European project « 5th PCRDT »
GARLIC AND EALTH
Second annual report
Partner P8 CIRAD-FLHOR
ADVANCEMENT OF THE
Works
Scientific and administrative
team
• L. FEREOL scientist
– Duties: somatic embryogenesis, histology and
flow cytometry
• S. CAUSSE technical assistant
– Duties: somatic embryogenesis, maintenance of
the material
• M. Roux-Cuvelier
– Duties: field trials
• R. Kahane
– Duties: management
Objectives
• Somatic embryogenesis
• Comparative characterisation of plantlets from
somatic embryos
Work plan
• Establishment of somatic embryogenesis
procedure for four varieties
– Rouge Reunion
– Messidrome
– Morasol
– Printanor
• Production and provision of somatic embryos via
callus and via cell suspensions of these varieties
Work plan (continue)
• Genetic evaluation of somatic embryos derived
plants by :
–
–
–
–
flow cytometry (by P8)
biochemistry (by P5)
molecular markers (by P1)
morphological and physiological analyses (by P8, P9)
Milestones of the first year
(= achieved;  partly achieved)
Provision of 200 plantlets from embryogenic calli « Rouge

Reunion »
Provision of 200 plantlets from embryogenic cell
suspension « Rouge Reunion »

Production of embryogenic callus « Messidrome »,

« Morasol »
Initiation of embryogenic cell suspension « Messidrome », 
« Morasol »
Production and germination of callus and cell suspension
derived somatic embryos

Milestones of the second year
Genetic characterisation of somatic embryos derived
plantlets « Rouge Reunion »

By flow ctytometry(P8), finger printing(P1), dry matter
and sulphur content(P5), morpho-physiology(P9)
Provision of 200 plantlets from embryogenic calli of
« Messidrome », « Morasol »
Provision of 200 plantlets from embryogenic cell
suspension of « Messidrome », « Morasol »
Genetic characterisation of these plants by flow cytometry



Milestones of the second year
(continue)
Production of embryogenic callus « Printanor »

Initiation of embryogenic cell suspension « Printanor »

Production and germination of callus and cell suspension
derived somatic embryos « Printanor »

Deliverables
DP 5 paper on: Evidence of a somaic embryogenesis
process and plant regeneration and acclimatisation in garlic
(Allium sativum L.)
L. FEREOL, V. CHOVELON, S. CAUSSE, N. MICHAUX
FERRIERE and R. KAHANE
DP13 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
S
U
B
M
I
Ted
In
P
R
O
G
R
ESS
RESEARCH ACTIVITIES
Different morphological and
histological aspect of garlic calluses
Compact non embryogenic
Semi-friable non embryogenic
Friable non embryogenic
Compact with embryogenic potentiel
Semi-friable with embryogenic potentiel
Friable with embryogenic potentiel
Established cell suspension
Macroscopical
aspect
Microscopical
aspect
Histological
aspect
Cell suspension before plating
Macroscopical
aspect
Microscopical
aspect
Histological
aspect
Plating after 16 days
Macroscopical
aspect
Histological
aspect
Plating after 30 days
Macroscopical
aspect
Histological
aspect
Plating after 45 days
Macroscopical
aspect
Histological
aspect
Media used for garlic somatic embryogenesis via callus and via cell
suspension
nutrient CIM1
s
CIM2
CMM
SM
EIM
EDM
EGM
Macro
nutient
s
CaCl2
Micro
nutrient
s
Vitamin
,Amino
acid,
organic
substa
nces
Growth
regulat
ors
N6 (m)
N6 (m)
N6
N6 (m)
N6 (m)
BDS
BDS
440
440
166
440
440
Heller
Heller
Heller
Heller
Heller
MS
MS
Vit B5
Pro 230
Myo
100
Malt ext
100l
2,4-D 1
AIA 0.2
ANA
0.2
Kin 0.1
Vit B5
glu 150
Myo
100
Malt ext
100
2,4-D
0.1
kin 0.5
Vit B5
Vit
glu 150 Morel
Myo
100
Malt ext
100
ANA
0.5
BAP
2.5
60g
Vit B5
Pro 230
Myo
100
Malt ext
100
2,4D0.5
AIA 0.2
ANA
0.2
Kin 0.1
45g
Vit B5
glu 150
Myo
100
Malt ext
100
2,4D0.3
BAP
0.1
Sucros
e
Vit B5
Pro 230
Myo
100
Malt ext
100
2,4D0.5
AIA 0.2
ANA
0.2
Kin 0.1
60g
45
45
45
20
Growth of cell suspension
cultures
pack cell volume (ml)
14
12
10
RR
MES
MOL
PRI
8
6
4
2
0
0
1
2
3
4
culture period (weeks)
5
6
multiplication rate every 2
weeks
Effect of age of cell suspension
culture on its multiplication rate
3
2,5
2
RR
MES
MOL
1,5
1
0,5
0
2
4
6
months old
8
10
nb embryos/ml suspension
Effect of age of cell suspension
culture on its regeneration
45
40
35
30
25
20
15
10
5
0
RR
MES
MOL
2
4
6
8
month old of suspension
10
nb embryos/ml suspension
Effect of cell density at plating
on number of embryos
regenerated
60
RR
MES
MOL
PRI
40
20
0
RR
MES
MOL
PRI
0,01
9
6
19
26
0,05
0,1
0,3
25
37
18
15
28
11
32
41
24
38
52
31
cell density (ml cell/20 cm2)