1. T-DNA Mutagenesis T-DNA Mutagenesis

2. Transfer-DNA Mutagenesis: a chemical or physical treatment that creates changes in DNA sequence which can lead to mutation strains that are passed on to the next generation.

3. -PURPOSE: To create loss of function mutations in order to determine the function of a gene.

4. -HOW DOES THIS WORK: Vector transmission by way of Agro  plant is randomly inserted into the nuclei chromosomal sites. T-DNA T-DNA Flanking Region LBRB KAN Pdi2

5. -Agro? Agrobacteria tumefaciens is a bacteria found on certain plants that were found to cause tumors on wounded plant areas. Found to contain Ti (Tumor inducing) plasmid that creates a mutation in the plants genomic sequence. The Ti plasmid’s ability to integrate itself into a DNA sequence was isolated and the tumor inducing quality was taken out.

6. -LOSS OF FUNCTION MUTATIONS: is when a mutation is created in such a way that death does not occur so as to observe the effects on the plant by the loss of a certain gene. In other words, a gene is knocked out and the plant is grown and observed for any differences between the mutant strain and the control strain. Thus facilitating (understanding) the function of that knocked-out gene.

7. T-DNA in the Arabidopsis Genome T-DNA – from Ti plasmid in the Agrobacteria tumefaciens.. Uses the insertional quality to carry foreign genes into the plant genome. R1 primer Wild Type Primer F1 Pdi2 A LB T-DNA

8. -VALID TEST RESULTS: To achieve valid data from the gel results. Homozygous cells must be used, not Heterozygous. T-DNA T-DNA Flanking Region LBRB KAN Pdi2 Genotype - Segregation

9. -HOMOZYGOUS: 2 of the same (genes). On gel, homozygous will only produce one band on either the wild type control side or the T-DNA control side, not both. Needed for accurate results.

10. -HETEROZYGOUS: different genes. On gel, will produce a band on both the WT control side and the T-DNA control side. Not used for verification of T-DNA.

11. -GEL ELECTROPHORESIS: 1. Verify if sample is homozygous, 2. Verify that T-DNA knockout is not there, 3. Verify that T-DNA is there and inserted. WT A1 A2 T DNA + - WT+ A1 A2 WT - WT A1 A2 WT+ - WT A1 A2 TDNA+ -

13. WT A1 A2 TDNA + - WT A1 A2 WT+ -

14. -disrupted genes do not make RNA. That function has been knocked out. -F1 and R2 primer will make WT. -F1 and LB primer will make T-DNA knockout. R1 primer Wild Type Primer F1 Pdi2 A LB T-DNA

15. Future Advances in Biotechnology Mutagenesis Research: BEFORE AFTER