Cytochemical characterization and ultrastructural organization in calluses of the agarophyte Gracilariopsis tenuifrons (Gracilariales, Rhodophyta).
ABSTRACT The culture and physiology of red macroalgae calluses are well documented. To date, however, no report has either performed a cytochemical analysis or characterized the ultrastructural organization of calluses at different stages of development and under the effect of plant growth regulators. Therefore, to undertake such analyses, this work studied the red seaweed Gracilariopsis tenuifrons (Bird et Oliveira) Fredericq et Hommersand. Morphology studies suggested three types of calluses: a) terminal callus having an irregular amorphous shape and filamentous projections originating from the cortical region of the thallus; b) apical callus growing on apical branches and having an elongated semispherical shape; and c) intercalary callus developing along the intermediary region of the thallus and having the appearance of small declivities with irregular edges. The abundance of intercalary calluses over terminal and apical calluses is most likely a result of a major cortical surface that would support the cellular growth required to generate calluses. Callus development was initially observed as a matrix of cellular disorganization with filamentous projections; then, the cellular mass seemed to become more compact with spherical uncolored aspect. The presence of starch grains in the inner part of the explant could be explained by absorption from the culture medium and by proper biosynthesis during callus development. Cell wall reaction to staining suggested cellulose and agar composition with acidic polysaccharides. Results suggest that none of the three morphological types of calluses showed any significant differences on the basis of either cytochemistry or ultrastructural organization.
Micron 42 (2011) 80–86
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Cytochemical characterization and ultrastructural organization in calluses of the
agarophyte Gracilariopsis tenuifrons (Gracilariales, Rhodophyta)
Zenilda Laurita Bouzona,∗, Eder Carlos Schmidta, Ana Carolina de Almeidab, Nair S. Yokoyac,
Mariana Cabral de Oliveirab, Fungyi Chowb
aUniversidade Federal de Santa Catarina, Depto. de Biologia Celular, Embriologia e Genética, Centro de Ciências Biológicas, CP 476, 88049-900, Florianópolis, SC, Brazil
bInstituto de Biociências, Universidade de São Paulo, CP 11461, CEP 05422-970, São Paulo, SP, Brazil
cInstituto de Botânica, Secretaria de Estado do Meio Ambiente, São Paulo, CP 4005, CEP 01061-970, São Paulo, SP, Brazil
a r t i c l ei n f o
Received 8 June 2010
Received in revised form 27 July 2010
Accepted 27 July 2010
a b s t r a c t
The culture and physiology of red macroalgae calluses are well documented. To date, however, no report
has either performed a cytochemical analysis or characterized the ultrastructural organization of cal-
luses at different stages of development and under the effect of plant growth regulators. Therefore, to
undertake such analyses, this work studied the red seaweed Gracilariopsis tenuifrons (Bird et Oliveira)
Fredericq et Hommersand. Morphology studies suggested three types of calluses: a) terminal callus hav-
ing an irregular amorphous shape and filamentous projections originating from the cortical region of the
thallus; b) apical callus growing on apical branches and having an elongated semispherical shape; and
c) intercalary callus developing along the intermediary region of the thallus and having the appearance
of small declivities with irregular edges. The abundance of intercalary calluses over terminal and apical
calluses is most likely a result of a major cortical surface that would support the cellular growth required
to generate calluses. Callus development was initially observed as a matrix of cellular disorganization
with filamentous projections; then, the cellular mass seemed to become more compact with spherical
uncolored aspect. The presence of starch grains in the inner part of the explant could be explained by
absorption from the culture medium and by proper biosynthesis during callus development. Cell wall
reaction to staining suggested cellulose and agar composition with acidic polysaccharides. Results sug-
gest that none of the three morphological types of calluses showed any significant differences on the
basis of either cytochemistry or ultrastructural organization.
© 2010 Elsevier Ltd. All rights reserved.
natural beds and harvested from mariculture systems around
the world as raw material for agar extraction (Oliveira Filho,
1984). Tissue culture techniques used to improve agar quality
and mariculture methods have important applications in seaweed
micropropagation and domestication of selected strains (Aguirre-
Lipperheide et al., 1995). In addition, these tools are essential
requirements for genetic engineering studies (Stevens and Purton,
1997). Studies reporting on the induction of calluses and callus-
like structures under controlled plant growth regulators have been
widely applied to commercial seaweed mariculture in order to
Fuller and Gibor, 1987; Bradley and Cheney, 1990; García-Reina
et al., 1991; Liu and Kloareg, 1991; Robledo and García-Reina,
∗Corresponding author. Tel.: +55 48 3721 5149.
