Influence of environmental and genetic factors on the alliin content of garlic bulbs
O. Huchette 1, R. Kahane2, J. Auger3, I. Arnault3, C. Bellamy1
1. DIJON CEREALES / COOPD’OR, R&D Department, INRA, BV 86510, 21065 Dijon
Cedex, France
2. CIRAD-FLHOR, TA50/PS4, bd de la Lironde, 34398 Montpellier Cedex 5, France
3. IRBI, Université François Rabelais, Avenue Monge, 37200 Tours, France
Keywords : Allium sativum, sulphur compounds, genotype, soil, light spectrum
Abstract
The influence of environmental conditions and genotype x environment effects on
the alliin content of garlic bulbs were studied. The objective was to improve the quality of
garlic bulbs with regard to alliin content. Field trials were carried out for 2 years in France
and Spain with increasing sulphur fertilisation in soil: 0-50-100-200 or 400 kg SO4/ha
were investigated on 3 varieties in 2000 (Printanor, Messidrôme and Morasol) and only
on Printanor in 2001. S fertilisation affected alliin content on Printanor in France, but was
not confirmed in Spain where alliin contents were 3 to 5 fold higher than in France. These
results were correlated to the soil sulphur in the two areas as Spanish soil was always
higher in water soluble and total sulphur than French soil. However, the influence of other
factors could not be excluded. Experiments were also carried out in controlled conditions
(in vitro and greenhouse) to study the influence of sulphur fertilisation combined to other
environmental factors such as light, carbohydrate availability and temperature. A strong
genotypic effect was confirmed in both controlled conditions with light spectral quality
having a large effect on alliin content on the in vitro bulbs. Temperature did not have a
large effect on alliin, but did interact with sucrose concentration to affect alliin
accumulation in the bulbs. We also found an interaction between S fertilisation and
genotype in greenhouse, and genotype by light spectrum interaction, in vitro. Our results
confirm that multiple factors affect alliin accumulation in garlic, and that these factors
should be considered when growing garlic for flavour or therapeutic value.
INTRODUCTION
Garlic (Allium sativum L.) is among the earliest domesticated plants and is
cropped world-wide. It is well-known for its health benefits. Numerous therapeutic
properties have been reported, i.e. antifungal, antibacterial, antiviral, antithrombotic,
antitumor, hypotensive, hypoglycaemic, hypolipidemic (Augusti, 1996; Sato, 2000).
Moreover, therapeutic value related to cardiovascular diseases, cholesterol metabolism,
atherosclerosis (Kik et al, 2001), and cancer (Le Bon and Siess, 2000) were recently
described. These benefits can be linked to garlic’s organosulfur compounds, particularly
the cysteine sulfoxides and thiosulfinates. Among them is S-allyl-L-cysteine sulfoxide, or
alliin, which derives from the related dipeptide -glutamyl-S-allyl-cysteine (GLUAlCS)
and accumulates in high concentration in garlic. Alliin goes on to produce diallyl
thiosulfinate, or allicin, which is formed following enzymatic transformation when the
bulb is cut or crushed. Allicin is the characteristic compound of garlic flavour, but is
unstable and degrades over time (Block, 1985 ; Block et al, 1993).
The influence of environmental factors, such as temperature, daylength and
carbohydrates has been often reported on bulb induction and development in garlic
1
(Takagi, 1990; Nagakubo et al, 1993; Kahane et al, 1997). In onion, light spectrum
quality is of primary importance for bulb formation (Lercari, 1982; Kahane et al, 1992a).
However, environmental factors not only influence bulb formation but also the flavour
quality, as observed on onion (Randle, 1997; Randle and Lancaster, 2002). With garlic,
only the influence of sulphate nutrition on different flavour components has been studied
(Freeman and Mossadeghi, 1971). Thus, this work aims at exploring the influence of both
environmental and genetic factors on garlic bulb alliin content as a specific flavour
quality trait also related to its health-value. To that aim, five experiments were carried out
on four cultivars from three different physiological groups (Messiaen et al, 1993) in
natural conditions and in the greenhouse using different levels of sulphur nutrition. In
addition, the influence of temperature, carbohydrates availability, and light spectrum were
studied in vitro.
