Effects of plant growth regulators on growth

and morphogenesis in tissue culture of

Chondracanthus chamissoi (Gigartinales,
Rhodophyta)
Nair S.Yokoya, Marcela vila, Maria
I.Piel, Fabiola Villanueva & Angelica
Alcapan
Journal of Applied Phycology
ISSN 0921-8971
Volume 26
Number 2
J Appl Phycol (2014) 26:819-823
DOI 10.1007/s10811-013-0130-4

1 23

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Author's personal copy

J Appl Phycol (2014) 26:819823
DOI 10.1007/s10811-013-0130-4

Effects of plant growth regulators on growth and morphogenesis

in tissue culture of Chondracanthus chamissoi (Gigartinales,
Rhodophyta)
Nair S. Yokoya & Marcela vila & Maria I. Piel &
Fabiola Villanueva & Angelica Alcapan
Received: 26 May 2013 / Revised and accepted: 22 August 2013 / Published online: 27 September 2013
# Springer Science+Business Media Dordrecht 2013

Abstract Chondracanthus chamissoi (C. Agardh) Ktzing

(Gigartinales, Rhodophyta) is an edible species and commercialised for carrageenan production in Chile. Investigations on
growth and development are needed to improve its cultivation; therefore, this study aims to evaluate the effects of plant
growth regulators (PGRs) on its growth and morphogenesis.
PGRs tested were two auxins [indole-3-acetic acid (IAA) and
2,4-dichlorophenoxyacetic acid (2,4-D)] and one cytokinin
(benzylaminopurine (BA)) in concentrations of 0.5, 5.0 and
50.0 M. These PGRs were added to seawater enriched with
half strength of von Stosch solution and were gelled with
0.6 % agar, and treatment control (without PGR addition)
was conducted simultaneously. Apical and intercalary segments were used as initial explants. Each treatment was tested
with six replicates of five axenic explants, and statistical
analyses were performed. After culturing in a solid medium
for 10 weeks to induce growth and callus formation, explants
were cultured in liquid medium with the same experimental
conditions for 10 weeks. Effects of auxins (IAA and 2,4-D)
and the cytokinin BA on growth rates of apical segments of C.
chamissoi were not significant, while low concentration of
IAA stimulated the growth of intercalary segments. On the
other hand, high concentrations of BA and IAA stimulated the
callus formation in apical and intercalary segments, respectively. In liquid medium, PGR did not have a significant effect
on growth rates of apical segments, while 2,4-D in concentrations from 0.5 to 50.0 M stimulated growth of intercalary

N. S. Yokoya (*)
Ncleo de Pesquisa em Ficologia, Instituto de Botnica,
Av. Miguel Estfano, 3687, 04301-012 So Paulo, Brazil
e-mail: nyokoya@hotmail.com
M. vila : M. I. Piel : F. Villanueva : A. Alcapan
Institute of Science and Technology, University Arturo Prat,
Ejercito 443, Puerto Montt, Chile
M. vila
e-mail: marcela.avila@unap.cl

segments, and formation of lateral branches was stimulated by

low 2,4-D in apical segments. These results suggest that PGRs
have a regulatory role on callus formation and growth of
specific explants of C. chamissoi. Furthermore, the formation
of lateral branches stimulated by auxin could be used for
seedling production under controlled conditions and could
improve the micropropagation and cultivation of C. chamissoi
in the Chilean coast.
Keywords Auxins . Chilean seaweeds . Chondracanthus .
Cytokinins . Gigartinales . Seaweed tissue culture

