Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol154.Issue2.2000:
Differences in the contents of total sugars, reducing sugars, starch
and sucrose in embryogenic and non-embryogenic calli from
Medicago arborea
L.
Ana Belen Martin
a, Yolanda Cuadrado
a, Hilario Guerra
b, Piedad Gallego
a,
Oscar Hita
a, Luisa Martin
d, Ana Dorado
c, Nieves Villalobos
a,*
aDepartamento de Fisiologı´a Vegetal,Facultad de Biologı´a,Uni6ersidad de Salamanca, A6da del Campo Charro s/n(Edificio de la F.de Farmacia),E-37007Salamanca,Spain
bDepartamento de Fisiologı´a Vegetal,Facultad de Farmacia,Uni6ersidad de Salamanca,E-37007Salamanca,Spain cDepartamento de Estadistica,Facultad de Economia y Empresa,Uni6ersidad de Salamanca,E-37007Salamanca,Spain
dLaboratorio de Fisiologı´a Vegetal,Facultad de Biologı´a,Uni6ersidad Complutensede Madrid,E-28040Madrid,Spain Received 21 May 1999; received in revised form 17 November 1999; accepted 22 November 1999
Abstract
The total sugars, reducing sugars, starch and sucrose in embryogenic and non-embryogenic calli from explants (cotyledons, petioles, hypocotyls and leaves) obtained from Medicago arborea L. seedlings were evaluated. Total sugars were the major components in the calli and no significant differences between embryogenic and non-embryogenic calli were observed. In contrast, important differences between the embryogenic and non-embryogenic calli were observed for reducing sugars, the highest levels being observed in embryogenic calli. The highest starch levels were found in non-embryogenic calli developed in MS medium. During the development of somatic embryogenesis very low starch levels in the callus were found. During the first months of culture, no significant differences in the sucrose content were found between calli that produced embryos and those that did not. The most important differences in sucrose were seen between calli transferred to medium F0, which had the greatest embryogenic capacity, and those transferred to medium F6, which inhibited embryogenesis. In the latter case, an increase in sucrose was observed. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Medicago arborea; Total sugars; Reducing sugars; Starch; Sucrose; Embryogenic calli; Non-embryogenic calli
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1. Introduction
Medicago arborea L. subsp. arborea is an ever-green shrub that may be considered a forage plant in dry areas.
One of the possible expressions of the totipotent character of plant cells and tissues is their ability to regenerate into plants via embryogenesis and
for organogenesis. In vitro plant tissue culture normally requires an external source of carbon. This is usually supplied in the medium as sucrose [1,2]. A part of the sucrose in the medium must first be degraded extracellularly and the resulting hexoses are then taken up by the cells [3]. Sucrose is an important hexose storage source and is nor-mally the main carbon source for the synthesis of polysaccharides, after their hydrolysis by sucrose
synthase [4] and/or invertase [5]. Sucrose
hydroly-sis, in the cytosol, is carried out by both acid and alkaline soluble invertases [5] to provide, as they become necessary, the hexoses used as major sub-strates for the synthesis of structural and storage polysaccharides [6,7].
Abbre6iations: 2,4-D, 2,4-dichlorophenoxyacetic acid; DW, dry
weight; F0, kinetin-free MS medium with 2,4-D (0.5 mg l−1); F6, MS medium supplemented with 2,4-D (0.5 mg l−1) and KIN (0.5 mg l−1); H8, MS medium containing 2,4-D (2 mg l−1) and KIN (2 mg l−1); KIN, kinetin; MS, medium of Murashige and Skoog (1962). * Corresponding author. Tel.: +34-23-29-4471; fax: + 34-23-29-4515.