MATERIAL AND METHODS
Plant materials
Three French garlic cultivars from different varietal Groups were supplied as
virus-free certified cloves by INRA Avignon, France, and studied: Morasol from Group I,
Printanor from Group II and Messidrôme from Group III (Messiaen et al, 1993). A
Spanish variety (Morado de Cuenca), similar to Morasol, was provided by the University
of Cordoba, Spain.
Experimental environments
1. Field experiments. Two field trials were set up in two locations, in the South of
France, and in Spain, over two years. Three accessions were studied in 2000 (Morado de
Cuenca, Messidrôme and Printanor), and one in 2001 (Printanor). Growing protocols
were similar in both locations (Table 1). The experiment included 5 sulphur treatments
and 4 replications in a factorial arrangement using a randomised block design. Each plot
comprised 100 plants spaced at 10 cm on a line and 30 cm between lines (2,5 m x 1,2 m).
A control plots were placed between experimental plots and border plants surrounded the
treatments. Sulphur fertilisation in the form of dry calcium sulphate (gypsum) was applied
in two times (plantation and onset of bulbing) at rates of 0-50-100-200- and 400 kg/ha.
Plants were harvested per plot at maturity (above 30° Brix of the juice). After a drying
period, bulbs were weighed, extracted fresh and ground into powder.
2. Greenhouse experiment. This experiment was set up under insect-proof conditions in
France in 2002. The three cultivars Printanor, Messidrôme and Morasol were planted in
boxes with washed river sand (12 cloves per box) on the 1st March. Three boxes were
planted for Printanor and Morasol, one for Messidrôme. Water and 50-100 ml per day of
a fertilising solution of: 779 mg/l Ca(NO3)2,4H2O, 556 mg/l KNO3, 73 mg/l
(NH4)2,H2PO4, 246 mg/l MgSO4,7H2O, 1 ml/l Fe-EDTA and micro nutrients (1.47 mg/l
H3BO3, 1.85 mg/l MnCl2, 1.04 mg/l ZnSO4, 0.24 mg/l CuSO4, 0.05 mg/l H2MoO4)
were supplied by drip irrigation. Sulphur was supplied at 0, 1x and 4x that of the original
fertility solution. Garlic was harvested when two to three leaves remained green. After
one week of drying at room temperature, three bulbs per replicate were sampled (five for
Messidrôme), weighed and prepared for analysis.
3. In vitro experiments. The plant material was introduced in vitro and propagated
following a cyclic multiplication previously reported for onion (Kahane et al, 1992b) to
2
produce enough material for three experiments. Temperature, sucrose (30 and 60 g/L) and
spectral quality (red vs. far-red light) were varied in vitro for each cultivar during bulbing.
Following a cold induction period (2 months at 3°C - photoperiod 10-24 hrs - white
light), bulb formation was initiated in a warm environment (22-24°C as standard
temperature) under long photoperiods (16-24 hours), with or without far-red light.
Analyses
1. Soil sulphur. Soil analysis were performed in 2001 by INRA Arras (France) from soil
samples collected in experimental plots in France and Spain without any additional
application of gypsum (CaSO4). Total and water soluble sulphur were analysed before
planting and at two dates during the cultural period (14 May, 20 June).
2. Sampling for HPLC analysis. Three to five bulbs per replicate were sampled from the
greenhouse experiment and crushed in a methanol / acidified water mixture in order to
stop the enzymatic reaction. For the experiments in vitro, all bulbs were used for analysis
(16 bulbs minimum per treatment). For the field experiments, 1 g of dried garlic powder
was extracted in 10 ml of solvent where as 1 g of fresh bulb tissue was extracted in 3ml of
solvent (Kamenetsky et al, in press). The extract was then derivatized for the Pico-Tag ®
HPLC analysis and diluted for the HPLC analysis using the ion-pairing method.
