J Appl Phycol (2014) 26:729746

DOI 10.1007/s10811-013-0122-4

Production of clonal planting materials from Gracilaria

changii and Kappaphycus alvarezii through tissue culture
and culture of G. changii explants in airlift photobioreactors
Hui-Yin Yeong & Siew-Moi Phang & C. R. K. Reddy &
Norzulaani Khalid

Received: 26 May 2013 / Revised: 15 August 2013 / Accepted: 16 August 2013 / Published online: 27 September 2013
# Springer Science+Business Media Dordrecht 2013

Abstract Global demand for seaweed resources has increased

due to their emergent use as sources of biopharmaceuticals,
nutraceuticals and biofuels. These high-valued products make
possible the use of micropropagation techniques that may be
more costly than conventional mariculture. This study reports
the successful tissue culture of Kappaphycus alvarezii (Doty)
Doty ex P. C. Silva and Gracilaria changii (B. Xia & Abbott)
Abbott, Zhang and Xia. Callus induction of K. alvarezii was
successfully developed following an explant sterilisation protocol. Callus formation and regeneration of K. alvarezii was
observed in solidified Provasolis enriched seawater medium.
Different culture conditions such as agar concentration, growth
hormones, nutrients, irradiance and enrichment media were
investigated to determine the suitable conditions for explant
culture of G. changii. Proliferations of adventitious shoots
were induced under the most suitable culture conditions. G.
changii explants were successfully cultured in airlift photobioreactors, with no decrease in the carbohydrate content in the
G. changii explants. This micropropagation technique can
provide a useful alternative system for seedling production of
economically important seaweeds.

H.<Y. Yeong (*) : S.<M. Phang

Institute of Ocean and Earth Sciences (IOES), University of Malaya,
50603 Kuala Lumpur, Malaysia
e-mail: yeong@um.edu.my
S.<M. Phang : N. Khalid
Institute of Biological Sciences, University of Malaya,
50603 Kuala Lumpur, Malaysia
C. R. K. Reddy
Discipline of Marine Biotechnology and Ecology, CSIRCentral
Salt and Marine Chemicals Research Institute,
Bhavnagar 364002, India

Keywords Tissue culture . Callus induction . Formation and

regeneration . Explant culture . Plant growth regulators . Red
seaweeds . Kappaphycus . Gracilaria . Micropropagation .
Algae biotechnology

Introduction
World production of seaweed biomass has increased greatly
from 1.995 million t (fresh weight) in 1970 to 19 million t
(fresh weight) in 2010 (FAO 2007, 2012) to meet the demands
for phycocolloid production, food and the emerging seaweedbased pharmaceutical, nutraceutical and biofuel industries. In
2010, 95.5 % of world seaweed biomass was produced
through mariculture (FAO 2012). This led to the rapid development of seaweed cultivation technology aimed at the advancement of industrial scale production of biomass. Efforts
have been focussed on developing techniques for consistent
supply of high-quality seed stock, strain improvement and
efficient mass culture of high yielding commercial strains.
Gracilaria and Kappaphycus are the two economically
important red seaweeds in the world trade market especially
for phycocolloid production. Gracilaria contributes 6080 %
of the worlds agar (Schramm 1991; Armisen 1995; Bixler
and Porse 2011). In 2009, agar production reached 12,500 t
annually from the processing of 57,500 t of raw material
(Bixler and Porse 2011). Approximately 71 % of the worlds
carrageenophyte resources, about 120,000 dry t.y1, is harvested
from Kappaphycus alvarezii (Doty) Doty ex P. C. Silva
(McHugh 2003) and has a market value of US $240 million
annually. In Malaysia, Kappaphycus farming is found mainly in
Sabah_s waters, and recently, a small-scale Kappaphycus farm
(tambalang variety) was established at Pangkor Island, west
coast Peninsular Malaysia (Phang 2006; Phang et al. 2010).
There is a total 22 taxa of Gracilaria in Malaysian waters
(Lim and Phang 2004; Phang 2006). Gracilaria changii (B.

730

Xia & Abbott) Abbott, Zhang and Xia, being one of the most
abundant agarophytes in Malaysia, contains high amounts of
agar (12 to 25 % dry weight) and agarose (13 to 16 % dry
weight) with high gel strength (up to 563 g.cm2 for agar and
950 g.cm2 for agarose) (Phang et al. 1996) and high amounts
of -carotene and essential fatty acids (20:5 3) (Phang et al.
1996; Marinho-Soriano et al. 2001; Chu et al. 2003), bioactive
compounds (Wong et al. 1994), as well as possesses antiinflammatory, gastroprotective and anti-ulcerogenic properties
(Shu et al. 2013). Extensive research has been reported by the
Algae Research Laboratory of the University of Malaya on
this species including its genetic diversity (Sim et al. 2007;
Yow et al. 2011, 2013), proteomics (Wong et al. 2006), functional genomics (Chan et al. 2004; Song et al. 2013),
transcriptomics (Teo et al. 2007, 2009; Ho et al. 2009; Siow
et al. 2012, 2013), genetic transformation (Gan et al. 2003,
2006) and protoplast regeneration (Yeong et al. 2008) contributing to prospective utilization of this alga for both applied and
basic research purposes. Thus, G. changii has great potential
for commercial development in Malaysia. Furthermore, the
success in transformation of G. changii (Gan et al. 2003,
2006) and protoplast isolation and regeneration of G. changii
(Yeong et al. 2008) makes possible the commercialisation of
G. changii especially through bioprocess development for the
pharmaceutical, nutraceutical and phycocolloid industries.
In general, tissue culture techniques developed for seaweed
micropropagation are basically established from tissue culture
techniques of higher plants. The first report of seaweed tissue
culture (Chen and Taylor 1978) involved the red alga
Chondrus crispus. Since then, many species of red and brown
algae have been studied for callus induction and thallus regeneration. However, seaweed tissue culture is still in the state
of development and lags far behind that of higher plants
(Reddy et al. 2008; Baweja et al. 2009). Parameters such as
growth media, plant growth regulators and nutrients and culture conditions such as temperature, irradiance and day light
cycle can influence the success of callus induction. Previous
studies showed success in the use of tissue culture techniques
for the production of planting materials from Kappaphycus.
Dawes and Koch (1991) reported the first successful tissue
culture of K. alvarezii and then further established propagule
production for laboratory and field trials (Dawes et al. 1993,
1994). Reddy et al. (2003) demonstrated better growth rates
(1.51.8 times higher) for tissue culture plantlets compared to
field plants during the field trial (Reddy et al. 2003).
Successful production of K. alvarezii plantlets from explants
culture (Hurtado and Biter 2007) and improved production
rate of explants for commercial nursery stocks (Yunque et al.
2011) have been reported. However, with Gracilaria spp., the
production of planting materials through tissue culture remains at the laboratory stage. Successful callus inductions
have been reported for Gracilaria verrucosa (Gusev et al.
1987; Kaczyna and Megnet 1993), Gracilaria textorii (Huang

J Appl Phycol (2014) 26:729746

and Fujita 1997), Gracilaria vermiculophylla (Yokoya et al.

