Somatic Embryogenesis in Garlic: Genetic Characterization and Mass Propagation Protocol

1. « European project « 5th PCRDT » GARLIC AND HEALTH Second annual report Liverpool, 17-21 February 2003 Partner P8 CIRAD-FLHOR • L. FEREOL: scientist • S. CAUSSE: technical assistant • M. Roux-Cuvelier: field technician • R. Kahane: manager

2. Objectives • Somatic embryogenesis • A mass propagation protocol, relying on in vitro regenerated plants, will be developed (byP8). This method will be based on the production of embryogenic calluses and embryogenic cell suspension cultures from four varieties which represent different physiologicalgroups in garlic. • Genetic characterisation of plantlets from somatic embryos • Particular attention should be paid on the genetic integrity of the regenerated plants.They will be evaluated by: • flow cytometry for determining the ploidy level (by P8). • biochemistry (dry-matter and sulphur contents, by P5). • molecular markers for finger printing (AFLP, by P1). • morphological and physiological analyses

3. Milestones

4. Deliverables

5. Delays • Milestones • Rouge Reunion • 2nd field evaluation (P8) and sulphur analys will be realised in 2003 • All the others milestones are achieved • Deliverables • DP 13 • This paper will be submitted in 2003

6. Resarch activities during the fourth reporting period Embryogenesis, genetic characterisation • Rouge Reunion • 2nd field evaluation (P8), biochemistry (P5) • Messidrôme, Morasol and Printanor from callus • 2nd field culture and evaluation (P9) • flow cytometry (P8), AFLP (P1), biochemistry (P5) • Messidrôme, Morasol and Printanor from cell suspension • 1st field evaluation • flow cytometry (P8), AFLP (P1), biochemistry (P5) • Further improvement of the embryogenesis procedure, maintainance of the suspension cultures, histologicals studies • Bulb mass production in vitro from embryo derived plantlets and furthe development of these bulblets into bulbs

7. Embryogenic cell suspension culture of garlic (Allium sativum) as method for mass propagation and convenient material for genetic improvement L. Fereol*1, V. Chovelon2, S. Causse1 and R. Kahane1 1. CIRAD, TA50/PS4, bd de la Lironde, 34398 Montpellier cedex 5, France 2. INRA, Pathologie végétale, BP 94, 84143 Montfavet, France

8. Materials and methods (I) • Cultivars • Rouge Reunion • Messidrome • Morasol • Printanor • Explants • Primordial leaves

9. Materials and methods (II) • Culture procedures • Callogenesis Basal parts of young leaves from desinfected cloves were used as explant. Embryogenic callus were obtained according to method developed by Fereol et al (2002). • Embryogenic cell suspension cultures Cell suspension cultures were initiated from less than one year old cultures of friable embryogenic calluses. These calluses were cultured in 6 X 10 ml multi-well dish containing 6 ml of liquid medium (SM, table 1) base on N6 modified salts (chu, Wang et al. 1975). The cultures were incubated at 24 - 26°C in the dark with continuing agitation at 100 rpm.

10. Callus induction Morphology Histology Medium

11. Induction of friable callus Morphology Histology Medium

12. Comparative callus induction from different cultivars

13. Cell suspension culture aspect Morphology Histology « SM » Medium

14. Pack cell volume from 150mg of friable callus of different cultivars

15. Growth rate of the PCV of the cell suspension culture function of the culture period

16. Regeneration aspect after plating on semi-solid embryo induction medium Morphology Histology « EIM » Medium

17. Conversion aspect into plantlets Morphology Histology Medium

18. Embryogenesis features and kinetic of regeneration