concentrations of
D
-glucose subjected to the same procedure.
Sucrose levels were evaluated following the method described by Paek et al. [16]. A solution
100 ml of invertase 10 mg of commercial inver- tase in 10 ml of 0.2 M sodium acetate buffer, pH
4.5 plus 100 ml of distilled water were added to 100 ml of ethanolic extract, the solution was heated
in a water bath at 55°C for 10 min and then, 200
m l of Somogyi reagent was added and the mixture
was boiled for 20 min. Then, 200 ml of Nelson reagent were added, plus 2.4 ml of distilled water.
Finally, the mixture was shaken and absorbance was measured at 540 nm. Sucrose concentration
was determined by calculating the difference be- tween the measurement of sugars obtained when
the enzyme was added to the extract and the measurement of reducing sugars initially present in
the extract.
2
.
3
. Extraction and e6aluation of starch This was carried out following the method of
Gordon et al. [17], with slight modifications. Callus 0.5 g were boiled in 10 ml of ethanol
for 2 or 3 min. After boiling, the mixture was filtered under vacuum using Albet no. 1305 paper.
The residue was dried in an oven at 30°C until constant weight was attained. The material was
ground in a mortar to a fine powder, which was used for starch evaluation.
The powder obtained from each sample was divided into two equal parts and placed in cen-
trifuge tubes. Distilled water 4 ml was added and the tubes were allowed to incubate for 1 h at
100°C. After cooling, 1 ml of a solution of amy- loglucosidase was added to one of the tubes. Amy-
loglucosidase was prepared by dissolving 25 mg in 0.05 M sodium acetate buffer, pH 4.5 and cen-
trifuging at 3000 × g for 5 min. The supernatant, before being brought up to 25 ml with the same
buffer, was passed through 10 ml Bio-cel gel columns Bio-Rad. The control tube received 1 ml
of 0.05 M sodium acetate buffer, pH 4.5 was added. Both tubes, covered with aluminium foil,
were incubated for 8 h at 50°C. The tubes were then allowed to cool and centrifuged for 10 min at
3000 × g.
For starch evaluation, 0.5 ml of each superna- tant was incubated for 1 h at 30°C with 2 ml of an
enzyme solution prepared with 8 ml of glucose-6- phosphate-dehydrogenase G-6-PDH and 20 ml of
hexokinase in 50 ml of enzymic buffer the latter consisted of a mixture of 3.4 g of imidazole, 4.06 g
of MgCl
2
· 6H
2
O, 800 ml of distilled water and 0.2 g of bovine serum albumin. The mixture was
adjusted to pH 6.9 with HCl and was taken up to a volume of 1 l with distilled water, then adding
750 mg of NAD and 600 mg of ATP freshly prepared. The mixture was kept at 4°C until
used. After the incubation time, absorbance was determined at 340 nm. The results were deter-
mined according to straight line equation obtained with different starch concentrations, subjected to
treatment with amyloglucosidase and evaluated as described above.
As blanks the following were used: a 2 ml of enzymic buffer and 0.5 ml of distilled water; b 2
ml of enzymic solution and 0.5 ml of extract obtained without amyloglucosidase; c 2 ml of
enzymic solution, 0.25 ml of distilled water and 0.25 ml of the amyloglucosidase solution; d 2 ml
of distilled water and 0.5 ml of extract incubated with amyloglucosidase; and e 2 ml of enzymic
solution and 0.5 ml of a solution of potato starch SIGMA 0.1 g in 100 ml of distilled water.
2
.
4
. Statistical analysis Results were analysed statistically by analysis of
variance using the SPSS programme in the version SPSS 8.0 for Windows. When analysis of variance
showed treatment effects P B 0.01 and P B 0.05, the least significant difference Fisher LSD test
was applied to make comparisons between means at the 0.01 and 0.05 levels of significance. Each
datum shows the average of three replicates; ex- periments were carried out in triplicate.