1999), Gracilaria chilensis (Collantes et al. 2004), Gracilaria
tenuistipitata , Gracilaria perplexa (Yokoya et al. 2004),
Gracilaria corticata (Kumar et al. 2007) and Gracilariopsis
tenuifrons (Yokoya 2000), but no callus formation and regeneration into new thalli has been reported for G. changii
although ball-shaped friable callus-like structures were observed once (Tan 1999).
The production of clonal planting materials for commercially important seaweeds is not only to increase the seedling
stock production for cultivation but can also provide a constant supply of selected breeds with desired characteristics.
Tissue culture techniques allow the production of disease-free
(particularly virus-free) plants and rapid propagation of species which are difficult to multiply by conventional vegetative
means. Malaysia as a Kappaphycus farming country has a
need to improve the current seaweed farming system by use of
high-quality planting materials to ensure the stability of production and the sustainable development. Our objective in this
study was to develop seaweed tissue culture techniques for
producing clonal planting materials for the two most promising seaweeds, namely K. alvarezii and G. changii. The effect
of six plant growth regulators, sources of nitrogen and phosphorus, a polyamine, pH, irradiance and lightdark cycle, on
callus formation was investigated. In addition, the culture of
explants of G. changii was investigated using two types of
airlift photobioreactors.

Materials and methods

Healthy Gracilaria changii plants were collected from mangrove areas of Morib, Selangor, west coast Peninsular
Malaysia (024548.5N, 101268.6E) during low tide
while K. alvarezii plants were collected from the
Kappaphycus farm at Semporna, Sabah, East Malaysia and
kept cool in an ice-chest with some seawater for transport back
to the laboratory. In the laboratory, the samples were cleaned
of mud and epiphytes using a soft brush and filtered seawater.
The samples were maintained as unicultures in tanks of filtered and aerated seawater in both the hatchery and laboratory
(252 C, 25 mol photons m2 s1 irradiance, 12-h:12-h
light dark cycle, 282 ppt seawater salinity) for about
4 weeks before they were used in the studies.
Thalli of G. changii or K. alvarezii were thoroughly
cleared of epiphytes using forceps and autoclaved seawater
under a dissecting microscope. The thalli were then cut into
5 cm pieces and rinsed several times with autoclaved artificial
seawater (Instant Ocean; Atkinson and Bingman 1997)
followed by sonication in autoclaved artificial seawater for
3 min before treatment with 1 % w/v PVP-I for 30 sec. The
thalli segments were then immersed in autoclaved artificial
seawater with an antibiotic mixture containing 0.100 g L1

J Appl Phycol (2014) 26:729746

kanamycin, 0.300 g L1 penicillin G, 0.020 g L1 polymyxin

B sulphate, 0.001 g L1 nalidixic acid, 0.020 g L1 cefotaxine
and 1.000 g L1 streptomycin sulphate for 12 h (Yeong et al.
2008). Sterility was tested using LB broth (Merck, Germany)
and Marine Agar 2216E (Difco) medium.
Tissue culture
The sterilised thalli were cut into short explants (length
~5 mm) using a sterile surgical blade and inoculated in the
desired culture media as listed in Tables 1 and 2. Different
culture media were prepared according to the experimental
requirements, and autoclaved sterile artificial seawater
(Instant Ocean; Atkinson and Bingman 1997) was used as
base medium. Five explants were inoculated in a 90-mm Petri
dish for solid media, or in a 200-mL culture vessel for liquid
media (25 C, 25 mol photons m2 s1 irradiance, 12-h:12-h
light dark cycle, unless specified otherwise). Fresh media
were supplied weekly throughout the culture period.
Observations of callus and adventitious branch induction were
made weekly up to day 56.
In order to determine the suitable tissue culture media and
conditions for callus induction in the G. changii explants
culture, the effect of agar (liquid media and solid media using
0.8 % w/v Bacto Agar), enrichment liquid media as Provasolis
enriched seawater (PES) media (James 1978) or Guillards
(f/2) marine water enrichment solution (GMWES) (Guillard
1975), plant growth regulators (PGRs) 2,4-dichlorophenoxy
acetic acid (2,4-D) (Technology Laboratories), indole-3-acetic
acid (IAA) (Sigma), -naphthalene acetic acid (NAA)
(Sigma), zeatin (Sigma), nutrient supplementation (phosphate
as K2PO4, sulphate as K2SO4, nitrogen as NH4Cl and urea),
polyamines, irradiance, lightdark cycle and pH was investigated (Table 1). For K. alvarezii explant culture, the effect of
agar (liquid media and solid media using 1.0 % w/v Bacto
Agar), enrichment liquid media as PES medium, PGRs (single
PGR (2,4-D) or combination of PGRs (2,4-D and kinetin) was
investigated (Table 2). All studies were conducted in three
replicates (minimum) and incubated at 25 C in a growth
chamber (Protech Model GC-1050).
Simultaneous studies on callus induction
Thalli of G. changii and K. alvarezii were sterilised using the
optimized sterilization protocol described above. The
sterilised thalli were cut into short explants (length ~5 mm)
using a sterile surgical blade, and ten explants were inoculated
into 1.0 % Bacto agar solidified PES medium in each 90-mm
petri dish. Both explant cultures were placed in an incubator
with 25 mol photons m2 s1 irradiance, 12-h:12-h light
dark cycle at 25 C. The experiments were repeated three
times using three different batches of seaweed samples, and
about 60100 explants were tested each time for each species.

731

These experiments were conducted at the same time for the

two species, and all protocols carried out were the same in
order to compare the responses of the two species to the same
conditions for callus induction.
Effect of airlift photobioreactor on explant culture of G.
changii
Two types of airlift photobioreactors, namely a 5-L tubular
airlift photobioreactor (working volume 3 L) and a 1-L spherical airlift photobioreactor (working volume 500 mL), were
used (Fig. 1). PES liquid culture media with or without plant
growth regulators (0.1 mg L1 2,4-D+0.1 mg L1 kinetin)
were used as the base media. Healthy axenic thalli sections of
G. changii were cut into short explants (lengths ~10 and
~20 mm) using a sterile surgical blade. Approximately
0.002 g mL1 of explants of two lengths (~10 and ~20 mm)
with equal weight per volume was inoculated into the 5-L
airlift photobioreactor, while only ~10-mm explants were
inoculated into the 1-L airlift photobioreactor. The airlift
photobioreactors were placed under an irradiance of
25 mol photons m2 s1 with 12-h:12-h light dark cycle
at 25 C. Fifty percent of the culture medium was replaced
with fresh medium weekly. Observations of adventitious
branch generation were made weekly up to day 72. After
72 days of culture, G. changii explants were harvested for
carbohydrate extraction. Carbohydrate contained in G.
changii explants were extracted in 2 N HCl and quantified
using the phenolsulphuric acid method (Kochert 1978).

