Patent
The invention provides three novel disarmed strains of Agrobacterium tumefaciens bacteria useful for the transformation of plants. The invention provides three engineered A. tumefaciens Chry5 strains or bacterial cells thereof which comprise the Chry5 strain chromosomal background and a disarmed pTiChry5 vector, and methods of using said bacterial strains or cells for transformation of fungal or plant cells, in particular dicot or monocot plant cells, including soybean, maize, wheat, and sugarcane cells. The invention further relates to the transgenic plants created by these methods.
1. An engineered Agrobacterium tumefaciens cell comprising a Chry5 strain chromosomal background and a fully disarmed pTiChry5 vector, wherein the disarmed pTiChry5 vector has both TL-DNA and TR-DNA regions removed, wherein the fully disarmed pTiChry5 vector comprises a nucleic acid sequence at least 90% identical to SEQ ID NO: 3.
2. An engineered Agrobacterium tumefaciens cell comprising a Chry5 strain chromosomal background and a fully disarmed pTiChry5 vector, wherein the disarmed pTiChry5 vector has both TL-DNA and TR-DNA regions removed, wherein the fully disarmed pTiChry5 vector comprises a nucleic acid sequence at least 70% identical to SEQ ID NO: 3.
3. An engineered Agrobacterium tumefaciens cell comprising a Chry5 strain chromosomal background and a fully disarmed pTiChry5 vector, wherein the disarmed pTiChry5 vector has both TL-DNA and TR-DNA regions removed, wherein a representative sample of said engineered Agrobacterium tumefaciens cell is deposited as ATCC Accession No. PTA-124005.
4. A variant or mutant of the engineered Agrobacterium tumefaciens cell of claim 1 , wherein the variant or mutant cell comprises a fully disarmed pTiChry5 plasmid comprising the nucleic acid sequence of SEQ ID NO: 3.
5. The variant or mutant cell of claim 4 , wherein the variant or mutant cell has a mutation in its bacterial chromosome.
6. The variant or mutant cell of claim 5 , wherein the mutation is in a gene on the bacterial chromosome or wherein the mutation is an insertion in the bacterial chromosome.
7. The engineered Agrobacterium tumefaciens cell of claim 1 , further comprising at least one additional nucleic acid molecule, wherein the at least one additional nucleic acid molecule is a booster plasmid, helper plasmid, virulence-enhancing plasmid, and/or a binary vector.
8. A method for producing a transgenic host cell comprising the steps of:
(a) providing the engineered Agrobacterium tumefaciens cell of claim 1 , further comprising a transgenic T-DNA region; and
(b) contacting said engineered Agrobacterium tumefaciens cell with a host cell, under conditions that permit the Agrobacterium cell to transform the host cell.
9. The method of claim 8 , wherein the host cell is a plant cell.
10. The method of claim 9 , wherein said plant cell is a cell from a plant tissue selected from the group consisting of: embryogenic plant tissue, organogenic plant tissue, vegetative plant tissue, callus tissue, and reproductive tissue; or wherein the plant cell is a cell from a plant part selected from the group consisting of: pollen, ovule, immature plant embryo, mature plant embryo, seed, seedling, root, cotyledon, stem, node, internode, bud, leaf, shoot apical meristem, floral meristem, flower bud, inflorescence, and cultured plant material.
11. The method of claim 9 , wherein said plant cell is a dicotyledonous plant cell.
12. The method of claim 11 , wherein said dicoytyledonous plant cell is from soybean ( Glycine max ).
13. The method of claim 9 , wherein said plant cell is a monocotyledonous plant cell.
14. The method of claim 13 , wherein said monocotyledonous plant is maize.
15. The method of claim 8 , wherein the transgenic T-DNA region comprises at least one plant-expressible gene of interest and/or at least one regulatory gene of interest and/or a nucleic acid molecule encoding for at least one genome editing nuclease.
16. A method for modifying a target site in the genome of a host cell, comprising using the engineered Agrobacterium tumefaciens cell of claim 1 to introduce into the host cell:
(a) a first nucleic acid comprising at least 16 contiguous nucleotides, wherein the at least 16 contiguous nucleotides have at least 90% identity with a target site in the genome of the host cell, and further comprising a transgene; and
(b) a second nucleic acid molecule comprising a nucleotide sequence encoding a nuclease for site-directed cleavage at a nuclease cleavage site in the genome of the host cell adjacent to the nucleotide sequence in the genome of the host cell that corresponds to the at least 16 contiguous nucleotides of (a),
under conditions wherein expression of the second nucleic acid molecule can produce the nuclease and the nuclease can cleave at the nuclease cleavage site in the genome of the host cell and modify the target site in the genome of the host cell.
17. The method of claim 16 , wherein the target site in the genome comprises at least a fragment of a native gene and wherein the first nucleic acid molecule comprises a donor DNA molecule, which comprises at least 16 contiguous nucleotides at least 90% identical to a genomic nucleic acid sequence, and further comprises a modified nucleic acid molecule comprising a nucleic acid sequence modified from the native gene; whereby the modified nucleic acid molecule is integrated at the target site in the genome of the host cell.
18. The method of claim 16 , wherein the host cell is a plant cell.