1. Dean W. Gabriel GM Citrus with Potential to Control Greening Integrated Plant Genetics, Inc. Alachua and Plant Pathology Department University of Florida, Gainesville

2. Strategies for greening disease control o No practical resistance breeding. o Eradication impossible. o Chemical control of insects may require 25 sprays per year. o Genetically modified (GM) citrus (add a resistance gene).

3. APHIS/PPQ intercepted the vector 40 times in US ports between 1985-98. APHIS/PPQ intercepted the vector 40 times in US ports between 1985-98. Citrus greening, likely trapped in dooryard citrus, nationwide. 6 year lag from psyllid establishment to greening diagnosis.

4. Canker bacteria Greening bacteria Why don't chemicals work on greening?

5. Nucleus How to make a "GMO" 1. Start with DNA cloning vector. 2. Add gene of interest + plant selection gene. 3. Add cloned genes on vector to specialized bacterial pathogen. 4. Contact pathogen with target plant cell. 5. The pathogen injects the cloned genes into the plant cell, and the genes are guided to the nucleus by DNA vector. 1 2

6. 1. In a test tube, construct a gene encoded on vector DNA. 2. Add the vector DNA to a specialized bacterial pathogen. 3. Contact wounded citrus with the pathogen; the vector DNA is injected by the pathogen into the plant cell. --Essenberg & Richardson  Transformation process for GM Citrus

7. Bacteria with cloned genes applied to cut end of citrus stem section. Antibiotic selection applied.  Transformation process for GM Citrus Greenhouse testing Grafting onto rootstock 1 2

8. 4 weeks --- 26% juvenile stem pieces survive; 1/2 of these are transformed

9. Mature Valencia & Hamlin regenerated

10. Juvenile grapefruit after grafting

11. May, 2008: Growth of transgenic citrus trees, each with 1 of 3 IPG DiseaseBlock genes.

12. Current status: GM citrus trees currently being evaluated by IPG. Technology (IP) is co-owned by UF.

13. Citrus has natural defenses Transgenes that interfere with defense are more susceptible...

14. Citrus has natural defenses Harder to prove are transgenes that enhance defense... a negative result (determined by PCR). To date, negative. IPG DiseaseBlock® added

15. Transgenic tobacco as proxy... to date negative IPG DiseaseBlock® added

16. GMOs can be accepted in the U.S. Transgenic and nontransgenic papaya in Hawaii. Work by: D. Gonsalves, Cornell U.; S. Ferreira and R. Manshardt, U.Hawaii; M. Fitch, USDA; J. Slightom, Pharmacia

17. Transgenic 'UH Rainbow' to be graded in a commercial packinghouse.

19. The Regulatory Process--- Who's minding the Store? NIH mandated IBC review USDA-APHIS facility review USDA-APHIS field-release approval USDA-APHIS shipping authority approval USDA-APHIS permission to commercialize or non-regulatory status (public comment, 12-24 mo)

20. The Regulatory Process EPA experimental use permit (if > 10 acres) EPA determines if limits needed on % gene product (public comment, 24-30 mo.) EPA product registration (public comment) FDA review (public comment (12-18 mo.) Earliest time to market: 2015

21. Integrated Plant Genetics, Inc. 12085 Research Drive Alachua, Florida 32615 USA http://www.ipgenetics.com gabriel@ipgenetics.com IPG Research Sponsored by: Southern Gardens Citrus USDA-APHIS NSF Fischer USA Goldsmith Plants