Methods and Vectors for Producing Transgenic Plants

Abstract

Methods of, and compositions for, assembling one or more transcription units in a genome without a linked selectable marker or other unwanted transcription unit are provided. Also provided methods of, and compositions for, assembling one or more transcription units in a genome with a reduced frequency of vector backbone.

1 - 27 . (canceled) 
     
     
         28 . A recombinant nucleic acid molecule comprising:
 a first DNA segment comprising a first transcription unit flanked on both sides and adjacent to a first and a second shorter transcribable nucleic acid sequence in opposite orientation to each other and located between a first left T-DNA border region and a first right T-DNA border region;   a second DNA segment comprising a second transcription unit flanked on both sides and adjacent to a third and a fourth shorter transcribable nucleic acid sequence in opposite orientation to each other and located between a second left T-DNA border region and a second right T-DNA border region, wherein said third shorter transcribable nucleic acid sequence is operably linked to a plant-expressible promoter, wherein a fifth shorter transcribable nucleic acid sequence is located on the opposite side of, and adjacent to, said second right T-DNA border region and in opposite orientation to said fourth shorter transcribable nucleic acid sequence; and   wherein said first, second, third, fourth, and fifth shorter transcribable nucleic acid sequences are homologous to a portion of said second transcription unit; and   a third transcription unit comprising a non-lethal negative selectable marker gene located between said first DNA segment and said second DNA segment;   wherein said T-DNA border regions are capable of enabling the transfer of said segments into a plant genome.   
     
     
         29 . The recombinant nucleic acid molecule of  claim 28 , wherein said first, second, third, fourth, and fifth shorter transcribable nucleic acid sequences are each at least 75 contiguous nucleotides in length, and share greater than 90% sequence identity to each other and to a portion of said second transcription unit. 
     
     
         30 . The recombinant nucleic acid molecule of  claim 28 , wherein said first, second, third, fourth, and fifth shorter transcribable nucleic acid sequences are each at least 21 contiguous nucleotides in length, and share greater than 90% sequence identity to each other and to a portion of said second transcription unit. 
     
     
         31 . The recombinant nucleic acid molecule of  claim 28 , wherein said first and second DNA segments are not identical. 
     
     
         32 . The recombinant nucleic acid molecule of  claim 28 , wherein said non-lethal negative selectable marker gene is a phytoene synthase gene. 
     
     
         33 . The recombinant nucleic acid molecule of  claim 28 , wherein the transcription of said second and fourth shorter transcribable nucleic acid sequences in a single transcript triggers the silencing of said second transcription unit. 
     
     
         34 . The recombinant nucleic acid molecule of  claim 28 , wherein the transcription of said first and third shorter transcribable nucleic acid sequences in a single transcript triggers the silencing of said second transcription unit. 
     
     
         35 . The nucleic acid molecule of  claim 28 , wherein the transcription of said fourth and fifth shorter transcribable nucleic acid sequences in a single transcript triggers the silencing of said second transcription unit. 
     
     
         36 . The nucleic acid molecule of  claim 28 , wherein said first and second shorter transcribable nucleic acid sequences are homologous to a portion of a sequence of interest within said first transcription unit. 
     
     
         37 . The recombinant nucleic acid molecule of  claim 28 , wherein said first transcription unit comprises a sequence of interest and said second transcription unit comprises a selectable marker gene. 
     
     
         38 . The nucleic acid molecule of  claim 37 , wherein said sequence of interest comprises a nucleic acid sequence that causes the targeted expression or over-expression of an exogenous nucleic acid sequence. 
     
     
         39 . The nucleic acid molecule of  claim 37 , wherein said sequence of interest comprises a protein coding sequence. 
     
     
         40 . The nucleic acid molecule of  claim 37 , wherein said sequence of interest causes the inhibition of expression of an endogenous gene via gene silencing. 
     
     
         41 . A method of selecting for unlinked first and second DNA segments in a plant cell, comprising:
 a. transforming one or more plant cells with the recombinant nucleic acid molecule of  claim 37 ;   b. growing said transformed one or more plant cells;   c. selecting from said transformed one or more plant cells a plant cell comprising said first DNA segment and said second DNA segment and lacking said third transcription unit; wherein said transforming is mediated through a bacterial cell selected from the group consisting of  Agrobacterium  spp.,  Rhizobium  spp.,  Sinorhizobium  spp.,  Mesorhizobium  spp.,  Phyllobacterium  spp.,  Ochrobactrum  spp., and  Bradyrhizobium  spp.   
     
     
         42 . The method of  claim 41 , wherein said selecting further comprises selecting for said second DNA segment using said selectable marker gene. 
     
     
         43 . The method of  claim 41 , wherein said plant cell is a sugarcane, wheat, corn, soybean, cotton, or canola plant cell. 
     
     
         45 . The method of  claim 41 , wherein said non-lethal negative selectable marker gene is a phytoene synthase gene. 
     
     
         46 . The method of  claim 41 , further comprising regenerating a plant from said transformed one or more plant cells. 
     
     
         47 . The method of  claim 41 , wherein said first transcription unit provides a desirable trait selected from the group consisting of disease resistance, insect resistance, pest tolerance, herbicide tolerance, yield improvement, nutritional enhancement, and stress tolerance.