Results
Tissue culture of G. changii
In experiment 1, explants cultured in Guillards (f/2) GMWES
with 3.315 mg L1 2,4-D showed the highest percentage of
explants with adventitious branches (12.004.90 %). About
8.00 % of explants cultured in 1.105, 2.210 and 4.420 mg L1
2,4-D treatment produced adventitious branches. In the IAA
and NAA treatments, adventitious branches were only observed in 1.105 mg L1 IAA and 0.221 mg L1 NAA (6.67
6.67 %), while no branches formed at all other concentrations of IAA and NAA tested. Explants cultured in GMWES
only and with 0.221 mg L1 2,4-D also did not produce any
branches. Those explants without new branches were found to
have lost their pigmentation and became bleached by the end
of day 56. However, KruskalWallis ANOVA showed no
significant differences on the effect of plant growth regulators
on formation of adventitious branches in the explants (p >0.05)
(Fig. 2).
In experiment 2, five different concentrations of 2,4-D in
both 0.8 % w/v Bacto agar solidified GMWES medium and

2,4-D (1.0, 5.0, 10.0, 15.0 and 20.0)

2,4-D (1.0, 5.0, 10.0, 15.0 and 20.0)

2,4-D (1.0, 5.0, 10.0, 15.0 and 20.0)

2,4-D (1.0, 5.0, 10.0, 15.0 and 20.0)

2,4-D (0.221, 1.105, 2.210, 3.315 and 4.420)

2,4-D (0.221, 1.105, 2.210, 3.315 and 4.420)

NAA (0.221, 1.105, 2.210, 3.315 and 4.420)

IAA (0.221, 1.105, 2.210, 3.315 and 4.420)

2,4-D and kinetin (0.1 2,4-D+1.0 kinetin;

0.1 2,4-D+0.1 kinetin; 1.0 2,4-D+0.1 kinetin)
1. Seawater only 2,4-D and kinetin (10 2,4-D+0.001 kinetin;
2. PES only
1.0 2,4-D+0.01 kinetin; 0.1 2,4-D+0.1 kinetin;
0.01 2,4-D+1.0 kinetin; 0.001 2,4-D+10
kinetin and 0.01 2,4-D+1 kinetin)
1. Seawater only 2,4-D and kinetin (10 2,4-D+0.001 kinetin;
1.0 2,4-D+0.01 kinetin; 0.1 2,4-D+0.1 kinetin;
2. PES only
0.01 2,4-D+1.0 kinetin; 0.001 2,4-D+10
kinetin and 0.01 2,4-D+1 kinetin)
PES only
2,4-D and kinetin (0.01 2,4-D+1.0 kinetin;
0.1 2,4-D+0.1 kinetin; 10 2,4-D+0.001 kinetin)
PES only
2,4-D and kinetin (0.01 2,4-D+1.0 kinetin;
0.1 2,4-D+0.1 kinetin; 10 2,4-D+0.001 kinetin)
PES only
2,4-D and kinetin (0.01 2,4-D+1.0 kinetin;
0.1 2,4-D+0.1 kinetin; 10 2,4-D+0.001 kinetin)
PES only
2,4-D and kinetin (0.01 2,4-D+1.0 kinetin;
0.1 2,4-D+0.1 kinetin; 10 2,4-D+0.001 kinetin)
PES only
2,4-D and kinetin (0.01 2,4-D+1.0 kinetin;
0.1 2,4-D+0.1 kinetin; 10 2,4-D+0.001 kinetin)

GMWES only

PES only

GMWES only

GMWES only

GMWES only

GMWES only

GMWES only

GMWES only

GMWES only

2,4-D (0.221, 1.105, 2.210, 3.315 and 4.420)

GMWES only

Concentration of plant growth regulators

(mg L1)

Experiment Treatment Control

PES

PES

PES

PES

PES

PES

PES

GMWES

PES

GMWES

GMWES

GMWES

GMWES

GMWES

GMWES

GMWES

GMWES

12 h L:12 h D and
34.18 mol photons m2 s1
12 h L:12 h D and
34.18 mol photons m2 s1
12 h L:12 h D and
34.18 mol photons m2 s1
12 h L:12 h D and 34.18 mol
photons m2 s1
12 h L:12 h D and 18.85 mol
photons m2 s1

25 C

25 C
25 C

25 C
25 C
25 C
25 C
25 C

25 C

25 C

0.8 % w/v

Bactoagar
solidified media
Liquid media

K2PO4 (10, 100

and 200 mM L1)
K2SO4 (10, 100
and 200 mM L1)
NH4Cl (10, 100
and 200 mM L1)
Urea (10, 100 and
200 mM L1)
K2PO4 (10, 100 and
200 mM L1)
Liquid media

Liquid media

Liquid media

Liquid media

Liquid media

Liquid media

Liquid media

Liquid media

Liquid media

Liquid media

0.8 % w/v Bacto

agar solidified
media
Liquid media

25 C

25 C

25 C

25 C

08 h L:16 h D and 25 mol

photons m2 s1

25 C

Liquid media

12 h L:12 h D and 25
photons m2 s1
12 h L:12 h D and 25
photons m2 s1
12 h L:12 h D and 25
photons m2 s1
12 h L:12 h D and 25
photons m2 s1
12 h L:12 h D and 25
photons m2 s1

mol

mol

mol

mol

mol

12 h L:12 h D and 25 mol

photons m2 s1
12 h L:12 h D and 25 mol
photons m2 s1

mol

mol

mol

25 C

mol

Liquid media

Liquid media

12 h L:12 h D and 25
photons m2 s1
12 h L:12 h D and 25
photons m2 s1
12 h L:12 h D and 25
photons m2 s1
12 h L:12 h D and 25
photons m2 s1

Culture Lightdark cycle

condition (h) and irradiance
(mol photons m2 s1)
25 C

Nutrient/
polyamines/pH

Enrichment Culture medium

basal
(solid/liquid)
medium

Table 1 Treatments and parameters investigated in tissue culture studies of G. changii (experiment period=56 days)

732
J Appl Phycol (2014) 26:729746

PES only

PES only

PES only

PES only

PES only

PES only

PES only

PES only

PES only

PES only

PES only

PES only

PES only

Experiment Treatment Control

Table 1 (continued)

NAA, BAP and zeatin

(1.0 NAA+1.0 BAP+1.0 zeatin)
NAA, BAP and zeatin
(1.0 NAA+1.0 BAP+1.0 zeatin)

