1
GARLIC AND HEALTH PROJECT
EU identification number QLK1-CT-1999-00498
E-mail report February 1 - June 1 2003
(E-report 25)
2
General
1. Long time since we had contact via this e-mail report, however here we go again! The Liverpool
meeting is already in the past (February 17-21) and Rolf, Remi and I look upon a very fruitful meeting.
Thanks once again for organizing the meeting: Brian, Hamish, Meriel and Angela !!
2. The upcoming meeting is in Munich from September 29 – October 3 2003. The Monday and Friday
are considered as travelling days. You have already been contacted by the Munich group if I am not
mistaken.
3. The Munich meeting will be the starting point for the writing of three reports, namely the fifth annual
G&H scientific progress report, the overall consolidated scientific G&H report and the technological
implementation plan. In view of this I urge you to reserve considerable time in October-December for
the writing of these reports. In my perception we should have all reports finished by the end of
December and have a final farewell meeting in Berlin in January 2004, as the projects ends on
February 1 2004.
4. The projects ends on February 1 2004, so for you who do not yet know. It is really finished by this
date, so no budget will be available from the EU after this date.
5. The third annual scientific and financial report have been sent to the EU. The scientific report on
March 18 and the financial report on April 16. Until present I did not receive any reflections from the
Commission on our reports, most probably we will get comments on the reports in the second half of
this year.
6. The ISHS meeting in China:
It was a pity, although completely understandable, that the symposium was cancelled. However it
would have been a good opportunity to present our results on this meeting. The organizing committee
has posponed the symposium by one year. However this means that it will be held after our project is
finished. I have had contacts with Rosanna D‘Amario on the subject of reimbursement by the EU for
those in our project that participate in this meeting. She answered me that the matter had to be
discussed internally and that she would come back to me with more info in the second half of this
year.
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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
Deliverables:
DP 3: Garlic collection
DP 7: Chemical distinction (CSO), fertility and clonal identity
between garlic accessions of the collection.
M&D 2001:
Milestones:
Clonal identity screening in newly collected material
Maintenance (core) collection
Deliverables:
DP 10: Establishment of core collection and writing paper
DP 31: Maintenance core collection
M&D 2002:
Milestones:
DP 31: Maintenance of core collection and distribution of material
Deliverables:
None
M&D 2003:
Milestones:
Maintenance of core collection and distribution of material
done
done
done
done
done
done
done
done
done
E. Research
What has been done:
Garlic core collection was planted in November outside for cold treatment in order to obtain good
flowering and currently we see the young inflorescences developing.
What will be done:
A paper has been written on the establishment of the garlic core collection in joint cooperation with
Rina, Haim and Jacques and it has already been submitted (see dissemination).
Bottlenecks:
None
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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:
Axenic material available for transformation experiments:
Deliverables: No deliverables in the first year
done
done
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:
DP12a (added DP): paper on reliable regeneration of garlic
done (2002)
DP12b (original DP12): paper on reliable transformation of garlic
partly done (2002)
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:
Deliverables:
partly done
no deliverables planned
E. Research
What has been done:
A reliable transformation protocol was developed based on both GUS and GFP systems. Molecular
analyses of the transgenic plants indicated that T-DNA was stably integrated into garlic genome. The
garlic transformation procedure via the GFP system is summarised in Figure 1.
A construct was made in which the APS1 gene (ATP sulfurylase) from Arabidopsis thaliana was
fused with GFP (APS1-GFP and GFP-APS1). Young callus derived from root segments of cv.
Printanor was used in a new series of experiments to transform garlic with these two constructs. ATP
sulfurylase from shallot was also cloned and has been fully sequenced. Also this shallot ATP
sulfurylase gene was fused with GFP.
What will be done:
1
2
Finishing writing a manuscript on garlic transformation.
Regeneration of transgenic garlic plants overexpressing ATP sulfurylase in sulphur metabolic
pathway.
5
F. Bottlenecks
None
A
AA
B
BB
C
CC
D
DD
E
EE
FFF
G
F
FF
GG
GGG
Figure 1. Overview of garlic transformation procedure via GFP system. A and AA. Light and fluorescence
imagines (GFP2 filter) respectively, showing transient GFP expression in the cells at the surfaces of twomonth old callus derived from root segments of cv. Printanor; infection with AGL0 (pC1300intA-GFP) after
four days of co-cultivation. B and BB. Light and fluorescence imagines (GFP2 filter) respectively, showing
transient GFP expression in the cells at one large area of two-month old callus derived from root
segments of cv. Printanor; infection with AGL0 (pC1300intA-GFP) after four days of co-cultivation. C and
CC. Light and fluorescence imagines (GFP2 filter) respectively, showing transient GFP expression in the
cells at the surfaces of two-month old callus derived from root segments of cv. Messidrome; infection with
AGL0 (pC1300intA-GFP) after four days of co-cultivation. D and DD. Light and fluorescence imagines
(GFP2 filter) respectively, stable and uniform expression of GFP in hygromycin-resistant callus of cv.
Printanor after two months growth on selective medium. E and EE. Light and fluorescence imagines
(GFP3 filter) respectively, regenerated shoots of cv. Printanor. F, FF and FFF. Light and fluorescence
imagines (GFP2 and GFP3 filters) respectively, note that FF is a fluorescence imagine without the
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chlorophyll fluorescence blocking in young leaf parts of garlic plants. G, GG and GGG. Light and
fluorescence imagines (GFP2 and GFP3 filters) of non-transgenic (left) and transgenic garlic leaves (right)
from the greenhouse, respectively. Note that GG is a fluorescence imagine without the chlorophyll
fluorescence blocking and shows red fluorescence in non-transgenic cv. Printanor leaf (left) and greenish
yellow fluorescence in transgenic cv. Printanor leaf (right).
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;
PR activities: home page, brochure;
Annual report
Deliverables:
DC 1: first annual report
DC 2: PR activities: home page, brochure
M&D 2001:
Milestones:
Annual (third) and fourth project meeting
PR activities
Second annual scientific progress report
Deliverables:
DC 3: second annual progress report
M&D 2002:
Milestones:
Annual (fifth) and sixth project meeting
PR activities
Third annual scientific progress report
Deliverables:
DC 4: Mid term review
DC 5: Second annual progress report
M&D 2003:
Milestones:
Seventh and eight project meeting have taken place
PR activities
Final progress reports written (annual, TIP and consolidated)
Future collaborations will be developed
Deliverables:
done
done
done
done
done
done
done
done
done
done
done
done
done
done
partly done
partly done
to be done
to be done
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DC 6: Final progress reports
to be done
E. Activities
What has been done
1. The third annual G&H scientific and financial progress reports have been sent to the EU in March and
April 2003.
2. For the ISHS Allium symposium quite some work has been done, because we had a considerable
inpu. However the symposium was cancelled due uncertainty with respect to SARS. The planning is
that the symposium will be held in April next year. This is after our projects ends and concerning the
financial aspects I have had contacts with our EU scientific officer. As the matter is complicated she
will discuss it first internally and then contact us.
What will be done
1. The next meeting in Munich will be organized and the route map for the production of three G&H
reports (fifth scientific report, overall consolidated and technological implementation plan) will be
developed.
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
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P3:
University of Liverpool, 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.
Milestones and Deliverables of the fourth year
Deliverables
DP 23 Paper on Allium Biosynthesis
DP 24 Paper on Genes for key CSO synthesis enzymes
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Milestones
Hypothesis on limiting steps in alliin synthesis tested.
Heterologous expression of enzyme steps in alliin biosynthesis replaced by invitro expression
E Research
What has been done
The transformation of the tobacco cells to express the s-allyl cysteine synthase, cysteine
synthases and serine acetyl transferase was successful but expression of the enzymes did not occur
because of a silencing problem within the plant cell tissue cultures. This silencing related to the alc
promoter. As an alternative strategy the same enzyme proteins are being synthesized in vitro using the
pIVEX RTS 100 system from Roche Molecular Biochemicals. The advantages of this system is that
silencing is no longer a problem and the enzyme can be tested for activity without a complicated extraction
and purification procedure. Arabidopsis plants have also been transformed by dipping the flowering heads
in a suspension of Agrobacterium tumefasciens containing the separate genes for the above enzymes.