3. Results and discussion
In this study, after testing with a variety of media with different concentrations of growth reg-
ulators, we have chosen four media cultures MS, H8, F0 and F6 specified in Section 2 and four
types of explants cotyledons, petioles, hypocotyls and leaves from M. arborea L. subsp. arborea
seedlings. The culture of the explants in MS medium induced non-embryogenic calli with all
types of explants, during the 5 months of culture. The use of H8 medium induced embryogenic calli
when cotyledons, petioles and hypocotyls were used as explants. Use of petioles as explants pro-
duced the highest frequency of somatic embryos 36.1 9 2.6 in the third month of culture and the
highest average number of somatic embryos per callus 2.3 9 0.4 in the third month of culture.
When leaves were used as explants in H8 medium non-embryogenic calli were induced. Callus trans-
fer from medium H8 to medium F0 lacking KIN and with lower 2,4-D concentrations after 2
months of culture induced development except when leaves were used as explants of large em-
bryogenic efficiency per callus frequency of so- matic embryos per number of somatic embryos:
17.88, 27.49 and 0.40 in the fifth month of culture, or in the third month of transference, when cotyle-
dons, petioles and hypocotyls, respectively, were used as explants. Transfer of calli at 2 months of
culture from medium H8 to F6 did not induce embryogenic calli. The embryos obtained were
able to germinate and give rise to seedlings.
Embryogenic calli, on initiation of the culture, were characterised by their yellowish-white colour,
their friability and their slower growth rates. Non- embryogenic calli were green, hard and faster
growing.
In 1985, Rawal et al. [18] reported that carbohy- drate metabolism plays an important role in
organogenesis. With this in mind, in our studies, we considered that it could also play a fundamen-
tal role in embryogenesis and therefore we began our efforts by analysing total sugars to see
whether there might be significant differences in the different calli.
On studying total sugars, a striking observation was that these sugars were the major components
Table 1, accounting for about 90 of the total dry weight of the calli. This was logical since the
synthesis of structural polysaccharides must have taken place [19] along with that of starch, as
demonstrated in the same type of callus. Regard- ing these components, there were almost no differ-
ences between embryogenic H8 and F0 media and non-embryogenic MS and F6 media calli,
unlike that reported by Naidu and Kishor [20] for organogenesis. However, it is possible that al-
though no differences were seen in the total sugar content, there could be differences in the compo-
nent sugars, as was observed by Kikuchi et al. [21] on studying the content of neutral and acidic
sugars in the pectic fraction of the cell wall of embryogenic and non-embryogenic calli.
Reducing sugars are important in callus forma- tion and differentiation because they are necessary
for the formation of reserves and cell wall polysac- charides [22]. With the different media and ex-
Table 1 Variation in total sugar contentdry weight mg g
− 1
DW in calli kept in the different media, using cotyledons, petioles and hypocotyls from M. arborea L. seedlings as explants
Culture media Explants
Culture times months 4
3 5
2 1
Cotyledon 868.96 9 2.1
MS 903.00 9 1.8
903.70 9 8.2 904.00 9 7.8
919.05 9 8.5 903.00 9 7.5
892.84 9 8.8 892.76 9 8.8
890.10 9 3.7 862.90 9 2.5
Petiole 909.39 9 6.4
909.34 9 1.7 904.70 9 4.3
919.47 9 7.3 Hypocotyl
909.32 9 6.4 858.27 9 5.3
868.97 9 2.4 869.02 9 2.8
870.13 9 4.1 Leaf
848.21 9 2.6 915.34 9 1.7
917.22 9 6.5 917.41 9 8.1
H8 918.04 9 7.3
Cotyledon 911.26 9 3.9
923.12 9 6.1 921.92 9 5.8
919.24 9 4.1 917.23 9 4.7