Introduction
Seaweed tissue culture is an important tool for micropropagation,
a technique which produces, within a short period, a large
number of individuals that can be used as seedlings for seaweed
cultivation rather than collecting them from natural beds (Yokoya
and Yoneshigue-Valentin 2011) and, also, for application in
genetic engineering (Stevens and Purton 1997). To improve
tissue culture techniques, it is fundamental to understand the
hormonal regulation of seaweed growth and development and,
also, to characterise the hormone profiles in seaweeds (Yokoya
et al. 2010).
Endogenous plant hormones have been detected in green,
brown and red seaweeds as well as in some extracts of various
kelp species that are used commercially as growth stimulants
in agricultural crops (Stirk and Van Staden 2006). Besides,
endogenous cytokinins, including both isoprenoid and aromatic groups, were identified in 5 Chlorophyta, 7 Phaeophyta
and 19 Rhodophyta species from South Africa (Stirk et al.
2003). Endogenous cytokinins, auxins and abscisic acid were
identified and quantified in 11 red algae collected from the
Brazilian coast (Yokoya et al. 2010). However, the physiological functions of these hormones need to be elucidated in
order to understand their regulation role on seaweed growth,

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820

development and morphogenesis. This knowledge could be

useful in the commercial breeding and cultivation of important
seaweeds. Considering this aspect, Chondracanthus
chamissoi (C. Agardh) Ktzing (Gigartinales, Rhodophyta)
is an important species for Chilean and Peruvian industries for
carrageenan production, and it has been exploited to Asian
countries for human consumption (Avila et al. 2011; Bulboa
and Macchiavello 2006). Several studies on C. chamissoi
have reported different aspects of vegetative propagation
(Macchiavello et al. 2003; Fonck et al. 2008; Sez et al.
2008), sexual reproduction (Avila et al. 2011), reproductive
phenology and populational dynamics (Gonzlez et al. 1997;
Vsquez and Vega 2001), growth and development under
controlled conditions (Gonzlez and Meneses 1996; Bulboa
and Macchiavello 2001; Bulboa et al. 2010) and cultivation
attempts (Bulboa and Macchiavello 2006). However, micropropagation and tissue culture techniques have not been investigated in this species. Therefore, this study aims to evaluate the
effects of plant growth regulators (PGRs) on the growth and
morphogenesis in tissue culture of C. chamissoi.

Material and methods

Algal material Female gametophytes with cystocarps of C.
chamissoi (C. Agardh) Ktzing (Gigartinales, Rhodophyta)
were collected in October 2007, in Coliumo, Chile (3631 S,
7257 W). Reproductive specimens were collected using hookah divers and were transported in a cooler with gel pack to
keep the algal samples cold and humid until the laboratory of
the Institute of Science and Technology of Arturo Prat
University, in Puerto Montt, Chile. In the laboratory, mature
cystocarpic specimens were washed four times with filtered
seawater to eliminate epiphytes. The procedures to stimulate
carpospore liberation and culture methods were described by
Avila et al. (2011). Isolated tetrasporophytes were cultured in
half strength of Provasoli medium (Provasoli 1968), replaced at
weekly intervals, under light/dark cycle of 16:8 h, temperature
of 13 C, photon flux density of 10 mol photons m2 s1 and
salinity of 33 practical salinity unit (psu).
Experiments with plant growth regulators PGRs tested were
two auxins [indole-3-acetic acid (IAA) and 2,4-dichlorophenoxyacetic acid (2,4-D)] and one cytokinin (benzylaminopurine (BA)) in concentrations of 0.5, 5.0 and
50.0 M. These PGRs (Sigma, USA) were added to seawater
enriched with half strength of von Stosch solution (VSES/2
medium) as described by Oliveira et al. (1995) and modified
with the reduction of 50 % in the vitamin concentrations
(Yokoya 2000) and were gelled with 0.6 % agar (Equilab,
Chile). Treatment control (without PGR addition) was
conducted simultaneously.

