GARLIC AND HEALTH PROJECT - Wageningen UR：大蒜和卫生项目-瓦赫宁根大学

GARLIC AND HEALTH PROJECT - Wageningen UR：大蒜和卫生项目-瓦赫宁根大学 GARLIC AND HEALTH PROJECT EU identification number QLK1-CT-1999-00498 E-mail report September 1 - October 31 2002 (E-report 22) General 1. Meetings: Halfyear meeting in Montpellier: We had a productive half year meeting in Montpellier and the overall progress of the project is good. Remi and Leo thanks very much on behalf of all of us for organizing this nice meeting !! Please find the presentations of the meeting on our home page (username: project; password: rentrap). Annual meeting in Liverpool: The upcoming annual G&H meeting will be in Liverpool: please take note of the following the meeting will be from February 17-21 2003 and not from February 10 –14 2003. ISHS Allium symposium: The presentation scheme on one of the afternoons during the symposium: 13:30-14:15 key note Alliums & Health: Chris; 14:15-14:35 CVD: Rolf; 14:35-14:55 Cancer: Marie-Helene; 14:55-15:30 tea; 15:30-16:15: key note Allium gene transfer: Si-Jun; 16:15-16:35 Genetic resources: Rina; 16:35-16:55: S-metabolism: Meriel; 16:55- 17:15: Cultivation Odile. To all persons who give an oral presentation: please send me your abstract to the symposium before November 15. In this way I can send all our abstracts in one time to the organizers. 3. Preparation of the third annual scientific and financial report: Our annual meeting in 2003 will be two months earlier than in 2002: second half of February in stead of second half of April. To fulfill our duties to the EU and to have a good meeting we need to think about the preparation of the third annual reports especially the scientific part of it one. We thought it would be a good idea that you had a look with us how things can be done in an efficient way. Our proposal for the time-schedule and the partion of the writing of the annual G&H report. 1. Every participant has to take care of: a. the writing of an annual scientific progress report (see attached file: guidelines SC DEMO; page 9: annex II, 3. role of participants or first annual scientific progress G&H report). ` b. If applicable, fill in 5. exploitation and dissimination activities (annex II, page 10) and 6. ethical aspects and safety provisions (annex II, page 10). c. cost statement of the third year. The lay-out of the cost statement can be found in annex II to the general contract: part E-1; the cost statement summary and the details by category need to be duly completed. We like that all partners send the duly completed scientific reports before November 30 2002 to the various WP coordinators and to the theme coordinators. The cost statement can wait, because our financial year runs from February 1 2002 to January 31 2003, you can send the cost statement of the third year to the general coordinator by the end of January 2003. 2. The WP coordinators are writing: a. the annual scientific progress reports for the WPs (see page 8/9: annex II, 2.3 description of the workpackages). b. Furthermore they will write annex II, 5. exploitation and dissimination activities and annex II 6. ethical aspects and safety provisions (page 10) for their own WP on the basis of the info they obtained from their WP partners. The WP coordinators will send before December 16 2002 their reports to the theme coordinators and the general coordinator. 3. The theme coordinators will write for their own theme: a. annex I; section 2: project progress report (page 5). b. annex II; 2.2 project structure, planning and timetable (page 7), c. annex II; 5. exploitation and dissemination activities (page 10) d. annex II; 6. ethical aspects and safety provisions (page 10). 4. The general coordinator will take care of: a. annex I: section 1: project identification (page 4) b. annex II: progress report: page 6 c. annex II: 1. objectives and expected achievements (page 7) d. annex II: 2.1 project workplan (page 7) e. cost statements (see annex II to the general contract: Part E-1 and Part E-2). The theme coordinators and the general coordinator will finish the third annual G&H report and the cost statements before the end of January 2003 P1: Plant Research International, Wageningen, the Netherlands The group at Plant Research International is engaged in three WPs namely, WP1: Genetic Resources, WP2: Genetic Systems and WP9: Co-ordination. The progress in the various WPs will be dealt with consecutively. A. Workpackage : WP1 Genetic Resources B. Objectives: 1. Building-up a collection of garlic 2. Fingerprinting of the garlic collection, to identify the relationships between the various accessions and to develop and maintain a core collection C. Persons involved: Karin Burger, Ria Vrielink, Sjaak van Heusden, Chris Kik D. Milestones & Deliverables M&D 2000 Milestones: Determination of the clonal identity of the accessions present in the existing and collected garlic collections via AFLP fingerprinting done Deliverables: DP 3: Garlic collection done DP 7: Chemical distinction (CSO), fertility and clonal identity between garlic accessions of the collection. partly done M&D 2001: Milestones: Clonal identity screening in newly collected material done Maintenance (core) collection done Deliverables: DP 10: Establishment of core collection and writing paper partly done DP 31: Maintenance core collection done M&D 2002: Milestones: Maintenance of core collection and distribution of material done Deliverables: None E. Research What has been done: The garlic material from the Central Asia expeditions together with other garlic material which has been planted in October/November 2001 in the experimental garden outside Plant RI was harvested in the end of July and is stored. Subsequently a core collection was selected from it and this core collection will be maintained during the project (see also second annual report; role of participants: PRI). The core collection has been sent to Jacques Auger in the beginning of September for further analysis concerning the organo-S compounds. What will be done: Garlic cloves from the core collection will be planted outside in November for cold treatment. Bottlenecks: None Workpackage : WP2 Breeding Systems A. Objectives The development of a reliable transformation protocol for garlic using A. tumefaciens as a vector. B. Persons involved Si-Jun Zheng, Betty Henken, Frans Krens, Chris Kik C. Milestones & Deliverables M&D 2000 Milestones: Establishment of a micropropagated collection of garlic clones: done Axenic material available for transformation experiments: done Deliverables: No deliverables in the first year M&D 2001 Milestones: Callus lines presenting high levels of transient expression of uidA and GFP genes: done Highly transformable callus lines available for stable transformation experiments: done Regeneration of transgenic plants steadily expressing reporter and selectable genes: done Garlic transgenic plants available: done Deliverables: DP12: paper on regeneration (transformation) of garlic: done M&D 2002 Milestones: Regeneration of transgenic plants steadily expressing genes from the sulphur pathway; transgenic garlic plants with alterations in the sulphur metabolism available: to be done Deliverables: No deliverables in the third year E. Research What has been done: The manuscript entitled ‘The development of an efficient cultivar independent plant regeneration system from callus derived from both apical and non-apical root segments of garlic (Allium sativum L.)’ has been accepted by In Vitro Cell Dev. Plant and will be published in May/June 2003. Leaf samples of transgenic garlic were collected from the greenhouse for DNA isolation and detailed molecular characterization. ATP sulfurylase is currently being cloned from shallot. The PRI garlic transformation protocol and A. tumefaciens EHA105(pCAMBIA1301) have been sent to Xabier Barandiaran based on discussions during the Montpellier meeting. What will be done: 1 Paper on garlic transformation. 2 Transfer, via genetic modification, a (key)gene from the sulphur metabolic pathway into garlic. Bottlenecks: None A. Workpackage: WP 9 Co-ordination B. Objectives: To ensure that collaborative links between the partners in the project are established and function and that there is a good link with the EU. C. Persons involved Remi Kahane, Rolf Gebhardt and Chris Kik D. Milestones & Deliverables M&D 2000: Milestones: Start-up meeting, (first and) second project team meeting; done PR activities: home page, brochure; done Annual report done Deliverables: DC 1: first annual report done DC 2: PR activities: home page, brochure done M&D 2001: Milestones: Annual (third) and fourth project meeting done PR activities done Second annual scientific progress report done Deliverables: DC 3: second annual progress report done M&D 2002: Milestones: Annual (fifth) and sixth project meeting partly done PR activities to be done Third annual scientific progress report to be done Deliverables: DC 4: Mid term review done DC 5: second annual progress report done E. Activities What has been done 1. The half year meeting in Montpellier took place from Sept 30 – October 4 2002. It was a productive meeting and it was felt that the project is overall right on track. Please find more info on this meeting on our home page. 2. EU-FP6 matters (see also my e-mail of october 18 2002 on this subject). We submitted an EoI to the EU called SESUCA and also were integrated in an NoE, however based on the published preliminary shortlist for FP6 projects both projects did not made it and that’s a pity. What will be done 1. Preperation of the third annual G&H report: this report needs to be finished medio January 2003 as we will have our next annual G&H meeting in Liverpool on February 17-21 2003. Next deadlines are on November 30 when the partners have to send their contributions to the WP leaders and December 15 when the WP leaders have to send their contributions to the theme coordinators (please find more on this issue in the GENERAL section). 