GARLIC AND HEALTH PROJECT
EU identification number QLK1-CT-1999-00498
E-mail report January 1 - January 31 2003
(E-report 24)
General
1. The Liverpool meeting: I guess things are going quite well, the transport and hotel are well arranged
and furthermore the social events have been organized in some detail. Dear partners make sure that
you have checked that your Powerpoint presentation is in good shape (so send it to Meriel Jones
before the meeting; deadline: February 14) and take a back up of your presentation with you in the
form of overhead sheets. As we need to put the Powerpoint presentations on the G&H homepage, I
also need a copy of your presentation, so the best way in this respect would be that Meriel puts all
your contributions on one CD that I can take with me to Holland (you won’t believe how many CDs I
have already from previous meetings!).
2. The social event on Thursday can be also used for discussions in groups of partners. Make your
arrangement before the meeting then you can use your time most efficiently.
3. As this is an annual meeting please check your milestones and especially your deliverables for year 3
and if applicable year 2 (posponed M&Ds).
4. Final agenda G&H meeting Liverpool, February 17-21 2003
Monday, February 17
Arrival
Tuesday, February 18
8:15 –8:30
Welcome by our host Brian Tomsett
HEALTH (Chair: Rolf Gebhardt; Minutes: who??)
08:30 - 08:45
08:45 - 11:00
11:00 - 11:30
11:30 - 13:00
13:00 - 14:30
14:30 - 15:15
15:15 - 15:45
15:45 - 17:00
17:00 - 18:00
Introduction to the meeting: Rolf Gebhardt /Remi Kahane/Chris Kik
WP 5 Cardio-vascular diseases
5.1 inflammation: Angelika Vollmar cs
5.2 cholesterol & vessel wall: Rolf Gebhardt cs
Coffee
WP 6 Cancer: Marie Helene Siess cs
Lunch
WP 7 Pharmaceutics: Thomas Haffner cs
Tea
WP 8 Human intervention study: Hans Princen cs
General discussion health part: Rolf Gebhardt
Wednesday, February 19
GARLIC (Chair: Remi Kahane; Minutes: who??)
08:30 – 10:00
10:00 – 10:30
10:30 – 11.15
11:15 - 12:00
12:00 – 13:30
13:30 - 15:30
15:30 - 16:00
16:00 - 17:00
WP 4 Biochemistry: Brian Thomas cs
Coffee
WP 3 Cultivation: Odile Huchette cs
WP 1 Genetic Resources: Rina Kamenetsky cs
Lunch
WP 2 Breeding Systems: Xabier Barandiaran cs
Tea
General discussion plant part
Thursday, February 20
8:30 -10:30
10:30 - 11:00
11:00 – 13:00
13:00 - 14:00
14:00 - ?
Parallel plant and health session in which the various bottlenecks of the
previous years and solving of these bottlenecks will be discussed. Chairs:
Rolf Gebhardt and Remi Kahane
Coffee
General project discussion and further project business (Chair: Chris Kik):
1. What are the bottlenecks we see presently and what can we do about it
to achieve our goals (summary of parallel session): Rolf Gebhardt, Remi
Kahane
2. Finances.
3. Writing of the fourth annual scientific report, consolidated progress report
and technological implementation plan (TIP) in the second half of 2003.
4. ISHS symposium China: lay-out posters etc.
5. EU FP6 developments.
Lunch
Social event or group discussions
Friday, February 21
Departure
5. You can find the draft version of the third annual report on the G&H home page (username: project;
password: rentrap). I would be very pleased to have your comments on this version.
6. Please send your cost statements (CSs) for the third year (February 1 2002 – January 31 2003) to me
(Chris Kik), you can also take your CS with you to Liverpool. Please make sure that the signature on
the official format is in BLUE !!
7. The reimbursement of the EU of the second year we received some weeks ago, you will receive the
money in due time with a letter in which the details for the reimbursement will be explained.
8. The ISHS meeting in China:
We need to present next to our presentations also posters: 1 general, 2 plant and 2 health. These
posters should have a more or less uniformous lay-out. I will give more details on this in the Liverpool
meeting.
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
partly done
done
done
done
E. Research
What has been done:
Garlic cloves have been planted in November outside for cold treatment in order to obtain good
flowering.
What will be done:
In the next months this will be the writing of a paper on the establishment of the garlic core collection.
