Strains of agrobacterium modified to increase plant transformation frequency

Abstract

Agrobacterium strains that harbor transformation-enhancing genes on a plasmid capable of replication independently of the Agrobacterium chromosome, the Ti plasmid, and plant transformation binary vectors, and uses for these Agrobacterium strains are provided. Additionally, Agrobacterium strains that are deficient in DNA recombination functions that result in instability or rearrangement of plant transformation binary vectors, and that harbor transformation-enhancing genes on a plasmid capable of replication independently of the Agrobacterium chromosome, the Ti plasmid, and plant transformation binary vectors, and uses for these strains, are also provided. Further included are Agrobacterium strains that harbor transformation-enhancing genes integrated into the Agrobacterium chromosome at a locus that does not interfere with or otherwise compromise the normal growth and plant transformation ability of the Agrobacterium cells, and uses for these Agrobacterium strains. Plants made using these Agrobacterium strains are also described.

What is claimed is: 
     
         1 . A method for transforming a plant, comprising contacting a cell of the plant with an  Agrobacterium  strain having at least one pTi helper plasmid comprising a 14.8 KpnI fragment of pSB1 and a pTi plasmid having at least one disarmed T-DNA region, the T-DNA region comprising at least a right T-DNA border and exogenous DNA adjacent to the border, wherein the plasmids have differing origins of replication relative to each other. 
     
     
         2 . The method of  claim 1 , wherein the 14.8 KpnI VirBCDG fragment isolated from pSB1 in  Agrobacterium  strains has a deficiency in RecA function. 
     
     
         3 . A method for transforming a plant, comprising contacting a cell of the plant with a bacterium of the genus  Agrobacterium  having a 14.8 KpnI VirBCDG fragment of pSB1 and a pTi plasmid having at least one disarmed T-DNA region, wherein the 14.8 KpnI VirBCDG fragment has been integrated into a neutral integration site of a chromosome of the bacterium. 
     
     
         4 . The method for transforming a plant according to  claim 1 , wherein the bacterium further comprises a plasmid having a T-DNA region adjacent to at least one  Agrobacterium  T-DNA border, the plasmid having a replication origin of an IncP incompatibility group. 
     
     
         5 . The method for transforming a plant according to  claim 3 , wherein the bacterium further comprises a plasmid having a T-DNA region adjacent to at least one  Agrobacterium  T-DNA border. 
     
     
         6 . The method for transforming a plant according to  claim 3 , wherein the  Agrobacterium  strain is deficient in RecA functionality. 
     
     
         7 . The method for transforming a plant according to  claim 4 , wherein the T-DNA region contains three or more gene sequences. 
     
     
         8 . The method for transforming a plant according to  claim 4 , wherein the T-DNA region contains equal to or greater than 25,000 nucleotide base pairs. 
     
     
         9 . The method for transforming a plant according to  claim 4 , wherein the T-DNA region is inserted into a single location in the plant cell when the plant is transformed. 
     
     
         10 . The method for transforming a plant according to  claim 4 , wherein the T-DNA region comprises more than one gene sequence and the gene sequences have equal to or greater than 60% sequence homology. 
     
     
         11 . The method for transforming a plant according to  claim 4 , wherein the T-DNA region encodes one or more of an insecticidal protein, a herbicidal protein, or a mixture of insecticidal proteins and herbicide tolerance proteins. 
     
     
         12 . The method for transforming a plant according to  claim 4 , wherein the T-DNA region encodes a Cry1 Ca insecticidal protein, a Cry1F insecticidal protein, and a Cry1Ab1 insecticidal protein. 
     
     
         13 . The method for transforming a plant according to  claim 4 , wherein the T-DNA region encodes a Cry1Ca insecticidal protein, a Cry1F insecticidal protein, a Cry1Ab1 insecticidal protein, and an AAD-1 herbicide tolerance protein. 
     
     
         14 . The method according to  claim 1 , wherein the plant is a monocot. 
     
     
         15 . The method according to  claim 1 , wherein the 14.8 KpnI VirBCDG fragment is cloned into the Kpn I site of a pDAB9291 plasmid. 
     
     
         16 . The method according to  claim 1 , wherein the pTi helper plasmid is plasmid pMP90. 
     
     
         17 . The method according to  claim 1 , wherein the pTi helper plasmid is plasmid pTiC58Δ. 
     
     
         18 . The method according to  claim 15 , further comprising transforming the  Agrobacterium  strain using plasmid pDAB9292 DNA. 
     
     
         19 . The method according to  claim 1 , further comprising a step of selecting a transformed cell or a transformed tissue, after subjecting said cultured tissue to transformation. 
     
     
         20 . An  Agrobacterium  strain having at least one pTi helper plasmid comprising a 14.8 KpnI fragment of pSB1 and a pTi plasmid having at least one disarmed T-DNA region, wherein the plasmids have differing origins of replication relative to each other. 
     
     
         21 . The  Agrobacterium  strain of  claim 20 , wherein the  Agrobacterium  strain has a deficiency in RecA function. 
     
