1. Maximizing Growth and Productivity of Alfalfa Through Biotechnology Jay SandmanDavid Johnson Lauren Johnson Jonathon Reich

2. Research Objectives Minimize plant response to chronic, sub- lethal stresses Reduce pre-harvest leaf loss caused by senescence

3. Premise of Work Most stresses are sub-lethal and result in unnecessary cessation of plant growth Senescence and responses to non-lethal stresses are genetically controlled by the plant Reduction of plant responses to senescence and non-lethal stresses will release brakes on plant growth and maximize productivity

4. Two Modes of Senescence Senescence induced by biotic and abiotic stresses Genetically induced senescence

5. Senescence Associated Response to Sub-lethal Stresses 0 Plant Development Harvest Senescence Stress Initiated Senescence Abortion of Stress & Recovery

6. Non-Productive Response to Sub-lethal Stress Plant Development 0 Harvest Plant Biomass Growth Potential Stress Effects Actual Growth

7. Non-Stress related, Genetically Induced Senescence 0 Plant Development Harvest Senescence Genetically Induced Senescence

8. Project 1: An Autoregulated Senescence Inhibition System The system is based on the expression of chimeric transgene PSAG12-IPT The SAG12 promoter is senescence specific The IPT gene encodes the enzyme isopentenyl transferase which catalyzes the rate-limiting step in cytokinin biosynthesis Cytokinins inhibit the development of senescence

9. The Autoregulated Senescence Inhibition System Senescence SAG12IPT Isopentenyl TransferaseIsopentenyl Adenine (Cytokinin) Senescence Inhibit

10. In Vitro SAG12-IPT Expression 21 Days on Water Agar Transgenic Wildtype

11. Greenhouse SAG-12 IPT Expression WildtypeTransgenic

12. Greenhouse SAG12-IPT Expression

13. SAG12-IPT Field Expression in Breeder Seed Population Data is weighted mean from three harvests at 50% bloom stage during 2002.

14. Project 2: Altering Genetic Expression of Senescence Genes of interest –Deoxyhypusine Synthase (DHS) activates elF-5A –Eucaryotic Translation Initiation Factor-5A (elF-5A) facilitates mRNA translation

15. Regulatory Role of DHS and Factor-5A in Senescence Transcription Circuitry Execution Circuitry Senescence Stimuli Natural Senescence Biotic Stress Abiotic Stress Protease Lipase RNAse DNAse DHS Factor 5A

16. 0 Plant Development Harvest Senescence Stress Initiated Senescence Abortion of Stress & Recovery Reducing the Expression of DHS and elF-5A

17. Plant Development 0 Harvest Plant Biomass Growth Potential Stress Effects Wildtype Reduction of Non-Productive Responses to Sub-lethal Stresses Transgenic

18. Genetic Modification of Genetically Induced Senescence 0 Plant Development Harvest Senescence Wildtype Transgenic