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Advanced Techniques in Plant Polyamine Analysis for Cryopreservation Study

This work package aims to refine analysis techniques and investigate the role of polyamines in plant survival after cryopreservation. The project involves identifying polyamines and aromatic amines, determining their composition changes, and finding treatments to enhance plant tissue cryo-ability. The research focuses on the relationship between polyamines and plant tissue survival post-cryopreservation. Through detailed analysis and experimentation, the study aims to provide insights that can improve cryopreservation methods for various plant species.

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Advanced Techniques in Plant Polyamine Analysis for Cryopreservation Study

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  1. Workpackage 5 POLYAMINES (PAs) and AROMATIC AMINES (AAs)

  2. WP5: Partners Partner 1: Katholieke Universiteit Leuven (KULeuven), Belgium, Partner responsible Partner 5: Fruit Tree Research Institute (ISF), Italy Partner 2: University of Abertay Dundee (UAD), UK Partner 3: University of Derby (UD), UK

  3. WP5: Period 30 months: Starting date: 01-11-2002 Completion date: 30-04-2005

  4. WP5: Background Polyamines bind to important cellular polyanions as DNA, RNA, phospholipids and acidic protein residues and cell wall components; Polyamines are involved in various cell functions such as cell division, growth, differentiation etc; Polyamine metabolism is also strongly involved in plant response towards environmental challenges such as osmotic stress, salinity, hypoxia, cold stress and environmental pollutants; Polyamine biosynthesis in plants happens through ADC and/or ODC pathway.

  5. WP5: Objectives To refine techniques – polyamine analysis To scale down analysis methods To define changes in polyamine composition – different cryo-protocols; To determine relationship between polyamines and survival after cryopreservation To find treatments for improving the cryo-ability of plant tissues

  6. Analysis parameters in WP5 PAs: - DAP (diaminopropane) - PUT (putrescine) - SPD (spermidine) - SPM (spermine) AAs - PEA (phenethylamine) - TYR (tyramine) - DA (dopamine) - SAL (salsolinol) ADC and ODC

  7. PAs and AAs after 0.4 M Sucrose pretreatment

  8. CRYO of banana clumps + PVS-2

  9. Structures of AAs identified

  10. HPLC of PAs and AAs Best Column packing: Spherisorb S5 ODS 2 (5 µm) Columns lab: home-made, 100x3 mm Too short for complete resolution of 4 PAs and 4 AAs in one run However: Alltech ODS2 is not as good as Spherisorb (longer gradient required)

  11. HPLC Separation PAs and AAs

  12. HPLC PAs and AAs Banana

  13. LC-MS of P4 and Sucr treatment

  14. DAP stress effect with survival

  15. PUT

  16. SPD

  17. SPM

  18. PEA

  19. TYR

  20. DA

  21. SAL

  22. “Importance” of stress effects

  23. ODC and ADC

  24. ODC and ADC • At 2 d: ADC in controls enhanced • At 2 d: Very low ODC activity • From 15 d on: huge increase, mainly of ODC (about 17 x more than ADC at 21 days • PAs analysed after 2 weeks: synthesised mainly by ODC pathway

  25. Conclusions • Many of the changes of PAs and AAs are positively or negatively correlated with survival percentages of the cultivars • DAP: higher in bad cvs (more PA breakdown?) • SPM: higher in good survivers before treatment: better radical scavenger capacity? • PUT: Higher in good survivers: protection?

  26. Conclusions (2) • AAs: before treatment: positively correlated with survival • AAs: after treatment: negatively correlated with survival • Anti-oxidative protection before and during sucrose treatment? • Production of radicals in bad survivers?? • Many questions, but few answers yet!

  27. Suggestions for Cryo • Adding PAs (different concentrations) • Adding inhibitors (DFMO, DFMA, to be synthesised again) • Adding AAs • Adding inhibitors of AAs biosynthesis • All combinations of the above • Study of biosynthesis of AAs, function in radical scavenging (and/or production!) • Study of metabolism of AAs

  28. Polyamine levels in shoot apex from Apple cv. Annurca dehydrated with mannitol (results by P5) pmol polyamine / mg protein Mannitol Mannitol Silica gel Liq. N2 Control Mannitol Silica gel

  29. Polyamine levels in shoot apex from Apple cv. Annurca dehydrated with sucrose pmol polyamine / mg protein Control Sucrose Sucrose Silica gel Sucrose Silica gel Liq. N2

  30. Polyamine content of 5 Ribes genotypes. Explants were analysed 6 weeks after sub-culturing, using leaf and stem tissue. Means and standard error of the means are shown (n = 5 individual explants). Different letters indicate genotypic differences according to analysis of variance and least significant difference (P < 0.05).

  31. Polyamine analysis in Olive

  32. PAs in Garlic Stem Discs

  33. Polyamines in Garlic Stem Discs

  34. Main problems resolved / to be resolved Two quantitatively very important unknown peaks in chromatograms have been identified: Dopamine and Salsolinol ADC and ODC should be further studied Studies with DFMA and DFMO, and reversal of inhibition by PUT Idem for AAs

  35. Milestones in WP5 M5.1: Development of methods for polyamine analysis for banana tissues (month 6) M5.2: Development of methods for polyamine analysis for different plant species (month 18) M5.3: Determination of polyamine changes in relation with survival after cryopreservation (month 24)

  36. Deliverables in WP5 D5.1: Detailed description of methods (month 18) D5.2: Correlation between polyamines and cryopreservation (month 24) D5.3: Scientific publication (month 24)

  37. Acknowledgements of Leuven group • We acknowledge the excellent technical assistance of Marta Ledo, Johan De Rue, Tom Struyf, Hilde Verlinden, René De Boer (MS) and F. Compernolle (MS)

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