European project « 5th PCRDT » GARLIC AND HEALTH Third year progress report Half year meeting Montpellier 1-2 october 2002

ADVANCEMENT OF THE Works

Scientific and administrative staff L. FEREOL scientist –Duties: somatic embryogenesis, histology and flow cytometry S. CAUSSE technical assistant –Duties: somatic embryogenesis, histology and flow cytometry M. Roux-Cuvelier –Duties: field trials in Reunion R. Kahane –Duties: management

Objectives Setting a somatic embryogenesis procedure Comparative characterisation of plantlets from somatic embryos and from classic multiplication by meristem culture (as control)

Work plan Establishment of somatic embryogenesis procedure for four varieties: Rouge Reunion, Messidrome, Morasol, Printanor Production and provision of somatic embryos via callus and via cell suspensions cultures of these varieties Genetic evaluation of somatic embryos derived plants by : flow cytometry (by P8), biochemistry (by P5), molecular markers (by P1), morphological and physiological analyses (by P8, P9)

Milestones of the first year √ = achieved; partly achieved;  to be done)

Milestones of the second year Genetic characterisation of these plants by flow cytometry « Messidrome », « Morasol » Provision of 200 plantlets from embryogenic calli, and from embryogenic cells suspension cultures of « Messidrome », « Morasol » √ Genetic characterisation of somatic embryos derived plantlets « Rouge Reunion »: By flow ctytometry(P8), finger printing(P1), dry matter and sulphur content(P5), morpho-physiology(P9) √ √

Milestones of the second year (continue) Production and germination of somatic embryos from callus and cell suspension cultures « Printanor » √ √ Production of embryogenic callus, and Initiation of embryogenic cell suspension cultures « Printanor »

Milestones of the third year Promotion of the embryogenesis procedure Genetic characterisation of these plants by flow cytometry « Printanor » √ Provision of 200 plantlets from embryogenic calli, and from embryogenic cells suspension cultures of « Printanor »

Embryogenesis from callus Friable embryogenic callus Globular somatic embryos h Mature embryo MORPHOLOGY HISTOLOGY

Embryogenesis from cell suspension culture Embryogenic cell suspension culture established Conversion into plantlets and then bulbification Regeneration 30 days after plating

Regeneration of cell suspension after plating Globular embryos

Varietal effect on somatic embryogenesis from callus Induction of embryogenic tissues Somatic embryo induction Conversion into plantlets Significant effect on the different parameters of somatic embryogenesis from callus

Varietal effect on somatic embryogenesis from cell suspension culture Growth of the cell suspension Regeneration after plating Conversion into plantlets - No varietal effect on the growth rate - But a significative effect on regeneration and conversion into plantlets

2 X 4 X 2 X + 4 X Flow cytometric histogram Of diploid and tetraploid Somatic embryos

Genetic characterisation by flow cytometry Nbr. of plants tested Nbr. of diploid Nbr.of tetraploid MES control 12 0 MOL control 16 0 MES cal MOL cal MOL ecs100964

Deliverables DP 5: paper on: Evidence of a somatic embryogenesis process and plant regeneration and acclimatisation in garlic (Allium sativum L.) L. FEREOL, V. CHOVELON, S. CAUSSE, N. MICHAUX FERRIERE and R. KAHANE Plant Cell Report, september 2002 DP13: paper on: Establishment of garlic (Allium sativum L.) cell suspensions and plant regeneration L. FEREOL, V. CHOVELON, S. CAUSSE, D. TRIAIRE, N. MICHAUX FERRIERE and R. KAHANE in progress DP21: paper on the use of somatic embryogenesis for rapid multiplication of garlic in vitro. In progress

What will be done Promotion of the embryogenesis procedure –Histological studies –Looking for more convenient conditions for somatic embryogenesis –Maintenance of the material MES ecs, PRI ecal and PRI ecs: –Genetic characterisation of plants from these material, by flow cytometry Rouge Reunion ecal and ecs: Field evaluation Flow cytometry of the material after field culture