This will be done in close cooperation with Rina, Haim and Jacques.
Bottlenecks:
None
Workpackage : WP2 Breeding Systems
A. Objectives
The development of a reliable transformation protocol for garlic using A. tumefaciens as a vector.
B. Persons involved
Si-Jun Zheng, Betty Henken, Frans Krens, Chris Kik
C. Milestones & Deliverables
M&D 2000
Milestones:
Establishment of a micropropagated collection of garlic clones:
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:
Highly transformable callus lines available for stable transformation experiments:
Regeneration of transgenic plants steadily expressing reporter and selectable genes:
Garlic transgenic plants available:
Deliverables:
DP12: paper on regeneration (transformation) of garlic:
done
done
done
done
M&D 2002
Milestones:
Regeneration of transgenic plants steadily expressing genes from the sulphur pathway;
transgenic garlic plants with alterations in the sulphur metabolism available:
Deliverables: No deliverables in the third year
to be done
done
M&D 2003
Milestones:
Transformation and regeneration of garlic transgenic plants containing different genes
to be done
Involved in the sulphur metabolism, isolated by P2 and P3; integration and expression
analysis.
Deliverables:
DP 27:Paper on anti-sense alliinase research
?
DP 33: Paper on other S pathway genes on the production of flavour precursors (CSOs) to be done
content of garlic
E. Research
What has been done:
Root segments from cv. Printanor are used to induce callus. Young callus will be used in new
experiments of on garlic transformation. The ATP sulfurylase (APS1) from Arabidopsis thaliana has
fused with GFP (APS1-GFP and GFP-APS1). ATP sulfurylase from shallot has also been cloned and
is being competely sequenced at this moment. ATP sulfurylase is the first and sole entry point
enzyme for the sulphur metabolism in plants. Sulphur is one of the six macronutrients required by
plants and incorporated into a large variety of metabolites such as cysteine, methionine, sulfolipids,
sulfated glucosides and other co-enzymes. The protein displays a transit peptide for plastid targeting.
What will be done:
1
2
Write a paper on garlic transformation.
Transfer, via genetic modification, a (key)gene from the sulphur metabolic pathway into garlic.
Bottlenecks
None
A. Workpackage: WP 9 Co-ordination
B. Objectives:
To ensure that collaborative links between the partners in the project are established and function and
that there is a good link with the EU.
C. Persons involved
Remi Kahane, Rolf Gebhardt and Chris Kik
D. Milestones & Deliverables
M&D 2000:
Milestones:
Start-up meeting, (first and) second project team meeting;
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
partly done
done
partly done
done
done
to be done
to be done
to be done
to be done
DC 6: Final progress reports
to be done
E. Activities
What has been done
1. It has been arranged that we can present our G&H project at the ISHS symposium in Beijing in a
plenary afternoon session. Furthermore financial reimbursements have been arranged by the EU for
possibly 3 persons (Rolf, Odile and Meriel).
2. The process leading to the preparation of the third annual G&H report is almost finished
What will be done
1. Editing the draft version of the third G&H scientific annual report based on the comments of the
partners during the Liverpool meeting and send final version to the EU.
2. Sampling of cost statements for the third year from the partners during February 2003 and send them
to the EU.
Bottlenecks
None
P2:
Horticulture Research International, Wellesbourne, UK
A. WP 4: Garlic Sulphur Biochemistry.
B. Objectives:
Understand the rate-limiting processes in the accumulation of aliin and its subsequent conversion to
alliicin.
Identify the developmental control points in of cysteine sulphoxide synthesis and translocation
Characterisation of the alliinase gene family in garlic.
C. Persons involved:
Brian Thomas, Lol Trueman, Brian Smith, Linda Brown
D. Milestones & deliverables of the first year:
Deliverables:

DP. 17: First sulphur budget for garlic (P2)

DP. 18: Clones for alliinase (P2)

DP. 23: Publication on alliin biosynthesis and sulphur partitioning (P2, P3)
Milestones:

Analysis of second-year field experiment completed

Whole plant labelling studies completed

Expression studies on alliinase clones initiated
E. Research
No info received
P3: University of Liverpool, UK
A. WP 4: Garlic Sulphur Biochemistry.
B. Objectives:
Identify intermediates on the pathway leading to synthesis of alliin.
