Cryopreservation Protocols and Effects of Preculture Treatments on Garlic and Olive Crops

1. Partner 3 Report Susan M. Trigwell, Ayesha Siddika, Graham Souch, and Paul T. Lynch. University of Derby, U.K. Alan J. Hargreaves and Philip L.R. Bonner. Nottingham Trent University, U.K.

2. P3 mandate crops: Garlic (Allium sativum L.) Olive (Olea europaea L.) WP3 Sugars WP4 Membrane Components * WP6 * Cytoskeletal Proteins WP7 Oxidative Stress

3. Visits • Ayesha Siddika to UAD, April 2004, - headspace analysis • Paul Lynch to UAD, April 2004, - WP7 meeting • Susan Trigwell to IRD, May 2004, - sugar analysis

4. Garlic stem-disc cryopreservation protocol

5. Olive somatic embryo cryopreservation protocol

6. Olive somatic embryos – effect of preculture on post-thaw regrowth % change in fresh weight - 28d post-thaw

7. Why is Sucrose Best Preculture Treatment ? • Sucrose, mannitol and sorbitol - do not cross plasma-membrane • ABA-mediated membrane sucrose transporters • Cellular osmolyte • Stabilises cellular macromolecules during dehydration • Mannitol and sorbitol growth retardants

8. WP3: Sugars (P4)

9. WP3: Sugars - Results Olive somatic embryos – sucrose accumulation Values % mg/mg dry weight - mean of 5 replicates

10. WP3: Sugars - Results Garlic stem-discs • Successfully performed sugar analysis • Problems expressing data – due to exceptionally low weight of explant after freezing – anomalous ‘dry weight’ value in calculation

11. WP4: Membrane Components (P1)

12. WP4: Membrane Components- Results • Successful trial analyses • Large amount of material required for sterol analyses • Continue with fatty acid analysis of cryo-treated tissue extracts (garlic in progress - olive to follow) Olive somatic embryos & garlic stem-discs

13. WP7: Oxidative Stress (P2)

14. WP7: Oxidative Stress - Results Olive somatic embryos Poorer post-thaw regrowth on sorbitol or mannitol SOD activity Schiff’s base content Better post-thaw regrowth on sucrose GR activity

15. WP7: Oxidative Stress - Results Olive somatic embryos – headspace analysis • Methane Production - produced by reaction of DMSO with hydroxyl radical. Total methane production over 3 days was higher in sorbitol and mannitol pretreated somatic embryos.

16. Olive somatic embryos – proline content Concentration mmol/g tissue Increased post-thaw regrowth following sucrose preculture treatment is associated with increased proline content.

17. *WP6*: Cytoskeletal proteins (P3)

18. WP6: Cytoskeletal Proteins • immunofluorescent staining • immunoprobing of protein blots to detect tubulin modification and activation of cell-signalling pathways • in vitro transglutaminase assays

19. Immunofluorescent staining of garlic stem-disc longitudinal sections with anti-crosslink antibody non-frozen 1d post-thaw

20. 50 kD 50 kD Immunoprobing of protein blots of cryo-treated garlic stem-disc extracts– tubulin modification Anti-a tubulin (total) Anti-a tubulin (tyrosinated) Protein modification/degradation after freezing – varies with desiccation time Protein degradation after freezing

21. 250 160 75 50 30 Immunoprobing of protein blots of cryo-treated garlic stem-disc extracts – cell signalling Anti-ERK 250 160 ~200kD band – after desiccation step – time dependent? 75 50 30 Anti-pThreonine Novel 50kD protein detected – after freezing – desiccation time-dependent?

22. WP6: Cytoskeletal Proteins in vitro transglutaminase assays biotin cadaverine- incorporation protein- crosslinking compromised by other polyamine-incorporating enzymes present in cell extracts formation of the e(g-glutamyl)lysine peptide bond is TG - specific

23. WP8: New protocols Assess tubulin modification after different desiccation periods/rates. * WP6 * Cytoskeletal Proteins Molecular marker? Determine action of plant growth factors on the observed Threonine phosphorylation profile. Molecular marker? Decrease tubulin degradation.

24. WP8: New protocols Further reduce potential for free-radical damage by use of desferrioxamine/cation-free post-thaw recovery medium. WP7 Oxidative Stress Monitor damage reduction using assays developed in WP7

25. Publications and Outputs • Publications: S.M. Trigwell, P.T. Lynch, M. Griffin, A.J. Hargreaves, and P.L.R. Bonner (2004) An improved colorimetric assay for the measurement of transglutaminase (type II) e-(g-glutamyl) lysine cross-linking activity. Analytical Biochemistry330 164-166 W. Harris, P.T. Lynch, A.J. Hargreaves, and P.L.R. Bonner (2004) Cryopreservation of Helianthus tuberosus cell suspension cultures: The effect of preculture treatments on cytoskeletal proteins and transglutaminase activity. CryoLetters25 213-217

26. Publications and Outputs • Presentations: P.T. Lynch (2004) The Significance of Preculture Treatments on the Post-thaw Recovery of Somatic Embryos of Olive (Olea europaea L.). Asian Institute of Medicine, Science, and Technology, Malaysia.

27. Publications and Outputs • Presentations: A. Siddika, P.T. Lynch, A. Mehra, C. Benelli, and M. Lambardi (2004) The Effect of Sugar and Polyol Pretreatments on the Post-thaw Regrowth and Oxidative Status of Somatic Embryos of Olive (Olea europaea L.). Society for Low Temperature Biology Annual Meeting, London. P.T. Lynch, S. Trigwell, and A. Siddika (2004) CRYMCEPT:An EC Project Building on Multi-Disciplinary Fundamental Studies to Enhance Plant Cryopreservation Protocols.Society for Low Temperature Biology Annual Meeting, London.