Disarmed agrobacterium strains, ri-plasmids, and methods of transformation based thereon

Abstract

The present invention relates “disarmed” strain variants of Agrobacterium strain K599 (NCPPB 2659), “disarmed” plasmid variants of the Ri-plasmid pRi2659, and derivatives thereof, and methods employing these strains and plasmids in plant transformation.

We claim: 
     
       1. A method for producing a transgenic plant cell or a transgenic plant, comprising:
 a) providing bacteria of a transgenic non-pathogenic strain variant of Agrobacterium strain K599 (NCPPB 2659) which comprise a genome comprising a 16S-23S rRNA intergenic sequence comprising at least one sequence motif selected from the group consisting of SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, 13, and 14, wherein said strain variant is capable to infect plant cells, to mediate T-DNA transfer into plant cells, and to mediate T-DNA insertion into the plant genome, but is lacking hairy root phenotype inducing properties, and wherein said strain variant further comprises a transgenic T-DNA, 
 b) co-cultivating a plant, plant cell or plant tissue with said bacteria, and 
 c) isolating or selecting plant cells comprising stably integrated into their genome said transgenic T-DNA, and optionally 
 d) regenerating plants comprising stably integrated into their genome said transgenic T-DNA. 
 
     
     
       2. The method of  claim 1 , wherein said non-pathogenic strain variant comprises a non-pathogenic plasmid variant of the Ri-plasmid pRi2659. 
     
     
       3. The method of  claim 2 , wherein the non-pathogenic plasmid variant
 (a) encodes a virD2 protein, the amino acid sequence of which comprises the sequence of SEQ ID NO: 112, or 
 (b) comprises the sequence of SEQ ID NO: 24. 
 
     
     
       4. The method of  claim 1 , wherein said transgenic T-DNA comprises at least one plant expressible selectable marker gene. 
     
     
       5. The method of  claim 4 , wherein said plant cell, plant tissue, or plant is derived from a plant selected from the group consisting of monocotyledonous plants, dicotyledonous plants, and gymnosperm plants. 
     
     
       6. The method of  claim 5 , wherein said plant is from a genus selected from the group consisting of Medicago, Lycopersicon, Brassica, Cucumis, Solanum, Juglans, Gossypium, Malus, Vitis, Antirrhinum, Populus, Fragaria, Arabidopsis, Picea, Capsicum, Chenopodium, Dendranthema, Pharbitis, Pinus, Pisum, Oryza, Zea, Triticum, Triticale, Secale, Lolium, Hordeum, Glycine, Pseudotsuga, Kalanchoe, Beta, Helianthus, and Nicotiana. 
     
     
       7. A non-pathogenic strain variant of Agrobacterium strain K599 (NCPPB 2659) which comprises a genome comprising a 16S-23S rRNA intergenic sequence comprising at least one sequence motif selected from the group consisting of SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, 13, and 14, wherein said strain variant is capable to infect plant cells, to mediate T-DNA transfer into plant cells, and to mediate T-DNA insertion into the plant genome, but is lacking hairy root phenotype inducing properties. 
     
     
       8. A transgenic non-pathogenic strain variant of Agrobacterium strain K599 (NCPPB 2659) which comprises a genome comprising a 16S-23S rRNA intergenic sequence comprising at least one sequence motif selected from the group consisting of SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, 13, and 14, wherein said strain variant is capable to infect plant cells, to mediate T-DNA transfer into plant cells, and to mediate T-DNA insertion into the plant genome, but is lacking hairy root phenotype inducing properties, and wherein said strain variant further comprises a transgenic T-DNA. 
     
     
       9. The non-pathogenic strain variant of  claim 7 , wherein said strain variant comprises a non-pathogenic plasmid variant of the Ri-plasmid pRi2659. 
     
     
       10. The non-pathogenic strain variant of  claim 8 , wherein is said strain variant comprises a non-pathogenic plasmid variant of the Ri-plasmid pRi2659. 
     