NAA and BAP (1.0 NAA+1.0 BAP)

2,4-D, kinetin and zeatin (0.1 2,4-D+0.1

kinetin+1.0 Zeatin)
NAA and BAP (1.0 NAA+1.0 BAP)

2,4-D, kinetin and zeatin (0.1 2,4-D+0.1

kinetin+1.0 Zeatin)

2,4-D and kinetin (0.1 2,4-D+0.1 kinetin)

2,4-D and kinetin (0.01 2,4-D+1.0 kinetin;

0.1 2,4-D+0.1 kinetin; 10 2,4-D+0.001 kinetin)
2,4-D and kinetin (0.01 2,4-D+1.0 kinetin;
0.1 2,4-D+0.1 kinetin; 10 2,4-D+0.001 kinetin)
2,4-D and kinetin (0.01 2,4-D+1.0 kinetin;
0.1 2,4-D+0.1 kinetin; 10 2,4-D+0.001 kinetin)
2,4-D and kinetin (0.01 2,4-D+1.0 kinetin;
0.1 2,4-D+0.1 kinetin; 10 2,4-D+0.001 kinetin)
2,4-D and kinetin (0.01 2,4-D+1.0 kinetin;
0.1 2,4-D+0.1 kinetin; 10 2,4-D+0.001 kinetin)
2,4-D and kinetin (0.1 2,4-D+0.1 kinetin)

Concentration of plant growth regulators

(mg L1)

PES

PES

PES

PES

PES

PES

PES

PES

PES

PES

PES

PES

PES

1.0 % w/v Bacto

agar solidified
media

1.0 % w/v Bacto

agar solidified
Liquid media

1.0 % w/v Bacto

agar solidified
Liquid media

1.0 % w/v Bacto

agar solidified
media
Liquid media

Liquid media

Liquid media

Liquid media

Liquid media

Liquid media

Liquid media

Enrichment Culture medium

basal
(solid/liquid)
medium

25 C
25 C

0.0018 mg L1 spermine 25 C

0.0018 mg L1 spermine 25 C

mol

mol

mol

mol

mol
25 C

12 h L:12 h D and 25
photons m2 s1
12 h L:12 h D and 25
photons m2 s1
12 h L:12 h D and 25
photons m2 s1
12 h L:12 h D and 25
photons m2 s1
12 h L:12 h D and 25
photons m2 s1

12 h L:12 h D and 25 mol

photons m2 s1
25 C

12 h L:12 h D and 18.85 mol

photons m2 s1
12 h L:12 h D and 18.85 mol
photons m2 s1
12 h L:12 h D and 18.85 mol
photons m2 s1
12 h L:12 h D and 34.18 mol
photons m2 s1
12 h L:12 h D and 18.85 mol
photons m2 s1
12 h L:12 h D and 25 mol
photons m2 s1
12 h L:12 h D and 25 mol
photons m2 s1

25 C

25 C

25 C

25 C

25 C

25 C

Culture Lightdark cycle

condition (h) and irradiance
(mol photons m2 s1)

0.0018 mg L1 spermine 25 C

K2SO4 (10, 100 and

200 mM L1)
NH4Cl (10, 100 and
200 mM L1)
Urea (10, 100 and
200 mM L1)
pH (6.9, 7.1, 7.3,
7.5, 7.8, 8.1)
pH (6.9, 7.1, 7.3,
7.5, 7.8, 8.1)
0.0018 mg L1 spermine

Nutrient/
polyamines/pH

J Appl Phycol (2014) 26:729746

733

mol

mol

mol

25 C

25 C

PES
2,4-D and kinetin (0.1 2,4-D+1.0 kinetin; 0.1
2,4-D+0.1 kinetin; 1.0 2,4-D+0.1 kinetin)
a
12

PES only

PES
2,4-D (0, 5, 10, 15, 20)
PES only
a
11

PES

PES only
b

1.0 % w/v Bacto agar

solidified media
1.0 % w/v Bacto agar
solidified media

25 C

12 h L:12 h D and 25
photons m2 s1
12 h L:12 h D and 25
photons m2 s1
12 h L:12 h D and 25
photons m2 s1
12 h L:12 h D and 25
photons m2 s1
25 C

1.0 % w/v Bacto agar

solidified media
Liquid media
PES
PES only
a
10

Culture
condition
Nutrient/
polyamines/pH
Culture medium
(solid/liquid)
Enrichment
basal medium
Concentration of plant growth regulators (mg L1)
Control
Treatment
Experiment

Table 2 Treatments and parameters investigated in tissue culture studies of K. alvarezii (experiment period=56 days)

mol

J Appl Phycol (2014) 26:729746

Lightdark cycle (h) and
irradiance (mol photons m2 s1)

734

liquid GMWES medium were tested. Solid media with

3.315 mg L1 2,4-D produced the highest number of explants
with new branches (32.0012.00 %) while no branch formation was observed in liquid media without 2,4-D and with
0.221 mg L1 2,4-D (Fig. 3). All solid media either with or
without 2,4-D induced adventitious branches while in liquid
media, adventitious branch formation was only observed in
treatments with 1.105 mg L1 2,4-D. A significant difference
was found in the effect of media on the success of adventitious
branch formation (p <0.05) but not in the effect of different
concentrations of plant growth regulator.
Higher concentrations of 2,4-D (1, 5, 10, 15 and 20 mg L1)
in both 0.8 % w/v Bacto agar solidified GMWES and liquid
GMWES media were investigated in experiment 3. A higher
number of adventitious branches were observed in liquid media compared to solid media (Fig. 4). The highest percentage of
explants with branches was 66.67 % observed in liquid media
with either 5 mg L1 or 20 mg L1 2,4-D. No branch formation
was observed on solid media with 20 mg L1 2,4-D, while the
lowest percentage (6.67 %) of explants with new branches was
in liquid media without 2,4-D and solid media with 0, 1 and
10 mg L1 2,4-D.
In experiment 4, both PES and GMWES media with five
different concentrations of 2,4-D were used. GMWES with
either 5 or 20 mg L1 2,4-D produced the highest percentage
of explants with new branch formation (66.676.67 and
66.6717.64 %, respectively) followed by PES liquid media
with 1 mg L1 2,4-D (24.0019.39 %). PES liquid medium
without 2,4-D had the lowest percentage of explants with new
branch formation (4.004.00 %) (Fig. 5). A significant difference was found in the effect of enrichment media on the
percentage of explants with new branch formation (p <0.05),
but not in the effect of different concentrations of plant growth
regulators.
In experiment 5, three combinations of plant growth were
tested with GMWES as the basal medium. A combination of
0.1 mg L1 2,4-D and 0.1 mg L1 kinetin was the best with
100 % of the explant cultures producing adventitious branches
(Fig. 6). This was followed by 1 mg L1 2,4 D and 0.1 mg L1
kinetin (40.0023.09 %) and 0.1 mg L1 2,4-D and 1 mg L1
kinetin (26.6713.33 %). Treatments without plant growth
regulators (control) formed the lowest number of new
branches (6.676.67 %). However, there was no significant
difference in the effect of plant growth regulators on new
branch formation. Furthermore, no callus induction was observed in G. changii explants in all treatments.
When variation in the lightdark cycle ( 12-h:12-h L:D or
8-h:16-h L:D) was combined with different concentrations of
2,4-D and kinetin on G. changii callus induction (experiment
6), the use of PES enrichment medium as base medium
together with 2,4-D and kinetin increased adventitious branch
formations in all treatments. In the 12 h light 12 h dark cycle,
adventitious branch formation was observed in 100 % of the

