Different authors have reported the presence of starch in plant cells at the beginning of the in vitro
developmental process [1,8,9]. The possible role of this polysaccharide in these processes remains un-
clear, although it has been suggested that it could act as an energy source or as an essential osmotic
agent for development [10].
Here, we report the variations occurring in con- tent of total sugars, reducing sugars, starch and
sucrose in undifferentiated and embryogenic calli. The calli studied were obtained using four types of
explants from M. arborea L. subsp. arborea seedlings and four different culture media which
induced several responses 1 basal MS medium; 2 F6 medium both induced non-embryogenic
calli; 3 H8 medium and 4 F0 medium both induced embryogenic calli, except when leaves are
used as explants.
In the course of the study of somatic embryoge- nesis, the analysis of metabolic differences between
embryogenic and non-embryogenic calli could be of relevance. We initiate these studies by determin-
ing the total sugars, reducing sugars, starch and sucrose content in calli, with the aim to determine
a possible relationship between sugar contents and somatic embryogenesis.
2. Materials and methods
2
.
1
. Plant material and culture Explants were established from cotyledons, peti-
oles, hypocotyls and leaves of seedlings aseptically obtained from seeds of M. arborea L. subsp. ar-
borea. Seeds were germinated in Murashige and Skoog 1962 medium MS [11] without sucrose
and with the salt concentration reduced to one quarter. Seeds were kept in dark chambers at
25 9 1°C. When the plantlets had achieved a height of 2 cm they were transferred to chambers
with a 16 h photoperiod 20 mE m
− 2
s
− 1
. Plantlets were kept under these conditions for 3
weeks, after which they were used to obtain explants.
The different explants 5 mm slices in transec- tion of cotyledons, petioles, hypocotyls and leaves
were incubated under 16 h photoperiod at a light intensity of 950 Lx 20 mE m
− 2
s
− 1
with Os- ram36 white fluorescent lamps at 23 9 2°C. The
calli were generated either 5 months in MS [11] or 5 months in MS medium supplemented with 2 mg
l
− 1
of 2,4-D and 2 mg l
− 1
of kinetin KIN H8 medium. Some of the calli cultured for 2 months
in H8 medium were then transferred to MS medium supplemented with 0.5 mg l
− 1
of 2,4-D but lacking KIN F0 medium or to MS medium
supplemented with 0.5 mg l
− 1
of 2,4-D and 0.5 mg l
− 1
of KIN F6 medium. Subcultures were performed every 4 weeks. Calli were collected each
month after the start of incubation of the explants in the media and were used to study total sugars,
reducing sugars, starch and sucrose. The experi- ments were repeated three times, using three repli-
cates in every experiment.
2
.
2
. Extraction and e6aluation of total sugars, reducing sugars and sucrose
The method used for extraction was based on that of Nguyen and Paquin [12], with some
modifications. Calli 0.5 g from each culture medium were
rapidly washed in an aqueous solution of 60 mM polyethyleneglycol and then washed several times
with distilled water. They were then ground in a mortar with 5 ml of 95 ethanol and filtered in
vacuo with Albet no. 1305 filter paper. The filtrates were recovered. Residues were washed
again with 70 ethanol, filtered and both filtrates were mixed. Distilled water 3 ml were added to
the mixture, plus 4 ml of chloroform. The mixture was then shaken and stored for 14 h at 4°C and
the upper ethanolic phase were used for analysis.
To evaluate total sugars, the phenol – sulfuric acid method was used [13]. Briefly, 0.5 ml of
distilled water and 0.5 ml of 5 phenol were added to 100 ml of dry ethanolic extract. After
shaking, 2.5 ml of concentrated H
2
SO
4
was added. The mixture was left to stand for 30 min and
absorbance was read at 490 nm. Pure
D
-glucose was employed as standard.
Reducing sugars were evaluated by the method of Somogyi [14] and Nelson [15]. Distilled water
200 ml plus 200 ml of Somogyi reagent were added to 100 ml of ethanolic extract, once dried.
The mixture was boiled for 20 min and, after cooling, 200 ml of Nelson reagent plus 2.4 ml of
distilled water were added. The mixture was shaken to eliminate CO
2
and absorbance was mea- sured at 540 nm. The results were determined
according to standard line obtained with different
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