J Appl Phycol (2014) 26:819823

To obtain axenic explants, thallus segments (1015 mm

long) were cut from tetrasporophytes and were cultured for
48 h in solution composed by VSES/2 with penicillin
(3.75 mg mL1) and nystatin (3.125 mg mL1). Inside a
laminar flow chamber, apical and intercalary segments
(5 mm long) were isolated and washed for 10 s in a solution
of sterile seawater with 0.5 % sodium hypochlorite and
0.002 % of organic detergent (Detlab, Chile) and were washed
several times in sterile seawater to remove this solution.
Before inoculation, these explants were washed with solution
of ciprofloxacin (5 mg L1) in sterile seawater. These explants
were inoculated into 30 mL autoclaved VSES/2 solid medium
supplemented with PGR. Experiments were conducted under
the same laboratory conditions described for culturing isolated
tetrasporophytes. Each treatment was tested with six replicates
of five explants. After culturing in a solid medium for
10 weeks, explants were transferred into a liquid medium with
the same experimental conditions and were cultured for
10 weeks. This experimental period was chosen based on
the growth of C. chamissoi in laboratory conditions (Avila
et al. 2011). Medium was renewed weekly, and morphological
variations were recorded under a stereomicroscope (Stemi
DV4; Carl Zeiss, Germany) using the ImageJ software.
Specific growth rate was calculated as [ln (B f Bo1)t 1],
where B o is the initial fresh biomass, B f is the fresh biomass
after t days, and t corresponds to the experimental period
(Yokoya et al. 2007). Morphological data include the number
and length of lateral branches and the percentage of callus
formation (number of calluses per explants).
Statistical analysis Data were analysed by one-way ANOVA
and test of planned comparison, which allow performing
parametric comparisons between each levels of an experimental factor and a control, making the necessary adjustments to
correct the type I error generated by the extensive use of
the same set of data in multiple comparisons (Chambers and
Hastie 1992).

Results
Effects of auxins (IAA and 2,4-D) and the cytokinin BA on
growth rates of apical segments of C. chamissoi were not
significant, while low concentration of IAA stimulated the
growth of intercalary segments cultured in a solid medium
(Fig. 1(a, b)). On the other hand, high concentrations of BA
and IAA stimulated the callus formation in apical and intercalary segments, respectively (Fig. 1(c, d)).
Apical and intercalary segments of C. chamissoi developed
calluses in different thallus regions: apical calluses developed
in apical regions of branches (Fig. 2a), intermediate calluses
developed in intermediate parts of the explant (Fig. 2b, c) and

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Fig. 1 Effects of indole acetic

acid (IAA), 2,4dichlorophenoxiacetic acid (2,4D) and benzylaminopurine (BA)
on growth rates (a, b) and callus
formation (c, d) in apical (a, c)
and intercalary segments (b, d) of
C. chamissoi cultured in half
strength of von Stosch medium
with 0.6 % agar for 10 weeks,
under light/dark cycle of 16:8 h,
temperature of 13 C, photon flux
density of 10 mol
photons m2 s1 and salinity of
33 psu. Each data point is the
mean of six replicates. Significant
differences are indicated by
different letters

basal calluses developed in the basal (proximal) pole of the

explant (Fig. 2d, e).
In liquid medium, auxins and cytokinin did not have a
significant effect on the growth rates of apical segments
(Fig. 3(a)), but low 2,4-D concentration stimulated the highest
number of lateral branches (Fig. 3(c)). However, different
responses were observed in intercalary segments, where 2,4D in concentrations from 0.5 to 50.0 M stimulated the
growth rates (Fig. 3(b)), but PGR did not have significant
effects on the number of lateral branches (Fig. 3(d)).

