DNA vector;
- selecting a plantlet that contains either or both of the P-DNA or the T-DNA;
- growing the plantlet to reproductive maturity and cross-fertlizing the reproductively mature plant with an untransformed plant;
- and
- segregating progeny plants whose cells contain only a P-DNA from progeny plants that contain a T-DNA.
A method for producing a transgenic plant that does not contain a T-DNA, comprising
- co-transforming a plant tissue with a P-DNA vector and a T-DNA vector;
- growing plantlets from the transformed plant tissue by incubating the plant tissue on media that contains a substance that destroys cells which do not contain the P-DNA vector or the T-DNA vector;
- selecting a plantlet that contains either or both of the P-DNA or the T-DNA;
- growing the plantlet to reproductive maturity and then self-fertlizing the reproductively mature plant;
- and
- segregating progeny plants whose cells contain only a P-DNA from progeny plants that contain a T-DNA.
A method for producing a transgenic plant that does not contain a T-DNA, comprising
- co-transforming a plant tissue with a first P-DNA vector and a second P-DNA vector, wherein the first P-DNA vector comprises a polynucleotide of interest, and wherein the second P-DNA vector comprises a selectable marker gene;
- growing plantlets from the transformed plant tissue by incubating the plant tissue on media that contains a substance that destroys cells which do not contain either of the P-DNA vectors;
- selecting a plantlet that contains either or both of the P-DNA vectors;
- growing the plantlet to reproductive maturity and either cross-fertlizing the reproductively mature plant with an untransformed plant or self-fertilizing the reproductively mature plant;
- and
- segregating progeny plants, whose cells contain only the P-DNA from the first P-DNA vector, from progeny plants that contain in their genomes the P-DNA from the second P-DNA vector.
A nucleic acid comprising the sequence depicted in SEQ ID NO. 96, and is capable of transferring one polynucleotide into another polynucleotide.
A nucleic acid comprising a sequence that has 90% sequence identity to the sequence depicted in SEQ ID NO. 96.
A nucleic acid comprising the sequence depicted in SEQ ID NO. 97, and is capable of transferring one polynucleotide into another polynucleotide.
A nucleic acid comprising a sequence that has 90% sequence identity to the sequence depicted in SEQ ID NO. 97.
Dependencies are inferred from the claim text.