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 . A method for transforming a soybean plant cell, comprising:
 (a) contacting at least a first soybean plant cell with a cell of a  Rhizobia  bacterium other than  Agrobacterium  sp. comprising: (i) a first nucleic acid comprising a vir gene region of a Ti plasmid wherein the vir gene region acts to introduce a nucleic acid of interest into the plant cell in a VirD2-dependent manner; and (ii) a second nucleic acid comprising one or more T-DNA border sequence(s) operably linked to a nucleic acid of interest; and   (b) selecting at least a first soybean plant cell transformed with the nucleic acid of interest;   wherein the  Rhizobia  cell is from  Bradyrhizobium  spp.   
     
     
         2 - 4 . (canceled) 
     
     
         5 . The method of  claim 1 , wherein the first nucleic acid lacks left and/or right T-DNA border sequences. 
     
     
         6 . (canceled) 
     
     
         7 . The method of  claim 1 , wherein the  Rhizobia  cell is grown in the presence of acetosyringone or other compound that induces vir gene function prior to contacting the soybean plant cell. 
     
     
         8 . (canceled) 
     
     
         9 . The method of  claim 1 , wherein the  Rhizobia  cell is  Bradyrhizobium japonicum.    
     
     
         10 . The method of  claim 9 , wherein the  Rhizobia  cell is  B. japonicum  USDA 6 or  B. japonicum  USDA 110. 
     
     
         11 . The method of  claim 1 , wherein the soybean plant cell is comprised in an explant from a soybean 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 . (canceled) 
     
     
         14 . The method of  claim 1 , wherein the first and second nucleic acids are introduced into the bacterium by electroporation. 
     
     
         15 . The method of  claim 1 , wherein selecting the soybean plant cell transformed with the nucleic acid of interest is carried out in the absence of a selection agent. 
     
     
         16 . The method of  claim 1 , wherein selecting the soybean plant cell transformed with the nucleic acid of interest comprises culturing the soybean 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 , 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 soybean plant regenerated from the soybean plant cell transformed with the nucleic acid of interest. 
     
     
         23 . The method of  claim 1 , wherein the bacterium comprises at least a third nucleic acid comprising a further nucleic acid of interest and wherein the soybean plant cell is transformed with the third nucleic acid. 
     
     
         24 . The method of  claim 1 , further comprising regenerating a soybean plant from the soybean plant cell, wherein the soybean plant comprises the nucleic acid of interest. 
     
     
         25 . The method of  claim 24 , wherein regenerating the soybean plant from the soybean plant cell comprises inducing formation of one or more shoots from an explant comprising the soybean plant cell and cultivating at least a first shoot into a whole fertile soybean plant. 
     
     
         26 . The method of  claim 24 , wherein regeneration occurs by organogenesis. 
     
     
         27 - 38 . (canceled) 
     
     
         39 . The method of  claim 1 , further comprising growing the bacterium other than  Agrobacterium  sp. in an induction medium comprising carbon sources which 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.