Seeds have been collected for plants transformed with the s-allylcysteine synthase gene and the other
transformants are in the pipeline. The seeds are being germinated to produce plants prior to testing for the
expression of the gene.
A new person Dr Jonathan Milne has been appointed to replace Dr Jill Hughes as the HPLC
specialist. He has run a series of garlic extracts and markers to acquaint himself with the garlic
components in preparation for conducting the enzyme assays. These assays will be conducted directly on
the enzymes produced in vitro by Angela Tregova.
What will be done
Enzyme assays are being conducted on crude garlic plant extract to provide experience of the
assay and to be able to conduct the assay on extracts from the transformed Arabidopsis at a later date. If
these extracts fail to show any activity the extract will have to be purified by column chromatography and
fraction collection.
The use of alternative substrates for the glutathione transferase is also being re-examined since
the preliminary experiments by Jill Hughes did not yield any positive leads.
Bottlenecks
The role of glutathione transferase as part of the mechanism of synthesis of alliin still 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, D. Perrin, E. Vade).
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D. Milestones & deliverables :
Summary Year 3 :
Differential display of clones exposed in vitro to various mineral nutrition treatments (normal and S-, N- or
Se-enriched nutrition) (P4). Greenhouse experiments with two genotypes, one low and one high in S
content (from the core collection), optimal growth conditions (temperature, daylength) and sub-optimal
sulphur nutrition for producing S-enriched garlic bulbs (P4). Sampling of fresh plant material during the
cultivation treatments for CSO content analysis (P4 & P5).
DP. 19

DP. 22
Delayed year
4
DP. 34
In progress
DP19. Garlic powder from bulbs (greenhouse grown) with distinct sulphur
contents for biological experiments (P4)
Paper on the interaction genotype x environment on the sulphur content of
garlic plants grown in vitro, in greenhouse and in field conditions (P4, P5, P6)
Chemical distinction (CSO) between garlic accessions when cultivated in
various S regimes (greenhouse) and mineral nutrition regimes (in vitro) (P4,
P5)
Year 4 :
Greenhouse cultivation of garlic bulbs enriched in CSO (P4). Comparison between extreme genotypes,
low and high in S content (from the core collection) and comparison between virus-free and viruscontaminated garlic for sulphur composition CSO content (P4 & P5). Chemical analysis (P5) before and
after processing into powder (P4). Transfer to the Health partners (P4). => no need for the Health partners
after discussion on the meeting in Liverpool.
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.
E. Research :
1. What has been done:
a) In vitro :

Experiment in vitro n° III : this experiment has been set up to test :
-
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-nutrients of the culture medium.
After 9 weeks under cold conditions (3°C – 10 hours day – White light), the plants have been
placed under the different light conditions for bulbification on the 8 January, and observations on
the growing of the plants (stage and number of green leaves) in the different treatments have
been planed every 10 days until the harvest. Measurements on the size of the plants have been
made every 20 days.
The different treatments tested are remembered in the table 1.
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Table 1: Treatments and varieties tested in the experiment in vitro III.
Light
F
F+i
F
F+i
9.50 W/m²
9.50 W/m²
20 W/m²
10 W/m²
T°C
T°C 22-24°C
T°C 22-24°C
T°C 22-24°C
T°C 25-27°C
Medium S-
PRI
PRI
PRI
-
Medium TS
PRI
PRI
PRI
PRI
MOR
MOR
MOR
MES
MES
MES
Medium S++ PRI
PRI
PRI
MOR
MOR
MOR
S- = 0 meq SO42- in macro-nutrients – TS = 3 meq SO42- - S++ = 9 meq SO42-
After 47 days of growing, differences could already be observed between different treatments for
Morasol and Messidrôme (Table 2). These differences of growing confirm a strong effect of the
addition of far-red light on bulbification, but no significant effect of an addition of sulphur in the
medium.
Table 2 : Observations after 47 days of growing for Morasol and Messidrôme (effect of an addition
of sulphur in the medium combined to different light conditions)
T°C
Light conditions
MOR
F9
F9
F 20
F 20
F+i
F+i
MES
F 9 F 20
F+i
Medium
Stage of growing *
Number of leaves
Size
of
the
plants
including green parts
(cm)
TS
1.9
2.3
18.7
TS
1.8
2.4
18.4
S++
2.0
2.4
17.0
TS
2.6
2.3
18.1
S++
2.5
2.2
18.5
TS
1.9
2.5
19.5
TS
3.0
1.2
17.5
S++
1.8
2.4
18.5
TS
1.7
2.4
18.6
* stage 1 = growing – 2 = beginning of bulbification – 3 = maturing
Comments :
- Plants from Morasol and Messidrôme placed under Fluorescent light conditions (9.5 and 20
W/m²) were still in vegetation, although plants placed under incandescent light were beginning
to make a bulb. This confirms the strong and positive influence of the quality of the light on the
bulbification, but no influence of the quantity of light could be observed during the growing
stages.
- No influence of the addition of sulphur in the medium was observed on the growing of the
plants, independently of the light conditions.
The same influence of the treatments could be observed for Printanor (Table 3). But an effect of a
lack of sulfur in the medium could also be confirmed for this variety, although it could not be
observed, or slightly, in greenhouse in 2002.
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Table 3 : Observations after 47 days of growing for Printanor (effect of sulphur combined to
different light and temperature conditions)
T°C
Light conditions
22-24°C
F 900 F 900 F 900 F
2000
Medium
STS
S++ SStage of growing *
1.8
1.9
2.1
2.3
Number of leaves
2.1
3.2
2.9
1.6
Size of the plants 8.7
19.0 19.1 7.9
including green parts
(cm)
F
2000
TS
1.8
3.0
19.4
F
2000
S++
1.8
2.8
19.2
F+i
F+i
F+i
25-27°C
F+i
S2.4
1.8
9.4
TS
2.3
3.4
19.6
S++
2.3
3.4
19.8
TS
2.1
3.3
17.4
* stage 1 = growing – 2 = beginning of bulbification – 3 = maturing
SPRINTANOR
TS
F+i conditions – 22-24°C
S++
03/02/03
Comments :
- Plants from Printanor placed under incandescent light also began to make a bulb before the
other light conditions, independently of the sulphur medium. No influence of the quantity of
light was observed during the growing stages.
- No influence of the addition of sulphur in the medium was observed on the growing of the
plants, although an effect of a lack of sulphur could be observed independently of the light
conditions. Plants on the S- medium were smaller and began to make a bulb before the
others. They didn’t look very healthy in general.
Harvests began with Messidrôme on the 31/03 and ended on the 09/05 with Printanor. The
following measurements were realised : weight, diameter and high of the bulb, diameter of the
neck, observation on the look of the bulb. After 3 days of drying at ambient temperature, bulbs
were sent to Jacques Auger and Ingrid Arnault (P5) for S compounds analysis.
These analysis are in progress but the first results obtained are presented below (Tables 4 and 5).
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Table 4 : Weights of the bulbs at harvest for Morasol and Messidrôme
T°C
Light conditions
Medium
Date of harvest
MOR
F9
F9
F 20
F 20
F+i
F+i
MES
F 9 F 20
TS
S++ TS
S++ TS
S++
TS
07/05 07/05 09/05 09/05 31/03 31/03 08/04
+08/0
4
well 93.8 90.5 87.5 85.7 100 100
100
F+i
TS
TS
31/03 31/03
+08/0
4
100
100
% of firm and
formed bulbs
Mean weight per bulb (g) 0.439 0.477 0.446 0.421 0.301 0.305 0.345 0.350 0.251
Diameter of the bulb / 7.9
11.0 12.0 10.0 7.1
8.7
15.9 13.1 12.6
diameter of the neck
Comments :
- Plants from Morasol and Messidrôme placed under incandescent light were mature more
rapidly and all the bulbs formed under this light condition were firm and normal. No positive
effect of the quantity of light could be observed.
