1. ABOUT OUR NEW HPLC METHOD  We have identified a new compound in garlic HPLC profile, the S-allyl cystein (SAC) that has a potential therapeutic activity Garlic & Health P5 J. AUGER Allicin biosynthesis from γ-glutamyl-S-allyl-L-cysteine  We are now able to quantify 3 precursor compounds of Allicin γ-glutamyl-S-allyl-L-cysteine (GLUAlCS) S-allyl-L-cysteine sulfoxide (alliin) γ-glutamyl- transpeptidase Allicin Alliinase N O S NH 2 H HOOC COOH S-allyl-L-cysteine (SAC) S NH 2 COOH O H2O2H2O2  With this method we are now ready to compare the core collection Chromatogram of 4 standards (left) or of a garlic bulb in inhibited alliinase conditions (right) 0,00 0,05 0,10 0,15 0,20 0,25 0,30 4,00 8,00 12,00 16,00 20,00 24,00 28,00 Alliin GLUAlCS SACAllicin 0,00 0,10 0,20 0,30 0,40 0,00 4,00 8,00 12,00 16,00 20,00 24,00 28,00 SAC Alliin GLUAlCS GLUPeCS GLUPheAla

2. G&H April 2002, Leipzig Stimulating effect of i light on Alliin synthesis WP3 / cultivation, P4 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 FF+iF27 Alliin (mmol/mg FW) 30 60 120 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 FF+iF27 Mean bulb weight (g) S30 S60 S120 PRI MES Sucrose (in g/l) * * * * * COOPD'OR R&D

3. IN VITRO EXPERIMENTS : REP. OF SERIE I THE AIM OF THIS EXPERIMENT : to verify the results of the first experiment with an other hybrid, Morasol. Bulbing conditions: Sucrose (S30, S60 and S120). Cold induction period, 2 months, and F+i. Lichtwer HPLC method used  The sucrose 60g/l seems to induce more Alliin contrary to the first results, and this HPLC method is more suitable for its quantification alone. Garlic & Health P5 J. AUGER Alliin concentration (nmole/mg)

4. G&H April 2002, Leipzig In the absence of i light, erratic response to S WP3 / cultivation, P4 0,020 0,025 0,030 0,035 0,040 0,045 0,050 0,055 0,060 0,065 0 S3S6 S12 S 0,0 0,2 0,4 0,6 0,8 1,0 0 S3S6S12S Bulb fresh weight (g) MES MOR Alliin content (nmole/mg FW) COOPD'OR R&D

5. G&H April 2002, Leipzig Sulphur is limiting for Alliin storage in garlic bulbs WP3 / cultivation, P4 Mean bulb weight (g) 0,0 0,1 0,2 0,3 0,4 0,5 0,6 2S_F+i1S_F+i2S_F1S_F 0,0 0,2 0,4 0,6 0,8 1,0 2S_F+i1S_F+i2S_F1S_F Alliin content (nmole/mg FW) MORASOL PRINTANOR MESSIDROME MORADO COOPD'OR R&D

6. CONCLUSION OF IN VITRO EXPERIMENTS Among all the conditions tested, the better to induce more alliin in garlic bulbs are: Sucrose in the medium : 60g/l Cold induction period : 3 months Environmental factor : F+i light Sulphur in the medium : 6 Meq SO 3 Garlic & Health P5 J. AUGER

7. Mean bulb weight (g) Alliin content (nmole/mg FW) COOPD'OR R&D G&H April 2002, Leipzig S fertilization greatly influenced alliin content WP3 / cultivation, P4

8. Garlic & Health P5 J. AUGER 8 10 12 14 16 18 0100200400 PRINTANOR POWDERS 2001 France 4 Sulphur fertilisation levels Spain 5 Sulphur fertilisation levels Relation between alliin concentration (nmole/mg) and Sulphur fertilisation level (kg/ha) Alliin content in Spanish PRI is 3-6 times that in French PRI  Alliin content in Spanish PRI is 3-6 times that in French PRI It seems that the treatment 2000 had no effect on Alliin content.  Alliin content increases in France with the S fertilisation (+25%) In Spain, the maximum was obtained even with the lowest S fertilisation (+8%). 200 240 280 320 360 01002004002000

9. G&H April 2002, Leipzig Soil quality may explain bulb quality WP3 / cultivation, P4 Treatment (S0 4 unit)Sulphur analysis25/08/0021/02/0114/05/0120/06/0114/08/01 0Total217149189156208 Water soluble975,6810 100Total172187186 Water soluble9,7119 200Total168127213 Water soluble12,21615 400Total188114185 Water soluble20,82220 14/05/01 234 9,5 249 10,1 238 8,7 244 8,4 FranceSpain COOPD'OR R&D

10. %DM0100200400Mean2000 I32,9437,2134,5035,0534,9337,27 II -33,0035,7832,5333,7733,60 III35,0033,7431,6732,8333,3137,40 IV32,3933,2934,5035,3833,8928,58 Mean33,4434,3134,1133,9533,9534,21 Powder (g)0100200400Mean2000 I548698682656646405 II207815819603611330 III5901411471955857370 IV578793892912794738 Mean481929716782727461 Total (g)19233717286431261843 Total powder available (g)13473 COOPD'OR R&D G&H April 2002, Leipzig Powder production from Spain WP3 / cultivation, P4

11. %DM 0100200400Mean I42,12*42,2643,5540,8342,19 II40,1843,5042,3242,8442,21 III45,0042,6541,3340,0042,24 IV37,8840,2940,2340,5139,73 Mean41,3042,1741,8641,0441,59 * burned material with reduced alliin content Powder (g) 0100200400Mean I30502830292029202930 II32703230260032703093 III39503440275033503373 IV32003170353032103278 Mean33683168295031883168 G&H April 2002, Leipzig Powder production from France WP3 / cultivation, P4 Total (g)7000667055206130 Total powder available (g)25320

12. G&H April 2002, Leipzig WP3 / cultivation, P4 Deliverables P4 DP 2Chinese garlic powder to be analysed DP 6Garlic powder issued from various S-fertilisation in field condition DP 14 Chemical distinction between garlic accessions when cultivated in various sulphur nutrition regimes and under distinct temperatures in vitro DP 15 Physiological (growth traits) and chemical distinction (CSO) between accessions when cultivated in various sulphur nutrition regimes over two years DP 19 Chemical distinction (CSO) between accessions when cultivated in various sulphur (greenhouse) and mineral nutrition regimes (in vitro) 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 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 DP 34Chemical distinction (CSO) between garlic accessions when cultivated in various sulphur regimes (greenhouse) and mineral nutrition regimes (in vitro) COOPD'OR R&D