2. ISHS Allium symposium: we have a deadline on November 15 2002 when we need to send the abstracts for the oral presentations for the ISHS Allium symposium to Beijing. Si-Jun arranged for us that we will have an afternoon at the symposium to present our project orally (please find more on this issue on the GENERAL section) Bottlenecks None P2: Horticulture Research International, Wellesbourne, UK A. WP 4: Garlic Sulphur Biochemistry. B. Objectives: Understand the rate-limiting processes in the accumulation of aliin and its subsequent conversion to alliicin. Identify the developmental control points in of cysteine sulphoxide synthesis and translocation Characterisation of the alliinase gene family in garlic. C. Persons involved: Brian Thomas, Lol Trueman, Brian Smith, Linda Brown D. Milestones & deliverables of the first year: Deliverables: , 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) Milestones: , Analysis of second-year field experiment completed , Whole plant labelling studies completed , Expression studies on alliinase clones initiated E. Research No info received P3: University of Liverpool, UK A. WP 4: Garlic Sulphur Biochemistry. B. Objectives: Identify intermediates on the pathway leading to synthesis of alliin. Characterisation of genes with altered expression in tissues with differences in levels of flavour compound pathway flux C. Persons involved: Hamish Collin, Brian Tomsett, Meriel Jones, Rick Cosstick, Angela Tregova, Jill Hughes, D. Milestones and Deliverables of the first year Deliverables: DP16: Analytical methods for labeling and analysis. Milestones: Measurement of alkyl cysteine sulphoxides, pathway intermediates and gamma-glutamyl peptides in bulbing and sprouting plants completed, Optimal conditions for pulse labelling established. Milestones and Deliverables of the second year: Deliverables: DP 16: Pathway intermediates identified. Milestones: Radiolabelled intermediates identified using HPLC and HPLC-MS in comparison with chemically synthesized compounds Differential display sequences isolated and re-analyzed by Northern blot and in situ hybridization, where possible, with other Allium species. Milestones and Deliverables of the third year Deliverables: DP 23.Paper on alliin biosynthesis. DP 24 Genes for key CSO synthesis enzymes Milestones: Analysis of pattern of labeling in later stage intermediates completed. This labeling approach has been superceded by isolation and identification of key pathway enzymes by biochemical and molecular biological methods. Purification of PCR amplified cDNAand sequencing completed.This is no longer applicable since the differential display approach has been discontinued. See above for the altered milestones. E Research What has been done The earlier analysis of Northern Blots of tissue to identify the location of specific enzymes continues. The enzymes thought to be involved in the synthesis of alliin are cytosolic cysteine synthase, chloroplastic cysteine synthase, serine acetyl transferase and allylcysteine synthase. The presence of the mRNA of these enzymes in garlic tissue as detected by Northern Blots is an indication of enzyme activity. oThe Northern Blots of these enzymes are being examined in garlic cloves maintained at 7C, in cloves at ambient temperature and in the root and leaf of the vegetatively active plant. In addition tobacco cells have been transformed to contain cytosolic cysteine synthase, serine acetyl transferase and allyl cysteine synthase. The transformation has included the inducible alc promoter to express these enzymes. This will then allow the enzymes to be analyzed and to establish their substrate specificity. The last is to confirm that these enzymes are engaged in a specific function within the metabolic pathway leading to alliin synthesis. Seedlings grown from seed obtained from Rina were analyzed along with stored garlic cloves to assess the relative importance of the alliin and iso alliin pathways in tissue in different developmental states. The ratio of alliin to iso alliin of the seedling tissue was 2:1, the cold adapted clove 5-8:1 and in the clove at ambient temperature, it was 60:1. The pathway leading to isoalliin seems to be more active in the young growing tissue than in the dormant resting tissue. What will be done The use of alternative substrates for the glutathione transferase is currently under discussion since the preliminary experiments did not yield any positive leads. The Northern Blot analysis of different tissue types continues. The transformed tobacco tissue is being analyzed for evidence of expressed enzymes. Bottlenecks The role of glutathione transferase as part of the mechanism of synthesis of alliin needs to be resolved. P4: COOPD’OR, Dijon, France A. Workpackage N?3 B. Title of research : Cultivation C. Persons involved : P4 (O. Huchette, C. Bellamy, F. Regourd, D. Perrin). D. Milestones & deliverables : 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). DP. 22 Paper on the interaction genotype x environment on the sulphur content of garlic In progress plants grown in vitro, in greenhouse and in field conditions (P4, P5, P6) DP. 34 Chemical distinction (CSO) between garlic accessions when cultivated in various In progress S regimes (greenhouse) and mineral nutrition regimes (in vitro) (P4, P5) E. Research : 1. What has been done: a) In vitro : , Experiment set up in common with Véronique Chovelon (INRA Avignon, P9) and Leonidas Fereol (CIRAD Montpellier, P8) = test of the effect of different factors on in vitro bulblet production of garlic plants derived cell suspension cultures. Lasts harvests of the bulbs were made in September but a great number of plants of Printanor hadn’t yet bulbified. However, as the plants have been cultivated in the medium for 5 months, it was decided to stop the experiment. If possible, 5 to 7 bulbs per treatment and per variety were sent to Jacques Auger for CSO analysis. It couldn’t be made for 5 treatments of Printanor among 6. The rest of the bulbs harvested was observed and the most beautiful (probably viable) were kept and given to Véronique Chovelon (P9) for plantation in greenhouse. The following table summarises the number of plants harvested for the 3 genotypes Printanor, Morasol and Messidrôme : Printanor Morasol Messidrôme T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 nb 12/13/22/18/22/23/16/21/23/22/20/24/19/23/23/21/22/harvested / 24 34 24 34 24 24 24 24 24 22 20 24 23 24 23 24 24 total plants CSO 0 0 0 0 yes 0 yes yes yes yes yes yes yes yes yes yes yes analysis (P5) Bulbs kept 7 6 13 11 12 6 5 6 14 10 14 16 12 13 13 12 14 for plantation (P9) T1-T2-T3-T4-T5-T6 = 3 factors tested as Light : T1-T2 = fluorescent light (F) T3-T4-T5-T6 = F + i / Medium : T1-T2-T3-T4 = R medium (30 g/l sucrose) T5-T6 = B medium (60 g/l sucrose) / Induction period (cold treatment) : T1-T3-T5 The results obtained with this experiment in term of number of plants bulbified were discussed with Véronique Chovelon (P9) and Léonidas Fereol (P8) at a meeting organised just after the Meeting in Montpellier. The results of alliin content analysis should be soon available and allow to finish the synthesis. , An experiment in vitro was set up in Dijon at the beginning of October. This experiment aims at testing : - the effect of the light (quality but also intensity) on the alliin content of the garlic bulbs when the medium is enriched in sulphur. - the effect of a lack of sulphur in the macro-elements of the culture medium. This treatment should confirm or help to understand the results observed in the experiment set up in greenhouse in 2002 . The variety Printanor is mainly concerned by this experiment because a lot of plants of Morasol and Messidrôme bulbified spontaneously during the second micro-propagation. Only 96 plants of Morasol and 48 plants of Messidrôme could be used to test the effect of the light source and intensity. The protocol is presented below: In vitro experiment 3/2002 : PRIvarieties + MESGradient inSO4 + MORl 0 3 9meq(in macro-elements)3mediums (ref. 688) (S- TS S++) 22-24?C - P = 16 hrs - F 1month Cold induction3?C - P = 10 hrs - F2months 3environmentalfactors 22-24?C – P=16 hrs22-24?C – P=16 hrs22-24?C – P=16 hrs9 treatments for PRIF 800 luxF+i - 800 lux F 1600 lux(24 plants / treatment) MES = 3 treatments = 1 medium only MORl = 6 treatments = 2 media onlyb) In greenhouse : Experiment with 3 varieties (Printanor, Messidrôme and Morasol) exposed to 3 (16 plants / treatment)mineral nutrition treatments (0 S content in the macro-elements, normal S content = 2 meq and very high S content = 8 meq). , The results of the experiment have been analysed and presented at the Meeting organised in Montpellier at the beginning of October. The main results are presented below. Regularly observations of the plants showed no effect of the S nutrition treatment on the growing of the leaves and bulbs. At harvest the whole plants were weighted after the roots had been washed and dried. This harvest occurred on the 17 June for Messidrôme, on the 18 July for Morasol and on the 23 July for Printanor. After a few days of drying, bulbs were prepared and sent to Jacques Auger (P5, ) for analysis of the alliin content of each treatment and each variety. Concerning Printanor and Morasol, 3 bulbs per treatment and per replicate were prepared. As no replication could be made for Messidrôme during the Graph 1 : Average weight of the bulbs (g) per variety and per treatment 35 30 25S-20TS15S++10 5 0 MESMORPRI Variety growing season, five bulbs per treatment were prepared and sent for this variety. No effect of the treatment was observed on the weight of the bulbs harvested per variety and no real differences were observed between varieties (graph 1). But significant effects of the variety and S treatment were observed on the alliin content of the bulbs (graph 2). The effect of the S treatment was mainly observed for