E-mail address:[email protected] (N. Villalobos)
0168-9452/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 9 9 ) 0 0 2 5 1 - 4
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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, reducdetermin-ing 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
2591°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 2392°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 H2SO4was 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 CO2and absorbance was
mea-sured at 540 nm. The results were determined according to standard line obtained with different
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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
100ml of ethanolic extract, the solution was heated
in a water bath at 55°C for 10 min and then, 200
ml 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 20ml of
hexokinase in 50 ml of enzymic buffer (the latter consisted of a mixture of 3.4 g of imidazole, 4.06 g
of MgCl2· 6H2O, 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 (PB0.01 and PB0.05),
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
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when cotyledons, petioles and hypocotyls were used as explants. Use of petioles as explants pro-duced the highest frequency of somatic embryos
(36.1%92.6 in the third month of culture) and the
highest average number of somatic embryos per
callus (2.390.4 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 soso-matic 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 differentiaforma-tion 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 content/dry weight (mg g−1 DW) in calli kept in the different media, using cotyledons, petioles and
hypocotyls fromM.arboreaL. seedlings as explants
Culture media Explants Culture times (months)
4
3 5
2 1
Cotyledon 868.9692.1
MS 903.0091.8 903.7098.2 904.0097.8 919.0598.5 903.0097.5 892.8498.8
892.7698.8 890.1093.7
862.9092.5 Petiole
909.3996.4 909.3491.7
904.7094.3 919.4797.3
Hypocotyl 909.3296.4
858.2795.3 868.9792.4 869.0292.8 870.1394.1 Leaf 848.2192.6
915.3491.7 917.2296.5 917.4198.1
H8 Cotyledon 911.2693.9 918.0497.3 923.1296.1 921.9295.8
919.2494.1 917.2394.7
908.1294.7 Petiole
918.2499.1 921.1396.6 921.9297.8 922.0498.3 Hypocotyl 903.1294.3
Leaf 845.1293.4 853.2793.8 864.2194.9 864.9796.2 869.3295.3 921.1693.1
919.3196.8
– 923.0496.3
– Cotyledon
F0
Petiole – – 918.1394.7 920.0293.5 921.4294.8 Hypocotyl – – 923.1299.1 923.9893.9 925.0296.7 Leaf – – 866.4393.8 866.8393.5 868.2296.1 F6 Cotyledon – – 916.2494.1 916.8193.3 918.0495.1 917.9394.7 917.6497.3
915.0293.7 –
Petiole –
Hypocotyl – – 918.4798.7 917.0793.6 919.1294.3 Leaf – – 868.0895.5 868.9793.8 869.2393.4
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Table 2
Variation in percentage of reducing sugars/total sugars in calli kept in the different media, using cotyledons, petioles and hypocotyls fromM.arboreaL. seedlings as explants
Culture media Explants Culture times (months)
1 2 3 4 5
15.38 17.60
MS Cotyledon 28.37 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 Cotyledon 38.22 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 Cotyledon 19.76 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.
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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 starch/total sugars in calli kept in the different media, using cotyledons, petioles and hypocotyls from
M.arboreaL. 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 Cotyledon 23.38 3.34
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
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Table 4
Variation in percentage of sucrose/total sugars in calli kept in the different media, using cotyledons, petioles and hypocotyls from
M.arboreaL. seedlings as explants
Culture media Explants Culture times (months)
1 2 3 4 5
14.40 11.81
MS Cotyledon 9.16 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 Cotyledon 9.30 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 Cotyledon 9.88 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 Cotyledon 10.70 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
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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 (PB0.001) 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 (PB0.001).
4. Conclusions
There were no differences in total sugars
be-tween embryogenic and non-embryogenic calli.
The highest contents in reducing sugars were
obtained when the calli were embryogenic.
During the development of somatic
embryogen-esis the starch contents in the calli were very low.
In the embryogenic calli, the percentages of
sucrose were much lower than in the non-em-bryogenic calli.
Acknowledgements
This work was supported by a grant from the Direccio´n General de Investigacio´n Cientı´fica and Te´cnica, Spain, No. PB94-1403.
References
[1] B.S. Mangat, M.K. Pelekis, A.C. Cassells, Changes in the starch content during organogenesis in ‘in vitro’ cultured Begonia rex stem explants, Physiol. Plant 79 (1990) 267 – 274.
[2] T.A. Thorpe, In vitro’ organogenesis and embryogene-sis: physiological and biochemical studies, in: K. Roubelakis-Angelakis, K. Tran Thanh Van (Eds.), Morphogenesis in Plants, Plenum Press, New York, 1993, pp. 19 – 38.
[3] F.C. Botha, M.M. O’Kennedy, S. du Plessis, Activity of key enzymes involved in carbohydrate metabolism in
Phaseolus 6ulgaris cell suspension cultures, Plant Cell
Physiol. 33 (1992) 477 – 483.
[4] H.A. Ross, H.V. Davies, L.R. Burch, R. Viola, D. McRae, Developmental changes in carbohydrate con-tent and sucrose degrading enzymes in tuberising stolons of potato (Solanum tuberosum), Physiol. Plant 90 (1994) 748 – 756.