Fig. 2 Different callus types in

explants of C. chamissoi cultured
in half strength of von Stosch
solid medium (0.6 % agar) for
10 weeks, under light/dark cycle
of 16:8 h, temperature of 13 C,
photon flux density of 10 mol
photons m2 s1 and salinity of
33 psu. Apical callus (a),
intermediate calluses (b, c) and
basal calluses (d, e). Callus was
indicated by arrows. Scale =
1 mm

Discussion
Explants of C. chamissoi developed apical, basal and intermediate calluses without a tendency in relation to PGR type or
concentration. Gracilaria tenuistipitata Chang and Xia and
Gracilaria perplexa Byrne and Zuccarello (Gracilariales,
Rhodophyta) also developed the same types of calluses
(Yokoya et al. 2004). Formation of basal calluses is common in
different species of Rhodophyta, since they are usually induced
by wounding process (Gusev et al. 1987; Polne-Fuller and Gibor

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J Appl Phycol (2014) 26:819823

Fig. 3 Effects of indole acetic acid

(IAA), 2,4-dichlorophenoxiacetic
acid (2,4-D) and
benzylaminopurine (BA) on growth
rates (a, b) and formation of lateral
branches (c, d) in apical (a, c) and
intercalary segments (b, d) of C.
chamissoi cultured in half strength
of von Stosch liquid medium for
10 weeks, under light/dark cycle of
16:8 h, temperature of 13 C,
photon flux density of 10 mol
photons m2 s1 and salinity of
33 psu. Each data point is the mean
of six replicates. Significant
differences are indicated by
different letters

1987; Kaczyna and Megnet 1993; Huang and Fujita 1996, 1997;
Yokoya and Handro 1996; Yokoya et al. 1999). On the other
hand, the development of apical calluses was described for some
species as Solieria filiformis (Ktzing) Gabrielson (Robledo and
Garcia-Reina 1993), Meristotheca papulosa J. Agardh (Huang
and Fujita 1997), Gracilariopsis tenuifrons (Bird and Oliveira)
Fredericq and Hommersand (Yokoya 2000), Kappaphycus
alvarezii (Doty) Doty ex P.C. Silva (Reddy et al. 2003) and
two colour strains of Hypnea musciformis (Wulfen in Jacqu.)
J.V. Lamour. (Yokoya et al. 2003). Calluses of C. chamissoi
showed a low potential for regeneration of what were observed in
other species of Gigartinales (Gusev et al. 1987; Huang and
Fujita 1996). For micropropagation purpose, the formation of
lateral branches is an alternative morphogenetic response and
could be used for seedling production instead of thallus regeneration from calluses.
Our results showed that auxins have stimulatory effects on
the growth of intercalary segments of C. chamissoi; specifically, low concentration of IAA and 2,4-D in concentrations
from 0.5 to 50.0 M stimulated the growth in solid and liquid
media, respectively. IAA also stimulated the callus formation
in intercalary segments of C. chamissoi. These stimulatory
effects of auxins could be related to their function on processes of cell division and elongation (Krikorian 1995), with
increases in DNA, RNA and protein content, indicating
changes in gene expression (Hagen 1995). Similar responses
were also observed in Gracilaria vermiculophylla (Ohmi)

Papenfuss (Yokoya et al. 1999) and in G. tenuifrons

(Yokoya 2000). On the other hand, apical segments of C.
chamissoi showed different responses: high concentrations
of BA stimulated the callus formation, and low concentration of 2,4-D stimulated the formation of lateral branches.
Distinct responses between apical and intercalary segments
of C. chamissoi could be explained by the presence of
apical cells in apical segments, which result in a physiological and biochemical gradient along the thallus, and
consequently, this factor could influence the cell sensitivity
to PGR (Davies 1995). The stimulatory effect of BA (an
aromatic cytokinin) on callus formation is a result of its
role on cell division.
In conclusion, our results suggest that PGRs have a regulatory role on callus formation and growth of specific explants
of C. chamissoi . Furthermore, the formation of lateral
branches stimulated by auxin could be used for seedling
production under controlled conditions and could improve
the micropropagation and cultivation of C. chamissoi in the
Chilean coast.
Acknowledgments The authors thank supports from the FONDEF,
Chile (projects D06I1058 and D08I1067 to MA), and the CONICYT
(project MEC 80100021 to NSY). The first author thanks the Universidad
de Magallanes (Chile) for the stay as an invited researcher and grants
from the Fapesp and from the CNPq (Brazil). We also thank valuable
suggestions made by two anonymous reviewers who helped to improve
the manuscript.