Characterisation of genes with altered expression in tissues with differences in levels of flavour compound
pathway flux
C. Persons involved:
Hamish Collin, Brian Tomsett, Meriel Jones, Rick Cosstick, Angela Tregova, Jill Hughes,
D. Milestones and Deliverables of the first year
Deliverables:
DP16: Analytical methods for labeling and analysis.
Milestones:
Measurement of alkyl cysteine sulphoxides, pathway intermediates and gamma-glutamyl peptides
in bulbing and sprouting plants completed, Optimal conditions for pulse labelling established.
Milestones and Deliverables of the second year:
Deliverables:
DP 16: Pathway intermediates identified.
Milestones:
Radiolabelled intermediates identified using HPLC and HPLC-MS in comparison with chemically
synthesized compounds
Differential display sequences isolated and re-analyzed by Northern blot and in situ hybridization,
where possible, with other Allium species.
Milestones and Deliverables of the third year
Deliverables:
DP 23.Paper on alliin biosynthesis.
DP 24 Genes for key CSO synthesis enzymes
Milestones:
Analysis of pattern of labeling in later stage intermediates completed. This labeling approach has
been superceded by isolation and identification of key pathway enzymes by biochemical and molecular
biological methods.
Purification of PCR amplified cDNAand sequencing completed.This is no longer applicable since
the differential display approach has been discontinued. See above for the altered milestones.
E Research
What has been done:
HPLC analyses were done to know whether the transformed tobacco cells were expressing any allyl
cysteine synthase and cystreine synthase genes. This they did not because as we eventually found out the
cells that Angela’s had inherited from The John Innes Institute were silenced. The cells were transformed
quite successfully but they were not expressing the genes. This conclusion was arrived at after an
exhaustive series of tests. Since then we have obtained a new set of cell lines fro the JI and are busy
transforming them but the results will not be known for a little time.
What will be done:
We have had some rethinking on our approach and are now going to adopt ‘a belt and braces’ approach
to expressing the enzyme. In this context E. coli will be transformed to get the gene expressed which was
always an alternative but we are going to go down that route now on the assumption that the cells might
not behave a second time.We need to have expression of this enzyme preferably in plant cells but fasiling
that in the E. coli.
Bottlenecks:
Time is running short because we used the wrong cell lines for transformation, we hope that our new
approach will give if us the results needed.
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).
D. Milestones & deliverables :
Differential display of clones exposed in vitro to various mineral nutrition treatments (normal and S-, N- or
Se-enriched nutrition) (P4). Greenhouse experiments with two genotypes, one low and one high in S
content (from the core collection), optimal growth conditions (temperature, daylength) and sub-optimal
sulphur nutrition for producing S-enriched garlic bulbs (P4). Sampling of fresh plant material during the
cultivation treatments for CSO content analysis (P4 & P5).
DP. 22
Delayed
DP. 34
In progress
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)
E. Research :
1. What has been done:
a) In vitro :

Experiment set up in common with Véronique Chovelon (INRA Avignon, P9) and Leonidas
Fereol (CIRAD Montpellier, P8) = test of the effect of different factors on in vitro bulblet
production of garlic plants derived cell suspension cultures.
The results of percentage of bulbs formed were presented in Montpellier by Véronique Chovelon.
But the results of alliin content analysis (P5) were only available in November. These analysis
couldn’t be made on all the material because many treatments didn’t produce bulbs enough, so
they have to be considered on the 3 sites Dijon, Avignon and Montpellier at the same time. A
synthesis has been made by Véronique Chovelon (P9).

An experiment in vitro (n° III) was set up in Dijon at the beginning of October to test (mainly on
Printanor) :
-
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.
The plants were placed under cold conditions (3°C – 10 hours day – White light) during the first
week of November. They were removed and placed under the different light conditions for
bulbification on the 8 January.
Observations on the growing of the plants in the different treatments are planed every 10 days
until the harvest. Observations were then made on the 13 and on the 23 January (stage of the
plant and number of green leaves).
The different treatments tested are remembered in the following table 1.
Table 1: Treatments and varieties tested in the experiment in vitro III.