908.12 9 4.7 Petiole
918.24 9 9.1 921.13 9 6.6
921.92 9 7.8 922.04 9 8.3
Hypocotyl 903.12 9 4.3
Leaf 845.12 9 3.4
853.27 9 3.8 864.21 9 4.9
864.97 9 6.2 869.32 9 5.3
921.16 9 3.1 919.31 9 6.8
– 923.04 9 6.3
– Cotyledon
F0 Petiole
– –
918.13 9 4.7 920.02 9 3.5
921.42 9 4.8 Hypocotyl
– –
923.12 9 9.1 923.98 9 3.9
925.02 9 6.7 Leaf
– –
866.43 9 3.8 866.83 9 3.5
868.22 9 6.1 F6
Cotyledon –
– 916.24 9 4.1
916.81 9 3.3 918.04 9 5.1
917.93 9 4.7 917.64 9 7.3
915.02 9 3.7 –
Petiole –
Hypocotyl –
– 918.47 9 8.7
917.07 9 3.6 919.12 9 4.3
Leaf –
– 868.08 9 5.5
868.97 9 3.8 869.23 9 3.4
Table 2 Variation in percentage of reducing sugarstotal sugars in calli kept in the different media, using cotyledons, petioles and
hypocotyls from M. arborea L. seedlings as explants
Culture media Culture times months
Explants 1
2 3
4 5
15.38 17.60
MS 28.37
Cotyledon 21.57
10.51 Petiole
20.30 22.61
20.13 13.45
12.20 12.77
20.72 27.51
23.01 13.99
Hypocotyl 11.27
16.62 21.60
Leaf 17.03
8.61 20.18
32.22 H8
38.22 Cotyledon
37.96 32.01
28.77 34.49
35.33 Petiole
31.80 19.78
Hypocotyl 18.19
31.00 33.23
30.95 26.03
12.12 14.09
22.07 Leaf
16.81 9.84
F0 Cotyledon
– –
33.72 31.70
30.80 –
– 34.17
34.45 34.26
Petiole –
– 31.41
Hypocotyl 31.68
31.70 –
– 23.87
20.39 Leaf
9.22 –
– F6
19.76 Cotyledon
17.59 10.48
– –
27.69 Petiole
19.18 8.21
– –
30.16 Hypocotyl
22.98 12.04
– –
20.65 Leaf
16.59 9.24
plants, the content of these types of sugars was studied to check whether there were any important
differences between embryogenic and non-embry- onic calli. Table 2 shows the percentages of reduc-
ing sugars with respect to total sugar content. Reducing sugars ranged between 8.21 and 38.22,
depending on the explants, media, and culture times used. The highest contents were observed
when the calli were embryogenic, such as those obtained from cotyledons, petioles and hypocotyls
in H8 and F0 media. In F0 medium, a few somatic embryos per callus were also obtained from leaves
0.62 – 0.68 on finalising culture but, perhaps ow- ing to their low embryogenic capacity, the calli thus
formed only had a slightly increased percentage of reducing sugars in comparison with non-embryonic
calli obtained with the same explant in other media.
In the light of the results, it would appear that there are important differences in reducing sugar
contents and in the evolution of such sugars be- tween embryogenic and non-embryonic calli. Possi-
bly, the higher levels of this type of sugar could be due to lower starch contents Table 3 found in
embryogenic calli, in agreement with the findings of Naidu and Kishor [20] for organogenic calli. These
authors also demonstrated that organ-forming calli have higher reducing sugar contents, in agreement
with what we observed here for embryogenesis. In all cases, the highest percentage of reducing sugars
was obtained during the third month of culture. A characteristic feature of in vitro culture is the
rapid appearance of starch in callus cells [1,9], marked differences existing between embryogenic
and non-embryogenic cells [23]. In view of the results obtained upon evaluating
starch in the calli developed in basal MS medium Table 3, the high levels in the first 2 months of
culture, mainly when hypocotyls and leaves were used as explants, is striking. In general, starch
levels decreased sharply between the second and third month of culture; this decrease continued
very slowly as callus development progressed.
With respect to medium H8 Table 3, in 1- month-old calli, starch contents were lower than
those observed in calli of the same age using MS medium. A striking observation when H8 medium
was used was a rapid decrease in callus starch contents from the second month of culture. The
levels of this polysaccharide remained very low from the second to the fifth month of development.
