out glycerol and cells expressed their embryo- genic competence expression phase. Multicellu-
lar dense embryos appeared 3 days after transfer in the expression medium. The presence of glyc-
erol delays the first division of the embryogenic cell, making of this system a valuable tool to
investigate the early events taking place during somatic embryogenesis. In previous studies, we
have shown the presence of proteins related to somatic embryogenesis during the induction step
in leaf tissues [9,10] and in extracellular condi- tioned medium of leaf explants [11,12].
In this paper, we report the changes in total fatty acid composition in leaf tissues of a nonem-
bryogenic responsive genotype and a embryo- genic
responsive genotype
of Cichorium
submitted to embryogenic conditions. The acyl and lipid class composition was investigated in
Cichorium hybrid ‘474’ during the embryogenic process. Moreover, we labelled plant lipids with
[
14
C] glycerol and we carried out a chase experi- ment during which the labeling of polar and
nonpolar lipids was studied.
2. Materials and methods
2
.
1
. Plant material and condition culture A Cichorium hybrid ‘474’ Cichorium intybus
L. var. sati6um × Cichorium endi6ia L. var. latifo- lia was used as plant material. Plantlets were
obtained as described previously [11]. Three leaf fragments of 6-week-old plantlets were precul-
tured 5 days at 35°C in darkness in 20 ml of an agitated basal medium supplemented with 60
mM sucrose and 330 mM glycerol. Addition of glycerol allowed activation of the mesophyll cells
for embryogenic competence without cell divi- sions [8]. This glycerol pretreatment delayed the
first division of the embryogenic cells until trans- fer of the 5-day-old induced leaf fragments on an
expression medium devoid of glycerol. The same experience was realised with a non-embryogenic
genotype of chicory: Cichorium intybus L. var. Pe´ve`le.
2
.
2
. Lipid extraction Leaf fragments were collected, dried on filter
paper and boiled 4 min in 4 ml distilled water to ensure
inactivation of
lipolytic enzymes,
if present. Lipids were extracted using a chloro-
formmethanolwater solvent system as described by [13]. Total lipids were dissolved in 200 ml
chloroform and stored in a freezer.
2
.
3
. Lipid classes separation The lipids extracted by the above described
procedure were separated by thin-layer chro- matography TLC using pre-coated silica gel
20 × 20 cm plates sil G-25, Macherey – Nagel. Neutral lipids were separated using a solvant sys-
tem consisting of hexanediethyl etheracetic acid 78:20:4 vvv. Polar lipids were fractionated in
chloroformacetonemethanolacetic
acidwater 50:20:10:10:5 vvvvv. Lipids were located by
spraying the plates with a solution of primuline 0.001 in 20 acetone, and viewed under ul-
traviolet light. Each lipid classes were identified by cochromatography with authentic standards
Sigma. Individual lipids were then scraped from the plates, recovered by dynamical elution with
chloroformmethanolacetic acid 2:1:0.5 vvv and analyzed for their fatty acid composition as
described below. An appropriate amount of hep- tadecanoic acid was used as an internal standard.
2
.
4
. Fatty acid composition determination Fatty acid methyl esters were produced from
total lipid fraction and from each lipid classes. Lipids were heated at 65°C for 60 min in 4 ml of
95 ethanol containing 0.4 ml 33 KOH and extracted into hexane after acidification. Released
fatty acids were then methylated by heating for 3 min at 90°C in the presence of 14 boron trifl-
uoride in methanol. The fatty acid compositions were determined by analysing methyl esters using
gas chromatography GC. The fatty acid methyl esters were analyzed on a GC Shimadzu 14 A
with flame ionisation detector with an Alltech Carbowax, capillary column 0.32 mm × 30 m,
0.25 mm thick phase. Separation of fatty acids was achieved under the following conditions: in-
jection port 250°C; detector 250°C; temperature programme 120 – 210 °C at 4°Cmin. The peak
areas were calculated by a Shimadzu C-R5A chromatopac integrator and fatty acids were
identified by comparison with commercial stan- dards Alltech.
2
.
5
. Labelling of lipids At day 0 of the induction step, leaf fragments
were incubated in 20 ml of the medium described above supplemented with 330 mM glycerol and
9.25 KBq [U-
14
C] glycerol 5.59 GBq mmol
− 1
; Amersham Corp. At different days at the em-
bryogenic culture,
radiolabelled leaves
were rinsed with distilled water to remove exogenous
isotope. Lipid extraction and lipid classes separa- tion were performed described above. After visu-
alization under UV light, the bands were scraped off and their radioactivity was counted in a
Beckman
LS 2800
scintillation spectrometer
Beckman Instruments Inc. Irvine, CA.
3. Results