Messidrôme, forwhich the alliin content increases with the dose of sulphur in the mineral nutrition. For the 2 others, there was no significant difference between the normal and the high level of sulphur, but an absence of sulphur induced a decreased level of alliin in the bulbs. The levels of alliin content appear however not so strongly affected by an absence of sulphur in the macro-elements of the fertilising solution as expected. It could be linked to an absorption of atmospheric S by the plants, which could compensate the lack of sulphur component in the mineral nutrition. This result could be controlled in vitro where atmosphere can’t compensate as well the absence of this component. Graph 2 : Alliin (in nmol/mg fresh matter) per variety and S treatment 140 120 100 80 60 40 20 0 PRI_S-PRI_TSPRI_S++MOR_S-MOR_TSMOR_S++MES_S-MES_TSMES_S++ Concerning the effect of the variety, Printanor presented higher levels of alliin than the 2 others with a mean of 104.1 nmol/ mg fresh matter for the 3 treatments. The average alliin content was 79.7 nmol/ mg fresh matter for Morasol and 73.7 for Messidrôme. These values are much more higher than those found by Rémi Kahane with the experiment in greenhouse in 2001, which were between 18.3 for Printanor and 34.1 nmol/mg fresh matter for Messidrôme. And Printanor appears to be here the richest variety in alliin, contrary to the observations of the previous experiments in field and greenhouse. , One bulb per treatment, per replicate and per variety was also prepared for analysis of total sulphur content by Laurence Trueman (P2, HRI). These bulbs were sent as freeze-dried samples separated in roots, bulbs and leaves. Analysis should be soon in progress. 2. What will be done : a) In vitro : , Progress of the experiment in vitro : plants will be exposed to the cold treatment in November to induce bulbification. , Further micro-propagation of the genotypes Printanor, Messidrôme and Morasol for next experiments in 2003. b) In greenhouse : , A next experiment will be prepared on the effect of nitrogen combined to a high level of sulphur on the alliin content of garlic bulbs. , Bulbs providing from the experiment 2002 will be prepared for plantation in January. A part of them should be planted in insect-proof conditions. Another part should be planted in conditions with a risk of thrips. 3. What are the bottlenecks : A part of the experiments in vitro initially planed for this autumn are delayed because of a lack of plants. A lot of plants of Morasol and Messidrôme bulbified spontaneously during the second micro-propagation and they can’t be used in an experiment studying the effect of a factor on the bulb content. This problem had already been encountered by Rémi Kahane but it has actually no explanation. So another micro-propagation is in progress using 2 sources of material (old material and material introduced in vitro in 2002). This should allow next experiments in 2003 P5: Universite F. Rabelais, Tours, France The group is involved in three workpackages (WP1, WP3 and WP6). A- WP1 Genetic resources 1- Objectives: Screening CSO of garlic collections. 2- Persons involved: Jacques Auger, Ingrid Arnault, Nicole Mandon. 3- Milestones & deliverables of the second year: Milestones: screening CSO of garlic collection. Deliverables: DP7: Overview of the variation in CSO. DP10: Paper on the construction of the core collection 4- Research: What has been done: th We received on june 26 25 garlic accessions from Rina Kamenetski (P7) and 6 others on july th16 HPLC analysis of these 31 accessions is over. th We received from Véronique Chovelon (P9) on june 21and 25 july bulbs issued from embryogenic calli. HPLC analysis is also over. We received in middle of September 55 accessions from Chris Kik (P1). We prepared all bulbs for analysis and HPLC analysis is still going on. What will be done: To achieve the screening of garlic accessions from Chris Kik. B- WP3: Cultivation 1- Objectives: Study effects of environmental factors on CSO content 2- Persons involved: Jacques Auger, Ingrid Arnault, Nicole Mandon & Tristan Doussineau. 3- Milestones & deliverables of the second year Milestones: CSO analysis of fresh plant material exposed in vitro to various temperatures and mineral nutrition treatments CSO analysis of the products before and after dehydratation and processing into powder. Deliverables: DP 7: Chemical screening (CSO) of the genetic resources. DP14: Chemical distinction (CSO) between garlic accessions when cultivated in various sulphur nutrition regimes and under distinct temperature in vitro. Comparison with field-grown material grown. DP 15: Physiological (growth traits) and chemical distinction between accessions when cultivated in various sulphur nutrition regimes over 2 years. CSO content of powder. DP22: Paper on the interaction genotype x environment on the sulphur content of garlic plants grown in vitro, in greenhouse and in field conditions. DP30: Paper on the effect of combined sulphur, nitrogen and selenium nutrition on garlic growth, flavour precursor content and on biological potential of garlic disease prevention. DP34: Chemical distinction (CSO) between accessions when cultivated in various sulphur (green house) and mineral nutrition regimes. 4- Research: What has been bone: We achieved analysis of bulbs from greenhouse experiment (3 mineral nutrition treatments, 3 varieties). Odile Huchette (P5), Leonidas Fereol (P8), Véronique Chovelon (P9) sent on july and september a part of bulbs from in vitro experiment. We prepared samples for HPLC analysis according to our protocol. What will be done: AlCSO analysis of bulbs from in vitro experiment. C- WP5.1 and 5.2: Effect of garlic active compounds on atherosclerosis and lipid metabolism. We analyse CSO from diets experiments with incorporated garlic. Sonia Santo Espirito and Hans Princen (P14) sent in October some different diets. Research: What will be been bone: HPLC analysis of diets with incorporated garlic. D- WP6: Garlic and cancer 1-Objectives Assess the anti-carcinogenic potential of garlic in relation with the bioavailability and the metabolism of garlic sulphur compounds . 2- Persons involved: Jacques Auger, Ingrid Arnault, Nicole Mandon. 3- Milestones of the second year: Synthesis of allicin, ajoenes, alliin and purification of diallyl disulphide, allyl mercaptan and allyl methyl sulphide. Not deliverable for our group in this workpackage 4-research No request from participants. P6: University of Cordoba, Cordoba, Spain A. Workpackage: WP2 Breeding Systems B. Objectives: Biolistic genetic transformation of garlic C Persons involved: Xabier Barandiaran, Nieves Martín, José Garrido, Jesús Martín. D. Milestones & deliverables: Establishment of a micropropagated collection of garlic clones; axenic material available for transformation experiments. (Done) Selection of organogenic callus lines presenting high levels of transient expression of UidA and GFP genes; Highly transformable callus lines available for stable transformation experiments. Regeneration of transgenic plants steadily expressing reporter ands selectable genes; garlic transgenic plans available.(In progress) Regeneration of transgenic plants steadily expressing genes from the sulphur pathway; transgenic garlic plants with alterations in the sulphur metabolism available. DP12 Paper on transformation of garlic E. Research 1. What has been done. Regenerable cell suspensions received from P8 and treated with the biolistic device are being cultivated in presence of hygromicine. 8 weeks after starting the selection process differences among treated explants and controls are still unclear. However, GUS expression is still detectable in treated samples. Agrobacterium strain EHA 105 pCAMBIA and a detailed protocol have been received from P1. 2.What will be done. Agrobacterium strain EHA 105 pCAMBIA will be used to infect cell suspensions, basal plates and roots following the protocol provided by P1. 3. Bottlenecks. Calli selection with hygromicine seems to be a very long process. A. Workpackage: WP3 Cultivation B. Objectives: Evaluation of the effect of sulphur fertilization on yield and quality of field cultivated garlic. C. Persons involved: Xabier Barandiaran, Francisco Mansilla, Jesús Martín. D. Milestones and deliverables: DP22 Paper on the interaction genotype x environment on the sulphur content of garlic plants grown in vitro, in greenhouse and in field conditions (in progress) E. Research Field trials completed. P7: Volcani, Bet Dagan, Israel and P10: The Hebrew University of Jerusalem, Israel The research group is involved in WP1: Genetic Resources, WP2: Breeding Systems I. Workpackage: WP1 Genetic Resources II. Objectives: 1. Construction and maintenance of a core collection; 2. Screening for fertility; 3. Collection of a large number of garlic accessions and close wild relatives in order to expand the current available short- and long-day collections. III. Persons involved: Rina Kamenetsky, Haim D. Rabinowitch, Furkat Khassanov (Uzbek Subcontractor), Idit London, Ada Harazy, Marina Baizerman. IV. Milestones and deliverables of the third year: Milestones: Screening for fertility. Maintenance of core collection and distribution of material. Deliverables: Maintenance and distribution of garlic clones for further use in the project V. Research No info received I. Workpackage: WP2. Breeding Systems II. Objectives Study of the sexual hybridisation system and fertility restoration III. Persons involved Rina Kamenetsky, Haim D. Rabinowitch, Idit London, Marina Baizerman, Hani Zemah IV. Milestones and deliverables of the third year: Milestones: 1. Forcing selected garlic clones to flower. 2. Pollination and study of seed production, seed viability and germinability. 