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G&H February 2003, Liverpool
WP3 / cultivation, P4
P4
: persons involved
• Odile Huchette, research engineer
• Christèle Bellamy, research technician
• Didier Perrin and Eric Vade, research assistants
Influence of environmental and genetic factors on
the alliin content of garlic bulbs
COOPD'OR R&D
G&H February 2003, Liverpool
WP3 / cultivation, P4
Year
1
Milestones
 Introduction of 4 genotypes in vitro for micropropagation (P4)
 Sulphur content of the Chinese garlic powder (P5).
 Field trial in 2 locations (P4 & P6). CSO analysis (P5) before and after
processing into powder (P4). Transfer to the Health partners.
2
 Differential display of clones exposed in vitro to various environmental factors
(T°C, light) and S nutrition treatments (P4). CSO content analysis (P5).
 Preliminary experiment in greenhouse (P4). Sampling of fresh plant material
during the cultivation treatments for CSO content analysis (P5).
 Second year field trial (P4 & P6). CSO analysis (P5) before and after dehydration
and processing into powder (P4). Transfer to the health partners.
3
 Differential display of clones exposed in vitro to various mineral nutrition
treatments (P4). CSO content analysis (P5).
 Greenhouse experiment in various S regimes (P4). Sampling of fresh plant
material during the cultivation treatments for CSO content analysis (P5).
(  completed in January 2003, or  in progress, or  delayed)








G&H February 2003, Liverpool
WP3 / cultivation, P4
Year
3 : what was done
– Greenhouse experiment in various S regimes (P4)
• Sampling of fresh material during the cultivation treatment for CSO analysis
( P5)
• Freeze drying of samples (P4) for total sulfur analysis (P2)
– Micro-propagation of 4 genotypes for new experiments in
vitro (P4)
• Experiment III (October 2002 => April 2003) : Differential display of clones
exposed in vitro to various light conditions (intensity + quality) and S
nutrition treatments (0 S in macro-nutrients, normal and excess)
• Experiment IV (January 2003 => July 2003) : Differential display of clones
exposed in vitro to various mineral nutrition treatments (S combined with N)
COOPD'OR R&D
G&H February 2003, Liverpool
WP3 / cultivation, P4
Greenhouse conditions 2001
Greenhouse conditions 2002
PRI
Plantation in boxes with washed river sand
Mineral nutrition with drop irrigation
Alliin in nmol/mg fresh matter
G&H February 2003, Liverpool
WP3 / cultivation, P4
Greenhouse
experiment 2001
45.0
40.0
35.0
30.0
25.0
20.0
15.0
10.0
5.0
0.0
MO
MES
PRI
Alliin concentration of various varieties of garlic bulbs
grown in greenhouse
COOPD'OR R&D
G&H February 2003, Liverpool
WP3 / cultivation, P4
Greenhouse
experiment 2002
 3 varieties : PRI, MOR, MES
 3 Sulphur nutrition treatments:
0 (in the macro-elements) = Treatment S2 meq (normal) = Treatment TS