J Appl Phycol (2014) 26:729746

Fig. 1 The three types of airlift
photobioreactors used in the
experiment. a 5 L spherical airlift
photobioreactor. b 5 L tubular
airlift photobioreactor. c 1 L
tubular airlift photobioreactor

735

explants in PES liquid media with 10 mg L1 2,4-D +

0.001 mg L1 kinetin; 1 mg L1 2,4-D+0.01 mg L1 kinetin
and 0.1 mg L1 2,4-D+0.1 mg L1 kinetin while in a 8-h
light 16-h dark cycle, 100 % adventitious branch formation
was observed in PES liquid media with 0 mg L1 2,4-D+
0 mg L1 kinetin; 10 mg L1 2,4-D+0.001 mg L1 kinetin
and 0.01 mg L1 2,4-D+1 mg L1 kinetin (Fig. 7). Liquid
media without PES enrichment and plant growth regulators had the lowest percentage of adventitious branch
formation in both lightdark cycle treatments (33.33
17.64 % in 12-h:12-h L:D cycle and 40.0020.00 % in
a 8-h:16-h L:D cycle).
In experiment 7, a total of three concentrations of four
nutrients, namely phosphate (K2PO4), sulphate (K2SO4), nitrogen (NH4Cl) and urea, were tested together with three

different combinations of 2,4-D and kinetin in PES liquid

media. Adventitious branch formations were observed in G.
changii explants in selected treatments (Table 3). For the
treatments with a combination of phosphate (K2PO4) and
plant growth regulators, adventitious branch formations were
found with 0.01 mg L1 2,4-D+1 mg L1 kinetin with K2PO4
(10, 100 and 200 mM L1) with 23.8123.81, 28.5728.57
and 14.2914.29 % of explants with new branch formation,
respectively. For the treatments with a combination of sulphate (K2SO4) and plant growth regulators, adventitious
branch formations were found in all the treatments except in
the combinations of 10 and 100 mM L1 K2SO4 without 2,4D and kinetin and the combination of 200 mM L1 K2SO4
with 10 mg L1 2,4-D+0.001 mg L1 kinetin. The highest
percentage of explants with adventitious branch formations

736

J Appl Phycol (2014) 26:729746

Fig. 2 Effect of different concentrations of plant growth regulators (2,4D, IAA and NAA) in GMWES liquid media on the formation of adventitious branches in G. changii explants. No bar indicates no adventitious

branch formation was observed on G. changii explants. Data are shown

as mean valuestandard error (experimental period=56 days; no. of
replicates=5)

was found in the treatment with the combination of

10 mM L1 K2SO4 with 10 mg L1 2,4-D+0.001 mg L1
kinetin (47.6226.51 %). In the treatments on combination of
nitrogen (NH4Cl) and plant growth regulators, adventitious
branch formations were not found in the combinations of 10
and 100 mM L1 NH4Cl with 0.1 mg L1 2,4-D+0.1 mg L1
kinetin and in 200 mM L1 NH4Cl without plant growth

regulators. However, the highest percentage of explants with

new branch formations (42.8629.74 %) was found in the
treatment with the combination 200 mM L1 NH4Cl with
0.1 mg L1 2,4-D+0.1 mg L1 kinetin. For the treatments on
combination of urea and plant growth regulators, no specific
trend was observed. Adventitious branch formations were
observed in the combination of 10 mM L1 urea with and

Fig. 3 Effect of 0.8 % w/v Bacto agar solidified GMWES media (solid
gray bar) and liquid GMWES media (diagonally striped bar) with
different concentrations of 2,4-D on the formation of adventitious
branches in G. changii explants. No bar indicates no adventitious branch

formation was observed on G. changii explants. Data are shown as mean

valuestandard error. Different alphabets in superscript indicate significant differences (p <0.05) between samples means (KruskalWallis
ANOVA) (experimental period=56 days; no. of replicates=5)

J Appl Phycol (2014) 26:729746

737

Fig. 4 Effect of 0.8 % w/v Bacto

agar solidified GMWES media
(solid gray bar) and liquid
GMWES media (diagonally
striped bar) with different
concentrations of 2,4-D on the
formation of adventitious
branches in G. changii explants.
No bar indicates no adventitious
branch formation was observed
on G. changii explants. Data are
shown as mean valuestandard
error (experimental period=
56 days; no. of replicates=5)

without plant growth regulator. The highest percentage of

explants with branch formations was found in the combination
of 100 mM L1 urea, 0.01 mg L1 2,4-D and 1 mg L1 kinetin
(38.1019.05 %) (Table 3).
In experiment 8, effect of combination of pH and plant
growth regulators on G. changii callus induction under different irradiances was examined. PES liquid medium with
pH 6.9 to pH 8.1 was used as base medium with different
combinations and concentrations of the PGRs. Higher number
of explants with new branches were observed under higher
irradiance (34.18 mol photons m2 s1). A total of seven out
of 20 treatments at 34.18 mol photons m2 s1 irradiance had
more than 80 % explants with adventitious branches (Fig. 8).
Only 5 % explants under low light (18.85 mol photons
m2 s1) produced adventitious branches, although pH 7.5
increased the percentage of explants with adventitious
branches at both irradiances. One hundred percent of explants
Fig. 5 Effect of PES liquid media
(solid black bar) and GMWES
liquid media (diagonally striped
bar) with different concentrations
of 2,4-D on the formation of
adventitious branches in G.
changii explants. Data are shown
as mean valuestandard error.
Different alphabets in superscript
indicate significant differences
(p <0.05) between samples means
(KruskalWallis ANOVA)
(experimental period=56 days;
no. of replicates=5)

with branch formation were observed in treatments with

pH 7.5, 0.01 mg L1 2,4-D+1 mg L1 kinetin, 34.18 mol
photons m2 s1; pH 7.5, 10 mg L1 2,4-D+0.001 mg L1
kinetin, 34.18 mol photons m2 s1 and pH 7.8, no PGR,
34.18 mol photons m2 s1. A significant difference was
observed in the effect of irradiance on adventitious branch
formation in G. changii explants (p <0.05).
In experiment 9, the effect of combination of plant growth
regulators and polyamine on callus induction of G. changii
explants in liquid and 1.0 % agar solidified PES media was
tested. The highest percentage of explants with branch formation was found with 0.1 mg L1 2,4-D+0.1 mg L1 kinetin in
solidified medium (76.00 13.27 %) and followed by
0.1 mg L1 2,4-D+0.1 mg L1 kinetin+1 mg L1 zeatin in
solidified medium (73.339.85 %) and 0.1 mg L1 2,4-D+
0.1 mg L1 kinetin+0.0018 mg L1 spermine in solidified
medium (73.3312.46 %). The lowest percentage of explants

