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University of Abertay (P2) Partner Report

University of Abertay (P2) Partner Report. Jason Johnston, Erica Benson, Keith Harding, Isobel Pimbley, Jayanthi Nadarajan, Samantha Gale, Jon Green, Brian Grout. Presentation Outline. Part 1 - Project Management/Outputs Visits, exchanges & conferences Problems Publications

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University of Abertay (P2) Partner Report

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  1. University of Abertay (P2) Partner Report Jason Johnston, Erica Benson, Keith Harding, Isobel Pimbley, Jayanthi Nadarajan, Samantha Gale, Jon Green, Brian Grout

  2. Presentation Outline • Part 1 - Project Management/Outputs • Visits, exchanges & conferences • Problems • Publications • Part 2 - Work Pack Highlights • DSC (WP1 & 8) • Oxidative stress (WP7 & 8) • Ethylene (WP7 & 8) • DNA methylation (WP7)

  3. Exchanges, Visits & Conferences in 2005 • CRYMCEPT Visits • Paul Lynch (P3) to UAD in February • Jason Johnston to Writtle College in March • Jon Green to UAD in June • Conferences (September 2005) • Society For Low Temperature Biology, York 2-oral invited/contributed presentations by Jason Johnston/Jon Green/Brian Grout • International Association For Plant Tissue Culture, Perth, Australia “Biotechnology & Sustainable Development” Erica Benson (Plenary) Keith Harding (Contributed/Session Chair)

  4. Problems Challenges • Potential access problems to DSC & HPLC resolved • Nuclease P1 back in production ☺ • Growth room facilities marginal • WP7 leader & PI (Erica Benson) returned after absence of sick leave • Erica extends a big thank you to all CRYMCEPT colleagues for their support and particularly to Jason, Keith, Paul and Brian.

  5. Publications – books & chapters • Life In The Frozen State (2004), Fuller, B; Lane, N & Benson, EE 3 chapters authored by E. Benson WP7/WP8/WP9 • Benson, E., Johnston, J., Muthusamy, J., Harding, K. (2005) Physical and Engineering Perspectives of In Vitro Plant Cryopreservation, in Plant Tissue Culture Engineering, Dutta Gupta, S., Ibaraki, Y., Eds., Springer, in press. WP1 • Benson, E., Harding, K., Johnston, J. (2005) Cryopreservation of shoot-tips and meristems, in Cryopreservation and freeze-drying protocols, Day, J., Stacey, G., Eds., Methods in Molecular Biology Humana Press, in press. WP7/WP8/WP9 • Day, JG, Benson, EE, Harding K (2006) Cryopreservation of plants and algae. Molecular Biomethods Handbook 2nd Edition, Humana Press, Eds. Walker, JM and Rapley, R. In prep. WP8/WP7

  6. Publications – Journals • 3 refereed journal reviews WP7,8,9 • 1 analytical methods paper (DNA Methylation) WP7 • 1 cryopreservation international technology transfer paper WP8 • 2 cryopreservation original papers (Thermal Analysis) WP1 • Many more to come! • Total antioxidants paper submitted August, WP7 • In process of preparing data for joint CRYMCEPT partners publications. WP7/WP8

  7. P2 Compliance with Deliverables & Milestones • Delivery of Deliverables/Milestones either achieved or on target for completion by the end of the project. • Outputs in progress to support D/M targets • Project exit strategy to include preparation of outputs and data for delivery in 2006 at conferences and for invited chapter contributions and original papers.