E-mail address: firstname.lastname@example.org (Z.L. Bouzon).
1993). Thus far, documenting the cellular characteristics and
reorganization of calluses involved in tissue development and
regeneration has led to greater understanding of callus formation
and differentiation, as well as improving technical approaches to
mariculture systems. Specifically, the economically important red
macroalgae has been well documented insofar as the physiology
of their calluses. These species include Gracilaria vermiculophylla
(Ohmi) Papenfuss (Yokoya et al., 1999), Gracilaria tenuistipitata
var. liui Zhang & Xia and Gracilaria perplexa Byrne et Zuccarello
(Yokoya et al., 2004), Gracilaria chilensis Bird, McLachlan & Oliveira
(Collantes et al., 2004), Gelidiella acerosa (Forsskål) Feldmann &
G. Hamel (Rajakrishna Kumar et al., 2004), Hypnea musciformis
(Wulfen) J.V. Lamouroux (Bravin et al., 2006; Rajakrishna Kumar
et al., 2007), Gracilaria corticata (J. Agardh) J. Agardh (Rajakrishna
Kumar et al., 2007), Kappaphycus alvarezii (Doty) Doty ex P. Silva
(Hayashi et al., 2008) and Gracilaria domingensis (Kützing) Sonder
ex Dickie (Ramlov et al., 2009). Some reports have even shown the
ultrastructure of agarophytes, such as Gelidium floridanum W.R.
Taylor (Bouzon et al., 2005), and the carragenophytes H. musci-
formis (Bouzon, 2006; Ouriques and Bouzon, 2003) and K. alvarezii
0968-4328/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
Z.L. Bouzon et al. / Micron 42 (2011) 80–86
Fig. 1. Calluses of Gp. tenuifrons developed from intercalary explants cultured on ASP12-NTA solid medium for two months. Figures 1A-1C, terminal callus. Figures 1D-1F,
apical calluses. Figures 1G-1I, intercalary calluses.
(Schmidt et al., 2009). To date, however, no report has either per-
organization of calluses at different stages of development and
under the effect of plant growth regulators. Therefore, to under-
tenuifrons (Bird et Oliveira) Fredericq et Hommersand, which has
been exploited in northeast Brazil as an important raw material for
the agar extraction industry.
2. Materials and methods
2.1. Culture conditions
Unialgal cultures of female plants of Gp. tenuifrons (strain 39)
from the Germoplasm Bank of the Laboratory of Marine Algae Édi-
son José de Paula, University of São Paulo, Brazil, were kept as
(Edwards, 1970) at the proportion of 10g of fresh weight (FW) alga
14h photocycle, 60±5?mol photons m−2.s−1(Li-Cor light meter
Intercalary segments of 5mm from these unialgal cultures were
treated with antibiotic and fungal antibiotic antimycotic solution
(Sigma, St Louis, MO, USA; 10,000 units penicillin, 10mg strepto-
mycin, and 25?g amphotericin B per mL) for 48h, washed with
autoclaved seawater solution plus 0.5% sodium hypochlorite and
200?L Triton-X-100 for 20s, and washed five times with auto-
claved seawater. These sterilized segments were inoculated as
initial explants in ASP12-NTA synthetic medium (Iwasaki, 1961),
modified with the addition of 100?g of thiamine hydrochloride,
plus 2.5mg.L−1indole-3-acetic acid (IAA; auxin) and 0.5mg.L−16-
benzylaminopurine (BA; cytokinin) and 0.7% agar. Tissue culture
flasks with 10 explants each were placed at ±32 p.s.u., 25±1◦C,
14h photocycle, and 40±5?mol photons.m−2s−1. Contaminated
explants were removed weekly. Solid von Stosch solution medium
at 50 and 100% dilution and 0.5% agar solid medium were also
tested. Callus development was monitored for six weeks, and sam-
ples were processed for light microscopy (LM) and transmission
electron microscopy (TEM).
