Sanitary

1. G&H April 2002, LeipzigPlant Part PLANT COORDINATION Cultivation S Biochemistry Genetic Resources Breeding systems Alliin pathway studies Which diversity among garlic accessions and wild relatives ? Garlic material from P9 Increasing S content in garlic bulbs Genetic transformation - biolistic protocol on meristems - Agrobact. mediated protocol on callus Mass propagation (for any new accession) - embryogenesis - characterisation of the regenerated plants Field trials in SP and FR - comparisons - powder available In vitro propagation and bulbing protocol Garlic collection characterisation (finger printing) Construction of a cDNA library - differentiating betwen plants parts (roots, leaf, bulb), - stage of growth, - S nutrition (hydroponic culture) Identification of the Alliin intermediates - chemical synthesis - differential display of plant tissues - characterisation of genes with altered expression Sexual system - developmental physiology - florogenesis - fertility studies - molecular markers Flavour Sanitary components status analysis S compounds Flowering Health participants fertility analysis assessment

2. G&H April 2002, LeipzigWP1 / Genetic Resources • Deliverables • DP.3: Collected bulbs for screening for CSO, fertility and clonal identity (finger printing). • DP. 7: Overview of the variation in CSO, fertility and clonal identity in the already existing collections and establishment of the core-collection (P1, P5, P7 & P10). • DP. 31: Maintenance and distribution of elite garlic clones for further use in the project (P1, P4, P6 & P9). • DP. 10: Paper on the construction of the core collection (P1, P5, P7 & P10). Genetic Resources Which diversity among garlic accessions and wild relatives ? Garlic collection characterisation (finger printing) Flavour Milestones Year 3 Screening for fertility (P7), Maintenance of core collection and distribution of material (P1, P10). Year 4 Maintenance of core collection and distribution of material (P1, P10). Sanitary components status analysis Flowering fertility assessment

3. Breeding systems Garlic material from P9 Genetic transformation - biolistic protocol on meristems - Agrobact. mediated protocol on callus Mass propagation (for any new accession) - embryogenesis - characterisation of the regenerated plants Sexual system - developmental physiology - florogenesis - fertility studies - molecular markers G&H April 2002, LeipzigWP2 / Breeding Systems • Recent deliverables • DP. 1: Virus-free bulb material for introduction in vitro (P9). • DP. 4: Paper on morphological and physiological aspects of floral/topset initiation and development (P7, P10). • DP. 5: Paper on embryogenic callus induction in garlic sp. (P8). • DP11: Paper on environmental regulation of flower differentiation and flowering process (P7&P10) • DP12: Paper on transformation of garlic (P1&P6). • DP13: Paper on Establishment of garlic (Allium sativum L.) cell suspensions and plant regeneration.

4. G&H April 2002, LeipzigWP2 / Genetic Resources • Next milestones • Year 3 • Forcing selected garlic clones for flowering. • Establishing mechanical and/or chemical treatments to obtain normal flowering. • Pollination and study of seed production, seed viability and germinability (P7). • Producing self-pollinated and cross-pollinated populations within and between selected clones respectively (P10). • Regeneration of transgenic plants steadily expressing genes from the sulphur pathway; transgenic garlic plants with alterations in the sulphur metabolism available (P1, P6). • Multiplication of healthy plants from the 2 selected clones (1) & (2) 100 plants /clone; acclimatisation of regenerated plants (3) & (4) (P9). • Production of plants from two selected varieties (with extremes in sulphur content for biological studies): 200 plants/varieties (P9). • Embryogenesis: in vitro bulbing studies (P8, P4). • Genetic characterisation: field evaluations of all embryogenic regenerants (P8 and P9).

5. G&H April 2002, LeipzigWP2 / Genetic Resources

6. Cultivation Increasing S content in garlic bulbs Field trials in SP and FR - comparisons - powder available In vitro propagation and bulbing protocol S compounds Health participants analysis G&H April 2002, LeipzigWP3 / Cultivation • Deliverables • DP. 7: Chemical screening (CSO content) of the genetic resources (P5). • DP. 2: Chinese garlic powder to be analysed (P5). • DP. 15: Physiological (growth traits) and chemical distinction (CSO) between accessions when cultivated in various sulphur nutrition regimes over two years (P4, P6). Processing into powder (P4), CSO content (P5) and biological value (transfer to the Health participants). • DP. 14: Chemical distinction between garlic accessions when cultivated in various sulphur nutrition regimes and under distinct temperatures in vitro (P4). Comparison with field-grown material grown (P4 & P6). • DP. 34: Chemical distinction (CSO) between accessions when cultivated in various sulphur (greenhouse) and mineral nutrition regimes (in vitro; P4). • DP. 22: Paper on the interaction genotype x environment on the sulphur content of garlic plants grown in vitro , in greenhouse and in field conditions (P4, P5 & P9). • DP. 30: Paper on the effect of combined sulphur, nitrogen and selenium nutrition on garlic growth, flavour precursor content and on biological potential of garlic for disease prevention (P4, P5).