- As winter variety, Messidrôme is making easily bulbs under all the light conditions.
- No influence of the enrichment of the medium with sulphur could be confirmed at this level for
these 2 varieties.
Table 5 : Weights of the bulbs at harvest for Printanor
T°C
Light conditions
Medium
Date of harvest
22-24°C
F 900 F 900 F 900 F
2000
STS
S++ S06/05 06/05 06/05 09/05
F
2000
TS
09/05
25-27°C
F
F+i
F+i
F+i
F+i
2000
S++ STS
S++ TS
09/05 22/04 08 + 08 + 09/05
22/04 22/04
0
50.0 96.7 100.0 21.4
% of firm and
well 54.2 13.3 8.3
29.2 0
formed bulbs
Mean weight per bulb 0.459 0.291 0.297 0.447 0.245 0.237 0.388 0.519 0.476 0.380
(g)
Diameter of the bulb / 3.3
2.2
1.9
2.6
2.0
2.0
4.4
12.8 10.7 2.2
diameter of the neck
Comments :
- Only plants from Printanor placed under incandescent light at 22-24°C on the media with
sulphur formed more than 90% of mature and normal bulbs. Under all other conditions and
although a lot of plants hadn’t formed mature bulbs , harvest occurred as the leaves had
dried.
- Under favourable conditions (incandescent light at 22-24°C), a lack of sulphur in the medium
affects the maturity of the plants and the bulbification in a negative way. Opposite
observations have been made in the other light conditions.

Experiment in vitro n° IV: this experiment has been set up at the beginning of the year to test
the effect of sulphur combined to nitrogen on the alliin content of garlic bulbs. 9 different
culture media have been tested with Printanor, as it was still difficult to produce enough plants
in the other genotypes (Morado, Morasol and Messidrôme) for the whole experiment. So
Morado, Morasol and Messidrôme have been used on selected media only .
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The plants have been placed under light conditions (fluorescent + incandescent light) on the 07
April after 9 weeks of cold conditions (3°C – 10 hours day – White light). The combinations of
media and varieties tested are presented in the Table 6.
Table 6 : Treatments and varieties tested in the experiment in vitro IV.
Light
F+i
950 lux
Medium S- / N
PRI - MORl
Medium S- / 3N
PRI - MORl
Medium TS / N
PRI – MORl – MORd - MES
Medium TS / 2N
PRI – MORl – MORd - MES
Medium TS / 3N
PRI
Medium S++ / N/2
PRI - MORl
Medium S++ / N
PRI – MORl – MORd - MES
Medium S++ / 2N
PRI – MORl – MORd - MES
Medium S++ / 3N
PRI
S- = 0 meq SO42- in macro-nutrients – TS = 3 meq SO42- - S++ = 9 meq SO42N = normal dosis of nitrogen in macro-nutrients
Observations on the growing of the plants (stage and number of green leaves) in the different
treatments have been realised on the 14 April, 24 April, 05 May, 19 May and 28 May, with
measurements of the size of the plants on the 24 April, 05 Mai and 19 May. These
observations will be realised every 10 days until the harvest, with measurements on the size of
the plants every 20 days.
The harvest is planed for the end of June, with the same measurements and analysis than the
Experiment III.
b) In greenhouse : 2 experiments have been set up at the beginning of February. Two main objectives
with these experiments :
1. To confirm the effect of a high level of sulphur combined to nitrogen (3 levels) on the
alliin content of garlic bulbs of Printanor, Morasol and Messidrôme
2. To observe the growing of bulbs providing from the experiment 2002 (bulbs enriched
in sulphur and not), and control their alliin content at the end of the growing season.
For sanitary reasons, all the bulbs have been grown in insect-proof conditions and treated against pests
and diseases.

Experiment 1: The garlic cloves of Printanor, Morasol and Messidrôme were supplied by
Véronique Chovelon (P8 - INRA Avignon) and planted in boxes with washed river sand in
greenhouse (12 cloves per box) on the 03 February. Greenhouse conditions are : natural
daylength at 20-18°C.
They have been exposed to 3 mineral nutrition treatments as following : high S content (8 meq
SO4 in macro-nutrients) combined with 3 levels of nitrogen (normal dosis, double dosis and triple
dosis). Two boxes per treatment have been planted for each variety. Plants have been supplied
15
with 50 ml to 100 ml of nutritive solution every day by drop irrigation (the dosis was following the
stage of the plant).
The plan of the experiment is presented below :
MOR 1
MES 1
PRI 1
S++ N2
PRI 2
MES 2
MOR 1
PRI 1
MOR 2
PRI 1
MES 1
MOR 1
MES 1
MES 2
MOR 2
PRI 2
MOR 2
S++ N1
S++
N3
PRI 2
MES 2
Treatments were grouped for practical reasons of supply .
Photo
18/03/03
Emergence was observed around the 14 February and the growing of the plants has been
observed every 10 days since the 25 February (number of green and died leaves and high of the
plants including green parts). The growing of the bulbs has been measured as soon as observed.
Harvests are actually in progress : Messidrôme was harvested on the 16 May, harvest of Morasol
is in progress, and Printanor is still growing. The end of the experiment is planed for the end of
June or the beginning of July.

Experiment 2: The garlic cloves of Printanor, Morasol and Messidrôme were coming from the
Experiment in greenhouse 2002 on the effect of the sulphur nutrition (rest of the bulbs not
used for analysis). They have been planted in boxes with washed river sand in greenhouse
16
(12 cloves per box) on the 07 February. Greenhouse conditions are : natural daylength at 2018°C.
Plants have been supplied with 50 ml to 100 ml of a classical fertilising solution by drop irrigation
every day (the dosis was following the stage of the plant).
Emergence was observed around the 18 February and the growing of the plants has been
observed every 10 days since the 28 February (number of green and died leaves and high of the
plants including green parts). The growing of the bulbs has been measured as soon as observed.
Photo
18/03/03
The plan of the experiment is presented below. It is indicating the number of cloves available per
treatments 2002 (S-, TS and S++) and per variety for plantation.
S-
MOR 1
MOR 1
(12 pl.)
(12 pl.)
MOR 2
MOR 3
PRI
(12 pl.)
(12 pl.)
PRI
MOR 3
MOR 3
MOR 2
(6 pl.)
(12 pl.)
(12 pl.)
(12 pl.)
TS
(12 pl.)
S++
(12 pl. MOR 2
from
S- (12 pl.)
TS
et
S++)
MES
PRI
MOR 1
(12 pl.)
(12 pl.)
Harvests are actually in progress : Messidrôme was harvested on the 16 May, harvest of Morasol
is in progress, and Printanor is still growing. The end of the experiment is planed for the end of
June or the beginning of July.
17
2. What will be done:
a)
In vitro :
 Experiment IV in progress :
-
b)
Observations on the growing and bulbification of the plants till the harvest
Harvest at maturity (estimated at the end of June) and measurements on the bulbs harvested
Bulbs will be sent to P5 for S compounds analysis after 3 days of drying
In greenhouse : Harvests are in progress. Measurements will be made on the bulbs after 7 days of
drying (weight and diameter). Then they will be sent to P5 for S compounds analysis.
3. Bottlenecks
Plant material should be sent to P5 before the end of June to have the analysis made and the
results back before October or November. As the harvest is following the maturity of the plants, we
are not sure actually to be able to respect this planning.
P5: Universite F. Rabelais, Tours, France
The group is involved in three workpackages (WP1, WP3 and WP6) and we participate in WP5 and WP2.
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 will be done:
No analysis programmed.
A- WP2 Breeding systems
1- Objectives:
Screening CSO of garlic .
18
2- Persons involved:
Jacques Auger, Ingrid Arnault, Nicole Mandon.
3- Milestones & deliverables of the second year:
No deliverable
4- Research:
What will be done:
No bulb received from embryogenesis multiplication.
B- WP3: Cultivation
1- Objectives:
Study effects of environmental factors on CSO content
2- Persons involved:
Jacques Auger, Ingrid Arnault & Nicole Mandon
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 done:
We received from Odile Huchette - P4 (in may) bulbs from in vitro experiment III.
