Agrobacterium-mediated method for producing transformed plant

Abstract

The present invention aims to provide a novel Agrobacterium -mediated method for producing a transformed plant. The method of the present invention is an Agrobacterium -mediated method for producing a transformed plant, which comprises: (i) a coculture step for culturing an Agrobacterium -inoculated plant material with a coculture medium; and (ii) a regeneration step for culturing the tissue obtained in (i) with a regeneration medium, either without callus growth culture or after callus growth culture, to thereby regenerate a whole plant, wherein 1) said method comprises transformation enhancement, and wherein 2) said method does not comprise selection of transformed cells based on the properties of a nucleic acid to be introduced by Agrobacterium in any step from coculture to regeneration.

1 . An  Agrobacterium -mediated method for producing a transformed monocotyledon, which comprises:
 (i) a coculture step for culturing an  Agrobacterium -inoculated monocotyledon embryo with a coculture medium; and   (ii) a regeneration step for culturing the embryo obtained in (i) with a regeneration medium, either without callus growth culture or after callus growth culture, to thereby regenerate a whole transformed monocotyledon,   wherein 1) said method comprises transformation enhancement, and   wherein 2) said method does not comprise any selection step based on the properties of a nucleic acid to be introduced by  Agrobacterium  in any step from coculture to regeneration.   
     
     
         2 . The method according to  claim 1 , wherein 3) said method further does not comprise any step for culturing the cocultured embryo with a callus growth medium between the coculture step and the regeneration step. 
     
     
         3 . The method according to  claim 1  or  2 , wherein the selection of transformed embryo based on the properties of a nucleic acid to be introduced by  Agrobacterium  is selection with a selective drug and a resistance gene for the selective drug. 
     
     
         4 . The method according to  claim 1 , wherein the transformation enhancement in 1) is intended to improve the efficiency of introducing a gene of interest into monocotyledon cells, to improve the efficiency of inducing a callus from an immature embryo, or to improve the efficiency of regeneration from a transformed callus. 
     
     
         5 . The method according to  claim 1 , wherein the transformation enhancement in 1) is selected from the group consisting of:
 thermal treatment;   centrifugation;   thermal treatment and centrifugation;   pressurization;   addition of silver nitrate and/or copper sulfate to the coculture medium;   inoculation with  Agrobacterium  in the presence of a powder;   addition of carbenicillin to the medium in the callus growth and/or regeneration step following the coculture step;   addition of N6 inorganic salts to the callus growth medium; and   addition of cysteine to the coculture medium.   
     
     
         6 . The method according to  claim 1 , wherein said method comprises addition of a compound belonging to benzoic herbicides in the coculture step. 
     
     
         7 . The method according to  claim 6 , wherein the compound belonging to benzoic herbicides is of benzoic acid type, salicylic acid type or picolinic acid type. 
     
     
         8 . The method according to  claim 6  or  7 , wherein the compound belonging to benzoic herbicides is 3,6-dichloro-o-anisic acid or 4-amino-3,5,6-trichloropicolinic acid. 
     
     
         9 . The method according to  claim 1 , wherein the monocotyledon is selected from the group consisting of maize, rice and wheat.