3. HPLC Methods. Two HPLC methods were used: the ion-pairing method (Arnault et
al, 2003), which allowed the screening of alliin and allicin; and the Pico-Tag method®
(Auger et al, 1993), which is more sensitive than ion-pairing, but only allowed for the
quantification of alliin. The Pico-Tag method was only used for analyzing the in vitro
temperature study. Alliin content is only reported here due to space limitations.
Statistical analysis
Results of garlic bulb alliin content are reported as means and standard deviations
of two to four replicates per treatment in vitro. In the other experiments data were
analysed by ANOVA (p = 0.05) with the software SYSTAT 10 for main plot effects and
appropriate interactions.
RESULTS AND DISCUSSION
Influence of the genotype
In greenhouse, the three varieties tested presented significant differences in alliin
content (Figure 1). Printanor had indeed the highest level of alliin (104.3 nmol/ mg fresh
matter in average on all S-treatments), while Messidrôme had the lowest one (73.7 nmol/
mg fresh matter) and Morasol was intermediate (79.7 nmol/ mg fresh matter ). This
influence of the genotype was also observed in field trials, but only in France (Figure 2).
Results showed a higher quality of bulbs in terms of alliin content in spring varieties
(Printanor, Morasol), compared to the autumn one. These data confirm observations on
onion where flavour intensity varied among cultivars grown in a single and uniform
environment (Randle, 1997), but also with developing information in wild garlic
(Kamenetsky et al, in press).
Influence of environmental factors
In the field trials, all varieties tested in 2000 presented higher alliin content, on
average, when cultivated in Spain (102.2 nmol/mg) than in France (62.1 nmol/mg)
3
(Figure 2). In 2001, Printanor grown in Spain had about four fold high alliin than that
measured from plants in France (Table 2). Moreover, no significant difference was found
among varieties in Spain, contrary to what observed in France (Figure 2). Surprisingly,
no influence of sulphur fertilisation was observed in Spain (Table 2). This, however, may
be explained by the soil analyses (Table 3). High total and water soluble sulphur levels
were found in Spanish soil, suggesting that sulphur supply was perhaps saturated for the
plants (Randle and Lancaster, 2002). This could partially explain the lack of differences
observed on alliin content among varieties grown in Spain, and is in line with
observations on the flavour of onion cultivated on different soil types (Hamilton et al,
1998). However, the dramatic difference in alliin accumulation between the French and
Spanish trials suggests that other factors may also have an influence ultimate
accumulation.
In vitro, the influence of temperature was tested on Printanor in correlation with
the carbohydrate level in the medium (Figure 3), but no effect was found for temperature
when averaged over carbohydrate levels. However, carbohydrate availability affected the
level of alliin accumulated, but was dependent on the growing temperature of the plants.
At the lower temperatures, alliin accumulation was favoured with the higher carbohydrate
level, but at the higher temperature, the opposite was found. A strong effect of the light
spectral quality was found in the spring garlic varieties (Figure 4). The addition of far-red
light during the bulbing period increase alliin content by four-fold for Printanor and by
seven-fold for Morasol. However, no influence was observed on the autumn variety
Messidrôme, suggesting an interaction between genotype and environmental factors.
These experiments show that alliin accumulation in garlic is dependent both and the
variety utilized and the environmental conditions in which the plants are grown. To
maximize alliin, proper cultivar selection is required as well as growing the plants in the
most conducive environment.
ACKNOWLEDGEMENTS
Experiments were carried out within the framework of a program called "Garlic
and Health" funded by an European Union FP5 grant in the Quality of Life program
(QLK1-CT-1999-0498). The authors would like also thank Dr S. Ochatt and Dr. W.M.
Randle for reviewing the paper and correcting the English.
Literature Cited
Arnault, I., Christidès, J.P., Mandon, N., Kahane, R., Haffner, T. and Auger J.
2003. High performance ion-pair chromatography method for simultaneous analysis of
alliin, deoxyalliin, allicin and dipeptide precursors in garlic products using multiple mass
spectrometry and UV. J Chromatogr A 991: 69-75.
Auger, J., Mellouki, F., Vannereau, A., Boscher, J., Cosson, L., and Mandon, N.
