Cryopreservation of non-orthodox seeds using coffee species as model system

1. Cryopreservation of non-orthodox seedsusing coffee species as model system S. Dussert, IRD (P4)

2. 1. State of the art before Crymcept

3. Seed cryopreservation in coffee genebanks Harvest • One harvest per year only • Difficulty to treat a large number of accessions within a short period : necessity to pre-store the seeds for few weeks before cryopreservation • Rapid decline of seed tolerance to LN exposure during storage (80 accessions of C. arabica)Seed Science Technol. 2005 • Optimal water status for seedstorage before cryopreservation ? • WP3 + WP4 + WP7 Storage Desiccation Cooling Rewarming Rehydration

4. Harvest Storage 1. Optimal water status for seed cryopreservation Mechanisms ? WP1 Desiccation Cooling Rewarming Rehydration

5. Harvest • 2. Desiccation tolerance • Level of seed desiccation sensitivity highly variable between coffee species • Seed Science Res. 1999 • Polygenic determinism • J. Exp. Bot. 2004 • Associated to imbibitional membrane damage • Physiol. Plant. 2003 • Mechanisms ? • WP3 + WP4 + WP7 Storage Desiccation Cooling Rewarming Rehydration

6. Harvest Storage Desiccation Beneficial effect of a low cooling rate Beneficial effect of a high warming rate CryoLetters 1997 Thermal behaviour of lipidsnot studied in Crymcept Cooling Rewarming Rehydration

7. Harvest • Beneficial effect of post-thaw osmoconditioning, • pre-heating, • or slow rehydration • Uncontrolled rehydration certainly leads to membrane damage • Rapid-cold rehydration injury is not free- radical mediated • Cryoletters 2000 Physiol. Pl. 2003 • No yet studied in Crymcept Storage Desiccation Cooling Rewarming Rehydration

8. Harvest Storage Effect of combiningall optimized conditions ? CRYMCEPT Desiccation Cooling Rewarming Rehydration

9. 2. Main results at mid-term

10. Water status after harvest 0.5 g/g Unfreezablewater content 0.2 g/g  Harvest Storage Viability Desiccation Two options Cooling Viability Rewarming  Rehydration

11. Water status after harvest 0.5 g/g Glutath. redox status Glutathione Unfreezablewater content 0.2 g/g Ascorbate  Harvest Storage Desiccation Two options Cooling Rewarming  Rehydration

12. Water status after harvest 0.5 g/g Unfreezablewater content 0.2 g/g  Harvest Storage Desiccation Two options Cooling The loss of viability of seeds maintained at optimal water status for cryopreservation is associated with a very rapid decline in antioxidant capacity Rewarming  Rehydration

13. Cryo tolerance Water status after harvest 0.5 g/g Desiccationtolerance Unfreezablewater content 0.2 g/g  Harvest Storage Desiccation Two options Cooling Rewarming  Rehydration

14. Water status after harvest 0.5 g/g Lyso-PE Lyso-PI Unfreezablewater content 0.2 g/g Harvest   Storage Desiccation Two options Cooling Rewarming  Rehydration

15. Water status after harvest 0.5 g/g Unfreezablewater content 0.2 g/g Harvest   The very rapid loss of toleranceto cryopreservation during storage in the hydrated state is associated withde-esterification of membrane lipidsand lipid peroxidation Storage Desiccation Two options Cooling Rewarming  Rehydration

16. Harvest • 1. Optimal water status for seed cryopreservation • Yesterday WP1 presentation • Optimal water status for seed cryopreservation =unfreezable water content • Unfreezable water content is negatively correlated to lipid content • Optimal water status can be reached directly by equilibration in a 80%RH atmosphere Storage Desiccation Cooling Rewarming Rehydration

17. 2.a) interspecific variability for seed desiccation tolerance Harvest • First significant correlationsin 5 years of research on theinterspecific variability forseed desiccation tolerance !! • promising results • candidate genes for QTLinterpretation Storage Desiccation Cooling Principal component analysis: WP3+WP4 data Rewarming Desiccationtolerant : high PC, high PI, high di+oligo. Desiccationsensitive: high PE high PA Rehydration

18. 2.b) desiccation time Harvest Equilibrationin 80% RH Storage Unfreezablewater content Desiccation Cooling Equilibrium reached after 13 days Desiccation timeroutinely applieduntil now : 20 days Rewarming Rehydration Question raised from the previous observation of the loss of antioxidant capacity in seeds stored at unfreezable WC: is there an effect of desiccation time on tolerance to cryopreservation ?

19. 2.b) desiccation time Harvest Cryo-tolerance Ascorbate Storage Desiccation Cooling Equilibrium reached after 13 days Desiccation timeroutinely applieduntil now : 20 days Rewarming Rehydration

20. Effect of combining all optimized conditions ? • Seeds stored for 1 week at 0.5 g/g before desiccation • Seeds desiccated for 13 days under 80% RH • Seeds rehydrated in 100% RH at 27°C after rewarming and before culture under germination conditions Harvest Storage Desiccation Cooling Rewarming Highestsurvival percentage ever observed Rehydration

21. All WPs  WP8 - New cryopreservation protocol for coffee seeds At 0.5 g H2O.g-1 dw and 20°C for a maximal duration of 4 weeks after harvest 1. Storage Equilibration under 80% RH For 13 days 2. Desiccation 3. Cooling Slow (10°C.min-1 down to -80°C) 4. Rewarming Rapid (70°C.min-1) 5. Rehydration Slow(24 h under 100% RH)

22. Special thanks Mark Davey, Leuven: Glutathione Ascorbate, TBARS, MDA Jason Johnston, Dundee: TEAC Sylvie Doulbeau, Montpellier: Sugars Andréina Laffargue, Montpellier: Lipids