[5] W. Van den Ende, A. Van Laere, Purification and properties of a neutral invertase from the roots of Ci
-chorium intybus, Physiol. Plant 93 (1995) 241 – 248. [6] S.S. Sung, D.-P. Xu, C.C. Black, Identification of
ac-tively filling sucrose sinks, Plant Physiol. 89 (1989) 1117 – 1121.
[7] S. Venkataramana, K.M. Naidu, S. Singh, Invertases and growth factors dependent sucrose accumulation in sugarcane, Plant Sci. 74 (1991) 65 – 72.
[8] P.L. Swarnkar, S.P. Bohra, N. Chandra, Biochemical studies on initiation of callus in Solanum surattense, J. Plant Physiol. 126 (1986) 293 – 296.
[9] C. Branca, A. Torelli, P. Fermi, M.M. Altamura, M. Bassi, Early phases ‘in vitro’ culture tomato cotyle-dons: starch accumulation and protein pattern in rela-tion to the hormonal treatment, Protoplasma 182 (1994) 59 – 64.
[10] J.A. Stamp, Somatic embryogenesis in cassava. The anatomy and morphology of the regeneration process, Ann. Bot. 57 (1987) 451 – 459.
[11] T. Murashige, F. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plant 15 (1962) 473 – 497.
[12] S.T. Nguyen, R. Paquin, Me´thodes d’extraction et de purification des acides amine´s libres et des prote´ines de tissus ve´ge´taux, J. Chromatogr. 61 (1971) 349 – 351. [13] M. Dubois, K.J. Gilles, F. Smith, Colorimetric method
for determination of sugars and related substances, Anal. Chem. 28 (1956) 356 – 361.
[14] M. Somogyi, Notes on sugar determination, J. Biol. Chem. 195 (1952) 19 – 23.
[15] N. Nelson, A photometric adaptation of the Somogyi method for the determination of glucose, J. Biol. Chem. 153 (1944) 375 – 380.
[16] K.Y. Paek, S. Canderlerd, T.A. Thorpe, Physiological effects of Na2SO4 and NaCl on callus cultures ofBras
-sica campestris (Chinese cabbage), Physiol. Plant 72 (1988) 160 – 166.
[17] A.J. Gordon, G.J.A. Ryl, D.F. Mitchell, K.H. Lowry, C.E. Powell, The effect of defoliation on carbohydrate, protein and leg-haemoglobin content of white clover nodules, Ann. Bot. 58 (1986) 141 – 154.
[18] S.K. Rawal, U.N. Dwivedi, B.M. Khan, A.F. Mas-carenhas, Biochemical aspects of shoot differentiation in sugarcane callus: II. Carbohydrate metabolizing en-zymes, J. Plant Physiol. 119 (1985) 191 – 199.
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[19] U.D. Yeo, W.Y. Soh, H. Tasaka, N. Sakurai, S. Kuraishi, K. Takeda, Cell wall polysaccharides of callus and suspension-cultured cells from three cellulose-less mutants of barley (Hordeum6ulgareL.), Plant Cell
Phys-iol. 36 (1995) 931 – 936.
[20] K.R. Naidu, P.B.K. Kishor, Activities of hydrolytic en-zymes in callus cultures of tobacco during organoge´nesis, J. Biosci. 20 (1995) 629 – 636.
[21] A. Kikuchi, S. Satoh, N. Nakamura, T. Fujii, Differ-ences in pectic polysaccharides between carrot embryo-genic and non-embryoembryo-genic calli, Plant Cell Rep. 14 (1995) 279 – 284.
[22] K. Dimassi-Theriou, A. Bosabalidis, Effects of light, magnesium and sucrose on leaf anatomy, photosynthesis,
starch and total sugar accumulation, in kiwi fruit cul-tured in vitro, Plant Cell Tiss. Organ. Cult. 47 (1997) 127 – 134.
[23] G.L. Keller, B.J. Nikolau, T.H. Ulrich, E.S. Wurtele, Comparison of starch and ADP-glucose pyrophosphory-lase levels in nonembryogenic cells and developing em-bryos from induced carrot cultures, Plant Physiol. 86 (1988) 451 – 456.
[24] J.A.A. Fujii, D. Slade, R. Olsen, S.E. Ruzin, K. Reden-baugh, Alfalfa somatic embryo maturation and conver-sion to plants, Plant Sci. 72 (1990) 93 – 100.
[25] F.-M. Lai, B.D. McKersie, Regulation of starch and protein accumulation in alfalfa (Medicago sati6aL.)
so-matic embryos, Plant Sci. 100 (1994) 211 – 219.
