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

Somatic embryogenesis A mass propagation protocol, relying on in vitro regenerated plants, will be developed (by P8). This method will be based on the production of embryogenic calluses and embryogenic cell suspension cultures from four varieties which represent different physiological groups 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 Objectives

Milestones

Deliverables

Delays Milestones –Rouge Reunion 2 nd 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

Resarch activities during the fourth reporting period Embryogenesis, genetic characterisation –Rouge Reunion 2 nd field evaluation (P8), biochemistry (P5) –Messidrôme, Morasol and Printanor from callus 2 nd field culture and evaluation (P9) flow cytometry (P8), AFLP (P1), biochemistry (P5) –Messidrôme, Morasol and Printanor from cell suspension 1 st 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

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

Materials and methods (I) Cultivars –Rouge Reunion –Messidrome –Morasol –Printanor Explants –Primordial leaves

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 °C in the dark with continuing agitation at 100 rpm.

Callus induction Morphology Histology -macro-nutrients modified B5 (CaCl2 440mg) -micro-nutrients MS -vitamines B5 + myo-inositol 100 mg + malt extract 100mg -prolin 230mg -2,4-D 0.5mg, IAA 0.2mg, NAA 0.2mg, Kin 0.1 mg -Sucrose 60g, phytagel 3g Medium

Induction of friable callus Morphology Histology -macro-nutrients B5 (CaCl2 150mg) -micro-nutrients MS -vitamines B5 + myo-inositol 100 mg + malt extract 100mg -prolin 230mg -2,4-D 0.5mg, IAA 0.2mg, NAA 0.2mg, Kin 0.1 mg -Sucrose 60g, phytagel 3g Medium

Comparative callus induction from different cultivars Type of callus induction % Culture period (weeks) Cultivars RRMESMOLPRI callus Callus with embryogenic tissue Friable and embryogenic callus

Cell suspension culture aspect Morphology Histology -macro-nutrients N6 -micro-nutrients H -vitamines B5 + myo-inositol 100 mg + malt extract 100mg -glutamin 150mg -2,4-D 0.3mg, BAP 0.1 mg -Sucrose 45g, phytagel 0g « SM » Medium

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

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

Regeneration aspect after plating on semi-solid embryo induction medium Morphology Histology « EIM » Medium -macro-nutrients N6 -micro-nutrients H -vitamines B5 + myo-inositol 100 mg + malt extract 100mg -glutamin 150mg -2,4-D 0.1mg, Kin 0.5 mg -Sucrose 45g, phytagel 3g

Conversion aspect into plantlets Morphology Histology -macro-nutrients BDS -micro-nutrients MS -vitamines Morel -Sucrose 20g, agar-agar 7.5g Medium

Embryogenesis features and kinetic of regeneration