Light
F
F+i
950 - 1000 lux 950 - 1000 lux
T°C
Medium S-
F
F+i
2000 lux
950 - 1000 lux
T°C 22-24°C
T°C 22-24°C
T°C 22-24°C
T°C 25-27°C
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
Further micro-propagations of the genotypes Printanor, Messidrôme and Morasol were made
to set up another experiment (n°IV) at the beginning of January 2003. This experiment aimed
at testing the effect of sulphur combined to nitrogen on the alliin content of garlic bulbs. 9
different culture medias are tested, but only with Printanor, as it was still difficult to produce
enough plants in the genotypes Morasol and Messidrôme for the whole experiment. For these
varieties, treatments were chosen after discussion with the co-ordinator of the Plant Part.

The plants will be placed under cold conditions (3°C – 10 hours day – White light) during the first week of
February.
The protocol is presented below :
WP3 / P4:
4 /2002
in vitro
experiment
Gradient in SO4 and nitrogen
SO4
Nitrogen
0
N
3
3N
9
N 2N 3N
N/2
meq (in macro-elements)
9 medias
N 2N 3N
22-24°C - P = 16 hrs - F
1 month
Cold induction 3°C - P = 10 hrs - F
2 months
Varieties:
PRI
+ MES
+ MOR1
+ MORd
,
22-24°C – P=16 hrs
F+i - 950 lux
1 environmental
factors
9 treatments for PRI
(48 plants / treatment)
MES, MORd = 4 treatments
MORl = 7 treatments
(31 plants / treatment)
The media tested for Messidrôme, Morasol and Morado are detailed in the table 2.
Table 2 : 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 SO42-
b) In greenhouse : 2 experiments have been prepared to be set up at the end of January. Two main
objectives with these experiments :

To test the effect of nitrogen combined to a high level of sulphur on the alliin content
of garlic bulbs (3 levels of nitrogen) of Printanor, Morasol and Messidrôme

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 will be grown in insect-proof conditions and treated against pests and
diseases.
For the first experiment, the garlic cloves were supplied by Véronique Chovelon (P8 - INRA Avignon).
All the cloves will be planted at the end of January in boxes (L 60*l 40*h 30 cm – 12 cloves per box) with
washed river sand. Water and nutrients will be supplied with drop irrigation.
2. What will be done :
a)
In vitro :
 Progress of the experiment III in vitro : observations of the growing of the plants.
 Progress of the experiment IV in vitro : the plants will be exposed to cold conditions at the
beginning of February.
b)
In greenhouse : Set up and following of the 2 experiments planed.
3. What are the bottlenecks : None.
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 has been done:
HPLC analysis of 55 accessions from Chris Kik (P1) is finished.
What will be done:
No analysis programmed
A- WP2 Breeding systems
1- Objectives:
Screening CSO of garlic .
2- Persons involved:
Jacques Auger, Ingrid Arnault, Nicole Mandon.
3- Milestones & deliverables of the second year:
No deliverable
4- Research:
What has been done:
On november, we received from Michel Roux Cuvelier via P9 bulbs issued from embryogenic calli,cellular
suspension and vegetative multiplication. HPLC analysis of these bulbs is finished.
On december, we received a second experiment of embryogenic bulbs from Michel Roux Cuvelier.
What will be done:
HPLC analysis of the second experiment will be over in february 2003.
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:
All AlCSO analysis is finished.
What will be done:.
No analysis programmed.
D- WP6: Garlic and cancer
1-Objectives
Assess the anti-carcinogenic potential of garlic in relation with the bioavailability and the metabolism of
garlic sulphur compounds
.
2- Persons involved:
Jacques Auger, Ingrid Arnault, Nicole Mandon.
3- Milestones of the second year:
Synthesis of allicin, ajoenes, alliin and purification of diallyl disulphide, allyl mercaptan and allyl methyl
sulphide.
Not deliverable for our group in this workpackage
4-research
We sent 2 ml of allicin (10-2M) to M.H. Siess on 22th january.
P6: University of Cordoba, Cordoba, Spain
A. Workpackage: WP2 Breeding Systems
B. Objectives: Biolistic genetic transformation of garlic
C Persons involved: Xabier Barandiaran, Nieves Martín, José Garrido, Jesús Martín.
D. Milestones & deliverables:
Establishment of a micropropagated collection of garlic clones; axenic material available for
transformation experiments. (Done)
Selection of organogenic callus lines presenting high levels of transient expression of UidA and GFP
genes; Highly transformable callus lines available for stable transformation experiments. Regeneration of
transgenic plants steadily expressing reporter ands selectable genes; garlic transgenic plans available.(In
progress)
Regeneration of transgenic plants steadily expressing genes from the sulphur pathway; transgenic garlic
plants with alterations in the sulphur metabolism available.