8 meq = Treament S++
• Weekly observations :Growing of the leaves and bulbs
• Measurements at harvest :
- Weight of the bulbs
- alliin content (P5)
- total sulfur (P2) in progress
COOPD'OR R&D
G&H February 2003, Liverpool
WP3 / cultivation, P4
No
differences during the growing
season between the 3 treatments
PRINTANOR
16
MORASOL
16
14
Leaves S-
12
Leaves TS
12
10
Leaves S++
10
8
14
8
Bulb growing S-
6
6
Bulb growing TS
4
4
Bulb growing S++
2
2
0
0
5/3
25/3 14/4
4/5
24/5 13/6
dates
3/7
MESSIDRÔME
14
5/3
25/3 14/4
4/5
24/5 13/6
3/7
dates
12
10
01/03 : Plantation
17/06 : Harvest of MES
10-11/07 : Harvest of MOR
16-17/07 : Harvest of PRI
8
6
Bulb growing = diameter of
the bulb / diameter of the neck
4
2
0
5/3
25/3 14/4
4/5
dates
24/5 13/6
3/7
G&H February 2003, Liverpool
WP3 / cultivation, P4
Results at harvest
Alliin (in nmol/mg fresh matter)
Average weight of the bulbs (g)
 influence of the variety
MES_S++
MES_TS
MES_S-
MOR_S++
S++
MOR_TS
TS
MOR_S-
S-
PRI
PRI_S++
MOR
PRI_TS
MES
PRI_S-
140
120
100
80
60
40
20
0
35
30
25
20
15
10
5
0
 no significative influence of the S treatment, except for MES
COOPD'OR R&D
G&H February 2003, Liverpool
WP3 / cultivation, P4
Conclusion
of greenhouse
experiments
– Alliin contents in bulbs are 3 to 4 times higher in 2002
compared to 2001
– Printanor appeared in 2002 as the richest variety in
alliin, contrary to the previous results
– Garlic plants are not affected by an absence of sulfur in
the macro-nutrients
• absorption of S atmospheric to be confirmed in vitro in 2003
– Garlic plants are not significatively affected by the
sulfur nutrition alone, except for one variety tested
• influence of the nitrogen to be confirmed in vitro and in greenhouse in
2003
COOPD'OR R&D
G&H February 2003, Liverpool
WP3 / cultivation, P4
In vitro experiments 2001/02
• 4 genotypes exposed to various environmental factors...
–
–
–
–
quality of the light
sucrose concentration of the medium
temperature
cold induction period
• ... and various mineral nutritions
– sulfur
COOPD'OR R&D
G&H February 2003, Liverpool
WP3 / cultivation, P4
Main
results of in vitro experiments
Among all the conditions tested, the better to induce
more alliin in garlic bulbs :
•
•
•
•
Sucrose in the medium : 60g/l [0 - 60 - 120 g/l tested]
Cold induction period (2 to 3 months)
Environmental factor :
F+i light
Sulphur in the medium : 6 Meq SO3 [0 - 3 - 6 meq tested]
To be confirmed in 2003 :
•
•
•
•
Influence of the intensity of the light
Effect of higher dosis of sulphur (9 meq)
Effect of nitrogen combined to sulphur
Role of S atmospheric
COOPD'OR R&D
G&H February 2003, Liverpool
WP3 / cultivation, P4
Year
3
Year 4 : what is planed
Milestones
 Differential display of clones exposed in vitro to various mineral nutrition
treatments (P4). CSO content analysis (P5).
 Experiments III and IV