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when cotyledons, petioles and hypocotyls were used as explants. Use of petioles as explants pro-duced the highest frequency of somatic embryos (36.1%92.6 in the third month of culture) and the highest average number of somatic embryos per callus (2.390.4 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 soso-matic 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 differentiaforma-tion 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 content/dry weight (mg g−1 DW) in calli kept in the different media, using cotyledons, petioles and
hypocotyls fromM.arboreaL. seedlings as explants
Culture media Explants Culture times (months)
4
3 5
2 1
Cotyledon 868.9692.1
MS 903.0091.8 903.7098.2 904.0097.8 919.0598.5
903.0097.5 892.8498.8
892.7698.8 890.1093.7
862.9092.5 Petiole
909.3996.4 909.3491.7
904.7094.3 919.4797.3
Hypocotyl 909.3296.4
858.2795.3 868.9792.4 869.0292.8 870.1394.1 Leaf 848.2192.6
915.3491.7 917.2296.5 917.4198.1
H8 Cotyledon 911.2693.9 918.0497.3
923.1296.1 921.9295.8
919.2494.1 917.2394.7
908.1294.7 Petiole
918.2499.1 921.1396.6 921.9297.8 922.0498.3 Hypocotyl 903.1294.3
Leaf 845.1293.4 853.2793.8 864.2194.9 864.9796.2 869.3295.3 921.1693.1
919.3196.8
– 923.0496.3
– Cotyledon
F0
Petiole – – 918.1394.7 920.0293.5 921.4294.8
Hypocotyl – – 923.1299.1 923.9893.9 925.0296.7
Leaf – – 866.4393.8 866.8393.5 868.2296.1
F6 Cotyledon – – 916.2494.1 916.8193.3 918.0495.1
917.9394.7 917.6497.3
915.0293.7 –
Petiole –
Hypocotyl – – 918.4798.7 917.0793.6 919.1294.3
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Table 2
Variation in percentage of reducing sugars/total sugars in calli kept in the different media, using cotyledons, petioles and hypocotyls fromM.arboreaL. seedlings as explants
Culture media Explants Culture times (months)
1 2 3 4 5
15.38 17.60
MS Cotyledon 28.37 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 Cotyledon 38.22 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 Cotyledon 19.76 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.
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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 starch/total sugars in calli kept in the different media, using cotyledons, petioles and hypocotyls from M.arboreaL. 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 Cotyledon 23.38 3.34
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
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Table 4
Variation in percentage of sucrose/total sugars in calli kept in the different media, using cotyledons, petioles and hypocotyls from M.arboreaL. seedlings as explants
Culture media Explants Culture times (months)
1 2 3 4 5
14.40 11.81
MS Cotyledon 9.16 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 Cotyledon 9.30 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 Cotyledon 9.88 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 Cotyledon 10.70 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
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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 (PB0.001) 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 (PB0.001).
4. Conclusions
There were no differences in total sugars
be-tween embryogenic and non-embryogenic calli.
The highest contents in reducing sugars were
obtained when the calli were embryogenic.
During the development of somatic
embryogen-esis the starch contents in the calli were very low.
In the embryogenic calli, the percentages of
sucrose were much lower than in the non-em-bryogenic calli.
Acknowledgements
This work was supported by a grant from the Direccio´n General de Investigacio´n Cientı´fica and Te´cnica, Spain, No. PB94-1403.
References
[1] B.S. Mangat, M.K. Pelekis, A.C. Cassells, Changes in the starch content during organogenesis in ‘in vitro’ cultured Begonia rex stem explants, Physiol. Plant 79 (1990) 267 – 274.
[2] T.A. Thorpe, In vitro’ organogenesis and embryogene-sis: physiological and biochemical studies, in: K. Roubelakis-Angelakis, K. Tran Thanh Van (Eds.), Morphogenesis in Plants, Plenum Press, New York, 1993, pp. 19 – 38.
[3] F.C. Botha, M.M. O’Kennedy, S. du Plessis, Activity of key enzymes involved in carbohydrate metabolism in Phaseolus 6ulgaris cell suspension cultures, Plant Cell
Physiol. 33 (1992) 477 – 483.
[4] H.A. Ross, H.V. Davies, L.R. Burch, R. Viola, D. McRae, Developmental changes in carbohydrate con-tent and sucrose degrading enzymes in tuberising stolons of potato (Solanum tuberosum), Physiol. Plant 90 (1994) 748 – 756.