DP12 Paper on transformation of garlic
E. Research
1. What has been done.
Regenerable cell suspensions received from P8 and treated with the biolistic device are being cultivated in
presence of hygromicine. 20 weeks after starting the selection process differences among treated
explants and controls are clear. Transgenic calli expressing high levels of GUS have been selected.
Control. Hygromycin 25 mg/l
Bombarded calli. Hygromycin 25
mg/l
GUS activity. Selected calli.
2.What will be done.
Transgenic calli will be transferred to a regeneration medium in order to obtain transgenic plants.
3. Bottlenecks.
None
A. Workpackage: WP3 Cultivation
B. Objectives: Evaluation of the effect of sulphur fertilization on yield and quality of field cultivated garlic.
C. Persons involved: Xabier Barandiaran, Francisco Mansilla, Jesús Martín.
D. Milestones and deliverables:
DP22 Paper on the interaction genotype x environment on the sulphur content of garlic plants grown in
vitro, in greenhouse and in field conditions (in progress)
E. Research
Field trials completed.
P7: Volcani, Bet Dagan, Israel and P10: The Hebrew
University of Jerusalem, Israel
The research group is involved in WP1: Genetic Resources 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
GENERAL LIST OF DELIVERABLES:
Deliverable
Deliverable title
Delivery
date
Completion
Participant
N°
P1, P5, P7,
P10
DP. 3
Collected bulbs for screening for CSO,
fertility and clonal identity (fingerprinting).
9
DP. 4
Paper on morphological and physiological
aspects of floral/topset initiation and
development
Chemical distinction (CSO), fertility and
clonal identity between garlic accessions of
the collection
12
P7, P10
12
P1, P5, P7
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
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
+
(According to
DP. 7
DP. 10
DP. 11
DP. 20
DP. 25
DP. 26
DP. 31
+
(According to
original
program)
Published
+
(According to
original
program)
Done
In progress
DP. 32
DP. 37
Paper on environmental regulation of flower
differentiation and flowering process
Blueprints for fertility restoration. F1 seeds
for biochemical and molecular studies
36
P7
original
program)
In progress
48
P7
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
suspensions 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.
Persons involved :
FEREOL Léonidas, CAUSSE Sandrine, ROUX-CUVELIER Michel, COTE François, KAHANE Rémi.
Milestones and deliverables of The fourth year
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 o(in progress)
6) DP 28 Paper on bulb mass production from embryogenesis of elite clone
Research
The task of P8 within the project aims at the development of a mass propagation method relying on in
vitro regenerated plants from embryogenic 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: A fourth variety “Printanor” has been taken into consideration for adaptation of the protocol to this
cultivar.
1) What has been done
Var. Rouge de la Réunion
- production of embryos from embryogenic calli on semi-solid medium (ecal) and from cell suspension
(ecs).
- Germination of these embryos.
- Provision to P9, of plants from these embryos (ecal and ecs), for further in vitro development,
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 (18 and 27 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 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 of the procedure for maintaining cell suspension cultures and for plating
- Genetic characterisation, by flow cytometry, of Moraso E-cal, Morasol Ecs and Messidrome E-cal
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
All varieties
- Promotion of the embryogenic procedure for all the varieties experimented. Adaptation and modification
of experimental conditions, particularly macro nutrients medium, for improving the preliminary results
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
% callus
production
% callus with
embryogenic
tissue
0.3
Rouge Réunion
Messidrome
Morasol
Printanor
86
90
97
96
24
32
44
51
0.5
Rouge Réunion
Messidrome
Morasol
Printanor
93
95
95
98
35
39
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
globular embryos per
150 mg fresh weight
callus
37
77
88
92
Mean number of embryos
converted into plantlets per
150 mg fresh weight callus
16
33
51
68
14
22
38
Cell suspension culture of Messidrome, Morasol and Printanor
Culture suspension 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 (Tab. 3). 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. The optimum inoculums size was 2 g fresh
weight callus for 50 ml liquid medium. (These suspension cultures were maintained on SM medium (Tab.
4). They were visually distinguished by the presence of small cell aggregates.
