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
3
.
1
. Fatty acids in total lipids The fatty acid composition of total lipids in
leaves of Cichorium ‘‘474’’ submitted to somatic embryogenesis is shown in Fig. 1A. In the con-
trol leaves D0, linolenic acid 18:3 was the ma- jor fatty acid 57.7, followed by linoleic acid
18:2 18.3 and then palmitic acid 16:0 12.7. The presence of 18:3 is characteristic of
photosynthetic tissues. A considerable decrease of the 18:3 proportion was observed during the in-
duction step 38 at 5th day and the expression step 17 at 5 + 7th day. Three days after
transfer in the expression medium D5 + 3, 18:2 became major fatty acid and it reached a max-
imun level at day 5 + 7 43.7 when globular embryos were observed, while the proportion of
18:3 was reduced to 15.7. The saturated fatty acid proportion remained similar during the two
phases.
In the non-embryogenic genotype ‘Pe´ve`le’, at D0, the fatty acid composition of total lipid frac-
tion is similar to the one observed in the control leaves of Cichorium ‘474’ Fig. 1B. However,
throughout the 12 days of culture period, the relative
proportions of
total fatty
acids in
‘Pe´ve`le’ leaves were different from those obtained for the ‘474’ leaves. The principal difference con-
sisted in the changes in relative proportions of polyunsaturated acids. The increase of 18:2 pro-
portion is also lower for the ‘Pe´ve`le’ genotype than the embryogenic genotype. At day 5 + 7,
18:3 and 18:2 were present in equal proportion about 33.
3
.
2
. Fatty acids in neutral and polar lipids Fatty acid compositions in polar and neutral
lipid fractions of Cichorium ‘474’ leaves during somatic embryogenesis are presented in Table 1.
The principal changes in fatty acid content of polar lipids concerned the C18 unsaturated fatty
acids. At day 0, 18:3 was the major component and its proportion reached more than 55 in
polar lipids. Its proportion decreased while the amount of 16:0 increased during the induction
phase and the 18:2 one increased during the ex- pression phase. At day 12, the polar lipid frac-
tion contained more 18:2 and less 18:3.
Fig. 1. Changes in total fatty acid composition between two genotypes of chicory leaf tissues cultured in embryogenic
conditions. A: embryogenic genotype Cichorium ‘474’; B: non-embryogenic genotype chicory ‘Pe´ve`le’. Three leaf frag-
ments were incubated for 5 days in an induction medium containing 330 mM glycerol and transferred for 7 days in an
expression medium without glycerol. Individual fatty acids are expressed as percentage of the total fatty acids. Data are
mean of three separate experiments and the bars represent the standard deviation.
Table 1 Fatty acid composition of neutral and polar lipids in Cichorium ‘474’ leaf fragments during somatic embryogenesis
a
Lipid classes Fatty acid composition of total fatty acids
16:0 18:0
18:1 Days of culture
18:2 18:3
29.5 Neutral lipids
15.8 16.8
11.9 17.3
30.5 14.2
18.6 3
13.9 13.9
24.3 11.6
15.5 17.8
5 22.4
25.4 10.3
22.6 5+3
20.2 12.8
5+7 24.2
11.5 11.8
29.8 13.8
16.6 2.2
Polar lipids 3.0
19.1 55.6
19.8 3.6
4.6 3
28.4 39.8
23.3 5.5
5.2 5
21.7 37.4
5+3 24.2
4.5 6.3
29.7 30.5
24.2 4.1
10.5 5+7
38.0 21.3
a
Three leaf fragments were incubated for 5 days in an induction medium containing 330 mM glycerol and transferred for 7 days in an expression medium without glycerol. Neutral and polar lipids were separated by TLC. Data are means of three independent
replicates. Table 2
Distribution of the different lipid classes in Cichorium ‘474’ leaf fragments during somatic embryogenesis
a
Lipid classes Percentages of different lipid classes
Day 3 Day 5
Day 0 Day 5+3
Day 5+7 Neutral lipids
b
22.6 21.1
12.3 23.0
TAG 32.0
16.6 15.4
16.4 18.9
DAG 20.1
25.2 26.1
30.1 21.7
MAG 17.8
35.6 37.4
38.9 31.3
SE 37.5
Polar lipids
c
25.4 MGDG
23.4 30.0
17.2 10.2
18.6 24.3
18.5 DGDG
16.7 25.8
18.0 22.5
16.4 22.8
PC 24.5
10.2 12.0
PE 13.4
7.4 17.7
7.4 10.8
6.5 16.2
PI 14.2
20.4 7.0
11.9 16.7
Others compounds 13.9
a
Three leaf fragments were incubated for 5 days in an induction medium containing 330 mM glycerol and transferred for 7 days in an expression medium without glycerol. Data are means of three independent experiments.
b
Neutral lipids comprised triacylglycerols TAG, diacylglycerols DAG, monoacylglycerols MAG and steryl esters SE.
c
Polar lipids were composed of monogalactosyldiacylglycerol MGDG, digalactosyldiacylglycerol DGDG, phosphatidyl- choline PC, phosphatidylethanolamine PE and phosphatidylinositol PI. The other compounds were phosphatidic acid,
phosphatidylserine and another unidentified polar lipid.
Palmitic acid was the predominant fatty acid in the neutral lipid fraction. The principal change
was a progressive increase in the proportion of 18:2. Linoleic acid became the major fatty acid at
day 12. There was only a little change in the relative proportions of the principal saturated
fatty acids, 16:0 and 18:0.
3
.
