Novel agrobacterium-mediated plant transformation method

Abstract

The present invention relates to a novel transformation system for generating transformed plants with lower copy inserts and improved transformation efficiency. In particular, the invention relates to the use of Agrobacterium growth inhibiting agents during the Agrobacterium-mediated transformation process that suppress Agrobacterium growth and reduce T-DNA transfer to the target plant genome.

What is claimed is:  
     
         1 . A method of transforming a plant cell or plant tissue using an Agrobacterium mediated process comprising the steps of: 
 inoculating a transformable plant cell or tissue with Agrobacterium containing at least one genetic component capable of being transferred to the plant cell or tissue in the presence of at least one growth inhibiting agent;    co-culturing the transformable plant cell or tissue after inoculation in a media capable of supporting growth of plant cells or tissue expressing the genetic component, said media not containing a growth inhibiting agent;    selecting transformed plant cells or tissue; and    regenerating a transformed plant expressing the genetic component from the selected transformed plant cells or tissue.    
     
     
         2 . The method of  claim 1  wherein the presence of the growth inhibiting agent during inoculation reduces the T-DNA transfer process of the Agrobacterium.  
     
     
         3 . The method of  claim 1  wherein the growth inhibiting agent is selected from the group consisting of antibiotics, compounds containing a heavy metal ion, and proteins, nucleic acids, cell extracts, growth regulators, or secondary metabolites capable of inhibiting or suppressing the growth of Agrobacterium.  
     
     
         4 . The method of  claim 3  wherein the compound containing a heavy metal ion contains silver, potassium, manganese or cadmium.  
     
     
         5 . The method of  claim 4  wherein the heavy metal ion is silver.  
     
     
         6 . The method of  claim 3  wherein the growth inhibiting agent is silver nitrate.  
     
     
         7 . The method of  claim 6  wherein the concentration of silver nitrate is from about 5 μM to about 50 μM.  
     
     
         8 . The method of  claim 7  wherein the concentration of silver nitrate is about 20 μM.  
     
     
         9 . The method of  claim 3  wherein the growth inhibiting agent is silver thiosulfate.  
     
     
         10 . The method of  claim 9  wherein the concentration of silver thiosulfate is from about 5 μM to about 50 μM.  
     
     
         11 . The method of  claim 10  wherein the concentration of silver thiosulfate is 20 μM.  
     
     
         12 . The method of  claim 3  wherein the growth inhibiting agent is an antibiotic.  
     
     
         13 . The method of  claim 12  wherein the antibiotic is carbenicillin.  
     
     
         14 . The method of  claim 3  wherein the growth inhibiting agent is a nucleic acid capable of suppressing Agrobacterium cell growth and the T-DNA transfer process.  
     
     
         15 . The method of  claim 1  wherein the transformable plant cell or tissue is from a monocotyledonous plant.  
     
     
         16 . The method of  claim 1  wherein the transformable plant cell or tissue is from a dicotyledonous plant.  
     
     
         17 . The method of  claim 15  wherein the monocotyledonous plant is a cereal.  
     
     
         18 . The method of  claim 16  wherein the monocotyledonous plant is corn, wheat, or rice.  
     
     
         19 . The method of  claim 17  wherein the dicotyledonous plant is soybean, cotton, canola, or sunflower.  
     
     
         20 . The method of  claim 18  wherein the monocotyledonous plant is corn.  
     
     
         21 . The method of  claim 18  wherein the monocotyledonous plant is wheat.  
     
     
         22 . The method of  claim 18  wherein the monocotyledonous plant is rice.  
     
     
         23 . A method of transforming a plant cell or plant tissue using an Agrobacterium mediated process comprising the steps of: 
 inoculating a transformable plant cell or tissue with Agrobacterium containing at least one genetic component capable of being transferred to the plant cell or tissue;    co-culturing the transformable plant cell or tissue after inoculation in a media capable of supporting growth of plant cells or tissue expressing the genetic component, said media further containing a growth inhibiting agent;    selecting transformed plant cells or tissue; and    regenerating a transformed plant expressing the genetic component from the selected transformed cells or tissue.    
     
     
         24 . The method of  claim 23  wherein the presence of the growth inhibiting agent during co-culture reduces the T-DNA transfer process of the Agrobacterium.  
     
     
         25 . The method of  claim 23  wherein the growth inhibiting agent is selected from the group consisting of antibiotics, compounds containing a heavy metal ion, and proteins, nucleic acids, cell extracts, growth regulators, or secondary metabolites capable of inhibiting or suppressing the growth of Agrobacterium.  
     
     
         26 . The method of  claim 25  wherein the compound containing a heavy metal ion contains silver, potassium, manganese or cadmium.  
     
     
         27 . The method of  claim 26  wherein the heavy metal ion is silver.  
     
     
         28 . The method of  claim 25  wherein the growth inhibiting agent is silver nitrate.  
     