Table 4. Effect of cell density on PCV and regeneration potential
during maintenance phase, var. “Morasol”
Cell
density
(%)
Culture
period
(days)
1%
0
15
30
50
60
0
15
30
50
60
0
15
30
50
60
0
15
30
50
60
0
15
30
50
60
2%
3%
4%
5%
PCV rate (%)
mean
1,00
7,22
13,06
21,94
18,33
1,00
5,28
8,47
10,83
8,89
1,00
4,07
8,80
9,67
8,78
1,00
3,26
6,81
6,88
6,53
1,00
2,61
5,00
5,72
5,39
s.e.
0,00
0,86
1,25
2,67
2,79
0,00
0,43
0,97
1,39
2,28
0,00
0,29
1,38
0,84
0,99
0,00
0,31
0,57
0,35
0,43
0,00
0,25
0,56
0,49
0,61
Number of
embryos
mean
s.e.
0,60
0,89
8,60
2,88
37,40
11,63
20,40
4,39
12,40
6,91
8%
0
15
30
50
60
1,00
2,22
2,88
2,71
2,57
0,00
0,25
0,16
0,26
0,28
11,60
10,41
Table 3. Effect of callus inoculation concentration on PCV rate and regeneration
capacity, var. Morasol
Callus inoculation
concentration (%)
1
2
3
4
5
6
PCV rate (%)
Mean
s.e.
0,67
0,21
0,60
0,09
0,41
0,05
0,38
0,06
0,34
0,06
0,30
0,07
Number of embryos
mean
s.e.
47,32
368,75
136,17
387,50
121,41
443,75
100,78
443,75
172,00
725,00
92,42
487,50
Milestone: 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.
Table 5. Genetic characterisation, by flow cytometry, of
plants from ecal and from ecs of Messidrome and Morasol
Type of sample
MESSIDROME
- control
- E-cal
MORASOL
- control
- E-cal
- Ecs
Ploïdy level
2X
4X
12 plants
102
0
10
16
100
100
0
3
4
Flow cytometric histogram
Of diploid and tetraploid
Somatic embryos
4X
2X
2X
+
4X
Figure 1. Flow cytometric histogram of embrygenic derived
and control plants (multiplied by axillary bud culture)
Milestone : 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. Treatments concerned:
. cold pre-treatment
. light spectrum quality
. daylength
. sucrose concentration in the medium
Collect of the records every two weeks.
2) What will be done
var. Messidrôme, Morasol and Printanor
- Bulb mass production from embryogenesis and further development of of these bulblets into bulbs
- Continue development of the most appropriate procedure to promote somatic embryogenesis
- Histological studies continue to follow and understand the different stages of somatic embryogenesis
in Allium sativum.
- Maintenance and in vitro multiplication of embryogenic calli and tissue.
- Carrying on studies for establishing cell suspensions from embryogenic calli of these varieties and
sustaining an active multiplication.
- Studies to lead to convenient conditions to regenerate embryos from embryogenic calli and cell
suspension.
- Research of the best conditions for producing bulbs from somatic embryo plants.
3) BOTTLENECKS, DELAYS
Milestone: Field evaluation , 200 plants R (E-cal), 200 plants R (Ecs)
These works are partly fulfilled, information are reported by P9.
All the other milestones are achieved
Deliverables:
DP 13 : paper will be submitted soon
DP 21 : paper will be submitted first semester 2003
4) COORDINATION
O. Huchette, L. Féréol and R. Kahane met V. Chovelon at INRA Avignon (13-14/5/02) to discuss about
the protocol for bulbing of embryo derived plantlets. Environmental conditions and plant material is shared
into 3 sites (Avignon, Dijon and Montpellier, France).
P9: INRA Avignon- Montfavet, France
A. Workpackage : WP2 Breeding systems
B. Objectives :
Embryogenesis, genetic characterisation: P9 is involved in the embryogenesis program to assure final in
vitro development and bulbification, acclimatisation, cultivation and evaluation of regenerated plants
produced by embryogenesis, to control their integrity and conformity, in comparison with the original
materiel multiplied in field (Messidrôme, Morasol and Printanor)
Sanitary aspects: P9 is engaged for producing virus-free plants (Messidrôme, Morasol, Printanor, Rouge
Reunion) in insect-proof conditions for others partners, for indexing viruses (ELISA tests) and for
regenerating selected garlic clones provided by P7.