What will be done:.
ALCSO analysis of bulbs from in vitro experiment III.
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:
19
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, 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 have been cultivated
in presence of hygromicine. 30 weeks after starting the selection process differences among treated
explants and controls are clear. A great number of transgenic cell lines have been obtained. A number of
shoots have been regenerated from the selected embriogenic suspensions via indirect organogenesis.
The vast majority of these shoots showed gus expression (figure 1). However , many regenerants
showed abnormalities (figure 2.). We have not observed any evidence of somatic embryogenesis
following the protocol provided by P8.
20
Figure 1
Figure 2
2.What will be done.
We have consulted the problem about somatic embryogenesis with P8 . As a solution, they have provided
us a new protocol, significatively different than the former, so it means we have to go 8 months back. At
the same time we will start the new protocol, we will try to improve the regeneration levels of the selected
cell lines.
3. Bottlenecks.
The somatic embryogenesis protocol initially provided by P8 does not work in our conditions.
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.
21
P7: Volcani, Bet Dagan, Israel and P10: The Hebrew
University of Jerusalem, Israel
The research group is involved in WP1: Genetic Resources and WP2: Breeding Systems
I. Workpackage: WP1 Genetic Resources
II. Objectives:
1. Construction and maintenance of garlic core collection;
2. Screening for fertility in garlic;
3. Collection of a large number of garlic accessions and close wild relatives, in order to expand the
current available short- and long-day genepools.
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 to G&H partners.
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, seed production, seed viability and germinability.
3. Production of self-pollinated and cross-pollinated populations within and between selected clones.
Deliverables:
Scientific publication on environmental control of garlic floral development
V. Research
No info received
22
GENERAL LIST OF DELIVERABLES:
Deliverable
Deliverable title
DP. 3
Collected bulbs for screening for CSO, 9
fertility and clonal identity (fingerprinting).
DP. 4
Paper on morphological and physiological 12
aspects of floral/topset initiation and
development
Chemical distinction (CSO), fertility and 12
clonal identity between garlic accessions of
the collection
DP. 7
DP. 10
DP. 11
DP. 20
DP. 25
DP. 26
DP. 31
DP. 32
DP. 37
Delivery
date
Establishment of the G&H garlic core
collection and writing a paper
Paper on environmental regulation of flower
differentiation and flowering process
Paper on environmental control of garlic
floral development
Seed and plant populations
Paper on environmental control of garlic
floral development and forcing
Maintenance and distribution of garlic
clones for further use in the project
21
Paper on environmental regulation of flower
differentiation and flowering process
Blueprints for fertility restoration. F1 seeds
for biochemical and molecular studies
Completion
Participant
N°
P1, P5, P7, +
P10
(According to
original
program)
P7, P10
Published
P1, P5, P7
+
(According to
original
program)
Done
22
P1, P5, P7,
P10
P7, P10
34
P7, P10
In progress
44
48
P7, P10
P7, P10
In progress
In progress
21
P1, P10
36
P7
+
(According to
original
program)
In progress
48
P7
In progress
In progress
P8: CIRAD Montpellier, France
Workpackage n°: 2
Breeding systems
OBJECTIVES
Embryogenesis and characterisation:
A mass propagation protocol will be developed (by P8) based on the production of embryogenic cell
suspension cultures from somatic embryogenic calluses. This tool will be developed on four varieties
which represent different physiological groups in garlic, and should respect the genetic integrity of these
genotypes, in comparison with the original material vegetatively multiplied in field.
23
Persons involved :
FEREOL Leonidas, CAUSSE Sandrine, ROUX-CUVELIER Michel, COTE François, HUGON
Rémy, KAHANE Rémi.
Milestones and deliverables of first, second, third and fourth year
Year 1 (2000):
√ : achieved; ‫ ٱ‬: partly achieved; ● :will be done
1) production of callus with embryogenic tissue and culture from var. Rouge Reunion
2) Provision by P8 of 200 in vitro plantlets from embryogenic calli Rouge de la Réunion
R(E-cal).
3) provision by P8 of 200 in vitro plantlets from embryogenic cell suspensions Rouge de la
Réunion R(ECS)
4) production of callus with embryogenic tissue from var. Messidrome and Morado de
Cuenca.
5) initiation of cell suspension from embryogenic callus of these varieties.
√
√
√
√
√
6) Production and germination of somatic emryos from embryogenic calli (E-cal) and cell √
suspension (ECS) of these varieties..
B. YEAR 2 (2001)
√ : achieved; ‫ ٱ‬: partly achieved; ● :will be done
1) Genetic characterisation of plants, from embryogenesis « Rouge Reunion », by : √
cytometry (P8), finger printing (P1), dry matter and sulphur content (P5), morphophysiology (P9)
2) Provision of 200 in vitro plants from embryogenic calli (ecal) derived embryos of √
Messidrome (MES ecal) and 200 Morasol (Mol ecal)
3) Provision of 200 in vitro plants from cell suspensions (ecs) derived embryos of 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
5) production of callus with embryogenic tissue from var. Printanor
6) initiation of cell suspension cultures from embryogenic callus of this variety.
√
√
7) DP 5 Paper on Evidence of a somatic embryogenesis process and plant √
regeneration and acclimatisation in garlic (Allium sativum L.)
L. FEREOL*, V. CHOVELON**, S. CAUSSE*, N. MICHAUX-FERRIERE* and R. KAHANE*
Plant Cell Report (2002 ) 21: 197-203
24
C. YEAR 3 (2002):
√ : achieved; ‫ ٱ‬: partly achieved; ● :will be done
1) Provision of 200 in vitro plants from embryogenic calli (ecal) of Printanor (PRI ecal) √
(P8)
2) Provision of 200 in vitro plants from cell suspensions (ecs) of Pintanor (PRI ecs) (P8) √
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 (P8, P9)
4) Field evaluation , 200 plants R (E-cal), 200 plants R (Ecs), (P8, P9)
√
5) In vitro bulbing studies (P8, P9, P4)
6) Improvement of the embryogenesis procedure (P8)
7) Provision of cell suspension cultures of var. Messidrome and Morasol to partners P1
and P6 (P8)
8) DP 13 Paper on Establishment of garlic (Allium sativum L.) cell suspensions and
plant regeneration
L. FEREOL*, V. CHOVELON**, S. CAUSSE*, D. TRIAIRE, N. MICHAUX-FERRIERE* and R.
KAHANE*
√
√
√
√
√
D. Year 4 (2003)
√ : achieved; ‫ ٱ‬: partly achieved; ● :will be done
1)
Bulb mass production from embryogenesis and further development of of these √
bulblets into bulbs
2) Continue development of the most appropriate procedure to promote somatic √
embryogenesis
3) Histological studies continue to follow and understand the different stages of √
somatic embryogenesis in Allium sativum.
4) Maintenance and in vitro multiplication of embryogenic calli and cell suspension.
√
5) DP 21 Paper on Embryogenic cell suspension cultures of garlic (Allium sativum L.) as
method for mass propagation and convenient material for genetic improvement
L. FEREOL*1, V. CHOVELON2, S. CAUSSE1 and R. KAHANE1E of
(in progress)
What has been done
Var. Rouge de la Réunion
- production of embryos from embryogenic calli on semi-solid medium (ecal) and from cell suspension
(ecs).
- Germination of these embryos.
25
- Provision to P9, of plants from these embryos (ecal and ecs), for further in vitro development,
bulbing, acclimation 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 if necessary.
- Genetic characterisation, by flow cytometry, of 100 plants from E-cal and 100 from E-cs.