     
         22 . An  Agrobacterium  strain having transformation-enhancing properties comprising a 14.8 KpnI VirBCDG fragment isolated from pSB1 and a pTi plasmid having at least one disarmed T-DNA region. 
     
     
         23 . The  Agrobacterium  strain according to  claim 20 , wherein the bacterium further comprises a plasmid having a T-DNA region adjacent to at least one  Agrobacterium  T-DNA border. 
     
     
         24 . The  Agrobacterium  strain according to  claim 22 , wherein the bacterium further comprises a plasmid having a T-DNA region adjacent to at least one  Agrobacterium  T-DNA border. 
     
     
         25 . The  Agrobacterium  strain according to  claim 24 , wherein the  Agrobacterium  strain is deficient in RecA functionality. 
     
     
         26 . The  Agrobacterium  strain according to  claim 23 , wherein the T-DNA region contains three or more gene sequences. 
     
     
         27 . The  Agrobacterium  strain according to  claim 23 , wherein the T-DNA region contains equal to or greater than 25,000 nucleotides. 
     
     
         28 . The  Agrobacterium  strain according to  claim 23 , wherein the T-DNA region comprises more than one gene sequence and the gene sequences have greater than 60% sequence homology. 
     
     
         29 . The  Agrobacterium  strain according to  claim 23 , wherein T-DNA region encodes one or more of an insecticidal protein, a herbicidal proteins, or a mixture of insecticidal proteins and herbicide tolerance proteins. 
     
     
         30 . The  Agrobacterium  strain according to  claim 23 , wherein the T-DNA region encodes a Cry1Ca insecticidal protein, a Cry1F insecticidal protein, and a Cry1Ab1 insecticidal protein. 
     
     
         31 . The  Agrobacterium  strain according to  claim 23 , wherein the T-DNA region encodes a Cry1Ca insecticidal protein, a Cry1F insecticidal protein, a Cry1Ab1 insecticidal protein, and an AAD-1 herbicide tolerance protein. 
     
     
         32 . A nilA genomic locus of  Agrobacterium tumefaciens , wherein a polynucleotide sequence is integrated into the nilA genomic locus. 
     
     
         33 . The nilA genomic locus of  claim 32 , wherein the polynucleotide sequence comprises a vir gene. 
     
     
         34 . An  Agrobacterium  strain with a 14.8 KpnI VirBCDG fragment of SB1 integrated into a neutral integration site on the  Agrobacterium  chromosome. 
     
     
         35 . The  Agrobacterium  strain according to  claim 34 , wherein the neutral integration site is a nilA genomic locus. 
     
     
         36 . The  Agrobacterium  strain according to  claim 34 , wherein the  Agrobacterium  strain is deficient in RecA functionality. 
     
     
         37 . The  Agrobacterium  strain according to  claim 34 , wherein the  Agrobacterium  strain is  Agrobacterium tumefaciens.    
     
     
         38 . An  Agrobacterium  strain LB4404 comprising a 14.8 KpnI VirBCDG fragment of pSB1on a pTi helper plasmid and a pTi plasmid having at least one disarmed T-DNA region and has exogenous DNA adjacent to at least one  Agrobacterium  T-DNA border, wherein the plasmids have differing origins of replication relative to each other. 
     
     
         39 . A plant according to  claim 1 . 
     
     
         40 . The plant according to  claim 39 , wherein any genetic traits introduced to the plant by the transformation are stably produced in progeny of the plant. 
     
     
         41 . A plant according to  claim 4 , wherein the T-DNA region is stably incorporated into the plant DNA. 
     
     
         42 . The plant according to  claim 41 , wherein any genes encoded by the T-DNA region are expressed in the plant. 
     
     
         43 . The plant according to  claim 41 , wherein any genes encoded by the T-DNA region are stably produced in progeny of the plant. 
     
     
         44 . The plant according to  claim 41 , wherein the plant stably expresses Cry1Ca insecticidal proteins, Cry1F insecticidal proteins, Cry1Ab1 insecticidal proteins, and AAD1 herbicidal proteins. 
     
     
         45 . The plant according to  claim 44 , wherein the plant is maize. 
     
     
         46 .  Agrobacterium  strain LBA4404 comprising at least one vir gene from a 14.8 KpnI VirBCDG fragment isolated from pSB1 integrated into a neutral integration site on the  Agrobacterium  chromosome. 
     
     
         47 . A fertile transgenic corn plant, or progeny thereof, which expresses insecticidal amounts of Cry1Ca protein, Cry1F insecticidal protein, Cry1Ab1 insecticidal protein, and herbicide-tolerant amounts of AAD-1 protein, wherein the Cry1 Ca, Cry1F, Cry1Ab1, and AAD1 proteins are collectively expressed from a single locus of recombinant DNA stably incorporated in the genome of the plant. 
     
     
         48 . The fertile transgenic corn plant of  claim 47 , wherein the single locus of recombinant DNA is substantially free of vector backbone sequences from a pTi DNA plasmid.