A clear difference was seen when leaves used as explants: the starch content did not undergo the
spectacular decrease recorded for the other ex- plants and the polysaccharide levels remained high
even during the fifth month of culture.
On comparing the results obtained with MS and H8 media Table 3, the differences are important,
not only with regards to the starch contents at the beginning of culture but also with regards to the
variations in its levels over the 5 months of callus development. During the first month of culture,
the calli produced in MS medium had higher starch contents than those obtained with H8
medium. As culture time progressed, the faster decreases and the very low calli starch content,
induced in medium H8, were striking.
Callus transfer from medium H8 to F0 Table 3 revealed a decrease in the levels of the polysaccha-
ride 1 month after the calli had been transplanted from medium H8. However, unlike the situation
of calli in medium H8, a small increase in starch levels occurred as the calli developed in medium
F0 Table 3. Leaves, which in this medium were also unable to induce embryogenesis, produced
calli whose development with regards to starch content was completely different. Thus, a continu-
ous starch decrease was seen during the last 3 months of culture in medium F0, similar to that
observed for medium H8.
Transfer of calli at 2 months of culture from medium H8 to F6 Table 3 revealed, during the 5
months of culture, a higher starch content than that detected in embryogenic calli developed in F0
medium. The use of leaves as explants pointed to a different kind of behaviour since the levels of
starch were higher in the calli and decreased as culture progressed.
Study of the starch contents of calli induced and developed in different media, revealed two inter-
esting aspects 1 at the beginning of callus devel- opment there were higher levels of starch in
non-embryogenic calli formed in MS medium; 2 during the development of somatic embryogenesis,
callus starch levels were very low. The very slight increase detected in calli obtained in F0 medium
towards the end of development could be due to the accumulation of starch grains in developing
somatic embryos.
Several authors have reported that at the begin- ning of in vitro developmental processes cells have
high starch levels [1,8,9]. This was the case of calli obtained from M. arborea since during the first
month of development the starch content ranged between 9.05 and 44.5 of callus dry weight.
However, many of the authors consulted report higher starch levels in embryogenic than in non-
embryogenic calli [10,23]. Our results are not in agreement with those authors’ reports since during
the first month of development, the highest starch levels were detected in non-embryogenic calli ob-
tained in MS medium. In agreement with Branca
Table 3 Variation in percentage of starchtotal sugars in calli kept in the different media, using cotyledons, petioles and hypocotyls from
M. arborea L. seedlings as explants
Culture media Explants
Culture times months 4
3 5
2 1
Cotyledon 31.66
MS 32.70
16.22 14.08
13.29 16.71
19.13 20.27
21.89 18.55
Petiole 18.35
60.51 49.21
14.11 Hypocotyl
17.92 49.33
30.12 26.78
22.15 Leaf
49.48 7.26
3.13 3.40
H8 3.34
Cotyledon 23.38
2.48 2.67
3.26 4.23
9.96 Petiole
6.13 2.51
2.52 1.45
Hypocotyl 21.95
Leaf 19.11
19.15 15.26
13.29 10.56
3.30 1.88
– 3.45
– Cotyledon
F0 Petiole
– –
2.41 3.16
3.98 Hypocotyl
– –
2.11 2.36
3.08 Leaf
– –
7.74 6.35
4.21 Cotyledon
– –
F6 7.82
7.08 6.84
4.75 4.85
5.27 –
– Petiole
Hypocotyl –
– 6.49
5.41 4.97
Leaf –
– 17.40
14.77 13.22
Table 4 Variation in percentage of sucrosetotal sugars in calli kept in the different media, using cotyledons, petioles and hypocotyls from
M. arborea L. seedlings as explants
Culture media Culture times months
Explants 1
2 3
4 5
14.40 11.81
MS 9.16
Cotyledon 7.75
4.58 17.66
16.35 10.31
Petiole 6.22
4.96 11.38
12.50 7.72
Hypocotyl 5.50
3.05 15.55
Leaf 15.32
17.91 15.56
5.41 12.62
9.31 H8
9.30 Cotyledon
5.23 1.90
9.31 8.78
7.63 Petiole
5.45 4.46
12.51 12.19