3
. Identification of lipid classes in total lipids Total lipids were separated in neutral and polar
lipids and analysed by thin layer chromatography. Changes occurring in the major lipid components
are presented in Table 2. For polar lipids, galac- tolipids 55 were more abundant than phospho-
lipids in Cichorium ‘474’ control leaves. Mono- galactosyldiacylglycerol MGDG 30 and di-
galactosyldiacylglycerol DGDG 25.8 were the most abundant polar lipids. This result is in gen-
eral agreement with other reports on green leaves. The proportions of the two galactolipids declined
rapidly especially during the expression phase where, at day 5 + 7, MGDG and DGDG percent-
ages were 10.2 and 16.7, respectively. It is evi- dent that the galactolipid proportions decreased,
while the phospholipid proportions increased. The major phospholipid was phosphatidylcholine PC
which showed an increase of its ratio from 16.4 at day 0 to 24.5 at day 5 + 7. The other phos-
pholipids were phosphatidylethanolamine PE and phosphatidylinositol PI.
The greatest change in the neutral lipid was observed in the triacylglycerols content TAG. In
the control leaves, TAG were minor components of the neutral lipid 12.3. During the expression
phase, TAG showed a significant increase of its ratio 32.
3
.
4
. Lipid incorporation of
[
14
C
]
glycerol In order to investigate the incorporation of glyc-
erol into lipids, [
14
C] glycerol was supplied to culture medium of foliar fragments at day 0. Fo-
liar fragments were collected at days 3 and 5 of the induction step and days 5 + 3 and 5 + 7 of the
expression phase after transfer at day 5 onto a glycerol-free medium. Following fractionation into
neutral and polar lipids, the percentage of each classes was determined by total [
14
C] incorporated. Individual classes were separated by TLC and the
five major lipid components PC, PE, MGDG, DGDG and TAG were identified and quantified
by measuring their total radioactivity.
During the induction step, labeled lipid classes were almost exclusively phospholipids and galac-
tolipids Fig. 2. The two major labelled phospho- lipids were PC and PE. The percentage of
incorporation of labelled glycerol in PC increased to 45 at the end of the induction phase then
decreased during the expression phase. However, PC was always the most strongly labelled individ-
ual lipid throughout the culture period. PE was found to be markedly labelled during the expres-
sion step. A large enrichment of labelled com- pound was also seen for MGDG and DGDG,
known to be plastidial lipids. The detected ra- dioactivity was more important in DGDG than in
MGDG overall the culture. This result is in gen- eral agreement with other reports on non-green
plastids and could indicate the presence of pro- plastids. It could be noticed that this experience
revealed a similar pattern of incorporation among these two galactolipids.
During the induction phase the incorporation of labelled glycerol was lower for TAG than for
other lipid classes. The radioactivity drastically increased in TAG from 5 at day 5 up to 17 at
day 5 + 3. So, an important increase of radioactiv- ity in TAG was noted during the expression phase.
3
.
5
. Fatty acids in PE, PC and TAG The acyl composition of TAG and main mem-
brane lipids, PC and PE, was examined and the results are shown in Table 3. At day 0, there were
not significant differences in the acyl composition of PC and PE. Both fractions contained 16:0 and
18:2 as their major moieties. Changes in fatty acid patterns of PC and PE were similar during the
induction and expression phases. The percentage of 18:2 in both PE and PC was upper to 30 at
day 5 + 7. There was little difference in the relative proportions of saturated fatty acids in these two
phospholipids throughout the culture period. These phospholipids have a distinct composition
compared to the storage TAG. The triacylglycerol fraction contained relatively high amount of satu-
Fig. 2. Incorporation of [U-
14
C] glycerol into the main lipid classes in Cichorium ‘‘474’’ leaf tissues during somatic em-
bryogenesis. Three leaf fragments were incubated for 5 days in 20 ml of a medium containing 9.25 KBq [
14
C] glycerol and transferred for 7 days in a medium without glycerol. The
proportion of incorporated
14
C in each lipid class was calcu- lated in comparison with the addition of radioactivity in
TAG, MGDG, DGDG, PC and PE. The values represent the mean of three separate experiments.
Table 3 Fatty acid composition of different lipid classes in Cichorium ‘474’ leaf fragments during somatic embryogenesis
a
Lipid classes Fatty acid composition of total fatty acids
16:0 18:0
Days of culture 18:1
18:2 18:3
40.3 13.6
13.9 10.6
TAG 12.9
18.1 6.8
13.7 27.9
3 24.4
24.1 9.3
9.9 5
21.6 26.0
5+3 22.1
8.0 7.9
30.3 25.1
34.0 13.2
7.5 5+7
31.0 4.7
PE 27.3
9.8 18.0
19.5 15.6
27.4 6.2
11.3 3
35.4 13.2
27.9 8.0
14.3 5
25.4 18.6
5+3 27.4
10.0 12.9
27.8 11.4
28.4 11.5
9.3 32.3
5+7 6.9
25.9 5.9
PC 11.5
26.0 25.0
24.0 5.7
10.7 3
35.8 21.1
28.8 7.4
11.1 5
25.3 24.0
5+3 30.2
8.1 14.7
28.9 14.5
27.3 9.0
10.0 36.3
5+7 11.6
a
Three leaf fragments were incubated for 5 days in an induction medium containing 330 mM glycerol and transferred for 7 days in a expression medium without glycerol. Lipids were separated by TLC and fatty acid methyl esters were analyzed by GC. Data
are means of three independent replicates.
rated fatty acids 54 and low amount of polyun- saturated acids. In the control leaves, triacylglyc-
erols were mainly enriched in 16:0 40.3. At day 5 + 7, the 18:2 proportion was more important
and reached 31, 16:0 being the major fatty acid 34.
4. Discussion