     
         29 . The method of  claim 28  wherein the concentration of silver nitrate is from about 5 μM to about 50 μM.  
     
     
         30 . The method of  claim 29  wherein the concentration of silver nitrate is about 20 μM.  
     
     
         31 . The method of  claim 25  wherein the growth inhibiting agent is silver thiosulfate.  
     
     
         32 . The method of  claim 31  wherein the concentration of silver thiosulfate is from about 5 μM to about 50 μM.  
     
     
         33 . The method of  claim 32  wherein the concentration of silver thiosulfate is 20 μM.  
     
     
         34 . The method of  claim 25  wherein the growth inhibiting agent is an antibiotic.  
     
     
         35 . The method of  claim 34  wherein the antibiotic is carbenicillin.  
     
     
         36 . The method of  claim 25  wherein the growth inhibiting agent is a nucleic acid capable of suppressing Agrobacterium cell growth and the T-DNA transfer process.  
     
     
         37 . The method of  claim 23  wherein the transformable plant cell or tissue is from a monocotyledonous plant.  
     
     
         38 . The method of  claim 23  wherein the transformable plant cell or tissue is from a dicotyledonous plant.  
     
     
         39 . The method of  claim 37  wherein the monocotyledonous plant is a cereal.  
     
     
         40 . The method of  claim 39  wherein the monocotyledonous plant is corn, wheat, or rice.  
     
     
         41 . The method of  claim 38  wherein the dicotyledonous plant is soybean, cotton, canola, or sunflower.  
     
     
         42 . The method of  claim 40  wherein the monocotyledonous plant is corn.  
     
     
         43 . The method of  claim 40  wherein the monocotyledonous plant is wheat.  
     
     
         44 . The method of  claim 40  wherein the monocotyledonous plant is rice.  
     
     
         45 . A method of transforming a plant cell or plant tissue using an Agrobacterium mediated process comprising the steps of: 
 inoculating a transformable plant cell or tissue with Agrobacterium containing at least one genetic component capable of being transferred to the plant cell or tissue in the presence of at least one growth inhibiting agent;    co-culturing the transformable plant cell or tissue after inoculation in a media capable of supporting growth of the plant cells or tissue expressing the genetic component, said media further containing a growth inhibiting agent;    selecting transformed plant cells or tissue; and    regenerating a transformed plant expressing the genetic component from the selected transformed cells or tissue.    
     
     
         46 . The method of  claim 45  wherein the presence of the growth inhibiting agent during inoculation and co-culture reduces the T-DNA transfer process of the Agrobacterium.  
     
     
         47 . The method of  claim 45  wherein the growth inhibiting agent is selected from the group consisting of antibiotics, compounds containing a heavy metal ion, and proteins, nucleic acids, cell extracts, growth regulators, or secondary metabolites capable of inhibiting or suppressing the growth of Agrobacterium.  
     
     
         48 . The method of  claim 47  wherein the compound containing a heavy metal ion contains silver, potassium, manganese or cadmium.  
     
     
         49 . The method of  claim 48  wherein the heavy metal ion is silver.  
     
     
         50 . The method of  claim 47  wherein the growth inhibiting agent is silver nitrate.  
     
     
         51 . The method of  claim 50  wherein the concentration of silver nitrate is from about 5 μM to about 50 μM.  
     
     
         52 . The method of  claim 51  wherein the concentration of silver nitrate is about 20 μM.  
     
     
         53 . The method of  claim 47  wherein the growth inhibiting agent is silver thiosulfate.  
     
     
         54 . The method of  claim 53  wherein the concentration of silver thiosulfate is from about 5 μM to about 50 μM.  
     
     
         55 . The method of  claim 54  wherein the concentration of silver thiosulfate is 20 μM.  
     
     
         56 . The method of  claim 47  wherein the growth inhibiting agent is an antibiotic.  
     
     
         57 . The method of  claim 56  wherein the antibiotic is carbenicillin.  
     
     
         58 . The method of  claim 47  wherein the growth inhibiting agent is a nucleic acid capable of suppressing Agrobacterium cell growth and the T-DNA transfer process.  
     
     
         59 . The method of  claim 45  wherein the transformable plant cell or tissue is from a monocotyledonous plant.  
     
     
         60 . The method of  claim 45  wherein the transformable plant cell or tissue is from a dicotyledonous plant.  
     
     
         61 . The method of  claim 59  wherein the monocotyledonous plant is a cereal.  
     
     
         62 . The method of  claim 61  wherein the monocotyledonous plant is corn, wheat, or rice.  
     
     
         63 . The method of  claim 60  wherein the dicotyledonous plant is soybean, cotton, canola, or sunflower.  
     
     
         64 . The method of  claim 62  wherein the monocotyledonous plant is corn.  
     
     
         65 . The method of  claim 62  wherein the monocotyledonous plant is wheat.  
     
     
         66 . The method of  claim 62  wherein the monocotyledonous plant is rice.