- Field evaluation, plants R (E-cal), P8
- Field evaluation , plants R (E-cs), P8
Var. Messidrome and Morasol
- Callus induction on mars 2000
- Induction of callus and embryogenic tissues
- Induction of embryos from embryogenic calli
- Germination of these embryos
- Maintenance and in vitro multiplication of embryogenic tissue
- Choosing the most appropriate procedure to promote embryogenic calli and maintenance of
embryogenesis
- Improvement of the embryogenic procedure. Improving the mean number of somatic embryos per
150 mg fresh weight callus (30 and 70 respectively for Messidrome and Morasol)
- Studies on the procedure to initiate friable embryogenic calli of these cultivars
- Reliable protocol for providing of embryogenic friable callus, convenient to initiate cell suspension
- Studies on the procedure to initiate cell suspension from friable embryogenic calli of these cultivars
- Provision to P6 of garlic embryogenic calli
- Communication to P1 and P6 of the procedure for garlic somatic embryogenesis
- Repetition of the experiments concerning initiation of embryogenic callus
- Histological studies: demonstration of unicellular origin of the somatic embryos
- Initiation of cell suspension from embryogenic callus
- Provision to P9 of some plants from embryos (E-cal and Ecs) for further in vitro development,
bulbing, acclimation and field evaluation
- In vitro development by P8 of one part of these plants issued from somatic embryogenesis via calli
and cell suspensions
- Provision of embryogenic cell suspension cultures of Morasol to P6, Morasol and Messidrome to P1
- Communication to P6 and P1 of the procedure for maintaining cell suspension cultures and for
regeneration after plating
- Genetic characterisation, by flow cytometry, of Moraso E-cal, Morasol E-cs, Messidrome E-cal and
Messidrome E-cs
Var. Printanor
- Callus induction
- Induction of callus and embryogenic tissues
- 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
- Induction of embryos from cell suspensions
- Germination of these embryos
- 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, bulbing,
acclimation and field evaluation
- Genetic characterisation, by flow cytometry, of Printanor (E-cal and Ecs)
26
What will be done
var. Messidrôme, Morasol and Printanor
- Maintenance and in vitro multiplication of embryogenic calli and cell suspension culture.
BOTTLENECKS, DELAYS
Milestones:
All the other milestones are achieved
Deliverables:
DP 21 : paper will be submitted first semester 2003
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 is shared
into 3 sites (Avignon, Dijon and Montpellier, France).
Research ACTIVITIES
The task of P8 within the project aims at the development of a mass propagation method relying on in
vitro regenerated plants from embryogenic calluses (E-cal) and from cell suspension (ECS) of four
varieties :
Rouge de la Réunion , 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: “Printanor” this fourth variety has been taken into consideration for adaptation of the protocol to
this cultivar
Callus production
A low level of 2,4-D increased the percentage of garlic explants producing callus (Tab. 1) and confirmed
previous report (Barandiaran, 1999a). A good compromise in callus production between quantity and
quality was obtained with 0.5 mg l-1 2,4-D from both types young leaf or root explants.
Table 1. Effect of various 2,4-D concentrations in the culture medium CIM2 on callus production
and on induction of embryogenic tissue (n=40)
2,4-D
concentration
Variety
%
production
callus %
callus
embryogenic
tissue
0.3
Rouge Réunion
Messidrome
Morasol
Printanor
86
90
97
96
24
32
44
51
0.5
Rouge Réunion
Messidrome
93
95
35
39
with
27
Morasol
Printanor
95
98
52
65
1
Rouge Réunion
Messidrome
Morasol
Printanor
73
68
79
88
19
18
29
36
1.5
Rouge Réunion
Messidrome
Morasol
Printanor
55
40
62
76
15
7
18
27
Embryo induction
After two months on culture medium EIM, up to 92% of the embryogenic callus formed globular somatic
embryos (Tab. 2). They were observed from different row of leaf. The best combination of growth
regulators for both embryogenic events and embryo number were 2,4-D/Kinetin (0.1/0.3 or 0.1/0.5 mg l-1).
Calllus of about 150 mg fresh weight developed a mean number of 33-68 globular embryos, up to 40% of
which converted into plantlets (Tab. 2).
Table 2. Embryo production and conversion
Variety
Rouge
Réunion
Messidrome
Morasol
Printanor
Percentage
of
embryogenic callus
developing
embryos
82
Mean
number
of Mean number of embryos
globular embryos per converted into plantlets per
150 mg fresh weight 150 mg fresh weight callus
callus
37
16
77
88
92
33
72
68
18
32
38
Cell suspension culture
Suspension culture from friable embryogenic callus
The establishment of cell suspension cultures from friable callus required approximately 4 months. A 2fold increase of PCV was observed every two weeks . The embryogenic cells observed in these
suspension cultures from embryogenic tissue were round, small and with dense cytoplasm. No oxidation
or browning was observed even after one year sub-culturing.
Initiation of the cell suspension cultures
- Effect of the concentration of callus inoculated in the liquid medium (Fig. 1)
The PCV rate decreased from 0.33 to 0.17 as the callus concentration increased from 1 to 6%. The
lowest concentration produced the highest PCV rate, and the lowest regeneration capacity. This capacity
increased as the callus concentration increased from 1 to 5%, and then decreased for 6%. For the low
concentrations, the cell clusters had more space and nutrients, conditions more convenient for cell
multiplication, but less in favour of cell differentiation. A 5% callus concentration induced the lowest PCV
rate and the highest regeneration capacity (Fig. 1).
28
Fig. 1 Effect of callus inoculation concentration on pcv rate and
1000
regeneration capacity
0,5
900
0,4
800
0,4
700
0,3
600
0,3
500
0,2
400
0,2
300
0,1
200
0,1
100
0,0
Number of Embryos / ml
PCV
PCV rate (pcv obtained /
initial callus concentration)
0,5
0
1%
2%
3%
4%
5%
6%
Initial callus concentration
- Effect of the initial cell density
The initial cell density significantly affected the PCV rate of the cell suspension cultures (Fig. 2). For
identical culture periods, increasing the cell density from 1% to 8% decreased the PCV rate. The optimal
growth phase was reached within 50 days with an initial cell density of 1 to 3%. From 4 to 8%, the optimal
growth phase was reached within 30 days. Low cell densities were best for mass cell production.
Nevertheless, the induction of somatic embryo was the highest for 3-4% cell density (Fig. 2). Such an
initial density was particularly suitable for long term suspension cultures, because the optimal PCV rate
was delayed 30 days after culture starting and the regeneration potential was better(Fig. 2).
There may be several possible explanations for the observed effects of low initial cell densities of subcultures. One possible reason is that the cultured cells altered the medium in a way that allows cell
differentiation. Below the minimum density there are too few cells to affect this alteration (Ammirato, 1983;
Hari, 1980).
29
25
2500
20
2000
15
1500
10
1000
5
500
0
Number of embryos / ml of PCV
PCV rate
Fig, 2 Effet of initial cell densities (after 60 days of culture)
0
1%
2%
3%
4%
5%
8%
Initial cell densities
- Effect of a periodic medium refreshment (Fig. 3)
Renewal medium period significantly affected the PCV rate of cell suspension cultures. When comparing
the refreshing intervals over a culture period of 42 days, the PCV rated highest for 14 days (Fig. 3).
Nevertheless concerning this interval period (14 days), we observed a reduction of growth rate after one
month of culture duration despite renewal of the medium. We would recommend medium refreshment
periods of 14 days, and 28 days sub-culture periods. Moreover, this period of medium refreshment lead to
the highest regeneration capacity (Fig. 3).
30
18
1800
16
1600
14
1400
12
1200
10
1000
8
800
6
600
4
400
2
200
0
Nomber of embryos / ml of PCV
PCV rate
Fig. 3 Effet of interval period medium refresment
0
7 jours
14 jours
21 jours
28 jours
interval period medium refreshment
genetic characterisation of somatic embryos derived plantlets, flow cytometry
Flow cytometry is an easy and powerful technique for genome size evaluation. It is much more convenient
than chromosome count for routine ploidy analysis because it is very quick and it can be used with any
kind of plant tissue, even callus or cell suspension. We have tested the ploidy level of the final products of
somatic embryogenesis derived from callus or from cell suspension, by analysing 100 acclimatized
plantlets from E-cal and from E-cs of each variety (Tab. 5, Fig. 1). Some plantlets with abnormal level of
ploidy were detected. About 4% of tetraploids were detected by flow cytometry. Whatever this percentage
is not high, it reveals a potential site of abnormalities. However, the ploidy level has to be checked again
on the same plants along the years to acertain the stability of these polyploidizations, and to observe the
morphological or biochemical consequences.