3. Producing self-pollinated and cross-pollinated populations within and between selected clones, respectively. Deliverables: Paper on environmental control of garlic floral development V. Research No info received GENERAL LIST OF DELIVERABLES: Deliverab Completion le Partici-pant Deliverable title Delivery N? date DP. 3 Collected bulbs for screening for CSO, fertility and 9 P1, P5, P7, + clonal identity (finger printing). P10 (According to original program) DP. 4 Paper on morphological and physiological aspects of 12 P7, P10 Done floral/topset initiation and development DP. 7 Chemical distinction (CSO), fertility and clonal 12 P1, P5, P7 + identity between garlic accessions of the collection (According to original program) DP. 10 Establishment of the G&H garlic core collection and 21 P1, P5, P7, In progress writing a paper P10 DP. 11 Paper on environmental regulation of flower 22 P7, P10 In progress differentiation and flowering process DP. 20 Paper on environmental control of garlic floral 34 P7, P10 In progress development DP. 25 Seed and plant populations 44 P7, P10 - DP. 26 Paper on environmental control of garlic floral 48 P7, P10 In progress development and forcing DP. 31 Maintenance and distribution of garlic clones for 21 P1, P10 + further use in the project (According to original program) DP. 32 Paper on environmental regulation of flower 36 P7 In progress differentiation and flowering process DP. 37 Blueprints for fertility restoration. F1 seeds for 48 P7 - biochemical and molecular studies P8: CIRAD Montpellier, France P8 : CIRAD-MONTPELLIER, FRANCE Workpackage n?: 2 Breeding systems Title of research : Embryogenesis – mass propagation Persons involved : FEREOL Leonidas, CAUSSE Sandrine, ROUX-CUVELIER Michel, COTE François, HUGON Rémy, KAHANE Rémi. Milestones and deliverables of first, second and third year Year 1 (2000): 1) provision by P8 of 200 in vitro plantlets from embryogenic calli Rouge de la Réunion R(E-cal). 2) provision by P8 of 200 in vitro plants from embryogenic cell suspensions Rouge de la Réunion R(ECS) 3) production of callus with embryogenic tissue from var. Messidrome and Morado de Cuenca. This last cultivar had been replaced by Morasol because it was field contaminated by viruses. 4) initiation of cell suspension from embryogenic callus of these varieties. 5) Production and germination of somatic emryos from embryogenic calli (E-cal) and cell suspension (ECS). YEAR 2 (2001) : 1) Genetic characterisation of plants, from embryogenesis ? Rouge Reunion ?, by : cytometry (P8), finger printing (P1), dry matter and sulphur content (P5), morpho-physiology (P9) 2) Provision of 200 in vitro plants from embryogenic calli (ecal) of Messidrome (MES ecal) and 200 Morasol (Mol ecal) 3) Provision of 200 in vitro plants from cell suspensions (ecs) of Messidrome (MES ecs) and 200 Morasol (Mol ecs) 4) Acclimatisation and evaluation (P9) of 200 plants MES(ecal), 200 MOL(ecal), 200 MES (ecs) and 200 MOL (ecs) - weaning and hardening in green house of these plants - genetic characterisation of these plants, flow cytometry YEAR 3 (2002): 1) Provision of 200 in vitro plants from embryogenic calli (ecal) of Printanor (PRI ecal) 2) Provision of 200 in vitro plants from cell suspensions (ecs) of Pintanor (PRI ecs) 3) Acclimatisation and evaluation (P9) of 200 plants PRI (ecal) and 200 PRI (ecs) - weaning and hardening in green house of these plants - genetic characterisation of these plants, flow cytometry 4) Plants from embryogenic calli “Rouge de la Reunion)” - Field evaluation , 200 plants R (E-cal), P8 - Genetic characterisation: flow cytometry (P8), finger printing (P1), dry matter and sulphur content (P5), morpho physiology (P8) 5) Plants from cell suspensions “Rouge de la Reunion)” - Field evaluation , 200 plants R (Ecs), P8 - Genetic characterisation: flow cytometry (P8), finger printing (P1), dry matter and sulphur content (P5), morpho physiology (P8) Research The task of P8 within the project aims at the development of a mass propagation method relying on in vitro regenerated plants from embryogenic calli (E-cal) and from cell suspension (ECS) of three varieties : - ? Rouge de la Reunoin ?, tropical varietal group, provided by P9 (INRA Montfavet in France). - ? Messidrôme ?, temperate varietal group 3,provided by P9 (INRA Montfavet in France). - ? Morasol ?, temperate varietal group 1, provided by P9 (INRA Montfavet in France). - Change: A fourth variety “Printanor” has been taken into consideration for adaptation of the protocol to this cultivar. 1) What has been done var . ? Rouge de la Reunion ? - production of embryos from embryogenic calli on semi-solid medium (ecal) and from cell suspension (ecs). - Germination of these embryos. - Provision to P9, of plants from these embryos (ecal and ecs), for further in vitro development, bulbification, acclimatation and field evaluation. - In vitro development by P8 of one part of these plants from somatic embryogenesis. - Maintenance in vitro of some of these plants from somatic- embryos for provision to P9, later, on october 2000 if necessary. Results of embryogenic callus induction according to histological studies, and production of embryos. Cultivars % explant % explant with % of embryogenic Mean number of with callus embryogenic tissue calli developing embryos for 150 mg embryos fresh weight of callus Rouge Réunion 85.7 35 70 12 - Genetic characterisation, by flow cytometry, of 100 plants from ecal and 100 from ecs. Sources of embryos Nb of Nb of plantlets diploid as Nb of plantlets with abnormal quantity plantlets the control of ADN compared to the control tested plantlets Ecal 146 146 20 Ecs 119 119 8 - Field evaluation , plants R (E-cal), P8 - Field evaluation , plants R (Ecs), P8 Var. ? Messidrome ? and ? Morasol ? - Callus induction on mars 2000 - Induction of callus and embryogenic tissues from these cv. - Induction of embryos from embryogenic calli - Germination of these embryos Results of embryogenic callus induction according to histological studies, and production of embryos. Cultivars % explant % explant with % of embryogenic Mean number of with callus embryogenic tissue calli developing embryos for 150 mg embryos fresh weight of callus Messidrome 95 28 68 18 Morasol 95 52 75 27 - Maintenance and in vitro multiplication of embryogenic tissue. - Look on the most appropriate procedure to promote embryogenic calli and maintenance of this one. - Improvement of the embryogenesis procedure on these cultivars. Increasing of mean number of somatic embryos per 150 mg fresh weight of embryogenic callus (18 and 27 respectively for Messidrome and Morasol). - Studies on the procedure to initiate friable embryogenic calli of these cultivars. - Obtention of embryogenic friable callus, convenient to initiate cell suspension. - Studies on the procedure to initiate cell suspension from embryogenic calli of these cultivars. - Provision to P6 of garlic embryogenic calli from cultivar ? Messidrome ? and ? Morasol ?. - Communication to P1 and P6 of the procedure for garlic somatic embryogenesis. - Repetition of experimentations concerning initiation of embryogenic callus of cultivars ? Messidrome ? and ? Morasol ?. - Histological studies: Demonstration of possible unicellular origin of the somatic embryos. - Initiation of cell suspension from embryogenic callus of these varieties. - Provision to P9, of some plants from embryos (E-cal and Ecs), for further in vitro development, bulbification, acclimatation and field evaluation. - In vitro development by P8 of one part of these plants from somatic embryogenesis via calli and cell suspension. - Provision of embryogenic cell suspension cultures of garlic “cv. Morasol” to P6, “cv. Morasol” and “cv. Messidrome” to P1. - Communication to P6 of the procedure for maintaining cell suspension cultures and for plating - Genetic characterisation, by flow cytometry, of “Moraso, E-cal”, “Morasol, Ecs” and “Messidrome E- cal”. Genetic characterisation by flow cytometry, Ploïdy level 2X 4X Undetermined MESSIDROME - control 12 plants 0 0 - E-cal 102 10 0 MORASOL - control 16 0 0 - E-cal 100 3 0 - Ecs 100 4 0 Var. ? Printanor ? - Callus induction - Induction of callus and embryogenic tissues from these cv - Induction of embryos from embryogenic calli - Germination of these embryos - Maintenance and in vitro multiplication of embryogenic tissue. - Initiation of cell suspension from embryogenic callus of this variety. - Induction of embryos from cell suspensions - Germination of these embryos 4 X2 X 2 X + 4 X - In vitro development by P8 of one part of these plants from somatic embryogenesis via calli and cell suspension. - Provision to P9, of plants from embryos (E-cal and Ecs), for further in vitro development, bulbification, acclimatation and field evaluation. All “var.” - Promotion of the embryogenesis procedure for all the cultivars tested. Adaptation and modification of experimental conditions, particularly macro nutrients medium, allow us to promote previous results. Callus production Table 2. Effect of various 2,4-D concentrations of CIM2 on callus production and induction of embryogenic tissue (n=40) 2,4-D Cultivars % callus % callus with concentration production embryogenic tisssue 0.3 Rouge Reunion 86 24 Messidrome 90 32 Morasol 97 44 Printanor 96 51 0.5 Rouge Reunion 93 35 Messidrome 95 39 Morasol 95 52 Printanor 98 65 1 Rouge Reunion 73 19 Messidrome 68 18 Morasol 79 29 Printanor 88 36 1.5 Rouge Reunion 55 15 Messidrome 40 7 Morasol 62 18 Printanor 76 27 We confirmed that a low level of 2,4-D increases the percentage of garlic explants producing callus -1(Barandiaran, 1999a). Our best result was obtained with 0.5 mg l 2,4-D for both types of explants (young leaf and roots). Embryo induction After two months on EIM, up to 92% of the embryogenic callus formed globular somatic embryos (Tab. 3). They were observed from different row of leaf. -1The best combinations of growth regulators were 2,4-D/Kinetin (0.1/0.3 or 0.1/0.5 mg l) for both embryogenic events and embryo numbers. Calllus of about 150mg fresh weight develop a mean number of 33-68 globular embryos, about 40% were converted