2.2. Light microscope (LM)
(pH 7.2) phosphate buffer overnight. Subsequently, the samples
were dehydrated in increasing series of ethanol aqueous solutions.
After dehydration, the samples were infiltrated with Historesin
(Leica Historesin, Heidelberg, Germany). Sections 5?m in length
Z.L. Bouzon et al. / Micron 42 (2011) 80–86
Fig. 2. Light microscopy of the transversal sections of calluses of Gp. tenuifrons. (A) Sections stained with TB-O. The cell wall (CW) shows metachromatic reaction. Observe
the metachromatic granulations in the apical and peripherical cells (arrows). (B) Sections stained with PAS. PAS-positive floridean starch grains (S) in the cytoplasm. The
arrows indicate positive reaction with the mucilage of apical cells. (C) Sections stained with CBB. Positive reaction with the nuclei (arrows). (D) Magnification of previous
figure showing positive reaction with the pit connections (arrows).
were stained with different histochemical techniques and were
investigated with an Epifluorescent (Olympus BX 41) microscope
equipped with the image Q Capture Pro 5.1 Software (Qimaging
Corporation, Austin, TX, USA).
2.3. Cytochemical staining
LM sections were stained as follows: Periodic acid-Schiff (PAS)
used to identify neutral polysaccharides (Gahan, 1984); Toluidine
Blue (TB-O) 0.5%, pH 3.0 (Merck Darmstadt, Germany) for acid
Candless, 1973); Coomassie Brilliant Blue (CBB) 0.02% in Clarke’s
solution (Serva, Heidelberg, Germany) for proteins (Gahan, 1984).
Controls consisted of applying solutions to sections without the
staining component (e.g., omission of periodic acid application in
the PAS reaction).
2.4. Transmission electron microscope (TEM)
For observation under the TEM, the calluses were fixed with
2.5% glutaraldehyde in 0.1M sodium cacodylate buffer (pH 7.2)
plus 0.2M sucrose overnight. The material was post-fixed with
1% osmium tetroxide for four hours, dehydrated in a graded
acetone series and embedded in Spurr’s resin. Thin sections
were stained with aqueous uranyl acetate followed by lead cit-
rate, according to Reynolds (1963). Four replicates were made
for each experimental group; two samples per replication were
then examined under TEM JEM 1011 (JEOL Ltd., Tokyo, Japan, at
3.1. Culture and morphology
After two months of culture, the survival of initial explants was
zero under solid von Stosch enrichment solution medium, and 95%
of survival was observed with solid ASP12-NTA synthetic medium.
Explants inoculated in 0.5% agar solid medium did not show callus
formation, and lateral branches were observed which penetrated
into the solid medium.
However, callus production was observed in the initial inter-
calary explants under solid ASP12-NTA synthetic media and 0.7%
nent calluses: a) terminal calluses produced from the sectioned
jections originating from the cortical region of thallus (Fig. 1A, B
and C); b) apical calluses growing on apical branches with elon-
gated semispherical shape (Fig. 1D, E and F); and c) intercalary
calluses developing along the intermediary region of the thallus
and having the appearance of small declivities with irregular edges
(Fig. 1G, H and I).
3.2. Observations under LM and histochemistry
When the calluses were stained with Toluidine Blue (TB-O),
they showed a metachromatic reaction in the cell wall, indicat-
ing the presence of acidic polysaccharides, while the cytoplasm
was orthochromatic. In the cytoplasm of the apical and periph-
Z.L. Bouzon et al. / Micron 42 (2011) 80–86
Fig. 3. Transmission electron microscopy (TEM) micrographic images of calluses of Gp. tenuifrons. (A) Detail of elongated chloroplast (C). Observe the presence of fibrillar
region, or genophore (G), and plastoglobuli (P). Starch grains juxtaposed to chloroplast. Note the electron-dense microfibrils in the cell wall. (B) Magnification of the previous
figure showing the presence of phycobilisomes (arrows). (C) Detail of the chloroplast showing the regular shape of thylakoids (arrows) and starch grains with diverse shape.
(D) Detail of pit connection between the callus cells. Note the striated pit plug (arrows).
erical cells of calluses, metachromatic granulations could also be
observed (Fig. 2A).