1993. Analysis of Allium sulfur amino acids by HPLC after derivatization.
Chromatographia 36: 347-350.
Augusti, K.T. 1996. Therapeutic values of onion (Allium cepa L.) and garlic
(Allium sativum L.). Indian J. Exp. Biol. 34: 634-640.
Block, E. 1985. The chemistry of garlic and onions. Sci. Am. 252 (3): 114-119.
Block, E., Naganathan, S., Putman, D. and Zhao, S-H. 1993. Organosulfur
chemistry of garlic and onion: Recent results. Pure Appl. Chem. 65(4): 625-632.
4
Freeman, G.G., and Mossadeghi, N. 1971. Influence of sulphate nutrition on the
flavour components of garlic (Allium sativum) and wild onion (A. vineale). J Sci Food
Agric 22: 330-334.
Hamilton, B.K., Yoo, K.S., Pike, L.M. 1998. Changes in pungency of onions by
soil type, sulphur nutrition and bulb maturity. Scientia Hortic. 74: 249-256.
Kahane, R., Teyssendier de la Serve, B., and Rancillac, M. 1992a. Bulbing in
long-day onion (Allium cepa L.) cultured in vitro: comparison between sugar feeding and
light induction. Ann. Bot. 69: 551-555.
Kahane, R., Rancillac, M., and Teyssendier de la Serve, B. 1992b. Long-term
multiplication of onion (Allium cepa L.) by cyclic shoot regeneration in vitro. Plant Cell
Tissue Organ Cult 28: 281-288.
Kahane, R., Schweisguth, B., and Rancillac, M. 1997. Trophic versus
environmental factors controlling in vitro bulb formation in onion and garlic
micropropagated plants. Proc. First International Symposium on Edible Alliaceae. Acta
Hortic. 433: 435-443.
Kamenetsky, R., London Shafir, I., Khassanov, F., Kik, C., Van Heusden, A.W.,
Vrielink-Van Ginkel, M., Burger-Meijer, K., Auger, J., Arnault, I., and Rabinowitch, H.D.
2003. Diversity in fertility potential and organo-sulphur compounds among garlics from
Central Asia. Biodivers. Conserv. (in press)
Kik, C., Kahane, R., and Gebhardt, R. 2001. Garlic and health. Nutr. Metab.
Cardiovasc. Dis. 11 (supppl. to 4): 57-65.
Le Bon, M.H., and Siess, M.H. 2000. Organosulfur compounds from Allium and
the chemoprevention of cancer. Drug Metab. Drug Interact. 17 (1-4): 51-79.
Lercari, B. 1982. The promoting effect of far-red light on bulb formation in the
long day plant Allium cepa L. Plant Sci. Lett. 27: 243-254.
Messiaen, C.M., Cohat, J., Leroux, J.P., Pichon, M., Beyries, A. 1993. Principes
de sélection. p. 139-163. In: INRA (ed.), Les allium alimentaires reproduits par voie
végétative. Paris, France.
Nagakubo, T., Nagasawa, A., Ohkawa, H. 1993. Micropropagation of garlic
through in vitro bulblet formation. Plant Cell Tissue Organ Cult 32: 175-183.
Randle, W.M. 1997. Genetic and environmental effects influencing flavor in
onion. Proc. First International Symposium on Edible Alliaceae. Acta Hortic. 433: 299311.
Randle, W.M., and Lancaster, J.E. 2002. Sulphur compounds in Alliums in
relation to flavour quality. p. 329-356. In: H.D. Rabinowitch and L. Currah (eds), Allium
Crop Science: Recent advances. CAB International.
Sato, T., and Miyata, G. 2000. The nutraceutical benefit, Part IV: Garlic. Nutrition
16: 787-788.
Takagi, H. 1990. Garlic (Allium sativum L.). p. 109-146. In: J.L. Brewster and
H.D. Rabinowitch (eds.), Onions and Allied Crops, Vol. III (6), CRC Press. USA.
5
Tables
Table 1 : Garlic crop characteristics from 2000 and 2001 field trials. Three garlic cultivars
were tested : Printanor (PRI), Morado (MC) and Messidrôme (MES).