 Greenhouse experiment in various S regimes (P4). Sampling of fresh plant
material during the cultivation treatments for CSO content analysis (P5).

 Complementary experiment in greenhouse in year 4
4
 Greenhouse cultivation of garlic bulbs enriched in CSO (P4). Comparison
between extreme genotypes, low and high in S content (from the core collection )


 Greenhouse cultivation of bulbs coming from the experiment 2002
 Comparison between virus-free and virus-contaminated garlic for sulphur
composition CSO content (P4 & P5).
 Chemical analysis (P5) before and after processing into powder (P4). Transfer to
the Health partners (P4).
(  completed in January 2003, or  in progress, or  delayed)
COOPD'OR R&D

G&H February 2003, Liverpool
WP3 / cultivation, P4
In vitro experiment III-2003
3 varieties
: PRI
+ MES
+ MORl
3 mediums
(S- TS S++)
Gradient in SO4
0
3
(in macro-elements)
9
meq
22-24°C - P = 16 hrs - F
1 month
Cold induction 3°C - P = 10 hrs - F
2 months
Effect of various light
conditions (intensity +
quality) and S nutrition
22-24°C – P=16 hrs
F 950 lux
22-24°C – P=16 hrs 22-24°C – P=16 hrs
F+i - 950 lux
F 2000 lux
G&H February 2003, Liverpool
WP3 / cultivation, P4
In vitro experiment IV -2003
varieties: PRI
meq (in macro-elements) + MES
+ MORl
+ MORd
N 2N 3N
N/2 N 2N 3N9 9 medias
22-24°C - P = 16 hrs - F
1 month
Gradient in SO4 and nitrogen
0
3
9
SO4
Nitrogen
N 3N
Cold induction
3°C - P = 10 hrs - F
2 months
Effect of S nutrition
combined to N nutrition
22-24°C – P=16 hrs F+i - 950 lux
Sulfur
Nitrogen
variety
SN
3N
PRI
MORl
PRI
MORl
N
PRI
MORl
MORd
MES
TS
2N
PRI
MORl
MORd
MES
S++
3N
N/2
PRI
PRI
MORl
N
PRI
MORl
MORd
MES
2N
PRI
MORl
MORd
MES
3N
PRI
G&H February 2003, Liverpool
WP3 / cultivation, P4
In
vitro experiments 2003
 Observations :
- stage of the plant (1= growing / 2=beginning of bulbification
/ 3=maturation / 4=mature bulb)
- number of green leaves
- height of the leaves
 Measurements at harvest :
- weight, size and observation of the bulbs
- height of the leaves
- alliin content (P5)
COOPD'OR R&D
G&H February 2003, Liverpool
WP3 / cultivation, P4
In vitro experiment III-2003
 Observation of the 03 February
PRI
F - 22-24°C P = 16h
SLight conditions
Medium
Nb of leaves
Height of the
leaves in cm
TS
F 950 lux
S++
F 2000 lux
F+i 950 lux
S2.1
TS
3.2
S++
3.1
S1.8
TS
3.1
S++
3.1
S2.3
TS
3.3
S++
3.4
9.5
16.0
18.1
8.8
15.1
17.0
12.1
19.1
18.8
COOPD'OR R&D
G&H February 2003, Liverpool
WP3 / cultivation, P4
Greenhouse
(I) Effect of N combined to an excess of S
experiment 2003
(II) Cloves from experiment 2002
S++ 2N
STS
MOR 1
MES 1
PRI 2
MES 2
MOR 1
(12 pl.)
MOR 1
(12 pl.)
PRI 1
MOR 1
PRI 1
MOR 2
MOR 3
(12 pl.)
PRI
(12 pl.)
MES (12
pl. S- TS
and S++)
MOR 2
(12 pl.)
PRI 1
MES 1
MOR 1
MES 1
PRI
(6 pl.)
MOR 3
(12 pl.)
MOR 3
(12 pl.)
MOR 2
(12 pl.)
MES 2
MOR 2
PRI 2
MOR 2
PRI
(12 pl.)
MOR 2
(12 pl.)
S++
S++ N
S++ 3N
PRI 2
MOR 1
(12 pl.)
MES 2
3 varieties and 3 treatments :
-S++ N = excess of sulfur, N
-S++ 2N = excess of sulfur, 2xN
-S++ 3N = excess of sulfur, 3xN
COOPD'OR R&D
Measurements :
- growing of the leaves (I-II) and
bulbs (I)
- weight and CSO content of the
bulbs (P5) at harvest (I-II)
G&H February 2003, Liverpool
WP3 / cultivation, P4
Aliin per treatment
(in nmol/mg fresh matter)
Effect of virus contamination
on AlCSOs in garlic bulbs
Average weight of the bulbs (in g)
140
60
120
50
100
40
80
30
60
40
20
20
10
0
healthy
LYSV
OYDV
Mean bulb
weight (g)
Healthy
LYSV
OYDV
42,36
32,30
15,04
COOPD'OR R&D
0
healthy
Concentration
(nmol/mg)
Alliin
Allicin
61,32
79,19
110,46
11,87
17,92
10,04
LYSV
Mean content per
bulb (nmol)
Alliin
Allicin
2 597 212
2 557 549
1 661 088
502 780
578 853
151 047
OYDV
G&H February 2003, Liverpool
WP3 / cultivation, P4
Deliverables P4
• DP 2 
Chinese garlic powder to be analysed
• DP 6 
Garlic 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 34 
Chemical distinction (CSO) between garlic accessions when cultivated in
various sulphur regimes (greenhouse) and mineral nutrition regimes (in vitro)
G&H February 2003, Liverpool
WP3 / cultivation, P4
garlic powder available from
the experiment 2002 in greenhouse
2 varieties : Morasol, Printanor
3 treatments :
• S- : no sulphur in the macro-nutrients
• TS : 2 meq S in the fertilising solution
• S++ : 8 meq S in the fertilising solution
Variety
PRI
PRI
MOR
PRI
PRI
MOR
PRI
PRI
MOR
Treatment
SSSTS
TS
TS
S++
S++
S++
Repetition
1-3
2
1-2-3
1
2-3
1-2-3
1-2
3
1-2-3
COOPD'OR R&D
nb of bulbs
freeze-dried
2
1
4
1
2
4
2
1
4
Weight available (g)
observation
12.86
6.03
10.62
7.87
10.78
12.45
8.38
3.88
10.79
further grinding needed
further grinding needed
further grinding needed
-