11.43 Hypocotyl
4.23 3.05
Leaf 16.23
13.75 12.07
7.88 3.10
– –
F0 9.88
Cotyledon 5.53
2.61 Petiole
– –
8.02 5.25
3.72 Hypocotyl
– –
11.50 4.09
2.89 –
– 13.13
Leaf 11.15
5.31 –
– F6
10.70 Cotyledon
12.47 11.52
– –
9.25 Petiole
9.53 9.89
– Hypocotyl
– 12.71
13.22 12.95
– –
9.34 9.33
Leaf 7.30
et al. [6], we also believe that starch is accumulated under all conditions during the first days of callus
development. It is clear that the decrease in starch levels in embryogenic calli has already occurred
after 1 month of development. For example Mangat et al. [1] observed that starch rapidly disappears
during organogenesis. These authors suggested that since organogenesis requires much energy, this
energy could come from the degradation of starch. This degradation gives rise to the formation of
glycolytic intermediates that, when catalysed oxida- tively, supply high levels of ATP that can be used
by cellular metabolism.
We believe that the explanation offered by Man- gat et al. [1] could also be applied to somatic
embryogenesis. The formation and development of somatic embryos is a process that logically requires
a considerable amount of energy. This energy could come from starch catabolism as the somatic em-
bryos develop. This in turn would account for the lower starch levels detected in embryogenic calli.
The slight increase in the starch contents of embryogenic calli observed during the last 3 months
of development Table 3 could be due to the formation of small starch grains in the somatic
embryos themselves. In fact, in somatic embryos from Medicago sati6a L., starch is accumulated as
the main source of carbon [24]. According to the literature and the results ob-
tained in the present study, starch seems to play a crucial role in the induction of any type of callus [10]
and, above all, in the development of somatic embryogenesis.
Normally, sucrose is the main carbon source for the synthesis of starch in seeds and it also plays an
important role in starch formation in calli. We observed lower levels of starch in embryogenic than
in non-embryogenic calli. Our aim in analysing sucrose in calli was to determine whether, as hap-
pens with starch, there were any differences in the contents of this important disaccharide between
embryogenic and non-embryonic calli. Considering the different media and explants, sucrose was seen
to represent between 1.90 and 17.66 of the total amount of sugars Table 4. The highest percentages
of the disaccharide were found in calli obtained in MS medium. The percentage of sucrose decreased
in all calli as culture time progressed, except when they were transferred from H8 to F6 medium. In the
embryogenic calli, the percentages of sucrose were much lower than in the non-embryogenic ones.
This seems to be consistent with what has been reported by Lai and McKersie [25], who considered
that sucrose must be used as a carbon source for the in vitro formation of somatic embryos. How-
ever, during the induction period, no appar-
ent changes were seen between embryo-producing calli and non-embryogenic calli. The decrease in
sucrose contents, which in general is detected in all calli, may be due — as suggested by Sung et al. [6]
— to their hydrolysis, to provide the hexoses used as substrate for respiration and the synthesis of
structural polysaccharides and starch formation. By contrast, large differences are seen between the
results obtained in the calli transferred to F0 medium, which has the greatest embryogenic ca-
pacity, and medium F6, which inhibits the em- bryogenesis
induced by
medium H8.
This inhibition seems to coincide with an increase in
sucrose contents in the calli. With the exception of sucrose during the first 2
months of culture, in all cases the statistical stud- ies conducted disclosed a highly significant triple
interaction P B 0.001 between culture time, ex- plant and medium. In the case of sucrose during
the first 2 months of culture, all the double inter- actions
culture time – explant,
culture time –
medium and
explant – medium were
highly significant P B 0.001.
4. Conclusions
