Markerless Transformation

Abstract

Methods for identification of successful transformation without a need for in vitro selection are provided. Direct detection of nucleotide sequences of interest is described which eliminate the need for use of ancillary nucleotide sequences.

1 - 8 . (canceled) 
     
     
         9 . A method for producing a transgenic plant comprising a nucleotide sequence of interest inserted into its genome, where said method comprises:
 a) transforming a plant tissue or plant cell with said nucleotide sequence of interest, which comprises a gene of interest; wherein said plant tissue or plant cell is not transformed with an ancillary nucleotide sequence comprising a selectable marker;   b) regenerating said plant tissue or plant cell without selection for a selectable marker following said transforming to produce a transgenic plant; and   c) directly detecting the presence or absence in said transgenic plant of the nucleotide sequence of interest, an encoded product of said nucleotide sequence of interest, or a metabolic product of said encoded product;   wherein said transgenic plant comprises the nucleotide sequence of interest introduced by said transforming; and   wherein said transforming does not introduce a transposable element into said plant tissue or plant cell.   
     
     
         10 . The method of  claim 9 , wherein directly detecting comprises the use of a method selected from the group consisting of southern hybridization, polymerase chain reaction, and western hybridization for the nucleotide sequence or encoded protein product, and HPLC for a metabolic product of the encoded product. 
     
     
         11 . The method of  claim 9 , wherein directly detecting comprises the use of a method selected from the group consisting of southern hybridization for the nucleotide sequence of interest and HPLC for a metabolic product of the encoded product. 
     
     
         12 . The method of  claim 9 , wherein directly detecting comprises the use of a method selected from the group consisting of polymerase chain reaction for the nucleotide sequence of interest and HPLC for a metabolic product of the encoded product. 
     
     
         13 . The method of  claim 9 , wherein directly detecting comprises the use of a method selected from the group consisting of western hybridization for the encoded product and HPLC for a metabolic product of the encoded product. 
     
     
         14 . The method of  claim 9 , wherein directly detecting comprises the use of HPLC for a metabolic product of the encoded product. 
     
     
         15 . The method of  claim 9 , wherein said directly detecting comprises pooling samples from a group of cells. 
     
     
         16 . The method of  claim 9 , wherein said directly detecting comprises pooling samples from a group of plants. 
     
     
         17 . The method of  claim 9 , wherein said plant tissue is a cotyledon. 
     
     
         18 . The method of  claim 9 , wherein said transforming involves a binary vector. 
     
     
         19 . The method of  claim 9 , wherein said regenerating is carried out in RZ1/2C medium, ROC medium, or a combination thereof. 
     
     
         20 . The method of  claim 9 , wherein said transforming does not involve an  Agrobacterium.    
     
     
         21 . A method for producing a transgenic plant comprising a nucleotide sequence of interest inserted into its genome, where said method comprises:
 a) transforming a plant tissue or plant cell with said nucleotide sequence of interest, which comprises a gene of interest; wherein said plant tissue or plant cell is not transformed with an ancillary nucleotide sequence comprising a selectable marker;   b) regenerating said plant tissue or plant cell without selection for a selectable marker following said transforming; and   c) directly detecting the presence or absence in said transgenic plant of the nucleotide sequence of interest, an encoded product of said nucleotide sequence of interest, or a metabolic product of said encoded product;   wherein said transgenic plant comprises the nucleotide sequence of interest introduced by said transforming; and   wherein said transforming does not introduce a transposable element into said plant tissue or plant cell.   
     
     
         22 . The method of  claim 9 , wherein directly detecting comprises the use of a method selected from the group consisting of southern hybridization, polymerase chain reaction, and western hybridization for the nucleotide sequence or encoded protein product, and HPLC for a metabolic product of the encoded product. 
     
     
         23 . The method of  claim 9 , wherein directly detecting comprises the use of a method selected from the group consisting of southern hybridization for the nucleotide sequence of interest and HPLC for a metabolic product of the encoded product. 
     
     
         24 . The method of  claim 9 , wherein directly detecting comprises the use of a method selected from the group consisting of polymerase chain reaction for the nucleotide sequence of interest and HPLC for a metabolic product of the encoded product. 
     
     
         25 . The method of  claim 9 , wherein directly detecting comprises the use of a method selected from the group consisting of western hybridization for the encoded product and HPLC for a metabolic product of the encoded product. 
     
     
         26 . The method of  claim 9 , wherein said directly detecting comprises pooling samples from a group of cells. 
     
     
         27 . The method of  claim 9 , wherein said directly detecting comprises pooling samples from a group of plants. 
     
     
         28 . The method of  claim 9 , wherein said plant tissue is a cotyledon.