[5] W. Van den Ende, A. Van Laere, Purification and properties of a neutral invertase from the roots of Ci-chorium intybus, Physiol. Plant 93 (1995) 241 – 248. [6] S.S. Sung, D.-P. Xu, C.C. Black, Identification of
ac-tively filling sucrose sinks, Plant Physiol. 89 (1989) 1117 – 1121.
[7] S. Venkataramana, K.M. Naidu, S. Singh, Invertases and growth factors dependent sucrose accumulation in sugarcane, Plant Sci. 74 (1991) 65 – 72.
[8] P.L. Swarnkar, S.P. Bohra, N. Chandra, Biochemical studies on initiation of callus in Solanum surattense, J. Plant Physiol. 126 (1986) 293 – 296.
[9] C. Branca, A. Torelli, P. Fermi, M.M. Altamura, M. Bassi, Early phases ‘in vitro’ culture tomato cotyle-dons: starch accumulation and protein pattern in rela-tion to the hormonal treatment, Protoplasma 182 (1994) 59 – 64.
[10] J.A. Stamp, Somatic embryogenesis in cassava. The anatomy and morphology of the regeneration process, Ann. Bot. 57 (1987) 451 – 459.
[11] T. Murashige, F. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plant 15 (1962) 473 – 497.
[12] S.T. Nguyen, R. Paquin, Me´thodes d’extraction et de purification des acides amine´s libres et des prote´ines de tissus ve´ge´taux, J. Chromatogr. 61 (1971) 349 – 351. [13] M. Dubois, K.J. Gilles, F. Smith, Colorimetric method
for determination of sugars and related substances, Anal. Chem. 28 (1956) 356 – 361.
[14] M. Somogyi, Notes on sugar determination, J. Biol. Chem. 195 (1952) 19 – 23.
[15] N. Nelson, A photometric adaptation of the Somogyi method for the determination of glucose, J. Biol. Chem. 153 (1944) 375 – 380.
[16] K.Y. Paek, S. Canderlerd, T.A. Thorpe, Physiological effects of Na2SO4 and NaCl on callus cultures of
Bras-sica campestris (Chinese cabbage), Physiol. Plant 72 (1988) 160 – 166.
[17] A.J. Gordon, G.J.A. Ryl, D.F. Mitchell, K.H. Lowry, C.E. Powell, The effect of defoliation on carbohydrate, protein and leg-haemoglobin content of white clover nodules, Ann. Bot. 58 (1986) 141 – 154.
[18] S.K. Rawal, U.N. Dwivedi, B.M. Khan, A.F. Mas-carenhas, Biochemical aspects of shoot differentiation in sugarcane callus: II. Carbohydrate metabolizing en-zymes, J. Plant Physiol. 119 (1985) 191 – 199.
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[19] U.D. Yeo, W.Y. Soh, H. Tasaka, N. Sakurai, S. Kuraishi, K. Takeda, Cell wall polysaccharides of callus and suspension-cultured cells from three cellulose-less mutants of barley (Hordeum6ulgareL.), Plant Cell
Phys-iol. 36 (1995) 931 – 936.
[20] K.R. Naidu, P.B.K. Kishor, Activities of hydrolytic en-zymes in callus cultures of tobacco during organoge´nesis, J. Biosci. 20 (1995) 629 – 636.
[21] A. Kikuchi, S. Satoh, N. Nakamura, T. Fujii, Differ-ences in pectic polysaccharides between carrot embryo-genic and non-embryoembryo-genic calli, Plant Cell Rep. 14 (1995) 279 – 284.
[22] K. Dimassi-Theriou, A. Bosabalidis, Effects of light, magnesium and sucrose on leaf anatomy, photosynthesis,
starch and total sugar accumulation, in kiwi fruit cul-tured in vitro, Plant Cell Tiss. Organ. Cult. 47 (1997) 127 – 134.
[23] G.L. Keller, B.J. Nikolau, T.H. Ulrich, E.S. Wurtele, Comparison of starch and ADP-glucose pyrophosphory-lase levels in nonembryogenic cells and developing em-bryos from induced carrot cultures, Plant Physiol. 86 (1988) 451 – 456.
[24] J.A.A. Fujii, D. Slade, R. Olsen, S.E. Ruzin, K. Reden-baugh, Alfalfa somatic embryo maturation and conver-sion to plants, Plant Sci. 72 (1990) 93 – 100.
[25] F.-M. Lai, B.D. McKersie, Regulation of starch and protein accumulation in alfalfa (Medicago sati6aL.)
so-matic embryos, Plant Sci. 100 (1994) 211 – 219.