Use of non-agrobacterium bacterial species for plant transformation

Abstract

The invention relates to methods for Rhizobia -mediated genetic transformation of plant cells, including soybean, canola, corn, and cotton cells. These include both VirD2-dependent and VirD2-independent methods. Bacterial species utilized include strains of Rhizobium sp., Sinorhizobium sp., and Mesorhizobium sp. Vectors for use in such transformation are also disclosed.

1 . (canceled) 
     
     
         2 . A method for transforming a plant cell, comprising:
 (a) contacting at least a first plant cell with a bacterium other than  Agrobacterium  comprising (i) a first nucleic acid required for conjugative transfer of DNA sequences independent of VirD2 function, and (ii) a second nucleic acid comprising a nucleic acid of interest; wherein the plant cell is a soybean, canola, corn, or cotton plant cell and wherein polypeptides encoded by the nucleic acid required for conjugative transfer act to transfer the nucleic acid of interest into the plant cell; and   (b) selecting at least a first plant cell transformed with the nucleic acid of interest.   
     
     
         3 . The method of  claim 2 , wherein the conjugative transfer is traA, tral, or mobA-dependent. 
     
     
         4 . The method of  claim 2 , wherein said first nucleic acid comprises oriT. 
     
     
         5 . The method of  claim 2 , wherein the first nucleic acid lacks left and/or right T-DNA border sequences. 
     
     
         6 . The method of  claim 2 , wherein the bacterium is a  Rhizobia  cell. 
     
     
         7 . The method of  claim 6 , wherein the  Rhizobia  cell is grown in the presence of acetosyringone or other compound that induces vir gene function prior to contacting the plant cell. 
     
     
         8 . The method of  claim 6 , wherein the  Rhizobia  cell is selected from the group consisting of:  Rhizobium  spp.,  Sinorhizobium  spp.,  Mesorhizobium  spp.,  Phyllobacterium  spp.  Ochrobactrum  spp. and  Bradyrhizobium  spp. 
     
     
         9 . The method of  claim 8 , wherein the  Rhizobia  cell is  Rhizobium leguminosarum.    
     
     
         10 . The method of  claim 9 , wherein the  Rhizobia  cell is  R. leguminosarum  bv.  trifolii, R. leguminosarum  bv.  phaseoli  or  Rhizobium leguminosarum . bv.  viciae.    
     
     
         11 . The method of  claim 2 , wherein the plant cell is comprised in an explant from a plant seed, seedling, callus, cell suspension, cotyledon, meristem, leaf, root, or stem; and the explant is contacted with the bacterium. 
     
     
         12 . The method of  claim 11 , wherein the explant comprises an embryonic meristem; callus; cell suspension; cotyledon; or tissue from leaves, roots, or stems. 
     
     
         13 . The method of  claim 2 , wherein the nucleic acid required for conjugative transfer independent of VirD2 function is introduced into the bacterium by electroporation. 
     
     
         14 . The method of  claim 2 , wherein the first and second nucleic acids are introduced into the bacterium by electroporation. 
     
     
         15 . The method of  claim 2 , wherein selecting a plant cell transformed with the nucleic acid of interest is carried out in the absence of a selection agent. 
     
     
         16 . The method of  claim 2 , wherein selecting a plant cell transformed with the nucleic acid of interest comprises culturing the plant cell in the presence of a selection agent, wherein the nucleic acid of interest confers tolerance to the selection agent or is operably linked to a further nucleic acid that confers tolerance to the selection agent. 
     
     
         17 . The method of  claim 16 , wherein the selection agent is glyphosate, kanamycin, bialaphos or dicamba. 
     
     
         18 . The method of  claim 17 , wherein the nucleic acid of interest or further nucleic acid encodes EPSP synthase. 
     
     
         19 . The method of  claim 18 , wherein the EPSP synthase protein is CP4. 
     
     
         20 . The method of  claim 16 , wherein the selection agent is glyphosate. 
     
     
         21 . The method of  claim 1  or  2 , wherein the nucleic acid of interest is not physically linked to a selectable marker gene. 
     