into plantlets (table 3). Table 3. Embryo production and conversion Cultivars % of Mean number of Mean number of embryogenic globular embryos embryos converted to calli developing for 150 mg fresh plantlets for 150 mg embryos weight of callus fresh weight of callus Rouge 82 37 16 Reunion Messidrome 77 33 14 Morasol 88 51 22 Printanor 92 68 38 Cell suspension culture of “cv. Messidrome, Morasol and Printanor” Culture suspension from friable embryogenic callus Establisment of cell suspension from friable callus has been required approximately 4 months. An increase of PCV X 2 was observed every two weeks. The embryogenic cells observed in these suspension cultures from embryogenic tissue was round, small and densely cytoplasmic. No oxidation or browning was observed in these embryogenic cell suspensions after one year of subculture. The optimum inoculum size was 1.5 g of callus fresh weight for 50 ml of liquid medium. These suspension cultures were maintained on SM medium (table 1). They were visually distinguished by the presence of small cell aggregates . Bulbification Set up of an experiment for establishing a procedure to promote bulbs production in vitro from somatic embryo plants. Treatments concerned: . cold pre-treatment . light quality . daylength . sucrose concentration in the medium Collect of results every two weeks Deliverables - DP 5 Paper on: Evidence of a somatic embryogenesis process for plant regeneration in garlic (Allium sativum L.). L. FEREOL, V. CHOVELON, S. CAUSSE, N. MICHAUX-FERRIERE and R. KAHANE (published in “Plant Cell Report”, august 2002) - DP 13 Paper on: Establishment of embryogenic cell suspensions cultures of garlic (Allium sativum L.) and plant regeneration. L. FEREOL*, V. CHOVELON, S. CAUSS*, D. TRIAIRE, N. MICHAUX-FERRIERE and R. KAHANE (in progress, to be published end 2002) 2) What will be done var. ? Messidrôme ?, ? Morasol ? and ? Printanor ? - Continue development of the most appropriate procedure to promote somatic embryogenesis. - Histological studies continue to follow and understand the differents stages of somatic embryogenesis in Allium sativum. - Maintenance and in vitro multiplication of embryogenic calli and tissue. - Carrying on studies for establishing cell suspensions from embryogenic calli of these varieties and sustaining an active multiplication. - Studies to lead to convenient conditions to regenerate embryos from embryogenic calli and cell suspension. - Research of the best conditions for producing bulbs from somatic embryo plants. 3) Bottlenecks,delays 4) Coordination O. Huchette, L. Féréol and R. Kahane met V. Chovelon at INRA Avignon (13-14/5/02) to discuss about the protocol for bulbing of embryo derived plantlets. Environmental conditions and plant material will be shared into 3 sites (Avignon, Dijon and Montpellier). P9: INRA Avignon- Montfavet, France A. Workpackage : WP2 Breeding systems B. Objectives : Embryogenesis, genetic characterisation: P9, as assistant contractor of P8, is involved in the embryogenesis program to assure final in vitro development and bulbification, acclimatisation, cultivation and evaluation of regenerated plants produced by embryogenesis, to control their integrity and conformity, in comparison with the original materiel multiplied in field (Messidrôme, Morasol and Printanor) Sanitary aspects: P9 is engaged for producing virus-free plants (Messidrôme, Morasol, Printanor, Rouge Reunion) in insect-proof conditions for others partners, for indexing viruses (ELISA tests) and for regenerating selected garlic clones (meristem culture: Israel VIII.I.96 and VIII.81.97) provided by P7. C. Persons involved : V. Chovelon, JP Leroux. D. Milestones & deliverables of the year 2002 : Deliverables: , DP1 virus-free bulbs materiel for introduction in vitro (done) , DP5 paper on embryogenic callus induction in garlic: done in collaboration with P8, accepted for publication in Plant Cell Report (in May 02). , DP13 paper on regeneration of garlic plants issued from embryogenic suspension cultures (writing in progress). Milestones: Embryogenesis: - Garlic variety “Rouge Reunion”: acclimatisation in greenhouse, growth and harvest of 200 plantlets regenerated from embryogenic callus and 200 plantlets regenerated from cell suspension; sending bulbs to P8 at Reunion island (done in June 2001). - Garlic varieties “Messidrôme” and “Morasol”: in vitro development of plantlets regenerated from embryogenic callus and from cell suspension. For each variety, acclimatisation in greenhouse of 200 plantlets regenerated from embryogenic callus and 200 plantlets regenerated from cell suspension (work in progress). - In vitro bulbification studies (work in progress). - Genetic characterisation: field evaluations of all embryogenic regenerants (work in progress). Sanitary aspects: - Production of virus-free plants from Messidrôme, Printanor, Morasol and Rouge Reunion for other partners (20 plants/variety/partner P4, P5, P6, P8): harvest was done in June 2001, and bulbs were sent to the different partners between October 01 and January 02. New production in October 2001 to June 2002. - Virus elimination (meristem culture) on 2 selected garlic clones (VIII.I.96) and (VIII.81.97) provided by P7; multiplication of healthy plants (work in progress). - Virus-free plants obtained by regeneration from selected clones (3) and (4) (work in progress). Research : E. 1- what has been done: Embryogenesis 1- Rouge Reunion: plants issued from embryogenic calli and cell suspensions Appropriate in vitro conditions of temperature and day length were definite for this tropical variety during the first year of the project. Plants acclimatisation was realised at different seasons. Plants were cultivated under greenhouse, and transferred under tunnel in March 01. - AFLP analyses were realised by P1 on plants issued from calli and cell suspensions and plant multiplied by cloves, as reference. The autoradiogram shows that the patterns are almost equal with one exception in plant multiplied by cloves in which there is an extra fragment present. - the ploïdy level was checked by flow cytometry on 120 plants from calli and 120 from cell suspensions: all the plants were diploids and any abnormal ploïdy level was detected (P8). - Normal phenotypic notations were observed during the culture; with however less vigorous plants and bulbs obtained from cell suspensions. - 565 bulbs from calli and 272 bulbs from cell suspensions were sent in June 01 to the Reunion Island (P8) for field evaluation. All the material planted was lost because of a failure in the temperature regulation of the greenhouse and very hot temperature during the culture. - Supplementary plants cultured in field under insect-proof tunnel were harvested and sent in May and June 2002 to Mr Roux at CIRAD Reunion (267 bulbs obtained from cell suspensions, 36 bulbs obtained from callus and 78 bulbs vegetatively multiplied) - New plantation was realised in field under insect-proof tunnel in July 02. - Different observations were given from plantation to harvest (weight clove mean, height plants mean, leaves number mean, vigour and colour of plants) showing a good and homogeneous development of plants from embryogenesis, however less developed and less vigorous than plants from vegetative multiplication. - Alliine and allicine contents will be analysed by P5 on bulbs after harvest. 2- First field evaluation: Messidrôme and Morasol plants issued from callus or cell suspensions Morasol and Messidrôme plants issued from callus and Morasol plants issued from cell suspensions were in vitro obtained, cultured in pot under greenhouse. and transferred in field under an insect-proof tunnel in March 02: 166 Messidrôme-cal, 145 Morasol-cal and 125 Morasol-ECS bulbs were harvested in June 02. - Ploïdy level was tested by flow cytometry (table 1) on 100 plants of each variety in June 02: 10% Messidrôme plants from callus, 3% Morasol plants from callus and 4% Morasol plants from cell suspensions are tetraploïd. - Notations of plants in vegetation (plant development, vigour, height, leaves colour) and after harvest (bulbs shape and weight) were realised: very heterogeneous plants were observed during the first field culture, but no particular difference was observed between diploïd and tetraploïd plants. All the bulbs obtained from diploïd and tetraploïd plants will be planted under tunnel in October 02, after clove homogeneous calibration, for a second field evaluation. Table 1: flow cytometry results Plants origin plants Ploïdy level number 2X 4X Messidrôme (reference) 12 100% 0% Morasol (reference) 16 100% 0% Messidrôme/callus 100 90% 10% Morasol/callus 100 97% 3% Morasol/cell suspension 100 96% 4% - Sulphur content tests on bulbs after harvest (P5): five bulbs of each origin (Morasol-cal, Morasol-ECS and Messidrôme-cal) were sent to P5 and tested by HPLC method in comparison with five bulbs of Morasol and Messidrôme vegetatively multiplied. The results showed that: 1) there is a great difference of dipeptides concentration between garlic from embryogenesis (cal and ECS) and garlic vegetatively multiplied: dipeptides concentrations are highest on plants issued from embryogenesis (table 2) and there proportions are reversed (table 3). 2) Alliin concentrations (table 2) are quasi similar on the different bulbs with a little superiority on Messidrôme-cal (57) and Morasol vegetatively multiplied (61). 