1. WP3. Sugars S. Dussert, IRD (P4)

2. Intracellular ice crystalformation and growth Increase of cytoplasm viscosity during pre-treatment Cell dehydration Sucrose Stachyose Raffinose Overview of the roles of di- and oligo-saccharides Unabated respirationoxidative stress Increasedmembrane Tm Proteindenaturation Membrane fusion LC-to-gel phase transition Water replacement Glass formation HII phases, demixing Preferential exclusion

3. Deliverable 3.1 Mi.3.2 month 15 WP3 Objectives • To develop simple and efficient methods for the analysis of the soluble sugar contents in various types of explants: • Coffee seedsP4 • Banana meristemsP1 • Garlic stem discsP3 • Olive somatic embryosP3 • To study the relationship between the soluble sugar composition of the four plant material studied and their tolerance to desiccation and LN exposure • Sugar analyses completed Deliverable 3.2 • Data analysis, in combination with data from all WPs,finished and co-publication Deliverable 3.3 Mi.3.1 month 6 Mi.3.3month 24

4. WP3 method • Sugar analysis was performed by anion exchange chromatography (Dionex Chromatography Co.) • Sugar separation on a CarboPac PA-100 (Dionex) column • Isocratic eluant (NaOH, 150 mM) • Addition of an internal standard (lactose) for sugar content determination Standards Peak identification and verification of the possibility to use lactose as IS Banana sample

5. WP3 Progress at mid-term Deliverable 3.1. To develop simple and efficient methods for the analysis of the soluble sugars contents in various types of explants: • Coffee seedsP4 completed • Banana meristemsP1 completed • Garlic stem discsP3 completed (visit to P4) • Olive somatic embryosP3 completed (visit to P4) • Methodology: • Dried plant material sent to Montpellier, • Sugar extracted and analyzed in Montpellier • Main results : • Sugar composition can be measured in samples of 2mg only • Simple and convenient procedure for multi-location collaboration: • Extraction is performed in partners’ own lab • Dried extracts sent to Montpellier prior to analysis

6. WP3 Progress at mid-term Deliverable 3.2. To study the relationship between the soluble sugar composition of the four plant material studied and their tolerance to desiccation and LN exposure : • Coffee seedsP4 in progress • Banana meristemsP1 in progress • Garlic stem discsP3 in progress • Olive somatic embryos P3 in progress • Main results : 3 examples • Inter-specific variability for seed tolerance to desiccation in coffee • Genotype x treatment interaction in banana • Effect of the pre-treatment medium composition in olive

7. 9 coffee species Desiccation sensitivity model: Vi V = 1 + exp(-b ( WC-WC50)) Cryo - Cryo + WP3 main results – example 1: inter-specific variability in coffee

8. WP3 main results – example 1: inter-specific variability in coffee Seed content in di- and oligo-saccharides is high in all coffee species studied, but it is also highly variable No significant correlation between desiccation tolerance and sugar contentP = 0.12

9. Cell dehydration Increasedmembrane Tm LC-to-gel phase transition Water replacement HII phases, demixing Sucrose Stachyose Raffinose General overview of the roles of di- and oligo-saccharides To study the membrane injury hypothesis, all factors contributing to membrane biophysical properties have to be analyzed simultaneously: WP3+WP4 Statistics: multivariate analysis

10. WP3 main results – example 1: inter-specific variability in coffee Principal component analysis: WP3+WP4 data Desiccationtolerance : high PC, high PI, high di+oligo. Desiccationsensitivity: high PE high PA

11. WP3 main results – example 2: treatment / cultivar interaction in banana Cryo-ability of 8 banana cultivars: - beneficial effect of the pretreatment - high variability between cultivars Sugar content: - pre-treatment induces an important increase of meristem sugar content - no significant correlation with cryo-ability after pretreatment

12. WP3 main results – example 3: pre-treatment medium effect in olive Cryo-survival clearly depends on the sugar employed in the medium and its concentration

13. WP3 main results – example 3: pre-treatment medium effect in olive The pretreatment medium composition has a considerable effecton the sugar composition of somatic embryos

14. WP3 main results – example 3: pre-treatment medium effect in olive Sucrose accumulation is associated with an increase in the cryo-ability of olive somatic embryos

15. WP3 main results – example 3: pre-treatment medium effect in olive Specificity of sucrose for the acquisition of tolerance to desiccation and LN exposure ?

16. WP3 conclusions at mid-term • In all plant systems studied, a high level of tolerance to desiccation and cryopreservation was always associated with a high content indi- and oligo-saccharides • However, sugar composition analysis did not explainthe differences observed • between genotypes: e.g. banana • between species: e.g. coffee for tolerance to desiccation and cryopreservation • Thus, sugar content alone cannot be used for predicting the cryo-ability of a given new plant material

17. WP3 conclusions at mid-term • Trials carried out in olive somatic embryos leads to an important but understudied question : is there a specific role for sucrose ? • Simultaneous analysis of data coming from different WPs can provide important information on the specific role of each component (WP) studied • Given the fact that tolerance to cryopreservation is a complex trait, the use of multivariate statistics in Crymcept will certainly become necessary