European project « 5th PCRDT »

GARLIC AND HEALTH

Third year progress report

Half year meeting Montpellier 1-2 october 2002

ADVANCEMENT OF THE Works

Scientific and administrative staff

• L. FEREOL scientist– Duties: somatic embryogenesis,

histology and flow cytometry

• S. CAUSSE technical assistant– Duties: somatic embryogenesis,

histology and flow cytometry

• M. Roux-Cuvelier– Duties: field trials in  Reunion

• R. Kahane– Duties: management

Objectives

• Setting a somatic embryogenesis procedure

• Comparative characterisation of plantlets from somatic embryos and from classic multiplication by meristem culture (as control)

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 cultures of these varieties

• 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

Production and germination of somatic embryos « Rouge Reunion » from callus and cell suspension cultures

√

Provision of 200 plantlets from embryogenic calli, and from cell suspension cultures « Rouge Reunion »

√

Production of embryogenic callus, and initiation of embryogenic cell suspension cultures « Messidrome, Morasol »

√

Production and germination of somatic embryos from callus and from cell suspension culture « Messidrome, Morasol »

√

√ = achieved; partly achieved; to be done)�

Milestones of the second year

Genetic characterisation of these plants by flow cytometry « Messidrome », « Morasol »

Provision of 200 plantlets from embryogenic calli, and from embryogenic cells suspension cultures of « Messidrome », « Morasol »

√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)

√

√

Milestones of the second year (continue)

Production and germination of somatic embryos from callus and cell suspension cultures « Printanor »

√

√

Production of embryogenic callus, and Initiation of embryogenic cell suspension cultures « Printanor »

Milestones of the third year

Promotion of the embryogenesis procedure

Genetic characterisation of these plants by flow cytometry « Printanor »

√Provision of 200 plantlets from embryogenic calli, and from embryogenic cells suspension cultures of « Printanor »

Embryogenesis from callus

Friableembryogeniccallus

Globular somatic embryos

h

Matureembryo

MORPHOLOGY HISTOLOGY

Embryogenesis from cell suspension culture

Embryogeniccell suspensionculture established

Conversion into plantlets and then bulbification

Regeneration30 days after plating

Regeneration of cell suspension after plating

Globular embryos

Varietal effect on somaticembryogenesis from callus

induction of embryogenic tissues

0%

10%20%

30%

40%

50%60%

70%

80%

1

varieties

% e

mbr

yoge

nic

callu

s RR

MES

MOL

PRI

Induction of embryogenic tissues

Embryo induction

0102030405060708090

1

varieties

Mean

nbr

of s

.e./1

50 m

g of

ca

llus

RR

MES

MOL

PRI

Somatic embryo induction

Conversion of embryos into plantlets

0

1020

30

40

5060

70

80

1

varieties

% ge

rmin

atio

n

RR

MES

MOL

PRI

Conversion into plantlets

Significant effect on the different parameters of somaticembryogenesis from callus

Varietal effect on somaticembryogenesis from cell

suspension culture

Growth of cell suspension culture

1

2

3

1

Varieties

Grow

th ra

te

RR

MES

MOL

PRI

Growth of the cell suspension

Regeneration after plating

0

2040

60

80

100120

140

160

1

varieties

nbre

of s

.e. /

ml P

CV

RR

MES

MOL

PRI

Regeneration after plating

Conversion of embryos into plantlets

0102030405060708090

1

varieties

% o

f ger

min

atio

n

RR

MES

MOL

PRI

Conversion into plantlets

- No varietal effect on the growth rate- But a significative effecton regeneration and conversion into plantlets

2 X4 X

2 X+4 X

Flow cytometric histogramOf diploid and tetraploid

Somatic embryos

Genetic characterisation by flow cytometry

Nbr. of plants tested

Nbr. of diploid

Nbr.of tetraploid

MES control

12 12 0

MOL control

16 16 0

MES cal 102 93 9

MOL cal 100 97 3

MOL ecs 100 96 4

DeliverablesDP 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. KAHANEPlant Cell Report, september 2002

DP13: paper on: Establishment of garlic (Allium sativum L.) cell suspensions and plant regenerationL. FEREOL, V. CHOVELON, S. CAUSSE, D. TRIAIRE, N. MICHAUX FERRIERE and R. KAHANE

in progressDP21: paper on the use of somatic embryogenesis for rapid multiplication of garlic in vitro.In progress

What will be done

• Promotion of the embryogenesis procedure– Histological studies– Looking for more convenient

conditions for somatic embryogenesis– Maintenance of the material

• MES ecs, PRI ecal and PRI ecs: – Genetic characterisation of plants from

these material, by flow cytometry

• Rouge Reunion ecal and ecs:Field evaluationFlow cytometry of the material after field

culture