     
       11. The non-pathogenic strain variant of  claim 9 , wherein the non-pathogenic plasmid variant
 (a) encodes a virD2 protein, the amino acid sequence of which comprises the sequence of SEQ ID NO: 112, or 
 (b) comprises the sequence of SEQ ID NO: 24. 
 
     
     
       12. The non-pathogenic strain variant of  claim 10 , wherein the non-pathogenic plasmid variant
 (a) encodes a virD2 protein, the amino acid sequence of which comprises the sequence of SEQ ID NO: 112, or 
 (b) comprises the sequence of SEQ ID NO: 24. 
 
     
     
       13. The non-pathogenic strain variant of  claim 8 , wherein said transgenic T-DNA comprises at least one plant expressible selectable marker gene. 
     
     
       14. The method of claim 1, wherein said plant cell or plant tissue is from a plant of the family Leguminosae; or wherein said plant is from the family Leguminosae. 
     
     
       15. The method of claim 14, wherein said plant cell or plant tissue is from a plant of the genus Glycine; or wherein said plant is from the genus Glycine. 
     
     
       16. The method of claim 1, wherein said plant cell or plant tissue is from a Glycine max plant; or wherein said plant is a Glycine max plant. 
     
     
       17. The method of claim 14, wherein said plant cell or plant tissue is from a soybean plant; or wherein said plant is a soybean plant. 
     
     
       18. The method of claim 1, wherein said plant cell or plant tissue is from an Arabidopsis thaliana, Nicotiana tabacum, oilseed rape, soybean, corn (maize), wheat, linseed, potato, or tagetes plant; or wherein said plant is an Arabidopsis thaliana, Nicotiana tabacum, oilseed rape, soybean, corn (maize), wheat, linseed, potato, or tagetes plant. 
     
     
       19. The method of claim 1, further comprising obtaining a transgenic plant comprising said T-DNA. 
     
     
       20. The method of claim 19, further comprising obtaining a transgenic plant cell, a transgenic plant or progeny thereof from said transgenic plant. 
     
     
       21. The method of claim 20, further comprising selecting a transgenic plant or progeny thereof having an agronomically valuable trait conferred by the T-DNA; or further comprising obtaining a transgenic cell from said transgenic plant or progeny. 
     
     
       22. The method of claim 19, comprising:
 1) selfing the transgenic plant or crossing the transgenic plant with a second plant; and   2) selecting resulting progeny comprising the T-DNA.   
     
     
       23. The method of claim 22, comprising obtaining progeny comprising the T-DNA. 
     
     
       24. The method of claim 20, comprising selecting a plant having the T-DNA stably transformed into its genome. 
     
     
       25. The method of claim 1, further comprising:
 1) selfing the regenerated plant or crossing the regenerated plant with a second plant; and   2) selecting resulting progeny comprising the T-DNA.   
     
     
       26. The method of claim 25, comprising obtaining progeny comprising the T-DNA. 
     
     
       27. A method comprising obtaining progeny from a fertile transgenic plant obtained by the method of claim 1, which progeny comprise the T-DNA. 
     
     
       28. The method of claim 27, wherein the progeny are obtained by crossing the fertile transgenic plant with a second plant. 
     
     
       29. The method of claim 27, comprising obtaining seed from the progeny and obtaining further progeny plants comprising the T-DNA from the seed. 
     
     
       30. The method of claim 27, wherein the progeny obtained are selfed or crossed with a second plant, to obtain further progeny which comprise the T-DNA. 
     
     
       31. The method of claim 29, wherein seeds are obtained from the further progeny plants and plants comprising the T-DNA are recovered from said seed. 
     
     
       32. The method of claim 29, wherein the further progeny are selfed or crossed with a second plant to obtain additional progeny which comprise the T-DNA. 
     
     
       33. The method of claim 32, further comprising obtaining seed from the additional progeny.