738

J Appl Phycol (2014) 26:729746

Fig. 6 Effect of combination of

plant growth regulators (2,4-D
and kinetin) in GMWES liquid
media on the formation of
adventitious branches in G.
changii explants. Data are shown
as mean valuestandard error
(experimental period=56 days;
no. of replicates=3)

with new branches was observed in 1 mg L1 NAA+1 mg L1

BAP+0.0018 mg L1 spermine in solidified medium (8.00
6.11 %) (Fig. 9). In general, 2,4-D and kinetin were found to
induce higher branch formation compared with NAA and
BAP in both liquid and solid media. A significant difference
was observed in the effect of combination of plant growth
regulators and polyamines on branch formation in G. changii
explants (p <0.05).
In all experiments, no callus formation was observed, only
the production of adventitious branches. These branches were
the young shoots which were induced at the cut surface of the
explants and were derived from the cortical cell zone without
intermediate proliferation of undifferentiated tissue or callus.
The tips of these shoots or branches which were often bifurcated were observed after 3 to 4 weeks culture depending on
the experimental conditions. No branches were observed at
the basal region of explants.
Fig. 7 Effect of lightdark cycle
(12 h L:12 h D (diagonally
striped bar) and 8 h L:16 h D
(solid black bar)) and different
concentrations of plant growth
regulators (2,4-D and kinetin) in
PES liquid media on the
formation of adventitious
branches in G. changii explants.
Data are shown as mean value
standard error (experimental
period=56 days; no. of
replicates=3)

Tissue culture of K. alvarezii

Callus induction and formation was observed in K. alvarezii
explants cultured in 1.0 % w/v Bacto agar solidified PES
media, while no callus was observed in all the explants in
liquid PES media (experiment 10). After 30 days of culture,
callus induction and formation was observed in 68.57
12.23 % of K. alvarezii explants cultured in 1.0 % w/v
Bacto agar solidified PES media.
Five different concentrations of 2,4-D in 1.0 % w/v Bacto
agar solidified PES media and combinations of 2,4-D and
kinetin were used to induce callus (experiment 11). Callus
formation was observed in all explants cultured either with or
without plant growth regulator (Fig. 10). Percentage of explants
with callus formation ranged from 38.011.90 % in treatment
without any plant growth regulator to 40.700.77 % in treatment with 0.1 mg L1 2,4-D and 1 mg L1 kinetin (Fig. 11).

J Appl Phycol (2014) 26:729746

739

Table 3 Effect of combination of nutrient supplementation (K2PO4, K2SO4, NH4Cl and urea) and plant growth regulators (2,4-D and kinetin) on the
formation of adventitious branches in G. changii
Nutrient
supplementations

K2PO4

K2SO4

NH4Cl

Urea

Percentage of explants with adventitious branch formation (%)

No PGR

0.01 mg L1
2,4-D+1 mg L1 kinetin

0.1 mg L1
2,4-D+0.1 mg L1 kinetin

10 mgL1
2,4-D+0.001 mg L1 kinetin

10 mM L1
100 mM L1
200 mM L1
10 mM L1
100 mM L1
200 mM L1

33.3333.33
0.000.00
0.000.00
0.000.00
0.000.00
23.8123.81

23.8123.81
28.5728.57
14.2914.29
28.5716.50
9.524.76
19.0519.05

0.000.00
0.000.00
0.000.00
23.8123.81
4.764.76
4.764.76

0.000.00
0.000.00
0.000.00
47.6226.51
33.3320.76
0.000.00

10 mM L1
100 mM L1
200 mM L1
10 mM L1
100 mM L1
200 mM L1

23.8123.81
23.8123.81
0.000.00
19.0519.05
0.000.00
0.000.00

19.059.52
33.3333.33
33.3333.33
4.764.76
38.1019.05
0.000.00

0.000.00
0.000.00
42.8629.74
4.764.76
0.000.00
0.000.00

14.2914.29
9.529.52
28.5728.57
28.5721.87
0.000.00
28.5721.87

Data are shown as mean valuestandard error (experimental period=56 days; no. of replicates=3)

Simultaneous studies on callus induction in G. changii and K.

alvarezii
No callus was observed in all G. changii explants cultured, but
callus formation was observed in most of the K. alvarezii
explants (Table 4). The first filamentous callus was observed
6 days after the K. alvarezii explants were cultured on agar
solidified PES medium. Thirty days later, most of the K.
alvarezii explants (batch 1) produced calli. Two types of callus
pH6.9

pH7.2

Fig. 8 Effect of different concentrations of plant growth regulators (2,4D and kinetin) and pH on the formation of adventitious branches in G.
changii explants under different irradiances (34.18 and 18.85 mol

were obtained from the studies, filamentous callus and compact callus. Both callus types were induced at the cut surface of
the explants and derived from both cortical and medullary
cells. Filamentous callus was observed in the explant surface
exposed to the air and compact callus formed on the explant
surface immersed in solid culture medium. The growth of the
filamentous callus was in the form of a uniseriate branch
initially, followed by formation of disorganized cell mass that
went on to form callus clumps with prolific filamentous
pH7.5

pH7.8

pH8.1

photons m2 s1). Data are shown as mean valuestandard error (experimental period=56 days; no. of replicates=3)

740

J Appl Phycol (2014) 26:729746

Fig. 9 Effect of combination of

plant growth regulators (2,4-D,
kinetin, NAA, BAP and zeatin)
and polyamines (spermine) on the
formation of adventitious
branches in G. changii explants in
liquid PES media (diagonally
striped bar) or 1.0 % agar
solidified PES media (solid gray
bar) under different irradiances
(34.18 and 18.85 mol photons
m2 s1). Data are shown as mean
valuestandard error
(experimental period=56 days;
no. of replicates=10)