  8. P2 Work Pack Highlights Oxidative Stress and Thermal Analysis WP8 and WP9 Methods/Delivery To Wider Cryo-conservation Community

  9. Thermal Analysis: DSC (WP1 & 8)

  10. WP1 Highlights • Ribes meristems study 1 (Sherlock et al., 2005) • Use of silica-gel as an alternative to air-flow desiccation • DSC identified optimum time for achieving Tg

  11. WP1 Highlights • Ribes meristems study 2 (Benson et al., 2005) • Should 0.75 M sucrose be included in alginate during encapsulation? • Inclusion of sucrose • Increased osmotically inactive water during desiccation • Induced a Tg earlier during desiccation and at a higher water content (30% FW) • No effect on survival for species with ca 40% shoot recovery

  12. WP8 DSC Highlights • Neem somatic embryos (Benson et al., 2005) • Optimisation of encapsulation protocol • Tg after 4 hours of air-flow desiccation • Tg occurred at residual moisture 13%, 0.02 gH2O/gDW (Ribes 25-30%, 0.4 gH2O/gDW) •  sucrose concentration during pretreatment  osmotically inactive water

  13. Oxidative stress (WP7 & 8)

  14. Oxidative stress during sucrose acclimation of Ribes (WP7) • For Ben More, sucrose acclimation also substantially increased: • Total phenolics • SH groups • Flavanoids/anthocyanins (visual) • •OH 66-91 % 8-25% shoot recovery

  15. Manipulation of oxidative stress during sucrose acclimation (WP8) • ABAP = 2,2’-azobis-2-amidinopropane dihydrochloride • CF = metal cation free (except Ca2+) • No effect on shoot recovery • Antioxidant supplements (Trolox and desferrioxamine) also did not improve recovery

  16. Oxidative stress (WP8) • Manipulation of OS during recovery had no effect on survival: • Cation free medium ± antioxidants (desferrioxamine & Trolox) • Cation free medium  for control meristems, but  for stressed meristems • Cation free medium prevented polyphenol blackening, but temporarily!

  17. Oxidative stress (WP8) • Oxidative stress markers developed for Ribes for culture performance were successfully applied to Sitka spruce shoots.

  18. DNA methylation (WP7)

  19. Methylation changes during sucrose-acclimation 8-25% 20-40% 50-90 % 66-91% shoot recovery

  20. Do methylation changes persist during recovery? • Yes, but only short-term!

  21. Ethylene (WP7 & 8)

  22. Genotype comparison of ethylene production during cryo (WP7) • Ethylene an major hormone in plants • Radial cell expansion • Senescence • Mediator of stress responses • Contrasting ethylene production between Ribes genotypes with different % shoot recovery after cryo • Cause, consequence or coincidence?

  23. S-adenosyl-methionine (SAM) ACC synthase 1-aminocyclopropane-1-carboxylic acid (ACC) ACC oxidase Ethylene Ethylene biosynthetic pathway (WP8)  Aminoethoxyvinylglycine (AVG)

  24. Manipulation of ethylene during sucrose acclimation (WP8)

  25. Manipulation of ethylene during recovery (WP8) • Stimulation of ethylene production during recovery had no effect on shoot recovery • Stimulation of ethylene production by ACC only occurred in meristems that survived cryo • May indicate membrane damage in non-surviving meristems • AVG inhibited meristem growth • Ethylene maybe essential for meristem growth

  26. Model for cryo-injury

  27. Antioxidants Polyamines Phenolic comp. Membrane comp. DNA Methylation Genotype Tissue culture strategy Acclimation strategy Cryoprotectants Other primary damage Other secondary effects Cell death Dormancy or slow growth problems Primary damage - membrane damage  ethylene biosynthesis & action Meristem growth inhibited

  28. Conclusions • DSC effective at optimising cryo-protocols for tropical & temperates species, but cannot explain genotype survival differences for Ribes meristems • Physiological problem • Accumulation of antioxidants during pretreatment important for recovery • Tissue culture and pretreatment regimes required that enhance endogenous antioxidants

  29. Conclusions • DNA methylation changes during sucrose pretreatment • May have some genetic stability implications • May be required for survival! • Ethylene not involved in sucrose acclimation responses • Ethylene maybe essential for meristem growth

  30. Acknowledgements • Thanks to all partners • Paul Lynch for assisting with P2 and WP7 PI activities

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