Year, Location
Variety
Planting date
Harvest date
2000, France
MC
PRI
MES
MC
PRI
MES
PRI
PRI
12-13 January
12-13 January
23 December
16-17 November
22-23 November
22-23 November
15 January
15 January
10 July
10 July
28 June
16 June
26 June
15 June
13 July
August
2000, Spain
2001, France
2001, Spain
Treatments (*)
Kg/ha
0-50-100-200
0-50-100-200
0-100-200-400
0-100-200-400
(*) Sulphur fertilisation in the form of dry Calcium sulphate (gypsum) applied in two times (50% each)
Table 2 : Mean alliin content (in nmol/mg) obtained in 2000 and 2001 on field trials set
in France and Spain using Printanor (n=3). Statistical mean difference analysed by
ANOVA was characterised per location and expressed as significant with different letters,
as determined by a Newman-Keuls Test (p = 0.05).
Treatment
(S04 unit)
0
50
100
200
400
Mean
France 2000
France 2001
Spain 2000
Spain 2001
46.7 a
59.7 b
69.1 c
89.7 d
66.3
57.8 a
75.2 b
90.2 c
88.8 c
78.0
102.2
300.6 a
302.2 a
308.8 a
329.9 a
310.4
Table 3 : Sulphur analysis throughout the growing period of garlic on soil samples
collected in France and Spain in 2001. Data was collected in experimental plots without
any additional application of SO4. Data expressed in mg/kg dry soil.
Sulphur in mg / 25/08/00
kg dry soil
France
Spain
Total
217
226
Water soluble
9
27.1
14/05/01
France
189
5,6
20/06/01
Spain
234
9,5
France
156
8
6
Figures
ns
100
Alliin content in bulbs
(nmol/mg FW)
ns
ns
120
ns
ns
80
bc
ab
a
ns
60
40
20
104.3
PRI
MOR 79.7
MES
73.7
0
0S
78.0
2S
88.4
Sulphur fertilisation level
(in meq)
Variety
8S
91.2
Fig.1. Concentration of alliin in garlic bulbs harvested in the greenhouse in 2002.
Statistical mean difference within varieties were analysed by ANOVA and characterised
and expressed as significant with different letters, as determined by a Newman-Keuls Test
(p = 0.05). Non significant differences were indicated as ns.
Alliin content in
bulbs (nmol/mg)
120
106.8 ns
90.2 ns
100
81.2 a
80
60
109.7 ns
66.3 b
38.9 c
40
20
0
Spain
MES
France
MC
PRI
Variety
Fig.2. Mean alliin content (in nmol/mg) obtained in 2000 on field trials set in France and
Spain with Messidrôme (MES), Morado (MC) and Printanor (PRI). Alliin content was
measured in samples of garlic powder (n=3). Statistical mean difference analysed by
ANOVA was characterised per location and expressed as significant with different letters,
as determined by a Newman-Keuls Test (p = 0.05). Non significant differences were
indicated as ns.
7
Alliin content (nmol/mg FW)
0.25
0.20
0.15
S30
0.10
S60
0.05
0.00
22-24°C
27°C
Temperature
Fig. 3. Influence of the culture temperature in vitro on the alliin content of bulbs from the
variety Printanor (PRI) according to the sucrose level in the medium (30 and 60 g/l). Bulb
formation occurred under fluorescent light, after a cold induction period of 2 months.
Bars represent means ± S.E.
0.90 a
0.82 a
Alliin content in bulbs
(nmol/mg FW)
1.0
0.8
0.6
0.12 b
0.20 b
0.4
0.18 b
0.2
'
0.17 b
0.0
F+i
MOR
PRI
Variety
MES
F
Light condition
Fig. 4. Influence of the light spectrum in vitro on the alliin content of bulbs from varieties
Printanor (PRI), Morasol (MOR) and Messidrôme (MES). Bulb formation at 22-24°C
with 60 g/l sucrose, after a cold induction period of 2 months. Statistical mean difference
analysed by ANOVA was characterised and expressed as significant with a NewmanKeuls Test (p=0.05).
8