Soybean transformation method

Abstract

A method is disclosed for the Agrobacterium -mediated germline genetic transformation of soybean. The method is based on Agrobacterium -mediated gene delivery to individual cells in a freshly germinated soybean meristem, which cells can be induced directly to form shoots that give rise to transgenic plants. This method does not involve callus-phase tissue culture and is rapid and efficient.

1 - 10 . (canceled)  
   
   
       11 . A method of producing a transformed soybean cell comprising: 
 (a) initiating the germination of a soybean seed;    (b) substantially removing at least one cotyledon from the germinating seed;    (c) exposing at least a portion of the meristematic region in the germinating seed;    (d) exposing one or more cells in the meristematic region to a disarmed  Agrobacterium  vector comprising a heterologous genetic construct, 
 wherein said genetic construct comprises a selectable marker gene, and said genetic construct is transferred into at least one cell in the meristematic region;  
   (e) culturing the cells of the meristematic region in the presence of a selection agent to identify a transformed soybean cell that contains the genetic construct.    
   
   
       12 . The method of  claim 11 , wherein the selectable marker gene confers resistance to an antibiotic or a herbicide.  
   
   
       13 . The method of  claim 11 , wherein the selectable marker gene confers resistance to glyphosate.  
   
   
       14 . The method of  claim 11 , wherein the selectable marker gene encodes an EPSP synthase.  
   
   
       15 . The method of  claim 14 , wherein the EPSP synthase protein is the CP4 protein.  
   
   
       16 . The method of  claim 11 , wherein the selection agent is an herbicide.  
   
   
       17 . The method of  claim 16 , wherein the herbicide is glyphosate.  
   
   
       18 . The method of  claim 11 , wherein the selectable marker gene confers resistance to kanamycin.  
   
   
       19 . The method of  claim 11 , wherein the selectable marker gene encodes a neomycin phosphotransferase II (nptII) protein.  
   
   
       20 . The method of  claim 11 , wherein the selection agent is kanamycin.  
   
   
       21 . The method of  claim 11 , further comprising: 
 (f) inducing formation of one or more shoots from the meristematic region, wherein said shoot comprises at least one transformed cell, to produce a transgenic soybean shoot.    
   
   
       22 . The method of  claim 21 , wherein step (f) comprises exposing said meristematic region to at least one shoot inducing hormone.  
   
   
       23 . The method of  claim 22 , wherein the at least one shoot inducing hormone is selected from the group consisting of BAP, IBA, IAA, NAA, cytokinins, auxins, kinetins, glyphosate, and thiadiazorun.  
   
   
       24 . The method of  claim 21 , wherein step (f) comprises exposing said meristematic region to a media containing BAP and IBA.  
   
   
       25 . A method of producing a fertile transgenic soybean plant comprising: 
 (a) initiating the germination of a soybean seed;    (b) substantially removing at least one cotyledon from the germinating seed;    (c) exposing at least a portion of the meristematic region in the germinating seed;    (d) exposing one or more cells in the meristematic region to a disarmed  Agrobacterium  vector comprising a heterologous genetic construct, 
 wherein said genetic construct comprises a selectable marker gene, and said genetic construct is transferred into at least one cell in the meristematic region;  
   (e) culturing the cells of the meristematic region in the presence of a selection agent in a manner that identifies a transformed soybean cell that contains the genetic construct.    (f) inducing formation of one or more shoots from the meristematic region, wherein said shoot comprises at least one transformed cell, to produce a transgenic soybean shoot.    (g) cultivating the transgenic soybean shoot into a fertile transgenic soybean plant.    
   
   
       26 . The method of  claim 25 , wherein step (g) comprises inducing root formation from the transgenic soybean shoot.  
   
   
       27 . The method of  claim 25 , wherein step (f) comprises exposing said meristematic region to at least one shoot inducing hormone.  
   
   
       28 . The method of  claim 22 , wherein the at least one shoot inducing hormone is selected from the group consisting of BAP, IBA, IAA, NAA, cytokinins, auxins, kinetins, glyphosate, and thiadiazorun.  
   
   
       29 . The method of  claim 25 , wherein step (f) comprises exposing said meristematic region to a media containing BAP and IBA.  
   
   
       30 . The method of  claim 25 , wherein the selectable marker gene confers resistance to an antibiotic or a herbicide.  
   
   
       31 . The method of  claim 25 , wherein the selectable marker gene confers resistance to glyphosate.  
   
   
       32 . The method of  claim 25 , wherein the selectable marker gene encodes an EPSP synthase.  
   
   
       33 . The method of  claim 32 , wherein the EPSP synthase protein is the CP4 protein.  
   
   
       34 . The method of  claim 25 , wherein the selection agent is an herbicide.  
   
   
       35 . The method of  claim 34 , wherein the herbicide is glyphosate.  
   
   
       36 . The method of  claim 25 , wherein the selectable marker gene confers resistance to kanamycin.  
   
   
       37 . The method of  claim 25 , wherein the selectable marker gene encodes a neomycin phosphotransferase II (nptII) protein.  
   
   
       38 . The method of  claim 25  wherein the selection agent is kanamycin.  
   
   
       39 . The method of  claim 25 , wherein said fertile transgenic soybean plant is cultivated to produce seed.  
   
   
       40 . The method of  claim 39 , wherein said seed are cultivated to produce progeny soybean plants.  
   
   
       41 . The method of  claim 40 , wherein said progeny soybean plants are cultivated to produce progeny seed.  
   
   
       42 . The method of  claim 41 , wherein said progeny seed is cultivated to produce a subsequent generation of soybean plants.  
   
   
       43 . The method of  claim 11 , wherein the exposed meristematic region is wounded after step (c).  
   
   
       44 . The method of  claim 43 , wherein the wounding is caused by ultrasonic waves, a plasma blast discharge, or by puncturing the soybean explant with a needle, other sharp object, or an abrasive object.  
   
   
       45 . The method of  claim 25 , wherein the exposed meristematic region is wounded after step (c).  
   
   
       46 . The method of  claim 45 , wherein the wounding is caused by ultrasonic waves, a plasma blast discharge, or by puncturing the soybean explant with a needle, other sharp object, or an abrasive object.