The calluses stained with Periodic Acid-Schiff (PAS) exhibited a
cytoplasm with neutral polysaccharides, especially showing a high
of red algae (Fig. 2B). The mucilage of apical cells also showed a
PAS-positive reaction (Fig. 2B).
Finally, when stained with Coomassie Brilliant Blue (CBB), the
distribution of organelles or structures rich in protein and localized
just at the margin in cytoplasm (Fig. 2C). The cells were uni-, bi- or
dense reaction to proteins was observed (Fig. 2D).
3.3. Observations under TEM
When observed by transmission electron microscopy (TEM),
the callus cells located near the explants’ tissues showed a fine
structure of vegetative cells similar to that of Florideophyceae
Class, especially in chloroplast organization. The chloroplasts were
large, elongated and able to adjust themselves to the cell morphol-
ogy (Fig. 3A). Furthermore, the chloroplasts, which consisted of
an individual and flat thylakoid surrounded by a single periph-
eral thylakoid, assumed the typical internal organization of red
algae, having unstacked, evenly-spaced thylakoids (Fig. 3 A, B and
C). Electron-dense lipid droplets described as plastoglobuli were
observed between the thylakoids (Fig. 3A). Among the thylakoids,
a fibrillar region corresponding to a genophore was also observed
(Fig. 3A). Numerous evenly-spaced phycobilisomes were attached
on both sides of the outer thylakoids (Fig. 3B). Starch grains were
located in the middle of the cytoplasm with diverse shapes, dislo-
cating the chloroplasts to the cytoplasm margin (Fig. 3A and C).
connections. The pit plug filled the pit connection with a slightly
granular, electron-dense material. This plug was covered by two
membranes and was composed of protein which filled the chan-
nel between the daughter cells, resulting in partial cytokinesis.
However, even though pit connections normally maintain contact
between daughter cells, no cytoplasmic continuity is permitted
since a plug is usually deposited in the septal aperture shortly
after furrowing ceases (Fig. 3D). These connections are considered
characteristic structures of some red macroalgae.
The cells located far from the explants’ tissues demonstrated
modifications, such as reduction in the number of thylakoids and
alteration in their shape, which showed a corrugated organization
(Fig. 4 A, B, C, D and E). The same cells showed a large amount of
starch grains when compared with vegetative cells of the explants
The cell walls of calluses showed two different distributions
for microfibrils. The internal region is formed by electron-
dense microfibrils, having a compact, parallel organization
which is soaked into an electron-translucent amorphous matrix.
Z.L. Bouzon et al. / Micron 42 (2011) 80–86
Fig. 4. TEM micrographic images of calluses of Gp. tenuifrons. (A) Detail of callus cell with chloroplasts showing reduction of thylakoids and alteration of their shape. Observe
a large quantity of starch grains with diverse shape. (B), (C), (D) and (E) Detail of thylakoids show corrugated organization (arrows). Note some starch grains near the
The external region is thin with electron-dense microfibrils
(Figs. 3A, B and 4 B, D).
Callus regeneration was observed in all three types of cal-
luses. Two processes of regeneration were distinguished: direct
regeneration through the differentiation of callus cells and indirect
regeneration from adventitious plantlet differentiation.
The three morphological types of calluses identified in this
study have also been observed for other seaweed species: Solieria
filiformis (Kütz.) P.W. Gabrielso (Yokoya and Handro, 2002), G.
tenuistipitata var. liui and G. perplexa (Yokoya et al., 2004), and
Turbinaria conoides (J. Agardh) Kützing (Rajakrishna Kumar et
al., 2007). Nevertheless, our study showed a developmental pat-
tern other than that observed by Yokoya (2000) for the same G.
tenuistipitata species. Specifically, these authors observed apical
calluses located at apical regions, as well as lateral branches. Non-
intercalary and terminal calluses were also identified.
We found that the callus cells originated from cortical cells, a
result also observed by Yokoya and Handro (2002); however, other
cells (Gusev et al., 1987; Yokoya and Handro, 2002; Rajakrishna
Kumar et al., 2007).
