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University of Abertay (P2) Partner Presentation

This presentation discusses the roles and progress of WP7 in studying oxidative stress, DNA-adducts, and their relationship with critical physical parameters in cryopreservation. It also highlights the challenges faced in Year 2 and the publications and outputs of the project.

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University of Abertay (P2) Partner Presentation

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  1. University of Abertay (P2)Partner Presentation Jason Johnston, Erica Benson, Keith Harding, David Bremner, Jon Green, Brian Grout

  2. P2 CRYMCEPT Roles • WP7 leader. • Protocols for studying oxidative stress and DNA-adducts (WP7). • Relationship between critical physical parameters (DSC; WP1) and oxidative stress (WP7). • Role of oxidative stress in cryo-injury (WP7)  new protocols (WP8). • Dissemination of new cryopreservation knowledge (WP9)

  3. Exchanges & Visits in Year 2 • Jason Johnston & David Bremner to KUL in Jan 04 • Jon Green & Brian Grout to UAD in Apr & Jul 04 • Paul Lynch & Ayesha Sidika (P3) to UAD in Apr 04 • Stéphane Dussert (P4) to UAD in Apr 04 • Jan Geuns (P1) to UAD in Apr 04

  4. Problems in Year 2 • Principle investigator on sick leave since March • New teaching courses at UAD may reduce HPLC & DSC availability • Nuclease P1 no longer commercially available. • Ongoing building work at UAD • Commissioning of DSC delayed until Mar 04 • Dust and associated contamination problems • Loss of Ribes growth room • Project delays for Keith Harding & Jason Johnston

  5. P2 Publications & Outputs • Life In The Frozen State • Edited by B. Fuller, N. Lane & E. Benson • http://pages.britishlibrary.net/b.fuller/ • 3 chapters co-authored by E. Benson • 1 chapter co-authored by D. Bremner

  6. P2 Publications & Outputs • Harding, K. (2004) Genetic Integrity of Cryopreserved Plant Cells: A Review, Cryoletters, 25, 3-22. • Sherlock, G., Block, W., Benson, E. (2004) Thermal analysis of the plant encapsulation-dehydration cryopreservation protocol using silica gel as the desiccant, Submitted to Cryoletters. • Benson, E., Johnston, J., Muthusamy, J., Harding, K. (2004) Physical and Engineering Perspectives of In Vitro Plant Cryopreservation, in Plant Tissue Culture Engineering, Gupta, S., Ed., Kluwer Press, in preparation.

  7. P2 Publications & Outputs • Johnston, J., Harding, K., Bremner, D., Benson, E. (2004) Cryopreservation affects DNA methylation of Ribes sanguineum Pursh shoot-tip cultures, Society for Low Temperature Biology Annual Meeting, London. • Green, J., Johnston, J., Gale, S., Harding, K., Benson, E., Grout, B. (2004) Physiological and biochemical differences in plant tissue cultures: a possible basis for variation in cryogenic survival, Society for Low Temperature Biology Annual Meeting, London.

  8. Markers of Culture Health • Considerable biochemical differences occur between Ojebyn accessions with contrasting growth rates/habits.

  9. Markers of Culture Health • Are these accessions the same genotype? • Do these biochemical differences affect cryogenic survival? • Similar results obtained with Sitka Spruce shoot-tip cultures.

  10. Trolox and Cryogenic Survival • Trolox is a water soluble vitamin E analogue. • Successfully used as an antioxidant in medical studies, yet to be tested in plants. • Preliminary studies with Trolox at UAD: • No cytotoxic effects on shoot-tip cultures. • Alters medium pH. • Retains antioxidant activity after autoclaving and during storage at lab temperatures. • Cryogenic studies at UAD: • 3 concentrations applied at different stages of the cryopreservation process.

  11. Trolox & Cryogenic Survival • High Trolox concentrations reduced survival when present during first 7 days of recovery.

  12. Ethylene and Recovery • Recalcitrant genotype had greater ethylene production during recovery.

  13. Is the Medium Generating Hydroxyl Radicals? • Trolox could be acting as a pro-oxidant in medium, which may explain reduced survival.

  14. Trolox Supplement • Pretreatment and dehydration experiments with Trolox are in progress. • Published antioxidant cryogenic studies (i.e. GSH) have assumed the supplement is acting as an antioxidant; is this true?

  15. DSC • Experiments underway to determine the effect of sucrose loading in alginate on physical water properties and survival.

  16. WP8 • Antioxidant or pro-oxidant supplements. • Inhibitors or promoters of ethylene biosynthesis. • Penetration enhancers for medium supplements.

  17. Conclusions • Considerable progress has been made for WP7 studies with Ribes. • Trolox experiments near completion. • Future emphasis on ethylene and links with polyamines and DNA methylation. • Emphasis will also be placed on evaluating genetic stability (DNA methylation & hydroxyl adducts).

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