3) but Alliin proportions (table 3) are higher on bulbs vegetatively multiplied owing to the fact that dipeptides are lower. It is important to note the great weight difference of the analysed bulbs: 20 to 40g for bulbs from embryogenesis and 70g for bulbs vegetatively multiplied. These results must be confirmed next year on bulbs with similar weight. Table2: bulbs sulphur compounds concentrations by HPLC (nmole/mg) Qb alliineQb allicineQb GLUAlCSQb GLUPeCS 706157605148465043423936403232263025191620151397610concentration en nmole/mg0 MORcalMORecsMEScalMORceeMEScee Table3 : sulphur compounds proportion on bulbs analysed by HPLC 5560 4850 3535354031313027253024232320152010987610 0 MORcalMORecsMEScalMORceeMEScee 3- Printanor plants issued from embryogenic calli: 348 plants were transferred on R medium in January-February 02, for in vitro development and rooting. After 3 weeks at 5?C to initiate the bulbification, plants were acclimatised in greenhouse in April 02 and cultured in pots: 117 plants are still in vegetation and 126 bulbs are obtained. They will be planted in October 02 under an insect-proof tunnel for first field evaluation. 4- In vitro bulb production of garlic plants issued from cell suspension According to the results obtained during last years with the different varieties in vitro cultured, an experimentation was realised between February and September 2002 to define important factors involved in garlic bulb induction and enlargement, in order to homogenise, increase and speed up the bulbification rate of different temperate varieties, and to obtained well-formed bulbs. P9, P8 and P4 were involved in this essay. Three varieties (432 plants Morasol and Printanor, and 144 plants Messidrôme) and different in vitro factors were tested: - saccharose concentration in the culture medium: 30g/l or 60g/l saccharose. - cold treatment: half part of plants was cold treated during 5 weeks at 5?C, and half part of plants was kept during the same time at 24?C (P9). - quality of light: half part of plants (cold treated or not) was cultured under additional far-red light (P9 and P4). - photoperiod: length day 12 or 16h (P8) - temperature: 22?C/ night and 24?C/ day (P4, P8 and P9). Notations were realised each 15 days (plant development, bulb induction and bulb maturation). Bulbs obtained in vitro were harvested at maturity, measured, weighted 3 days after harvest and described (colour, aspect, shape). For each culture condition and each variety, when it was possible, 5 bulbs were sent to P5 for alliine and dipeptides analyses. At the end of August 02, the matured bulbs % was: 92% for Messidrôme, 65% for Morasol and only 36% for Printanor. The influence of the different parameters on bulb % was separately analysed at the end of the essay (2/09/02), and statistical method was applied (Khi2 test): - the different parameters did not have a particular influence on bulb maturation % for Messidrôme variety (up to 87% matured bulbs were obtained in all the different culture conditions). - Light quality: the additional far-red light had a great influence on Morasol bulbificaton (80% instead of 59% with only white light) and also on Printanor with however less bulbs obtained (44% with additional far-red instead of 31% with white light). (Morasol observed Khi2: 19.88 - Printanor observed Khi2: 80.1 - theoretical Khi2: 3.84) - culture medium: for Morasol and Printanor best results were obtained on medium B with higher sucrose concentration (60g/l) . Morasol: 83% bulbs obtained with B medium (60g) instead of 61% with R medium (30g) Printanor: 51% bulbs obtained with B medium (60g) instead of 28% with R medium (30g) (Morasol observed Khi2: 19.52 - Printanor observed Khi2: 22.84 - theoretical Khi2: 3.84) - photoperiod: only Morasol and Printanor were tested for this factor. The increase of the length day (16h) favoured widely the bulb induction and maturation: Morasol: 80% bulbs obtained at 16h instead of 58% at 12h Printanor: 52% bulbs obtained at 16h instead of 26% at 12h (Morasol observed Khi2: 22.26 - Printanor observed Khi2: 50.77 - theoretical Khi2: 3.84), In all the cases, the observed Khi2 was widely superior to the theoretical Khi2, we reject the hypothesis Ho “the factor do not influence the bulbification”, and we accept the fact that these three factors (light quality, culture medium and photoperiod) favour garlic bulbification. - Temperature: the cold treatment applied during 5 weeks at 5?C did not favour bulb formation for the 3 varieties. This result is very surprising according to the previous essay, but it probably link to a too short cold treatment time. The observed Khi2 was inferior to theoretical Khi2 (Morasol observed Khi2: 0.4 - Printanor observed Khi2: 1.38 - theoretical Khi2: 3.84), we accept the hypothesis Ho “the factor temperature do not influence the bulbification”, light qualities culture medium 100%100%94%92%91%87%83%80%80%80% 61%59%60%60%MES51%44%MOL40%40%31%PRI28% 20%20% 0%0%RBwhitewhite+red Photoperiod Cold treatment 80%100%92%80%90% 70%80%71%58%68%60%52%60%50%MES40%37%MOL40%32%26%30%PRI20%20% 10% 0%0%24癈5癈12h16h In conclusion: the best conditions for garlic in vitro bulbification are probably different following the different varietal groups: - for varietal group I (Morasol) and III (Messidrôme) witch are mediterranean and temperate varieties the mean factors are : an increased photoperiod (16h), a high sucrose concentration (60g/l) and an additional far-red light. - for varietal group II (Printanor) witch is a temperate variety with great dormancy, this 3 factors are important to increase bulbs formation (50% bulbs obtained with this 3 conditions instead of 25 to 30%), but this variety probably need more long cold treatment, and a new essay must be realised with 6 to 8 weeks at 5?C. All the bulbs obtained in this essay will be planted in pots in November 02 and transfered in soil under tunnel in March 03. Sanitary aspects 1- Production of virus-free plants - For each variety Messidrôme, Morasol and Printanor, 300 plants were cultured in field under insect- proof conditions. - Virological control by ELISA tests were performed about pathogen viruses (OYDV and LYSV from potyvirus group) and latent viruses (GCLV from carlavirus group and MbFV from allexivirus group) : all the varieties were free of potyviruses, but some one contained latent viruses: Messidrôme: all the plants contained GCLV Morasol: 83% contained Allexivirus Printanor: all the plants contained Allexivirus. - For each variety, the bulbs were harvested at maturity and dried at room temperature before sending to the different partners (September-October 02). - Remaining bulbs (300 cloves/variety) will be planted under tunnel to assure a new production for June 03. 2- Virus elimination by meristem culture on garlic clones selected by P7: Two garlic clones were selected by P7 for their fertility , and regenerated by meristem culture, after thermotherapy treatment to eliminate more easily pathogen and latent viruses. - plants well developed with roots and bulblets were obtained, acclimatised in September 01 and cultured in pots under greenhouse (Octob. 01-February 02). - all the plants were planted in field under an insect-proof tunnel in March 02. - virological control were realised in April 02 on plants in vegetation against pathogen (OYDV, LYSV) and latent viruses (GCLV, MbFV): all the plants were free of viruses. - matured bulbs were harvested in June 02: VIII.1.96: 37 bulbs (20 < weight bulbs < 75g) VII.81.97: 10 bulbs (12 < weight bulbs < 80g) For each clone two bulbs will be planted under tunnel by P9, and the other bulbs were sent to P7 for sexual seed production. 3- Virus elimination by meristem culture on garlic clones (3) and (4) selected by P9: In 2001, any new clones were selected by P7 from the core collection for meristem regeneration by P9. However, two clones were selected for their fertility by P9 in his own collection, and regenerated: - Clone (3): 19 plants in vitro cultured. - Clone (4): 30 plants in vitro cultured. Plants were acclimatised in greenhouse in September 02. 1- Wat will be done: Embryogenesis: 1- Rouge Reunion: bulbs are cultured at Reunion Island for field evaluation under tropical climate. Biochemistry analyses (dry mater and sulphur content) will be realised by P5 on bulbs after harvest. 2- Messidrôme, Morasol and Printanor plants issued from embryogenic callus: plants will be cultured in field under tunnel for a second field evaluation, flow cytometry and AFLP analyses will be realised in May 03, alliine content will be evaluated in July 03. 3- Messidrôme, Morasol and Printanor plants issued from cell suspension : bulbs plantation and first field evaluation, flow cytometry, biochemistry analyses. Sanitary aspects - Production of virus free material: new cloves will be planted and cultured in 2002/03. - clones (3) and (4): greenhouse culture (October 02 to February 03) and field plantation (March/June 03). 2- Bottlenecks: none P10: Hebrew University, Rehovot, Israel see P7 P11: University of Leipzig, Leipzig, Germany A. Workpackage : WP5.2 Cholesterol Metabolism B. Objectives Effect of garlic compounds on cholesterol metabolism and signaling transduction pathways. Persons involved Doris Kellert, Katrin Meyer, Katja Lerche, Frank Struck, Rolf Gebhardt Milestones and deliverables of the first year Milestones : - Characterisation of the molecular target within HMGCoA-reductase regulatory and signal transduction pathways. - Interaction of garlic flavonoids with cholesterol biosynthesis Deliverables : DH2, short report on flavonoids (needs to be written) Milestones and deliverables of the second year Milestones : - Studies on garlic with different sulphur content on cholesterol biosynthesis. Structure-function relationships concerning additional garlic organosulphur compounds and the AMP-dependent kinase pathway. - Influence of garlic constituents on MMP and TIMP production by endothelial cells Deliverables : DH6, Publication on the molecular interaction of garlic compounds on AMP-dependent kinase pathway (Almost finished) DH7, Publication on the influence of garlic constituents on MMP and TIMP gene expression (2 abstracts provided, manuscripts in preparation) Milestones and deliverables of the third year Milestones : - Influence of garlic constituents on MMP and TIMP production by endothelial cells as well as in blood and tissue samples from Leiden ApoE mice. Deliverables : DH11, DH12, publications Research No info