Such a spontaneous polyploidization discovered in plantlets derived from somatic embryogenesis might
be a serious drawback for the method as mass propagation of garlic. In order to established a true-to-type
in vitro process of garlic mass propagation, we will check the ploidy level at each step of embryo
regeneration.
31
Table 3. Genetic characterisation, by flow cytometry, of plants from ecal and from ecs of
Messidrome and Morasol
Type of sample
MESSIDROME
- control
- E-cal
- E-cs
MORASOL
- control
- E-cal
- Ecs
PRINTANOR
- control
- E-cal
- Ecs
Ploïdy level
2X
4X
Total
12 plants
102
68
0
10
1
12
112
69
16
100
100
0
3
4
16
103
104
1
90
97
0
10
3
1
100
100
Flow cytometric histogram
Of diploid and tetraploid
Somatic embryos
4X
2X
2X
+
4X
Figure 4. Flow cytometric histogram of embrygenic derived and control plants (multiplied by
axillary bud culture)
In vitro bulbing studies
Set up of a common experiment (P4, P8, P9) for establishing a procedure to promote bulb production in
vitro from somatic embryo plants.
32
Treatments concerned:
. cold pre-treatment
. light spectrum quality
. daylength
. sucrose concentration in the medium
Collection of the records every two weeks
P9: INRA Avignon- Montfavet, France
INRA station de Pathologie végétale, domaine St Maurice, BP 4, 84143 Montfavet cedex, France.
Workpackage 2: Breeding system
Objectives:
1- Embryogenesis, genetic characterisation:
A mass propagation protocol is developed by P8, based on the production of embryogenic cell
suspensions from somatic embryogenic callus. This tool is developed on three varieties from different
garlic physiological groups, and should respect the genetic integrity of these genotypes. P9 is involved in
the embryogenesis program to assure cultivation and evaluation (morphological and physiological status)
of regenerated plants produced by embryogenesis, to control their integrity and conformity, in comparison
with the original material multiplied in field.
2- Sanitary aspects:
P9 brings competencies for indexing viruses, for regenerating selected garlic clones and for producing
virus-free plants in insect-proof conditions for others partners (for biochemical and biological studies).
Persons involved:
V. Chovelon (research scientist), JP Leroux (research technician), M. Ricaud (field technician)
Milestones and Delivrables:
3
1- Embryogenesis : Genetic characterisation of plants from embryogenesis “Rouge done
2002 Reunion” : first field evaluation (P8), morpho-physiology (P8)
2- Embryogenesis: Genetic characterisation of plant from embryogenesis (MES-cal,
MOL-cal, MOL-ecs): first field evaluation (P9), flow cytometry (P8), sulphur content
(P5)
3- Embryogenesis: first in vitro bulbification studie (P8, P9, P4)
4- Embryogenesis : Regeneration and acclimatisation of 200 vitroplants Messi-ecs ;
200 vitroplants Printanor from embryogenic callus (PRI-ecal) and 200 vitroplants
Printanor from cell suspensions (PRI-ecs)
1- Sanitary aspects: Production of virus-free bulbs for other partners (3 varieties; 300
plants/variety)
2- Sanitary aspects: Field multiplication of virus-free plants obtained from 2 selected
clones (1), (2)
3- Sanitary aspects: Acclimatisation of virus-free plants obtained from 2 selected
clones (3), (4)
done
done
done
done
done
done
33
4
1- Embryogenesis : Genetic characterisation of plants from embryogenesis “Rouge partly done
2003 Reunion”: second field evaluation (P8), morpho-physiology (P8), dry matter and
sulphur content (P5)
2- Embryogenesis: Genetic characterisation of plant from embryogenesis :
- MES-cal, MOL-cal, MOL-ecs: second field evaluation (P9), flow cytometry (P8),
sulphur content (P5)
- Messi-ecs, Print-cal and Print-ecs: first field evaluation (P9), flow cytometry (P8),
sulphur content (P5)
3- Embryogenesis: second in vitro bulbification studie (P8, P9)
1- Sanitary aspects: Production of virus-free bulbs for other partners (3 varieties; 300
plants/variety)
2- Sanitary aspects: Field multiplication of virus-free plants obtained from selected
clones (3), (4)
N°
DP1
DP5
DP13
DP21
Deliverable title
Previous
delivery date
virus-free bulbs materiel for introduction in vitro
1
Paper on Evidence of a somatic embryogensis process and plant 12
regeneration and acclimatization in garlic (Allium sativum L.) L.
FEREOL*, V. CHOVELON**, S. CAUSSE*, N. MICHAUXFERRIERE*, R. KAHANE*. Plant Cell Report (2002) 21:197-203.
paper on Establishment of garlic (Allium sativum L.) cell 24
suspensions and plant regeneration. L. FEREOL*, V. CHOVELON**,
S. CAUSSE*, D. TRIAIRE, N. MICHAUX-FERRIERE*, R. KAHANE*.
paper on Embryogenic cell suspension cultures of garlic (Allium 36
sativum L.) as method for mass propagation and convenient material
for genetic improvment. . L. FEREOL*, V. CHOVELON**, S.
CAUSSE*, R. KAHANE*
partly done
partly done
partly done
partly done
State
done
Done
(published)
Partly done
Partly done
Research
What has been done:
EMBRYOGENESIS
1- Rouge Reunion: plants issued from embryogenic calli and cell suspensions
Plants were cultivated under greenhouse, and transferred under tunnel (March to June 01):
- AFLP analyses were realised (by P1)
- The ploïdy level was checked by flow cytometry :all the plants were diploids (P8).
- Normal phenotypic notations were observed during the culture; with however less vigorous plants and
bulbs obtained from cell suspensions.
- bulbs from callus (565) and bulbs from cell suspensions (272) were sent to the Reunion Island for
field evaluation (P8). All the material planted was lost because of a failure in the temperature
regulation of the greenhouse during the culture. Supplementary plants were sent in June 2002 to the
Reunion Island (267 bulbs obtained from cell suspensions, 36 bulbs from callus and 78 bulbs
vegetatively multiplied) and a new plantation was realised in July 02.
- First field evaluation: different notations 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 on bulbs after harvest (no info received from Reunion Island).
- Second field evaluation: (no info received received from Reunion Island).
34
2- Morasol, Messidrôme and Printanor plants issued from callus or cell suspensions
a- First field evaluation:
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 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 but no particular
difference was observed between diploïd and tetraploïd plants.
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:
- 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 and there proportions are reversed.
- Alliin concentrations are quasi similar on the different bulbs produced by embryogenesis or
vegetatively multiplied, with a little superiority on Messidrôme-cal (57) and Morasol vegetatively
multiplied (61).
- but Alliin proportions are higher on bulbs vegetatively multiplied (more than 50%) owing to the
fact that dipeptides are lower, whereas it is only at 35% in bulbs from embryogenesis.
- 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 in
2003 on bulbs with similar weight.
Printanor plants issued from callus and from cell suspensions were in vitro obtained.
- 220 Printanor-cal plants were acclimatised, cultured in pot under greenhouse and then transferred in
field under an insect-proof tunnel in March 03. Notations of plants in vegetation (plant development,
vigour, height, leaves colour) are realised until harvest in July 03.
- 271 Printanor-ecs plants were in vitro obtained in August 2002 (in vitro bulb essay 2002 results: 83
bulbs obtained, and 188 no bulbified plants): plants were transferred in field in March 2003, and
notations in vegetation (plant development, vigour, height, leaves colour) are realised until harvest in
July 03.
Messidrôme plants issued from cell suspensions were in vitro obtained.
- 93 Messidrôme-ecs plants were in vitro obtained in August 2002 (in vitro bulb essay 2002 results: 60
bulbs obtained, and 33 no bulbified plants): plants were transferred in field in March 2003, and
notations in vegetation (plant development, vigour, height, leaves colour) are realised until harvest in
July 03.
b- Second field evaluation of Morasol and Messidrôme plants issued from callus and Morasol plants
issued from cell suspensions.
