Methods and vectors for producing transgenic plants
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. A recombinant nucleic acid molecule comprising:
a first DNA segment comprising a first transcription unit located between a first left T-DNA border region and a first right T-DNA border region, wherein said first transcription unit is flanked on both sides and adjacent to a first and a second shorter transcribable nucleic acid sequence in opposite orientation to each other;
a second DNA segment comprising a second transcription unit located between a second left T-DNA border region and a second right T-DNA border region, wherein said second transcription unit is flanked on both sides and adjacent to a third and a fourth shorter transcribable nucleic acid sequence in opposite orientation to each other, wherein said third shorter transcribable nucleic acid sequence is operably linked to a plant-expressible promoter, and wherein said first, second, third, and fourth 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.
2. The recombinant nucleic acid molecule of claim 1 , wherein said first, second, third, and fourth 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.
3. The recombinant nucleic acid molecule of claim 1 , wherein said first, second, third, and fourth 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.
4. The recombinant nucleic acid molecule of claim 1 , wherein said first and second DNA segments are not identical.
5. The recombinant nucleic acid molecule of claim 1 , wherein said non-lethal negative selectable marker gene is a phytoene synthase gene.
6. The recombinant nucleic acid molecule of claim 1 , 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.
7. The recombinant nucleic acid molecule of claim 1 , 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.
8. The nucleic acid molecule of claim 1 , wherein said first and second shorter transcribable nucleic acid sequences are non-natural sequences.
9. The nucleic acid molecule of claim 1 , 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.
10. The recombinant nucleic acid molecule of claim 1 , wherein said first transcription unit comprises a sequence of interest and said second transcription unit comprises a selectable marker gene.
11. The nucleic acid molecule of claim 10 , wherein said sequence of interest comprises a nucleic acid sequence that causes the targeted expression or over-expression of an exogenous nucleic acid sequence.
12. The nucleic acid molecule of claim 10 , wherein said sequence of interest comprises a protein coding sequence.
13. The nucleic acid molecule of claim 10 , wherein said sequence of interest causes the inhibition of expression of an endogenous gene via gene silencing.
14. 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 10 ;
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 non-lethal negative selectable marker gene;
wherein said transforming is achieved 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.
15. The method of claim 14 , wherein said selecting further comprises selecting for said second DNA segment using said selectable marker gene.
16. The method of claim 14 , wherein said plant cell is a sugarcane, wheat, corn, soybean, cotton, or canola plant cell.
17. The method of claim 14 , wherein said non-lethal negative selectable marker gene is a phytoene synthase gene.
18. The method of claim 14 , further comprising regenerating a plant from said transformed one or more plant cells.
19. The method of claim 14 , 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 enhancements, and stress tolerance.