C. Persons involved :
V. Chovelon, JP Leroux.
D. MILESTONES
year
activities
State
done
done
done
partly done
3
Field multiplication of virus-free plants obtained from 2 selected clones (1), (2)
2002 Acclimatisation of virus-free plants obtained from 2 selected clones (3), (4)
Production of virus-free bulbs for other partners (3 varieties; 300 plants/variety)
Genetic characterisation of plants from embryogenesis “Rouge Reunion” : first field
evaluation (P8), morpho-physiology (P8), dry matter and sulphur content (P5)
Genetic characterisation of plant from embryogenesis (MEScal, MESecs, MOLcal,
MOLecs): first field evaluation (P9), flow cytometry (P8), sulphur content (P5)
partly done
Embryogenesis: in vitro bulbification studies (P8, P9, P4)
partly done
Regeneration and acclimatisation of 200 vitro plants Printanor from embryogenic callus done
(PRI ecal) and 200 vitro plants Printanor from cell suspensions (PRI ecs)
E. DELIVERABLES
N°
Deliverable title
DP1
DP5
virus-free bulbs materiel for introduction in vitro
Paper on Evidence of a somatic embryogensis process and plant
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 suspensions 24
and plant regeneration. L. FEREOL*, V. CHOVELON**, S.
CAUSSE*, D. TRIAIRE, N. MICHAUX-FERRIERE*, R. KAHANE*.
DP13
DP21
Previous
delivery date
1
12
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*
F –Research
No info received
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)
State
done
done
delayed:
writing in
progress
(end 2002)
delayed
writing in
progress
(1rst semester
2003)
Milestones and deliverables of the second year
Milestones :
- Studies on garlic with different sulphur content on cholesterol biosynthesis. Structure-function
relationships concerning additional garlic organosulphur compounds and the AMP-dependent kinase
pathway.
- Influence of garlic constituents on MMP and TIMP production by endothelial cells
Deliverables : DH6, Publication on the molecular interaction of garlic compounds on AMP-dependent
kinase pathway (Almost finished)
DH7, Publication on the influence of garlic constituents on MMP and TIMP gene expression (2
abstracts provided, manuscripts in preparation)
Milestones and deliverables of the third year
Milestones :
- Influence of garlic constituents on MMP and TIMP production by endothelial cells as well as in
blood and tissue samples from Leiden ApoE mice.
Deliverables : DH11, DH12, publications
Research
No info received
P12: INRA-Dijon, France
1. 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
2. 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) has left the lab March 2002
Duties: Metabolism and bioavailibility
of organosulfur compounds
Post-doctoral fellows (to be hired)
(12 person months)
Duties: Carcinogen metabolizing enzymes,
genotoxicity and metabolism
Research technicians
Marie-France Pinnert
(6 person months)
Patrick Tassin
(6 person months)
Duties: Immunohistological methods
for preneoplasic foci
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
In vivo effects of garlic extracts on several carcinogen metabolizing enzymes (Phase I and phase II).
Year 3
Determination of the bioavailability of allicin and garlic powder in rat by measuring the concentrations of
the metabolites in blood and in main organs in progress
Evaluation of the effects of subcellular fractions from rats treated with garlic extracts on the mutagenicity
of carcinogens using the Ames test in progress
Year4
Investigation of the antiinitiating effects of garlic extracts using the medium term hepatocarcinogenesis
model in the rat.
WP7
Year4
Identification and quantification of sulphur compounds in man by measuring their concentrations in blood
and urine after ingestion of a garlic preparation.
Determination of the levels of phase II enzymes (glutathion S-transferase) in human lymphocytes
Evaluation of the DNA alteration in human lymphocytes and the antimutagenic effects of urine
Deliverables
DH 8
DH 9
DH 10
DH 13
DH 14
DH 15
DH 20
DH 21
In vitro metabolism of diallyl disulfide and allicin (paper send to an editor)
Paper on In vitro effects of sulphur compounds on human P450 iso-enzymes
(writing in progress)
Paper on In vitro evaluation of the anti-geno-toxic properties of garlic compounds in Hep
G2 cells (writing process delayed)
In vivo metabolism of diallyl disulfide : paper published in Xenobiotica
Effects of garlic extracts on carcinogen-metabolising enzymes in the rat (in progress)
Publication of the in vivo anti-genotoxic properties of garlic in rat
Modulation of the mutagenicity in the Ames test
Performing intervention study with report and analysis of results
DH 22
DH 27
Short report on the results concerning biomarkers of cancer in human intervention
In vivo metabolism of garlic powder
study
Research
What has been done:
WP 6 Effects of ingestion of garlic powder on the mutagenicity of carcinogens
During 2002, we have shown that hepatic subcellular fractions from rats treated with garlic powders
slightly modified the genotoxicity of mutagens (aflatoxin B1 and heterocyclic amine) when compared with
control.