     
         22 . The method of  claim 21 , wherein the marker gene and the nucleic acid of interest genetically segregate in progeny of a plant regenerated from the plant cell transformed with the nucleic acid of interest. 
     
     
         23 . The method of  claim 2 , wherein the bacterium comprises at least a third nucleic acid comprising a further nucleic acid of interest and wherein the plant cell is transformed with the third nucleic acid. 
     
     
         24 . The method of  claim 2 , further comprising regenerating a plant from the plant cell, wherein the plant comprises the nucleic acid of interest. 
     
     
         25 . The method of  claim 24 , wherein regenerating a plant from the plant cell comprises inducing formation of one or more shoots from an explant comprising the plant cell and cultivating at least a first shoot into a whole fertile plant. 
     
     
         26 . The method of  claim 24 , wherein regeneration occurs by organogenesis. 
     
     
         27 . The method of  claim 24 , wherein the plant is selected from the group consisting of: a corn plant, a cotton plant, a soybean plant, and a canola plant. 
     
     
         28 - 32 . (canceled) 
     
     
         33 . A DNA construct competent for virD2-independent transfer from  Rhizobia  and lacking T-DNA border sequence, the construct comprising an oriT sequence operably linked to a nucleic acid of interest. 
     
     
         34 . The DNA construct of  claim 33 , further comprising a traA or mob sequence. 
     
     
         35 . A  Rhizobia  cell transformed with the DNA construct of  claim 33 , wherein the  Rhizobia  is selected from the group consisting of:  Rhizobium  spp.,  Sinorhizobium  spp.,  Mesorhizobium  spp.,  Phyllobacterium  spp.  Ochrobactrum  spp. and  Bradyrhizobium  spp. 
     
     
         36 . The  Rhizobia  cell of  claim 35 , wherein the  Rhizobia  cell is selected from the group consisting of:  Rhizobium  sp.,  Rhizobium  sp. NGR234,  Rhizobium leguminosarum  Madison,  R. leguminosarum  USDA2370,  R. leguminosarum  bv.  trifolii  USDA2408,  R. leguminosarum  bv.  phaseoli  USDA2668,  R. leguminosarum  2370G,  R. leguminosarum  2370LBA,  R. leguminosarum  2048G,  R. leguminosarum  2048LBA,  R. leguminosarum  bv.  phaseoli, R. leguminosarum  bv.  phaseoli  2668G,  R. leguminosarum  bv.  phaseoli  2668LBA,  R. leguminosarum  RL542C,  R. leguminosarum  bv.  viciae, R. leguminosarum  bv.  trifolii, Rhizobium etli  USDA 9032,  R. etli  bv.  phaseoli, Rhizobium tropici, Mesorhizobium  sp.,  Mesorhizobium loti  ML542G,  M. loti  ML4404,  Sinorhizobium  sp.,  Sinorhizobium meliloti  SD630,  S. meliloti  USDA1002,  Sinorhizobium fredii  USDA205,  S. fredii  SF542G,  S. fredii  SF4404 , S. fredii  SM542C,  Bradyrhizobium  sp.,  Bradyrhizobium japonicum  USDA 6, and  B. japonicum  USDA 110. 
     
     
         37 . The  Rhizobia  cell of  claim 36 , wherein the cell is a  Rhizobium leguminosarum  cell. 
     
     
         38 . The  Rhizobia  cell of  claim 36 , wherein the cell is a  R. leguminosarum  bv.  trifolii, R. leguminosarum  bv.  phaseoli  or  Rhizobium leguminosarum . bv.  viciae cell.    
     
     
         39 . The method of  claim 2 , further comprising growing the bacterium other than  Agrobacterium  sp. under conditions to minimize polysaccharide production during growth in induction medium. 
     
     
         40 . The method of  claim 39 , wherein the carbon source(s) used to minimize polysaccharide production during growth in induction medium is glucose in AB-TY medium, or L-arabinose and potassium gluconate in ATA medium.