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Workpackage number4

Workpackage number4. Membrane components. WP4: Partners. Partner 1: Katholieke Universiteit Leuven (KULeuven), Belgium, Partner responsible. Partner 4: Institut de Recherche pour le Développement (IRD), France. Partner 3: University of Derby (UD), UK.

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Workpackage number4

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  1. Workpackage number4 Membrane components

  2. WP4: Partners Partner 1: Katholieke Universiteit Leuven (KULeuven), Belgium, Partner responsible Partner 4: Institut de Recherche pour le Développement (IRD), France Partner 3: University of Derby (UD), UK Partner 2: University of Abertay Dundee (UAD), UK

  3. WP4: Period 30 months: Start date: 01-11-2002 Completion date: 30-04-2005

  4. WP4: Background Cell membranes are the primary site of freezing injury in plants; One of the major membrane components are phospholipids; Both fatty acid and polar head-group compositions of phospholipids are important parameters for stress resistance; Sterols are also important membrane components regulating membrane fluidity and permeability.

  5. WP4: Objectives To refine techniques – plant membrane sterol and phospholipid composition To scale down analysis methods To define changes of membrane components – different cryo-protocols To determine the relationship between membrane components and cryopreservation To find treatments which improve cryo-ability

  6. Analysis parameters in WP4 Sterols Phospholipids Phospholipid - fatty acids Glyceride – fatty acids Glycolipid and sphingolipid – fatty acids Free fatty acids

  7. Analysis methods (protocols) in WP4 Banana meristems Extracts of lipids Fraction 3 Fraction 2 Fraction 1 Glycolipids and sphigolipids Phospholipids sterols, glycerides Sterols Fatty acids Fatty acids Fatty acids Phospholipids

  8. Free fatty acid analysis Two methods Diazomethane method H2SO4 – MeOH method

  9. Banana materials Cultivar Genomic group Cryo-ability Cachaco ABB +++ Williams AAA ++ Obino l’Ewai AAB-p + Mbwazirume AAA-h - Medium - P4: MS medium, 100 µM BA, 3% sucrose - Sucrose: MS medium 10 µM BA, 0.4 M (13.7%)sucrose

  10. Cryopreservation P4 LS PVS2 LN RS MS (0.3) MS (2.22) Sucrose Shoot regeneration % of four banana cultivars after cryopreservation

  11. GC separation of free sterols in banana meristems cholesterol I.S. campesterol stigmasterol sitosterol

  12. Sterol analysis in banana B: Campesterol A: Cholesterol C: Stigmasterol D: Sitosterol

  13. Ratio of stigmasterol to sitosterol in banana meristems P4 Sucrose Sucrose/P4 Cac 1.05±0.28 2.44±1.25 2.33 Will 1.85±0.26 5.51±2.82 2.97 Obi 1.46±0.05 4.10±1.94 2.81 Mbw 0.53±0.02 2.11±1.12 4.01 Sitosterol Stigmasterol Cholesterol

  14. GC separation of fatty acids in banana meristems C14:0 C16:0 C16:1 I.S. C18:0 C18:1 18:1c11 C18:2 C18:3

  15. Bound and free fatty acids in banana meristems Unsaturated Saturated Glyceride –fatty acids Glycolipid and sphigolipid–fatty acids Phospholipid –fatty acids Free fatty acids

  16. Correlation between fatty acids and regeneration % after cryopreservation for banana meristems r = 0.707 at 5% level of significance

  17. Ratio of unsaturated / saturated fatty acids = (3 x C18:3 + 2 x C18:2 + C18:1 + C18:1c11 + C16:1) / (C14:0 + C16:0 + C18:0)

  18. Ratio of unsaturated / saturated fatty acids in banana meristems

  19. Conclusion from banana results Stigmasterol / sitosterol may be involved in cryo-process Glycerides may also be involved in cryo-process Ratio of unsaturated / saturated FAs bound to phospholipids and ration of unsaturated / saturated FFAs are important parameters for cryopreservation.

  20. Preliminary results in olive and garlic In olive, fatty acids are higher in somatic embryos than in shoots In garlic, for BFA, an increase in C18:1 after sucrose treatment ; for FFA, an increase in C18:1 and a decrease in C18:2, C18:3 after sucrose treatment. In garlic, low amount of sterols; an increase (3.5x) in ratio of stigmasterol / sitosterol after sucrose treatment.

  21. Ribes shoot-tips Fatty acid double bond index in Ribes shoot-tips before 0.75M sucrose pretreatment.

  22. Ribes shoot-tips Genotype tolerance to cryopreservation is related to the number of double bonds in fatty acids. • Relationship may be improved following analysis of pretreated tissues. • Fatty acid profile indicates likely end-products of lipid peroxidation (WP7) • Linolenic acid  MDA, ethylene, ethane • Linoleic acid  HNE, pentane

  23. Main problems resolved / to be resolved Phospholipids by HPLC: technical meeting Further analysis in olive, garlic materials Further analysis in Ribes plants

  24. Milestones in WP4 M4.1: Development of methods for analysis membrane components in banana (month 6) M4.2: Development of methods for analysis membrane components in different plant species (month 18) M4.3: Determination of essential membrane components changes associated with protection / tolerance towards cryopreservation (month 24)

  25. Deliverables in WP4 D4.1: Detailed description of analysis methods (month 18) D4.1: Detailed description of relationship between the parameters and cryopreservation (month 24) D4.1: Scientific publication (month 24)

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