All the bulbs obtained (from diploïd and tetraploïd plants) were planted under tunnel in November 02, after
clove homogeneous calibration, for a second field evaluation. Notations of plants in vegetation (plant
development, vigour, height, leaves colour) are realised until harvest in July 03
c- In vitro bulb production of garlic plants issued from cell suspension
Two essays were realised in 2002/2003 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.
35
- first experimentation (February to September 2002)
P9, P8 and P4 were involved in this essay. Three varieties were tested (Morasol, Printanor and
Messidrôme) on plants issued from cell suspensions. Different in vitro factors were tested: saccharose
concentration in the culture medium (30g/l or 60g/l); cold treatment (5 weeks at 5°C); quality of light (white
light or additional far-red light); photoperiod (12 or 16h). 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). The influence of the
different parameters on bulb percentage was separately analysed at the end of the essay (2/09/02) :
-
-
-
-
the different parameters did not have a particular influence on bulb maturation percentage 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).
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)
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
In these three 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 but it probably link to a too short cold treatment time. The
observed Khi2 was inferior to theoretical Khi2, we accept the hypothesis Ho “the factor temperature do
not influence the bulbification”.
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, these 3 factors are
important to increase bulb formation (50% bulbs obtained with these 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.
For each culture condition and each variety, when it was possible, 5 bulbs were sent to P5 for alliine and
dipeptides analyses by HPLC method (P5):
- on Morasol bulbs, alliin concentration was quasi similar in the different culture conditions, and high
dipeptids concentrations were obtained.
- on Printanor bulbs, alliin concentration was more important (up to 2 nmol/mg) and dipeptidss
concentrations were very heterogeneous and sometimes reversed.
The bulbs were planted in pots in October 2002 and transferred in field under insect-proof tunnel in March
03. Germination rate, notations in vegetation and after harvest are released.
-
Second experimentation (December 2002 to June 2003)
P9 and P8 are involved in this essay. Two varieties are tested (Morasol and Printanor) on plants issued
from cell suspensions. According to the previous results, only two in vitro factors are tested: cold
treatment (5 or 10 weeks at 5°C) and quality of light (white light or additional far-red light).
Saccharose concentration in the culture medium is 60g/l and photoperiod 16h.
36
Notations are realised each 15 days (plant development, bulb induction and bulb maturation). Harvested
bulbs are measured, weighted and described (colour, aspect, shape) and will be sent to P5 for Sulphur
analyses. The influence of the different parameters on in vitro bulbification will be analysed at the end of
the essay.
SANITARY ASPECTS
1- production of virus-free bulbs for other partners (300 bulbs/variety).
- Messidrôme (INRA, 1973), temperate group 3
- Morasol (INRA/TOPsemences, 1994), temperate group 1
- Printanor (INRA, 1983) temperate group 2
For each variety, 300 plants are cultured in field under an insect-proof tunnel to assure a new production
of virus-free bulbs:
- Messidrôme
300 plants (November 02 to June 03)
- Printanor
300 plants (December 02 to July 03)
- Morasol 300 plants (December 02 to July 03)
Notations in vegetation are made regularly during the culture (plant development, absence of viruses
symptoms).
2- Virus elimination by meristem culture on garlic clones (3) and (4)
In 2001, any new clones were selected by P7 from the core collection for meristem regeneration, 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, and transferred in field in March 03.
What will be done:
1- Rouge Reunion, Morasol, Messidrôme and Printanor plants issued from callus or cell
suspensions (1st and 2nd field evaluations):
- notations on bulbs after harvest (bulbs shape and weight) in July-August 03.
- Flow cytometry (100 plants/variety/origin) by P8 (May-June 03)
- Sulphur contents (5 bulbs/variety/origin) by P5 (June-July 03).
3-
In vitro bulb production of garlic plants issued from cell suspension (2nd experimentation):
harvest and notation of Printanor bulbs obtained (June 03)
Sulphur analyses (Morasol and Printanor bulbs)
Influence analyses of the two tested parameters (cold and light quality)
4-
production of virus-free bulbs (Messidrôme, Printanor and Morasol)
bulbs harvest (June-July 03)
storage at room temperature (August 03)
sending to the different partners (September-October 03).
5- Virus elimination by meristem culture on garlic clones (3) and (4)
-
Virological control by Elisa tests (June 03)
bulbs harvest (July 03).
Boottlenecks
None.
37
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
38
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
(16 person months)
Duties: genotoxicity
Dr. Raymond Bergès
(6 person months)
Duties: Hepatocarcinogenesis
Dr. Caroline Teyssier
(16 person months)
Duties: Metabolism and bioavailibility
of organosulfur compounds
Post-doctoral fellows (to be hired)
(12 person months)
Duties: Carcinogen metabolizing
genotoxicity and metabolism
enzymes,
Research technicians
Marie-France Pinnert
(6 person months)
Duties: Immunohistological methods
for preneoplasic foci
Patrick Tassin
(6 person months)
Duties: Animal care
Marie-France Vernevaut
(12 person months)
Duties: Enzymes assays and
Immunoblots
Christine Belloir
(16 person months)
Duties: genotoxicity tests
Joëlle Chevalier
(16 person months)
Duties: Metabolism of organosulphur 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
39
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
Year4
Investigation of the antiinitiating effects of garlic extracts using the medium term hepatocarcinogenesis
model in the rat (in progress).
WP7
Year4
Identification and quantification of sulphur compounds in man by measuring their concentrations in blood
and urine after ingestion of a garlic preparation (to be done).
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 (to be
done)
Deliverables
DH 8
DH 9
DH 10
DH 13
DH 14
DH 15
DH 20
DH 21
DH 22
DH 27
In vitro metabolism of diallyl disulfide, ajoene and allicin
In vitro effects of sulphur compounds on human P450 iso-enzymes (publication in preparation)
In vitro evaluation of the anti-geno-toxic properties of garlic compounds in Hep
G2 cells (publication in preparation)
In vivo metabolism of diallyl disulfide
Effects of garlic extracts on carcinogen-metabolising enzymes in the rat (publication on the
web site)
Publication of the in vivo anti-genotoxic properties of garlic in rat (publication in preparation)
Modulation of the mutagenicity in the Ames test
Performing intervention study with report and analysis of results
Short report on the results concerning biomarkers of cancer in human intervention
study
In vivo metabolism of garlic powder
Research
What has been done?
Evaluation of the antigenotoxic properties of garlic powders in vivo
The aim of this study was to assess the protection of garlic powders against carcinogen genotoxicity in
different tissues in the rat (liver, colon). Effects of garlic powders in liver have already been evaluated (see
2002 report). In the present experiment, rats were fed with garlic powders then they were treated by
genotoxic compounds which act in the colon. The level of DNA damage was determined using the comet
assay. Dimethylhydrazine (DMH) and methylnitrosourea (MNU) have been selected as colon genotoxic
compounds. They act through different mechanisms of action: DMH is an indirect-acting genotoxic
compound, i.e. it needs to be metabolised by cytochrome P450 2E1 to exert its genotoxic action; MNU is a
direct-acting compound, it does not need biotransformation to alter DNA.
The effects of garlic powders with different contents in alliin have been tested in this experiment.
40
MATERIALS AND METHODS
Printanor garlic bulbs were obtained from the 2001 field trail (french samples). Three garlic powders,
prepared by Rémi Kahane (P4) were tested: the first one was prepared from garlic cultivated in soil not
supplemented with SO4 (SA), the second one was prepared from garlic cultivated in soil enriched with 100
kg/ha SO4 (SB) and the third one was prepared from garlic cultivated in soil enriched with 200 kg/ha SO4
(SC). These powders contain increased amounts of alliin (ca 60, 75 and 90 nmoles/mg alliin, respectively,
results obtained from J. Auger, P5). Male Wistar rats, 4 week-old, were used. For the first week, they
received a control diet then they were separated into 4 groups. Three groups received 5% of garlic powder
in their diet for 2 weeks. The 4th group received the control diet (group C) till the sacrifice. DMH and MNU
were respectively administered 24 hours and 2 hours before the sacrifice. Colons were quickly removed
and the comet assay was immediately performed on isolated cells obtained after collagenase treatment.
