European project « 5th PCRDT »

1. European project « 5th PCRDT » GARLIC AND HEALTH Second year progress report Half year meeting 4-5 october 2001 Partner P8 CIRAD-FLHOR

2. Scientific and administrative team • L. FEREOL scientist • R. HUGON administrative officer • S. CAUSSE technical assistant

3. Objectives • Development of somatic embryogenesis • Morphological, physiological and Genetic characterisation of plantlets from somatic embryos

4. Research activities

5. Somatic embryo

6. Cell suspension

7. Work plan • Development of somatic embryogenesis procedure for four varieties • Rouge Reunion • Messidrome • Morasol • Printanor • Production and provision to P9 of somatic embryos obtained via callus and culture suspension of these varieties

8. Work plan (continue) • Characterisation of plants from somatic embryo by : • flow cytometry (by P8) • biochemistry (by P5) • molecular markers (by P1) • morphological and physiological analyses (by P8, P9)

9. Milestones of the first and second year • Embryogenesis via Calli • Production of calli with embryogenic tissue (on the four cv.) • Done for the cv. Rouge Reunion • Messidrome, Morasol and Printanor • Production and germination of embryos from these calli (of the four cv.) • Done for Rouyge Réunion, Messidrome and Morasol • Provision to P9 of 200 plantlets from somatic embryos via callus of three cv. • Done for cv. Rouge Reunion • Messidrome and Morasol,

10. Somatic embryogenesis development from young leaf explant % callus = % explant with callus % emb.callus= % explant showing embryogenic callus %callus with g.e.= %embryogenic callus showing globular embryos Mean nb of embryos= mean number of mature embryos converted in plantlets per 150mg of fresh weight productive callus

11. Some Histological aspect showing monocellular origin of these somatic embryo

12. Milestones of the first and second year (continue) • Embryogenesis via cell suspension • Initiation of cell suspension from embryogenic calli of the four cv. Rouge Reunion, Messidrome, Morasol and Printanor • Done for Rouge Reunion, Messidrome, Morasol • production of embryos from these cell suspension • Done for Rouge Reunion, Messidrome and Morasol

13. Semi friable nodular callus Compact nodular callus Friable embryogenic callus Suitable for cell suspenion Research to a suitable material for Initiation of cell suspension

14. Milestones of the first and second year (continue) • Cell suspension (continue) • Provision to P9 of 200 plantlets from somatic embryos via cell suspension of cv. Rouge Reunion, Morasol and Messidrome • Done for Rouge Reunion and Morasol

15. Milestones of the first and second year (continue) • Analyses and characterisation of plantlets coming from somatic embryos, cv. Rouge Reunion • Flow cytometry • Done • Molecular markers, AFLP analyses (by P1) • Done • Biochemistry (dry matter and sulphur contents by P5) • Will be done • Morphological and physiological notations (by P8 and P9) • Partly done

16. Deliverables • Paper DP 5: Regeneration and acclimatisation of garlic plants (Allium sativum L.) from embryogenic callus • L. Fereol, V.Chovelon, S.Causse and N.Michaux-Ferriere • In progress, to be submited soon

17. Paper DP 5 et prot a c b ge d pd e f ra sa g i h j a b ge c d ra i f h g Fig 2. Plant regeneration via somatic embryo from nodular callus of garlic (Allium sativum) (a) nodular callus with embryogenic tissue (et). (b)histological section of tissue with meristematic and embryonic cells. (c) Isolated embryogenic cells showing protein reserves (prot). (d-g) morphological (d) and histological (e-g) aspect of globular somatic embryos (ge). (e) pro globular embryo developed after 4 weeks in medium EIM. (f) globular embryo imbed in the peripheral layer of nods, protoderm (pd). (g) isolated globular embryo. (h) matured somatic embryo on the surface of an embryogenic callus, developed in medium EDM after 4 weeks of culture. (i) histological section of a mature embryo, showing shoot (sa) and root (ra) apex. (j) converted plantlets from somatic embryos. Bar = m ( ); mm ( ) g h