received P12: INRA-Dijon, France 2. Address Unité de Toxicologie Alimentaire, INRA, 17 rue Sully, 21065 Dijon Cedex, France Tel : 33 3 80693221 Fax : 33 3 80693225 E-mail : siess@dijon.inra.fr 3. Scientific team Research leader Dr. Marie-Hélène Siess (12 person months) Duties: Co-ordination of this task and carginogenic-metabolising enzymes. Scientists and engineers Dr. Anne-Marie Le Bon Dr. Raymond Bergès (16 person months) (6 person months) Duties: genotoxicity Duties: Hepatocarcinogenesis Dr. Caroline Teyssier Post-doctoral fellows (to be hired) (16 person months) (12 person months) Duties: Metabolism and bioavailibility Duties: Carcinogen metabolizing enzymes, of organosulfur compounds genotoxicity and metabolism Research technicians Marie-France Pinnert Patrick Tassin (6 person months) (6 person months) Duties: Immunohistological methods Duties: Animal care for preneoplasic foci Marie-France Vernevaut Christine Belloir (12 person months) (16 person months) Duties: Enzymes assays and Duties: genotoxicity tests Immunoblots Joëlle Chevalier (16 person months) Duties: Metabolism of organo- sulphur compounds Workplan WP6 Year 1 In vitro metabolism of sulphur compounds by human and rat subcellular fractions from liver In vitro effects of garlic compounds on human CYP P450 isoenzymes In vitro evaluation of the antigenotoxic properties of garlic compounds in Hep G2 cells Year 2 Determination of the bioavailability of diallyl disulfide in rat by measuring the concentrations of the metabolites in blood and in main organs. Evaluation of the antigenotoxic properties of garlic extracts in tissues of rat In vivo effects of garlic extracts on several carcinogen metabolizing enzymes (Phase I and phase II). Year 3 Determination of the bioavailability of allicin and garlic powder in rat by measuring the concentrations of the metabolites in blood and in main organs in progress Evaluation of the effects of subcellular fractions from rats treated with garlic extracts on the mutagenicity of carcinogens using the Ames test in progress Year4 Investigation of the antiinitiating effects of garlic extracts using the medium term hepatocarcinogenesis model in the rat. WP7 Year4 Identification and quantification of sulphur compounds in man by measuring their concentrations in blood and urine after ingestion of a garlic preparation. Determination of the levels of phase II enzymes (glutathion S-transferase) in human lymphocytes Evaluation of the DNA alteration in human lymphocytes and the antimutagenic effects of urine Deliverables DH 8 In vitro metabolism of diallyl disulfide, ajoene and allicin DH 9 In vitro effects of sulphur compounds on human P450 iso-enzymes DH 10 In vitro evaluation of the anti-geno-toxic properties of garlic compounds in Hep G2 cells DH 13 In vivo metabolism of diallyl disulfide DH 14 Effects of garlic extracts on carcinogen-metabolising enzymes in the rat DH 15 Publication of the in vivo anti-genotoxic properties of garlic in rat DH 20 Modulation of the mutagenicity in the Ames test DH 21 Performing intervention study with report and analysis of results DH 22 Short report on the results concerning biomarkers of cancer in human intervention study DH 27 In vivo metabolism of garlic powder Research No info received P13: University of Muenchen, Muenchen, Germany Workpackage: N? 5.1 Title of reasearch: The interaction of garlic with mediators of inflammation Persons involved: Prof. Dr. A. Vollmar; Dr. V. Dirsch; H.-P. Keiss Milestones & deliverables for the first year. 1. Influence of garlic on the expression of adhesion molecules and pro-inflammatory cytokines and their soluble forms. Milestones & deliverables for the second year. 2. Influence of garlic on pro-inflammatory cytokines in human nuclear cells. The potential role of TGF-,1 in the effect of garlic on atherosclerosis Milestones & deliverables for the third year: Milestones: 3. Influence of garlic on the expression of adhesion molecules and pro-inflammatory cytokines and their soluble forms. 4. Influence of garlic on pro-inflammatory cytokines in human nuclear cells. The potential role of TGF-,1 in the effect of garlic on atherosclerosis. 5. Analysis of interaction of garlic compounds with the group of selectins and their role in endothelial cell interaction and with the signal transduction factor NF-,B. Deliverables: 1. Influence of garlic on the expression of adhesion molecules and its relevance to the atherosclerotic process. (in preparation) 2. Influence of garlic on cytokines as important factors influencing the cellular mechanism of atherosclerosis. (in preparation) 3. Influence of garlic on the expression of selectins and signal transduction factor NF-,B. (in preparation) 4. Publication on the influence of garlic on the expression os selectins and signal transduction foctor NF-kappaB (delayed until 08/02) Research: What has been done: We examined if garlic powder extracts (GPE) are able to increase NO-release in endothelial cells by enhancing eNOS activity. For this purpose wed use a modified DAF-2 method, which was established in our lab. (Leikert, et.al; 2002). In brief, HUVECs were incubated with various amounts of GPE for 20h. Supernatants were 2+collected and subsequently incubated with Arginin,Ca-Ionophor A23187 and 4,5- diaminofluorescein (DAF). The liberated NO forms a fluorescent product with DAF, which can be determined by spectrofluorimetry. Garlic extracts on HUVECsGarlic extracts on HUVECs1.401.40 1.201.20 1.001.00 0.800.80SpainSpain0.600.60 0.400.40FranceFrance x-fold controlx-fold control0.200.20 0.000.00 µg/mlµg/mlControl5050050500Control5050050500 2000200120002001 Figure 1: Influence of GPE on endothelial NO release For this purpose we used mixed garlic powders from Spain and France of the year 2000 & 2001. 500 mg of the mixed powders were extracted in 1000 µl DMSO for 30 minutes and subsequently used for the experiments. 50 and 500 µg/ml garlic extract were used in our experiments. Spanish GPE had no influence on eNOS NO production. French GPE of year 2001 showed infact a decreased NO liberation. (Figure 1) Further we examined the possible cytotoxic effects of GPE or single garlic constituents on human leukemic T-cells by MTT-assay. We incubated Jurkat cells for 24 h with garlic constituents or GPE. Afterwards the cells were incubated with MTT wich is reduced by NADPH by living cells. The product of this reaction is a blue Formazan that can be quantified by UV spectroscopy. We used AMSO, AMSO and DADS in concentrations from 10 nM to 100µM. Only DADS showed 2at 100µM a moderate cytotoxic effect. AMSO and AMSO showed only very weak cytotoxicity. 2(Figure 2) 125 100 ******************** AMSO752 AMSO***DADS50 Percent living cells*** 25 0 CN 10nM 100nM 1礛Figure 2: Influence of garlic constituents on cell viability 10礛 100礛 10nM100nM 1礛 10礛For GPE we used the same extracts as described above. GPE from Spain and France showed 100礛10nMonly very weak cytotoxicity in concentrations of 50 and 500 µg/ml. (Figure3) 100nM 1礛 礛 10 100礛Act.125 100*** ********* 75 F2000 S2000 50S2001 Percent living cells ***25 0 CN /ml礸500 礸/ml50 Figure 3: Influence of GPE on cell viability /ml礸500 礸/ml50 /ml礸500 礸/ml50 Act. µgTo test our system we used 1 / Actinomycin (Act.) as positive control. We can not show data mlfor French GPE of the year 2001, due to bacterial and fungal contaminations, which developedafter 24h of incubation. What will be done: Test influence of garlic on endothelial NO liberation. Test influence of garlic on apoptotic cell death. Bottlenecks: None P14: TNO, Leiden, The Netherlands Workpackage 5.1 The interaction of garlic with mediators of inflammation. 5.2 The interaction of garlic with vessel wall and cholesterol metabolism. Title of research: Effect of garlic active compounds on atherosclerosis and lipid metabolism Persons involved: Dr. H.M.G. Princen; S. Espirito Santo M.Sc., Ing. R. Buytenhek Milestones & deliverables for the first year. No activities planned. No milestones and deliverables. - In October 2000 a Ph.D. student was appointed to the project, who is responsible for the daily excution of the project and the presentation of the data in the form of scientific papers. After extensive discussions with a number of participants (P4, P5, P11, P12, P13 and P15) a protocol was finalized for a combined lipid and atherosclerosis study. Milestones & deliverables for the second year. , 5.1 Effect of garlic preparations on expression ICAM-1, VCAM-1, ELAM, and E-selectin in cross sections of apoE3-Leiden mice and measurement of these adhesion molecules in plasma from the mice was not done, because the model was too severe and no beneficial efffect on atherosclerosis was observed. This would have been a task for P13. This is still possible in the second atherosclerosis study which is underway in the third year. Milestones accomplished: , 5.2 Effect of garlic preparations on plasma lipid and lipoprotein and hepatic lipid metabolism in apoE3- Leiden transgenic mice, a mouse model for hyperlipidemia and atherosclerosis. , 5.1 Additional work: Effect of garlic preparations on inflammation markers, platelet activation, blood pressure and atherosclerosis in apoE3-Leiden mice. This will be repeated in the second atherosclerosis study which is underway in the third year. Deliverables: DH 18: partly done. Effects on lipids and platelet activation are studied. Manuscript in preparation. Effects on markers for oxidation, inflammation, endothelial function and smooth muscle proliferation is underway in second atherosclerosis study. Milestones & deliverables for the third year. , 5.1 and 5.2 Effects of garlic preparations on markers for oxidation, inflammation, endothelial function is underway in second atherosclerosis study. Blood pressure will be repeated and platelet activation has been done in year 2. Studies on expression of MMPs and TIMPs in blood and tissue samples obtained from E3-Leiden mice can be done in the study which is