We had some problems for assessing the effect of the microsomal fraction on nitrosamine mutagenicity.
Experiments are underway for solving this problem.
WP 6 Determination of the bioavailability of garlic powder in rat by measuring the concentrations
of the metabolites in blood and in main organs.
The GC-MS analyses has shown the formation of allyl methyl sulphide (AMS), allyl methyl sulphoxide
(AMSO) and allyl methyl sulphone (AMSO2) in most tissues. AMSO2 is the most abundant and the most
persistent compound. At this moment we are calculating the concentrations of these metabolites in
different tissues and we are determining the pharmacokinetic parameters. We are going to analyse the
metabolites in aqueous phase.
WP7 : Human intervention study
Preparation of the protocol for the human intervention study with Hans. Selecting the endpoints for cancer.
We have decided to measure the following biomarkers of carcinogenesis :
- Measurement of DNA damage on lymphocytes by the comet assay
- Measurement of antioxidant enzymes: glutathion peroxidase, and superoxide dismutase (plasma
and red blood cells)
- Antimutagenic properties of urines of volunteers using the Ames test
We also plan to identify some metabolites of garlic sulfur compounds in plasma and urine
Bottlenecks
The manuscript : “In vitro and ex vivo metabolism of diallyl disulphide by rat liver enzymes and in vitro by
human liver enzymes has been send to an editor (Drug Metabolism and Disposition ) but has been
rejected
P13: University of Muenchen, Muenchen, Germany
Workpackage: N° 5.1
Title of reasearch:
Persons involved:
The interaction of garlic with mediators of inflammation
Prof. Dr. A. Vollmar; Dr. V. Dirsch; H.-P. Keiss
Milestones & deliverables for the first year.
1. Influence of garlic on the expression of adhesion molecules and pro-inflammatory cytokines and
their soluble forms.
Milestones & deliverables for the second year.
2. Influence of garlic on pro-inflammatory cytokines in human nuclear cells. The potential role of
TGF-1 in the effect of garlic on atherosclerosis
Milestones & deliverables for the third year:
Milestones:
3. Influence of garlic on the expression of adhesion molecules and pro-inflammatory cytokines and
their soluble forms.
4. Influence of garlic on pro-inflammatory cytokines in human nuclear cells. The potential role of
TGF-1 in the effect of garlic on atherosclerosis.
5. Analysis of interaction of garlic compounds with the group of selectins and their role in endothelial
cell interaction and with the signal transduction factor NF-B.
Deliverables:
1. Influence of garlic on the expression of adhesion molecules and its relevance to the
atherosclerotic process. (in preparation)
2. Influence of garlic on cytokines as important factors influencing the cellular mechanism of
atherosclerosis. (in preparation)
3. Influence of garlic on the expression of selectins and signal transduction factor NF-B. (in
preparation)
4. Publication on the influence of garlic on the expression os selectins and signal transduction foctor
NF-kappaB (delayed until 08/02)
Research:
No info received
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
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
1. What has been done
 Ongoing stability studies on film coated tablets containing freeze-dried [formulation 1] and standard
garlic powder in a formulation using pre-dried excipients [Formulation 3]. Results for
formulation 1 show significant advantages over conventional formulations – more than 1,5 till 2 times
prolongation of the stability (18 – 24 months instead of 12 months on average for standard quality) –
estimated on the allicin release potential. A 3 months testing period (testing and documentation) with
sufficient results has been added as recent results.
 Study medication human intervention study: The testing for release of the garlic powder from
Spain which is dedicated for the human study medication has been done almost completely. All main
pharmaceutical parameters are within the specification of the European Pharmacopoeia. Additional
tests for residues (heavy metals, pesticides, mycotoxins) has been started in externals contract labs –
results should be available within the next 2 weeks. These external results will have no influence on
the time table for the production of the study medication which have already been started by planning
the works in the galenic labs properly. Start of the production will be within the next two weeks – in
time!
2. What will be done
 Producing study medication
3.
What are the bottlenecks
-