When observed by transmission electron microscopy, it was
were observed near the explants, the chloroplasts showed a typical
structure of red algae. The number of parallel thylakoids is vari-
able, and this number mainly depends on the spatial location of the
lipid material with a reserve role. This structure was also described
by Wetherbee and Wynne (1973) in Polysiphonia novae-angliae
W.R. Taylor, by Pueschel (1988) in Hildenbrandia rubra (Sommer-
felt) Meneghini, and by Bouzon (2006) in Hypnea musciformis.
However, in the chloroplasts located in the peripherical cells
of the callus, the number of thylakoids was reduced, and their
shape was corrugated when compared to thylakoids present in the
explants. Consequently, the group of cells became more hyaline.
As observed from PAS reaction and by TEM, calluses are able
to synthesize their proper organic material based on the elevated
Z.L. Bouzon et al. / Micron 42 (2011) 80–86
amount of starch grains found in the callus cells. The localization
pattern of starch grains formed within cells was heterogeneous;
cells farther away from explants showed more starch grains than
cells near explants. This accumulation of starch grains seems to
be caused both by gradients of substances absorbed from cul-
ture medium and by physiological gradients manifested in the
The callus cell walls in Gp. tenuifrons showed a slightly
PAS-positive stain reaction, indicating the presence of cellulosic
compounds. The PAS assay is used to identify neutral polysaccha-
rides because it requires the presence of 1.2 glycol groups that are
oxidized to aldehydes by periodic acid (Trick and Pueschel, 1990).
The cell walls of Gp. tenuifrons reacted positively to TB-O, also sug-
gesting cellulosic and agar composition. TB-O staining produces a
violet metachromatic reaction in the cell walls of all sections of Gp.
tenuifrons, as observed in Chondrus crispus Stackhouse (Gordon and
Mc Candless, 1973), Chondria tenuissima C. Agardh (Tsekos et al.,
2009). This metachromatic reaction is used in histological studies
The violet metachromatic color with TB-O is produced by polysac-
charides with carboxyl and sulfated groups (McCully, 1968). Agar
interpretation is based on the assumption that the configuration of
each sulfated tertiary polyanion exposes the polymer, forming a
large amount of complex with the TB-O (Gordon and Mc Candless,
CBB histochemistry assays for proteins show a large number of
nuclei and pit connections. In fact, cell division in Florideophyceae
remains incomplete because of the permanence of pit connections.
To explain, during the final phase of cell division, a pore remains
plug (Pueschel, 1980). Using CBB, pit connections were observed
by Ramus (1971) in Griffithsia pacifica Kylin, by Tsekos (1983) in
in H. musciformis and by Schmidt et al. (2009) in K. alvarezii.
The frequencies of developed intercalary calluses were signifi-
cantly higher than terminal and apical calluses, probably resulting
from the major cortical surface that would support the cellu-
lar growth required to generate calluses. Cellular disorganization
seems to be an initial phase of callus development with filamen-
tous projections (two weeks). After the third week, the cellular
mass seems more compact with spherical uncolored aspect. Obvi-
ous pigmentation could not be observed, and amorphous mass
was retained in both terminal and intercalary calluses, whereas
apical calluses developed structures exhibiting a progressive pig-
mentation after two months of in vitro tissue culture. The rapid
differentiation of apical callus cells might be a response to direc-
tional polarity and apical meristematic cells that could influence
the early morphogenetic differentiation to pigmented cortex cells.
Filamentous projections were also observed in laboratory cul-
ture thalli of Gp. tenuifrons after mechanical cutting by razor blade.
These projections could be an attempt to attach the substrate or
a natural physiological response to wounding. Since culture thalli
are kept under semi-constant air turbulence every 30min with
no addition of plant growth regulators, projection development is
ephemeral with rapid cell differentiation and the appearance of a
pigmented cortex layer around the terminal section. This response
might be similar to that observed by Shintani (1988) and Perrone
and Cecere (1991) in S. filiformis after sectioned surfaces were
Finally, we concluded that the three morphological callus types
and ultrastructural organization. In spite of the reduced number
of chloroplasts, a large quantity of starch grains was observed in
the main callus cells, which was interpreted as resulting from the
translocation of nutrients from the culture medium.
Electron Microscopy (LCME), Federal University of Santa Catarina,
Florianopolis, Santa Catarina, Brazil, for the use their transmission
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