underway, but should only be done if there is a significant effect on atherosclerosis (this would be a task for P11). Deliverables: DH 18: remaining part is underway, data are expected mid 2003 DH 5+19: will depend on the outcome of the study that is underway. P14 will measure adhesion of monocytes to activated endothelium and plasma levels of soluble ICAM in E3-L mice. Other measurements (primarily immunohistochemistry and in situ hybridisation) can be done in the atherosclerosis study that is underway (this would be a task for P13). Samples will be made available. DH 24: underway, data are expected mid 2003 DH 3: this is a task for P13 Bottlenecks: Measurement of organic sulfur compounds in garlic powders. Measurement of organic sulfur compounds in diets to assess the stability. Choice of preparations. Research: An intermediate report on the second mouse study will be prepared for the next email report. The study is now 6 weeks underway and will take about 24 weeks. Workpackage 8: the human intervention study Persons involved: Dr. H.M.G. Princen (coordinator); Dr. K. Burgggraaf (CHDR); Dr. G.J. Blaauw (LUMC); S. Espirito Santo M.Sc.; in collaboration with P4, P11, P12, P13, P15 and other partners. There were no activities planned during the first and second year. In the third year preparations are made to conduct the intervention study in the fourth year. A synopsis and action list has been made and is presented below. Deliverables: DH 17: underway Synopsis human intervention trial Rationale: Beneficial effects of garlic on human health have been claimed for decades but these are not unambiguously supported. Therefore, a major collaborative EU project (‘Garlic and Health’) was started to further investigate this relationship. The program was tailored to develop of garlic species that contain the highest content of the compounds thought to be responsible for the health effects. Simultaneously, pre-clinical experiments were carried out to better define the role of garlic (compounds) on presumed pharmacological targets. An overview of all projects is available (see www.plant.wag-ur.nl/projects /garlicandhealth). In summary, it seems that garlic does not meaningfully influence cholesterol metabolism, but there are indications that garlic may have beneficial effects on the inflammatory processes that are characteristic for atherosclerosis. Therefore, a study is proposed to investigate the effects of garlic on biomarkers of arteriosclerosis in man in a population with known risk factors for cardiovascular disease, but without overt clinical symptoms. Objective: To investigate the effects of garlic on biomarkers of arteriosclerosis in man. The primary endpoint in the study is the change in C-reactive protein between the treatment groups versus placebo. Secundary efficacy parameters: endothelial function (biochemically); sensitivity leukocytes to inflammatory stimulus; indicators of lipid metabolism; blood pressure. Study Design: Parallel design with 3 groups treated with a garlic powder (French Printanor 2001) versus control. As a positive control simvastatin will be used. Garlic powder contains all compounds present in fresh garlic and is processed in such a way that sulfur-rich compounds (alliin) expected to have a beneficial effect on health are preserved. Planned Sample: Ninety subjects of either gender aged between 40-75 years with known risk factors not readily subject for therapeutic intervention for cardiovascular disease (such as overweight, smoking, women postmenopausal (note: effect HRT on CRP) etc.) and not using medication for this condition will be recruited for the study. Power calculation: The power to find a change of 30% in plasma CRP levels with 30 subjects per arm and α= 0.05 is 0.87. To find a change of 25% the power is 0.72. Preparations and Dosages: , Placebo , Simvastatin (proposal 40 mg/d) , Garlic preparation from the EU-sponsored breeding program (proposal 2-4 g/d) Main Parameters: - Pharmacodynamics: - clinical: BP, ECG/HRV - biochemical: CRP, vWF, fibrinogen, cytokines after whole blood stimulation with LPS (TNF-α or IL- 10). - safety: routine biochemistry (including cholesterol, HDL, triglycerides) and haematology (Hb, haematocrit, white blood cell count); liver function (ALAT); kidney function (creatinin); muscle (creatine kinase); others? Procedures (outlay): The volunteers will be recruited using advertisements. After a medical screening to assess eligibility, the subjects will complete a 2-week run-in period. At the end of the run-in period blood sampling (twice 10-14 days apart) and measurements will take place. Thereafter the subjects will be randomised to one of the treatment arms. They will return for blood sampling and measurements after approximately 45 (or 30?) days of treatment (twice) and after approximately 90 days of treatment (twice). During the intervention period, telephonic enquiries will be made regarding the health status of the subjects. Points on which a decision was made: , Characteristics of the study group: mild obese, smokers, women postmenopausal (note: effect HRT on CRP) OK , Positive control OK , Supplier intervention preparations (EU-preparation) OK , Necessity of double blood collection midway? Will be done. OK , Acronym for the study: proposal EUGI(I)S (European Union Garlic Inflammation (Intervention) Study). If anyone still has a better idea, please indicate this. All Points of action: , Dosage positive control: proposal 40 mg/d simvastatin Koos, Hans , Dosage: proposal 2-4 g garlic powder (to be discussed) Koos, Thomas, Hans , Size of the tablets? Preferably all powder for one day should be contained in 6-9 pills. Volunteers will ingest tablets 3 times per day before or during the meal (2-3 tablets per meal). Can we check compliance? Koos, Thomas, Hans , Amount needed. There is about 18 kg French Printanor 2001 available. This is minus the amount of 1 kg needed by Marie-Helene for rat experiments and 500 g to be sealed and stored at -80 C in porties of 50 g for further measurements. Is 18 kg enough to beat about 13 kg in pills? Calculation: 30 volunteers X 4 g/day X 100 days= 12kg + 10% extra + 13.2 kg. Thomas , Archiving of garlic powder. 500 g will be sealed and stored at -80 C in porties of 50 g for further measurements. Marie-Helene , Preparation of placebo and positive control. Can we somehow mask the smell of volatile compounds which appear in the breathing air and are excreted via perspiration? Thomas, Koos, Hans , Quality of the EU garlic preparation should be checked with respect to microbiology, aflatoxin B1, heavy metals, pesticides. Other points for purity, safety and quality control? Remi, Thomas , Composition of sulfur containing compounds should be made available. Remi, Jacques Measurement of total sulfur Lawrence Measurement of fructanes and flavonoids Remi Remi coordinator , Inclusion/exclusion criteria (e.g. people who dislike garlic) Koos, Hans , Records of smelling Koos , Preparation of protocol, approval medical ethical committee, CRF, information for volunteers etc. Koos , Ample blood collection for potentiallly additional measurements later. Will be added to the protocol. Koos , Collection of urine. Will be added to the protocol. Koos , Other measurements: Five specific measurements are estimated previously. A number of other interesting markers is given below. Please, indicate which other markers you think that could be of interest and indicate the type of blood (plasma/serum etc), precautions, handling and storage conditions. Action all Other interesting cardiovascular markers mentioned in proposal to EU: (s-VCAM, s-ICAM, s-Selectine) ox-LDL and isoprostanes PAI-1 ? SAA ? New options if a positive effect is observed in subsequent research: mRNA measurements in leukocytes on gene array IL-18; CD40-L Also mentioned in proposal to EU: Marie-Helene Metabolism of metabolites of garlic compounds in urine (and blood?) Cancer biomarkers: Phase II enzymes (glutathione S-transferase and quinone reductase) in plasma and lymphocytes. Better option glutathione peroxidase, SOD and catalase in red blood cells or in serum/plasma. Measurement of DNA damage in lymphocytes using comet assay Anti-mutagenic properties of urine using Ames test P15: Lichtwer Pharma AG, Berlin, Germany Workpackage WP 7: Pharmaceutics Persons involved Dr. Thomas Haffner (Research leader) Thomas Goerke (Research technician) A. Milestones & deliverables of the first year Milestones , Finished development process and formulation of novel garlic solid dosage form(s) with known content, stability, and in-vitro-dissolution characteristics (42 months) Deliverables , [DH. 33] Development of (a) novel/improved solid dosage form(s) providing improvements in in-vitro dissolution and stability [42 Months] , [DH. 34] Report on development process and characteristics on the new formulation(s) , [DH. 35] Publication/patent on the development of (a) novel/improved solid dosage form(s) B. Research 1. What has been done , Ongoing stability studies on film coated tablets containing freeze-dried [formulation 1] and standard garlic powder in a formulation using pre-dried excipients [Formulation 3]. Results for formulation 1 show significant advantages over conventional formulations – more than 1,5 till 2 times prolongation of the stability (18 – 24 months instead of 12 months on average for standard quality) – estimated on the allicin release potential. Formulation 3 starts promising within the first 6 months. , In the meantime we succeeded in producing garlic extracts (lab scale!) containing 5 – 7 % alliin by using only one single extraction step (very economic!). This means an up-concentration of 4 to 5 of alliin. This extract can be used to produce an “high end” product [see meeting in Leipzig = formulation 2]. The activities were set on hold giving priority to the production of study medication. 2. What will be done , Producing study medication 3. What are the bottlenecks