7. Cultivation Increasing S content in garlic bulbs Field trials in SP and FR - comparisons - powder available In vitro propagation and bulbing protocol S compounds Health participants analysis G&H April 2002, LeipzigWP3 / Cultivation • Milestones • Year 3 • Differential display of clones exposed in vitro to various mineral nutrition treatments (normal and S-, N- or Se-enriched nutrition) (P4). • Greenhouse experiments with two genotypes, one low and one high in S content (from the core collection), optimal growth conditions (temperature, daylength) and sub-optimal sulphur nutrition for producing S-enriched garlic bulbs (P4). • Sampling of fresh plant material during the cultivation treatments for CSO content analysis (P4 & P5) Done by HRI on hydroponics • Year 4 • Greenhouse cultivation of garlic bulbs enriched in CSO (P4). • Comparison between extreme genotypes, low and high in S content (from the core collection). • Comparison virus-free and virus-contaminated garlic for sulphur composition CSO content (P4 & P5). • Chemical analysis (P5) before and after powder processing (P4). • Transfer to the Health partners (P4). Too late !

8. S Biochemistry Alliin pathway studies Construction of a cDNA library - differentiating betwen plants parts (roots, leaf, bulb), - stage of growth, - S nutrition (hydroponic culture) Identification of the Alliin intermediates - chemical synthesis - differential display of plant tissues -characterisation of genes with altered expression G&H April 2002, LeipzigWP4 / Sulphur Biochemistry • Deliverables • DP. 8: Analytical methods for labelling and analysis (P2, P3) • DP. 9: A cDNA library from garlic (P2) • DP. 16: Pathway intermediates identified (P3) • DP. 17: First sulphur budget for garlic (P2) • DP. 18: Clones for alliinase (P2) • DP. 23: Publication on alliin biosynthesis and sulphur partitioning (P2, P3) • DP. 24: Genes for key CSO synthesis enzymes (P2,P3) • DP. 29: Publication on the characterisation of key enzymes in alliin biosynthesis (P2, P3, P5) • DP. 35: Publication on the regulation of alliinase expression (P2, P3) • DP. 36: Publication on the regulation of sulphur biochemistry in garlic (P2, P3, P5)

9. G&H April 2002, LeipzigWP4 / Sulphur Biochemistry L-Serine Acetyl Co-A • Milestones • Year 3 • Analysis of pattern of labeling in later stage intermediates completed (P3). • Analysis of second-year field experiment completed (P2). • Whole plant labeling studies completed (P2). • Publication on the developmental control of alliin synthesis submitted (P2). • Purification of PCR amplified cDNA and sequencing completed (P3). • Expression studies on alliinase clones initiated (P2). • Year 4 • Hypotheses on limiting steps in alliin synthesis tested. (P2, P3). • PCR products, and full-length cDNA clones isolated (P2, P3). • Heterologous expression in E. coli and/or yeast completed. (P3). SAT/CS OAS Sulfide Free CS Cysteine Cyanide Free ACS/CS 3-Cyano-L-Ala

10. G&H April 2002, LeipzigPlant Part • Requirements from the EU • Checking the better quality of a specific (European) garlic • additional garlic powder production from high S-fertilized field • garlic powder production from selected accessions (core collection) • correspondance between Alliin content and bio-extracts S compounds Health participants analysis Checking the quality effect on Health • dose effect • bio-avalability • nutritional recommendations

11. PLANT COORDINATION • Greenhouse production of garlic • PRI-HRI-Coopd’Or- • Cordoba-INRA Core collection Transgenic plants G&H April 2002, LeipzigPlant Part • Bulbing experiments in vitro • Coopd’Or-CIRAD-INRA • IVth International Allium Symposium (China April 2003) • all partners ? • united presentations; annual meeting there ?