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References
Avila M, Piel MI, Caceres JH, Alveal K (2011) Cultivation of the red alga
Chondracanthus chamissoi: sexual reproduction and seedling production in culture under controlled conditions. J Appl Phycol 23:529536
Bulboa C, Macchiavello J (2001) The effects of light and temperature on
different phases of the life cycle in the carrageenan producing alga
Chondracanthus chamissoi (Rhodophyta, Gigartinales). Bot Mar
44:371374
Bulboa C, Macchiavello J (2006) Cultivation of cystocarpic, tetrasporic
and vegetative fronds of Chondracanthus chamissoi (Rhodophyta,
Gigartinales) on ropes at two localities in northern Chile. Investig
Mar 34:151154
Bulboa C, Macchiavello J, Veliz K, Oliveira E (2010) Germination rate and
sporeling development of Chondracanthus chamissoi (Rhodophyta,
Gigartinales) varies along a latitudinal gradient on the coast of Chile.
Aquat Bot 92:137141
Chambers JM, Hastie TJ (1992) Statistical models. In: Chambers JM,
Hastie TJ (eds) Statistical models in S. Wadsworth & Brooks/Cole,
Pacific Grove
Davies PJ (1995) The plant hormone concept: concentration, sensitivity and
transport. In: Davies PJ (ed) Plant hormonesphysiology, biochemistry and molecular biology. Kluwer Academic, Dordrecht, pp 1338
Fonck E, Martinez R, Vsquez J, Bulboa C (2008) Factors that affect the
re-attachment of Chondracanthus chamissoi (Rhodophyta,
Gigartinales) thalli. J Appl Phycol 20:311314
Gonzlez J, Meneses I (1996) Differences in the early stages of development of gametophytes and tetrasporophytes of Chondracanthus
chamissoi (C. Ag.) Ktzing from Puerto Aldea, Northern Chile.
Aquaculture 143:91107
Gonzlez J, Meneses I, Vsquez J (1997) Field studies in Chondracanthus
chamissoi (C. Agardh) Ktzing: seasonal and spatial variations in lifecycle phases. Biol Pesq 26:312
Gusev MV, Tambiev AH, Kikova NN, Shelyastin NN, Aslanyan RR
(1987) Callus formation in seven species of agarophyte marine
algae. Mar Biol 95:593597
Hagen G (1995) The control of gene expression by auxin. In: Davies PJ
(ed) Plant hormonesphysiology, biochemistry and molecular biology. Kluwer Academic, Dordrecht, pp 228245
Huang W, Fujita Y (1996) Callus induction and thallus regeneration in
some species of red algae. Phycol Res 45:105111
Huang W, Fujita Y (1997) Callus induction and thallus regeneration of
the red alga Meristotheca papulosa (Rhodophyta, Gigartinales). Bot
Mar 40:5561
Kaczyna F, Megnet R (1993) The effects of glycerol and plant growth
regulators on Gracilaria verrucosa (Gigartinales, Rhodophyceae).
Hydrobiologia 268:5764
Krikorian AD (1995) Hormones in tissue culture and micropropagation.
In: Davies PJ (ed) Plant hormonesphysiology, biochemistry and
molecular biology. Kluwer Academic, Dordrecht, pp 774796
Macchiavello J, Bulboa C, Edding M (2003) Vegetative propagation and
spore recruitment in the carrageenophyte Chondracanthus chamissoi
(Rhodophyta, Gigartinales) in northern Chile. Phycol Res 51:4550
Oliveira EC, Paula EJ, Plastino EM, Petti R (1995) Metodologias para
cultivo no axenico de macroalgas marinhas in vitro. In: Alveal K,
Ferrario ME, Oliveira EC, Sar E (eds) Manual de Mtodos
Ficolgicos. Universidad de Concepcin, Concepcion, pp 429447

