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 method of selecting for unlinked first and second DNA segments in a plant cell, comprising:
a. transforming one or more plant cells with a nucleic acid molecule via Rhizobiaceae-mediated transformation, said nucleic acid molecule comprising:
a first DNA segment comprising a first transcription unit located between a first left border region and a first right border region;
a second DNA segment comprising a second transcription unit located between a second left border region and a second right border region, wherein said border regions are capable of enabling the integration of said segments into a plant genome; and
a third transcription unit comprising a non-lethal negative selectable marker gene, wherein said non-lethal negative selectable marker gene is located between said first DNA segment and said second DNA segment, and wherein said left and right border regions are T-DNA border regions;
b. growing said transformed one or more plant cells; and
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.
2. The method of claim 1 , wherein said plant cell is a sugarcane, wheat, corn, soybean, cotton, or canola plant cell.
3. The method of claim 1 , wherein said plant cell transformation 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.
4. The method of claim 1 , wherein said first transcription unit comprises a sequence of interest.
5. The method of claim 1 , wherein said second transcription unit comprises a selectable marker gene.
6. The method of claim 1 , wherein said non-lethal negative selectable marker gene is a phytoene synthase gene.
7. The method of claim 1 , wherein said T-DNA border regions are capable of enabling the transfer of said segments into a genome mediated by Agrobacterium or other Rhizobia.
8. The method of claim 1 , 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.
9. The method of claim 1 , further comprising selecting plant cells with a single copy of said first DNA segment.
10. The method of claim 9 , wherein said selecting plant cells with a single copy of said first DNA segment comprises measuring copy number by Southern blotting or PCR.
11. The method of claim 1 , wherein said method achieves transformation events with said first and said second DNA segments being unlinked at a frequency selected from the group consisting of about 0.5% or more, about 1% or more, about 1.5% or more, about 2% or more, about 2.5% or more, about 3% or more, about 5% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, and about 50% or more than the frequency achieved using a conventional vector having said first and said second DNA segments without said third transcription unit.
12. The method of claim 1 , wherein said non-lethal negative selectable marker gene is selected from the group consisting of a GGPP synthase gene, a GA 2-oxidase gene, an isopentenyltransferase (IPT) gene, a CKI1 (cytokinin-independent 1) gene, a ESR-2 gene, a ESR1-A gene, an auxin biosynthetic gene, a gene resulting in overexpression of ethylene biosynthetic enzymes, a VP1 gene, an AB13 gene, a LEC1 gene, and a Bas 1 gene.
13. A method of selecting for unlinked first and second DNA segments in a plant cell, comprising:
a. transforming one or more plant cells with a nucleic acid molecule via Rhizobiaceae-mediated transformation, said nucleic acid molecule comprising:
a first DNA segment comprising a first transcription unit located between a first T-DNA left border region and a first T-DNA right border region;
a second DNA segment comprising a second transcription unit located between a second T-DNA left border region and a second T-DNA right border region, wherein said T-DNA border regions are capable of enabling the integration of said segments into a plant genome; and
a third transcription unit comprising a non-lethal negative selectable marker gene, wherein said non-lethal negative selectable marker gene is located between said first DNA segment and said second DNA segment;
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; and
d. determining the linkage status of said first DNA segment and said second DNA segment in said selected plant cell and selecting for desired plant cells with unlinked said first DNA segment and said second DNA segment.
14. A recombinant nucleic acid molecule comprising:
a first DNA segment comprising a first transcription unit located between a first left border region and a first right border region;
a second DNA segment comprising a second transcription unit located between a second left border region and a second right border region, wherein said border regions are capable of enabling the integration of said segments into a plant genome, and wherein said left and right border regions are T-DNA border regions; and
a third transcription unit comprising a non-lethal negative selectable marker gene, wherein said non-lethal negative selectable marker gene is located between said first DNA segment and said second DNA segment.
15. The recombinant nucleic acid molecule of claim 14 , wherein said T-DNA border regions are capable of enabling the transfer of said segments into a genome mediated by Agrobacterium or other Rhizobia.
16. The recombinant nucleic acid molecule of claim 14 , wherein said second transcription unit comprises a selectable marker gene.
17. The recombinant nucleic acid molecule of claim 14 , wherein said non-lethal negative selectable marker gene is a phytoene synthase gene.
18. The method of claim 13 , said step d comprises:
i. regenerating a plant from said selected plant cell; and
ii. crossing said regenerated plant with another plant to produce one or more F1 progeny plants.