MTR Presentation Oxidative Stress WP7

1. MTR PresentationOxidative Stress WP7 Jason Johnston

2. Presentation Outline • Partners • WP objectives • Key results • Pretreatments (Olives and Ribes) • Desiccation (Coffee seed) • Genetic Stability (Ribes) • Collaborations • Problems • Progress towards milestones and deliverables

3. Partners • UAD • Erica Benson (WP leader), Jason Johnston, Keith Harding, David Bremner • Affiliated with Jon Green & Brian Grout, Writtle College • UD • Paul Lynch, Susan Trigwell, Graham Souch, Ayesha Sidika • IRD • Stéphane Dussert, Andréina Laffargue • DSMZ • Heinz Martin Schumacher • KUL • Jan Geuns, Mark Davey

4. Objectives • Profile oxidative stress and DNA methylation in cryopreserved germplasm. • Based on (1) apply antioxidants/analogues to cryopreserved systems to reduce free radical damage. • Combine DSC and oxidative stress studies to evaluate the critical physical parameters required to limit free radical damage.

5. Key Results fromPretreatment Experiments

6. Olive somatic embryos (P3) • Reduced post-thaw regrowth following sorbitol and mannitol pretreatments is associated with reduced SOD activity.

7. Olive somatic embryos (P3) • Increased post-thaw regrowth following sucrose pretreatment is associated with increased GR activity.

8. Olive somatic embryos (P3) • Reduced post-thaw regrowth following sorbitol and mannitol pretreatments is associated with increased Schiffs base content.

9. Ribes shoot-tips (P2) • Hydroxyl radical – differential genotype responses to pretreatment.

10. Ribes shoot-tips (P2) • Ethylene – differential genotype responses to pretreatment.

11. Common results • Both Ribes & Olives require induction of a stress response during pretreatment to survive LN2. • Increased endogenous antioxidants. • Free radicals may signal induced increased antioxidant content. • Measurement of antioxidants in Ribes required to test this hypothesis.

12. Key Results fromDesiccation Experiments

13. Non-orthodox coffee seed (P4) • Decline in viability of desiccated seeds when stored at +20oC.

14. Non-orthodox coffee seed (P4) • Decline in viability of desiccated seeds associated with reduced antioxidant content.

15. Non-orthodox coffee seed (P4) • Reduced desiccation and LN2 tolerance with storage in hydrated state is associated with increased MDA.

16. Coffee Seed Summary • Decline in seed viability when stored in desiccated state is associated with reduced antioxidant protection. • Decline in tolerance to desiccation and LN2 when stored in hydrated state is associated with increased MDA. • Are these relationships cause or coincidence?

17. Key Results fromGenetic Stability Experiments

18. Ribes shoot-tips (P2) • De-methylation of King Edward VII during cryopreservation.

19. Ribes shoot-tips (P2) • De-methylation related to increased RNA content.

20. DNA Methylation Summary • De-methylation may be an important regulatory mechanism for inducing a stress responses during pretreatment (i.e. antioxidants). • Does de-methylation occur in other Ribes genotypes? • Does de-methylation persist in vitro and in the field, and if so does this affect phenotype?

21. Collaborations • Graham Souch (P3) to UAD in Jul 03. • Jon Green & Brian Grout to UAD in Aug 03, Apr 04 & Jul 04.

22. Collaborations • Jason Johnston & David Bremner to KUL in Jan 04. • Paul Lynch & Ayesha Sidika (P3) to UAD in Apr 04.

23. Collaborations • Stéphane Dussert (P4) to UAD in Apr 04. • Jan Geuns (P1) to UAD in Apr 04.

24. Problems • WP leader on sick leave (Jan-Jun 03; March 04 onwards). • New teaching courses at UAD may reduce HPLC availability for DNA methylation analysis. • Nuclease P1 no longer commercially available. • Ongoing building work at UAD • Dust and associated contamination problems • Loss of Ribes growth room • Project delays for Keith Harding & Jason Johnston

25. Milestone 7.1 (Month 24) • Application of Antioxidants. • Ascorbate applied to potato shoot-tips (DSMZ) improved survival in one genotype but not others. • Desferrioxamine (Fe3+ chelator) applied to olive somatic embryos (UD), in progress. • Trolox (water-soluble vitamin E analogue) applied to Ribes shoot-tips (UAD), in progress. • Milestone in progress and on schedule.

26. Milestone 7.2 (Month 30) • Association between critical physical parameters and oxidative stress. • Planning phase. • Milestone in progress and on schedule.

27. Milestone 7.3 (Month 30) • Relationship between oxidative stress, DNA adducts and cryogenic survival. • Key DNA adducts being 5-methylcytosine and 8-OH-deoxyguanosine. • DNA methylation analysis complete for 1 Ribes genotype and 3 garlic cultivars, in progress for 4 Ribes genotypes and 1 potato cultivar. • Analysis of markers of oxidative stress in progress for 4 Ribes genotypes, 1 olive cultivar, and nine species of coffee seed. • Milestone in progress and on schedule.

28. Deliverable 7.1 (Month 24) • Detailed description of oxidative stress methodology. • Oxidative stress manual • Collation of oxidative stress protocols from all partners. • Phase I completed and on website for partner access. • Phase II will be updated by end of year 2 to include protocols developed during CRYMCEPT. • Deliverable in progress and on schedule.

29. Deliverable 7.2 (Month 30) • New protocols for analysis of oxidative stress and DNA methylation. • GC headspace analysis. • HPLC analysis of DNA methylation. • HPLC analysis of 8-hydroxy-2’-deoxyguanosine. • Total aqueous antioxidant colorimetric assay. • Deliverable in progress and on schedule.

30. Deliverable 7.3 (Month 30) • Enhanced Fundamental Knowledge. • Induction of a stress response during pretreatment may be essential for cryogenic survival. • Free radicals and DNA methylation may be important regulators of stress responses. • In progress and on schedule.

31. Conclusions • Considerable progress has been made in the understanding of how oxidative stress and DNA methylation relates to cryogenic survival. • Technical exchanges and visits have been a successful feature of WP7 collaborations to date.