No PGR
PCR A
PGR A & Spermine
PGRA & Zeatin
PCR B
PGR B & Spermine
PGR B & Zeatin

outgrowths. After 120 days, adventitious shoots were obtained

from the callus clumps. Regeneration and elongation of shoot
occurred and bifurcate apical shoot formation was observed
after 6 months. The observations are shown in Figs. 12 and 13.
However, none of the G. changii explants produced callus
even after day 56. The experiment was repeated three times
with different batches of seaweeds, and similar results were
obtained. A significant difference on the callus formation was
obtained between the two species tested, but not among the
three batches of experiments (p <0.05).
Fig. 10 Effect of different
concentrations of plant growth
regulator (2,4-D) in 1.0 % w/v
Bacto agar solidified PES media
on the formation of callus in K.
alvarezii explants. Data are
shown as mean valuestandard
error (experimental period=
56 days; no. of replicates=3)

No plant growth regulator

0.1mg L-1 2, 4-D + 0.1 mg L-1 kinetin
0.1mg L-1 2, 4-D + 0.1 mg L-1 kinetin + 0.0018 mg L-1 Spermine
0.1mg L-1 2, 4-D + 0.1 mg L-1 kinetin + 1 mg L-1 Zeatin
1mg L-1 NAA + 1 mg L-1 BAP
1mg L-1 NAA + 1 mg L-1 BAP + 0.0018 mg L -1 Spermine
1mg L-1 NAA + 1 mg L-1 BAP +1 mg L-1 Zeatin

Effect of airlift photobioreactor on explant culture of G.

changii
Branch formations were observed in all treatments in the
photobioreactors. After 72 days, explant biomass increased
in all treatments except in 500 mL PES medium without 2,4-D
and kinetin in the tubular airlift photobioreactor and 3,000 mL
PES medium without 2,4-D and kinetin in the spherical airlift
photobioreactor (Fig. 14). The decrease in biomass was due to
bleaching and death of some explants. No significant

J Appl Phycol (2014) 26:729746

741

Fig. 11 Effect of combinations of

plant growth regulators (2,4-D
and kinetin) in 1.0% w/v Bacto
agar solidified PES media on the
formation of callus in K. alvarezii
explants. Data are shown as mean
valuestandard error
(experimental period=56 days;
no. of replicates=3)

difference was obtained in the effect of airlift photobioreactor

on biomass of G. changii explants (p <0.05).
Carbohydrate content of G. changii explants on day 0 was
1.310.03 % of dry weight, and there was no significant
difference in the carbohydrate content of G. changii in both
types of airlift photobioreactors on day 72 (Fig. 15). Branch
formation in the explants is shown in Fig. 16.

Discussion
Tissue culture
In this study, different parameters including culture media,
plants growth regulators, nutrients, polyamines, irradiance
Table 4 Effect of callus induction treatments on G. changii and K.
alvarezii
Batch

Batch 1
Batch 2
Batch 3

Species

Kappaphycus alvarezii
Gracilaria changii
Kappaphycus alvarezii
Gracilaria changii
Kappaphycus alvarezii
Gracilaria changii

Percentage of explants with

callus formation (%)
Day 15

Day 30

96.671.67
0.000.00
28.759.16
0.000.00
90.002.94
0.000.00

100.000.00
0.000.00
71.2510.90
0.000.00
96.303.02
0.000.00

Data are shown as mean valuestandard error (experimental period=30 days;

no. of replicates=7)

level and light dark cycle were used to induce callus in the
G. changii explants. No callus induction was observed, but
adventitious branches were successfully formed on the cut
surfaces of explants in most treatments, showing the capacity
for regeneration after wounding. The presence of auxins and
cytokinins, either alone or in combinations and in different
concentrations, failed to induce callus in the G. changii explants. Similar observations were reported by Huang and
Fujita (1997) and Collantes et al. (2004). Huang and Fujita
reported that the use of IAA and BAP had no effect on callus
formation in the explants of G. textorii, while Collantes et al.
(2004) reported that the use of IAA and kinetin in PES
medium did not enhance callus formation in G. chilensis
explants as the callus formation was a result of the wounding.
However, a high callus induction rate was obtained in G.
verrucosa explants inoculated into modified ASP6F2 medium with the supplementation of a combination of IAA, indole3-propionic acid and N 6-(2-isopentenyl) adenine (Kaczyna
and Megnet 1993). Low concentration of IAA, 2,4-D or BA
(1.0 mg L1) was found to produce the maximum growth of
calli in the apical segments of G. vermiculophylla while
0.1 mg L1 IAA, 10 mg L1 2,4-D or 1.0 mg L1 IAA+
0.1 mg L1 BA enhanced the growth of calli from the intercalary segments of G. vermiculophylla (Yokoya et al. 1999).
Yokoya et al. (2004) reported that the use of different concentrations of IAA, 2,4 D and kinetin successfully stimulated
different types of calli in G. tenuistipitata and G. perplexa
and that different Gracilaria species or even different types of
cells responded differently to the plant growth regulators.
Tan (1999) reported that ball-like friable callus-like structures were obtained in liquid PES media containing a

742
Fig. 12 Callus induction and
formation of K. alvarezii. a
Filamentous callus was observed
on day 10 in the K. alvarezii
explants cultured in agar
solidified PES medium. b
Filamentous callus was observed
on day 15 in the K. alvarezii
explants cultured in agar
solidified PES medium. c
Filamentous callus was observed
on day 30 in the K. alvarezii
explants cultured in agar
solidified PES medium. d
Filamentous callus was observed
on day 45 in the K. alvarezii
explants cultured in agar
solidified PES medium. e
Filamentous callus was observed
in the K. alvarezii explants
cultured in agar solidified PES
medium. f Compact callus was
observed in the K. alvarezii
explants cultured in agar
solidified PES medium

J Appl Phycol (2014) 26:729746

combination of 2,4-D and kinetin. Similar treatments were

carried out in the present study, but no callus formation was
observed in G. changii explants. This may be because not all
parts of seaweed thalli respond similarly to the given plant
growth regulators as reported by Yokoya et al. (2004). Yokoya
et al. (2004) concluded that both auxins (IAA and 2,4-D) and
cytokinin (kinetin) play a regulatory role in the growth and
morphogenetic processes of G. tenuistipitata and G.
perplexa, and the differential sensitivity of the explants was
due to the seaweed-specific development system. The differential sensitivity of explants to plant growth regulators might
be due to a regulatory mechanism rather than the concentration of plant growth regulators (Trewavas 1982). Baweja et al.
(2009) pointed out the lack of attention on the factor of cellular
competence to plant growth regulators in seaweed tissue
culture studies and that more fundamental research should
be done to answer the question.
However, the use of combinations of auxin and cytokinin
successfully produced higher number of explants with adventitious branches or shoots in G. changii compared to the use of

single hormones or their omission from the medium. These

adventitious shoots originated from the cortical zone of the cut
surface of the explants, directly without an intervening callus
phase, and were followed by shoot elongation and bifurcation
at the apices of the shoots. Titlyanov et al. (2006) reported that
young fronds developed directly from the cut edges of the
meristematic fragments in the superficial cortical layers of
Palmaria palmata thallus.
In higher plants, shoot formation can be executed directly
or indirectly with an intervening callus phase in response to
the extra-cellular signals (Hicks 1994; George et al. 2008;
Elhiti and Stasolla 2012), which may be described as a competent phenomenon which has been indentified in three distinct phases: competence acquisition, canalization and morphogenesis. Elhiti and Stasolla (2012) explained that in the
first phase, cells within the explant acquire competence to
respond to inductive signals, then canalised into a shoot
development program which results in morphogenesis.
These responses may be linked to signals from the presence
of auxins and cytokinins (plant growth regulators) and also the