The comet assay was performed as described previously (see 1 st annual report). DNA damage is
expressed as Olive Tail Moment (OTM) defined as the fraction of DNA in the comet tail multiplied by the
tail lengh.
RESULTS
Garlic powders were not genotoxic by themselves since no increase of DNA alteration was observed in
rats treated by garlic (groups SA, SB and SC) when compared to rats receiving the control diet (group C)
(see figure).
The garlic powders significantly inhibited the genotoxicity of DMH in the colon of rats. The three garlic
powders induced the same effects (about 50% reduction), suggesting that the level of fertilization did not
influence the antigenotoxic properties of garlic. Garlic powders administration did not modify the
genotoxicity of the direct-acting genotoxic compound MNU in the colon.
These results suggest that garlic powders exert their antigenotoxic potency through modulation of drugmetabolizing enzymes. Prevention of DMH genotoxicity is consistent with the inhibition of CYP 2E1
induced by garlic ingestion that we previously observed in liver (see 2001 report).
OTM
(mean of 5 rats/group)
7.00
6.00
5.00
4.00
3.00
2.00
*
*
*
Figure : Effects of feeding garlic powder on DMH- and MNU-induded genotoxicity in rat colon.
C= control diet
1.00
SA= garlic powder prepared from garlic cultivated in soil not supplemented with SO4
SB= garlic powder prepared from garlic cultivated in soil enriched with 100 kg/ha SO4
0.00
garlic powder
prepared
from garlic
cultivated
soil enriched
with
200 kg/ha
SO4MNU-SC
C SC=SA
SB
SC
DMH-C
DMH-SA
DMH-SBinDMH-SC
MNU-C
MNU-SA
MNU-SB
MNU = methylnitrosourea ; DMH = dimethylhydrazine
* : significantly different from the respective control group (Dunnett’ test, p 0.05)
41
Investigation
of
the
antiinitiating
effects
of
garlic
extracts
using
the
medium
term
hepatocarcinogenesis model in the rat.
The objective is to determine the effects of consumption of garlic powders (from the field trials) on the
carcinogenicity of chemicals, using a medium term hepatocarcinogenesis model in the rat. The working
hypothesis is that the reduction of the genotoxic effects of chemicals by consumption of garlic (which has
been previously demonstrated using the Ames test and the Comet assay), results in an inhibition of their
carcinogenicity.
The effects of feeding rats with garlic powders are determined on the appearance of liver
preneoplastic foci. We use an hepatocarcinogenesis model initiated either by a diethylnitrosamine (NDEA)
or by aflatoxin B1 (AFB1). These chemicals are potent hepatocarcinogens. NDEA is activated by CYP 2E1
and AFB1 is activated by CYP 2B and detoxicated by glutathione S-transferase (GST) . We have shown
previously that garlic consumption decreased the activity of CYP 2E1 and increase the activity of GST.
Thereby , it is suggested that garlic consumption will reduce the carcinogenicity of these chemicals
3 garlic powders with different S levels (low, medium and high content) have been selected :
french trial 2001 PRI S fertlisation O kg/ha SO4 (allin content 58 nmoles /mg)
french trial 2001 PRI S fertlisation 200 kg/ha SO4 (allin content 90 nmoles /mg)
spanish trial 2001 PRI S fertlisation 400 kg/ha SO4 (allin content 330 nmoles /mg)
The hepatocarcinogenesis protocol which is used allows the detection of preneoplastic foci
expressing the placental form of glutathione S-transferase as an end point. Hepatocytes with altered DNA
are initiated by a non necrogenic dose of NDEA or AFB1. These hepatocytes are further stimulated by a
selection procedure ie, administration of acetylaminofluorene, together with a mitogenic stimulus (partial
hepatectomy), to form resistant hepatic foci by clonal expansion. The cells of the foci differ from normal
cells by morphological and histochemical characteristics and constitute markers of the preneoplastic
transformation. The placental form of glutathione S-transferase is strongly expressed in these
preneoplastic cells and therefore is used as a marker of preneoplastic foci.
Experimental
80 Male SPF Wistar rats are used in this protocol. They are divided in 8 groups as follows :
Treatments
NDEA
AFB1
Control
10 rats
10 rats
Garlic powder S 0
10 rats
10 rats
Garlic powder S 200
10 rats
10 rats
Garlic powder S 400
10 rats
10 rats
They are fed a purified control diet for one week before the initiation of carcinogenesis by 10 i.p.
injections of NDEA at the dose of 2.5 mg/kg body weight or by 10 i.p. injections of AFB1 at the dose of
0.025 mg/kg body weight. During these treatments rats are fed either the control diet or the same diet
containing 5 % of the garlic powders. After 3 weeks of treatment with garlic powders, all the groups are
fed with the control diet for 2 weeks. Then, the rats are submitted to a selection procedure by
acetylaminofluorene (AAF) treatment for 2 weeks. In the middle of this period, the rats are submitted to a
partial hepatectomy (PH). After this 2 weeks-phase of selection, rats are fed again one week with the
control diet and then are sacrificed.
42
NDEA
AFB1
SS
PH
0
weeks
1
2
3
4
5
6
7
8
9
Control diet
Diet with garlic powders
2-AAF 100 mg/kg diet
PH partial hepatectomy
S
sacrifice
Livers are removed and slices about 4 mm thick are cut in each liver and are frozen in isopentane
at – 150 °C . Serial liver sections are cut with a cryostat and used for the detection and the quantification
of preneoplastic foci.
The morphometric analysis of these preneoplastic foci are in progress. The first data are expected
at the begining of July.
What will be done:
The current planning is that the EUGIS study will start at the begining of June. We will receive the human
samples : lymphocytes, plasma , red cell and urine in September. Considering the delay of the EUGIS
study, it will be difficult to complete all the measurements of cancer end-points before the end of 2003.
Significant difficulties or delays:
We had some difficulties for performing the comet assay in rat colon. Preliminary trials were necessary to
find the conditions which allow the recovering of isolated cells in good health and to find the correct doses
and treatment times of genotoxic compounds. This experiment was therefore delayed with regard to the
initial planning.
P13: University of Muenchen, Muenchen, Germany
Workpackage:
Title of reasearch:
N° 5.1
The interaction of garlic with mediators of inflammation
Persons involved:
P13; Prof. Dr. A. Vollmar; Dr. V. Dirsch; Nancy Lopez; Thomas Roos
43
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.
1. Influence of garlic on pro-inflammatory cytokines in human nuclear cells. The potential role of
TGF-1 in the effect of garlic on atherosklerosis
Milestones & deliverables for the third year.
Milestones:
1. Influence of garlic on the expression of adhesion molecules and pro-inflammatory cytokines and
their soluble forms.
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.
3. 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 rebuttal)
2. Influence of garlic on cytokines as important factors influencing the cellular mechanism of
atherosclerosis. (in press)
3. Influence of garlic on the expression of selectins and signal transduction factor NF-B. (in
rebuttal)
4. Publication on the influence of garlic on the expression of selectins and signal transduction of NFB (in rebuttal)
Research:
What has been done:
1. The paper entitled “Garlic (Allium sativum L.) Modulates Cytokine Expression in
Lipopolysaccharide-Activated Human Blood Thereby Inhibiting NF-B Activitiy” has been accepted
in J. Nutr.
2. The model of rat vascular smooth muscle cells (VSMC) has been established in order to test the
effect of DADS on hypertrophy and hyperplasia
What will be done:
Test of DADS on VSMC
Bottlenecks:
None
44
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 apoE3Leiden 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
45
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
Research:
No info received
P15: Lichtwer Pharma AG, Berlin, Germany
Workpackage
WP 7, Participant 15 (Lichtwer Pharma AG, Berlin, Germany)
Title of research
Pharmaceutics
Persons involved
Dr. Thomas Haffner (Research leader)
Thomas Goerke (Research technician)
Silvia Striegl (Technician Galenic Department)
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)
 Production and delivery of study medication for a human intervention
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)
 Study medication human intervention study
B. Research
No info received