823
Polne-Fuller M, Gibor A (1987) Calluses and callus-like growth in
seaweeds: induction and culture. Hydrobiologia 151/152:131138
Provasoli L (1968) Media and prospects for the cultivation of marine algae.
In: Watanabe A, Hattori A (eds) Cultures and collection of algae.
Proceedings of the US Japanese Society of Plant Physiologists
Conference, Hakone, pp 6375
Reddy CRK, Raja Krishna Kumar G, Siddhanta AK, Tewari A (2003) In
vitro somatic embryogenesis and regeneration of somatic embryos
from pigmented callus of Kappaphycus alvarezii (Doty) Doty
(Rhodophyta, Gigartinales). J Phycol 39:610616
Robledo DR, Garcia-Reina G (1993) Apical callus formation in Solieria
filiformis (Gigartinales, Rhodophyta) cultured in tanks. Hydrobiologia
260/261:401406
Sez F, Macchiavello J, Fonck E, Bulboa C (2008) The role of secondary
attachment disc in vegetative propagation of Chondracanthus
chamissoi (Rhodophyta, Gigartinales). Aquat Bot 89:6365
Stevens DR, Purton S (1997) Genetic engineering of eucaryotic algae:
progress and prospects. J Phycol 33:713722
Stirk WA, van Staden J (2006) Seaweed products as biostimulants in
agriculture. In: Critchley AT, Ohno M, Largo DB (eds) World
seaweed resources. ETI Information Services Ltd, Wokingham, pp
132, DVD ROM
Stirk WA, Novak O, Strnad M, Van Staden J (2003) Cytokinins in
macroalgae. Plant Growth Regul 41:1324
Vsquez J, Vega JA (2001) Chondracanthus chamissoi (Rhodophyta,
Gigartinales) in northern Chile: ecological aspects for management
of wild population. J Appl Phycol 13:267277
Yokoya NS (2000) Apical callus formation and plant regeneration controlled by plant growth regulators on axenic culture of the red alga
Gracilariopsis tenuifrons (Gracilariales, Rhodophyta). Phycol Res
48:133142
Yokoya NS, Handro W (1996) Effects of auxins and cytokinins on agar
culture of Grateloupia dichotoma (Gigartinales, Rhodophyta).
Hydrobiologia 326/327:393400
Yokoya NS, Yoneshigue-Valentin Y (2011) Micropropagation as a tool
for sustainable utilization and conservation of populations of
Rhodophyta. Braz J Pharmacogn 21:334339
Yokoya NS, Kakita H, Obika H, Kitamura T (1999) Effects of environmental factors and plant growth regulators on growth of the red alga
Gracilaria vermiculophylla from Shikoku Islands, Japan.
Hydrobiologia 398/399:339347
Yokoya NS, Plastino EM, Artel R (2003) Physiological responses and
pigment characterization of two colour strains of the carrageenophyte
Hypnea musciformis (Rhodophyta). In: Chapman ARO, Anderson
RJ, Vreeland VJ, Davison R (eds) Proceedings of the 17th international seaweed symposium. Oxford University Press, New York, pp
425433
Yokoya NS, West JA, Luchi AE (2004) Effects of plant growth regulators
on callus formation, growth and regeneration in axenic tissue cultures of Gracilaria tenuistipitata and G. perplexa (Gracilariales,
Rhodophyta). Phycol Res 52:244254
Yokoya NS, Necchi Jnior O, Martins AP, Gonzalez SF, Plastino EM
(2007) Growth responses and photosynthetic characteristics of wild
and phycoerythrin-deficient strains of Hypnea musciformis
(Rhodophyta). J Appl Phycol 19:197205
Yokoya NS, Stirk WA, van Staden J, Novak O, Tureckova V, Pencik A,
Strnad M (2010) Endogenous cytokinins, auxins and abscisic acid in
red algae from Brazil. J Phycol 46:11981205