J Appl Phycol (2014) 26:729746

Fig. 13 Callus formation and
regeneration of K. alvarezii. a, b
Filamentous callus observed in
the explant surface exposed to the
air while the compact callus
formed on the explant surface
immersed in solid culture
medium. c Callus were induced at
the cut surface of both end of the
explants and derived from both
cortical and medullary cells. d
Callus followed by formation of
disorganized cell mass and
formed callus clumps with
prolific filamentous outgrowths. e
Regeneration of callus of K.
alvarezii. f Regeneration and
elongation of shoot from callus of
K. alvarezii

743

relationship between the level of endogenous plant hormones

and organogenesis, a subject which has yet to be further
understood in seaweeds (Kim et al. 2000; Reddy et al. 2008;
Fig. 14 Effect of PES liquid
media with and without plant
growth regulators (0.1 mg L1
2,4-D+0.1 mg L1 kinetin) on the
biomass of G. changii explant in
tubular and spherical airlift
photobioreactors on day 0
(diagonally striped bar) and
day 72 (solid gray bar) of culture.
Data are shown as mean value
standard error (experimental
period=72 days; no. of
replicates=3)

Baweja et al. 2009). Yokoya and Handro (2002) reported that

the physical state of the culture medium affected the regeneration process in Solieria filiformis: Liquid medium induced

744

J Appl Phycol (2014) 26:729746

Fig. 15 Effect of tubular and

spherical airlift photobioreactors
(1, 5 L) on carbohydrate content
of G. changii explants culture in
PES liquid media with plant
growth regulators (0.1 mg L1
2,4-D+0.1 mg L1 kinetin)
(diagonally striped bar) and
without (solid gray bar) plant
growth regulator. Data are shown
as mean valuestandard error
(experimental period=72 days,
no. of replicates=3)

regeneration of adventitious plantlets, while solid medium

induced growth of filaments. Similar observations were reported by Polne-Fuller et al. (1986) where solid medium
induced callus formation and liquid medium induced thallus
differentiation for Porphyra , Laminaria angustata and
Dictyosiphon foeniculaceus. However, the physical state of
the culture medium showed no significant effect on the regeneration process in G. changii explants in the present study.
Fig. 16 New branch formations
in G. changii explants. a New
branch formations in G. changii
explants harvested from PES
medium with 0.1 mg L1 2,4-D+
0.1 mg L1 kinetin in 3,000 mL
spherical airlift photobioreactors.
b New branch formations in G.
changii explants harvested from
PES medium with 0.1 mg L1
2,4-D+0.1 mg L1 kinetin in
3,000 mL tubular airlift
photobioreactors. c New branch
formations in G. changii explants
harvested from PES medium with
0.1 mg L1 2,4-D+0.1 mg L1
kinetin in 500 mL spherical airlift
photobioreactors. d New branch
formations in G. changii explants
harvested from PES medium with
0.1 mg L1 2,4-D+0.1 mg L1
kinetin in 500 mL tubular airlift
photobioreactors

Huang and Fujita (1997) showed that success in callus

induction varies with species; of the 14 species of red seaweeds tested, they found that G. textorii, Gloiopeltis tenax
and Laurencia undulata were the three species which had the
lowest callus induction rate. They also reported that most of
the species tested produced callus in ASP12NTA 1.5 % solid
medium regardless of the effect of plant growth regulators
(IAA, BAP).

J Appl Phycol (2014) 26:729746

When the same protocols were repeated with G. changii

and K. alvarezii, it was found that, although callus was not
induced in G. changii, more than 90 % of K. alvarezii explants produced callus after 30 days culture. The success of
callus induction in K. alvarezii explants indicated that there
was probably no significant problem in the callus induction
protocol, the personal skill and techniques as well as the
culture (incubation) environment. An important factor in seaweed callus formation is its response to injury or wounding
(Kumar et al. 2004, 2007). Callus formation in G. verrucosa
(Kaczyna and Megnet 1993) and G. corticata (Kumar et al.
2007) has been reported as a result of wound responses. In the
present study, callus formation was found in 68.5732.37 %
of K. alvarezii explants cultured in 1.0 % w/v Bacto agar
solidified PES medium for 30 days without any plant growth
regulator indicating that the wounding effect may be responsible for inducing callus formation in K. alvarezii explants.
However, the wounding effect (cutting) at both ends of the G.
changii explants did not induce any callus formation in G.
changii explants but produced adventitious shoots instead.
The failure to induce callus in G. changii may be due to the
microenvironment of its original habitat. G. changii inhabited
the mangroves while the Kappaphycus was farmed using
vegetative propagation on long lines. The Gracilaria grew
epiphytically on the roots of mangrove trees, and propagation
was probably through spores produced during the triphasic
life cycle. Perhaps microflora is present in the mangrove soil
which produces micronutrientsessential for the growth of
the seaweed, especially under artificial conditions. Mangrove
soils are sulphur rich as well, and the use of sulphate in
the culture medium could be an interesting study. The
present study can be used as guideline for further studies. Kappaphycus , on the other hand, has been cultivated artificially in Sabah for several decades through
cuttings and has probably developed the capacity for
totipotency.
In conclusion, callus formation and regeneration was successfully obtained for K. alvarezii explants. No callus formation was observed in G. changii explants; instead, proliferation of adventitious shoots was successfully obtained. The use
of airlift photobioreactors not only increased the biomass of G.
changii explants but also showed that there were no significant changes in the carbohydrate content of the explants
compared to the original plants. These findings will contribute
to successful production of clonal planting materials to support the expanding seaweed farming in Malaysia.
Acknowledgments This research was funded by the Ministry of Science, Technology and Innovation, Malaysia (E-Science Fund: 02-01-03SF0148 and 04-01-03-SF0664; Top-down Project: 06-02-02-003 BTK/
ER/016, Grant No: 39-02-03-9002/Oracle 8301902), the Malaysian
Toray Science Foundation (Japan) Grant (MTSF 1050-2009A) and the
University of Malaya Research Grants (UMRG Grant No: RG10010SUS, F0233/2004D, P0114/2006A).

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