J. Seckbach and D.J. Chapman (eds.

), Red Algae in the Genomic Age,

Cellular Origin, Life in Extreme Habitats and Astrobiology 13, 307341
DOI 10.1007/978-90-481-3795-4_17, Springer Science+Business Media B.V. 2010

DEVELOPMENTS IN BIOTECHNOLOGY OF RED ALGAE

C.R.K. REDDY, VISHAL GUPTA, AND BHAVANATH JHA

Discipline of Marine Biotechnology and Ecology, Central Salt
and Marine Chemicals Research Institute, Council of Scientific
and Industrial Research (CSIR), Bhavnagar, 364002, India

1. Introduction
The marine macroalgae represent one of the components of coastal ecosystem
and are of fundamental ecological importance as primary producers in the coastal
regions of ocean waters. Of the total marine macrophytic algae, red algae with
most ancient eukaryotic lineage are diverse in their habitats and cellular organizations, and comprise the highest number of species diversity over 6,000 followed by
brown algae and green algae with 1,700 and 1,200 species, respectively (Guiry and
Guiry, 2009). Red algae constitute the largest number of commercially valuable
species of the three macroalgal groups, and include species cultivated and harvested as a source of food, phycocolloids, phycosupplements (soil additives, fertilizers, and animal feed), pharmaceuticals, nutraceuticals, and cosmetics (Hanisak,
1998; McHugh, 2003). The most valued of all marine macroalgae is the edible
red alga Porphyra (or nori in Japanese), which is consumed as food throughout
the world and is the basis for the development of farming in Japan, Korea, and
China. Total production volume of Porphyra worldwide is 900,000 wet tons per
annum with a value of US$1.5 billion (Hanisak, 1998; Chopin and Bastarache,
2004). The other important utilization of red seaweeds is as a source of raw material for production of phycocolloids; the gelling, thickening, emulsifying, binding,
stabilizing, clarifying, and protecting agents known as carrageenans and agars.
Kappaphycus, Eucheuma, and Chondrus are the principal sources of raw material for production of carrageenan and cultivated extensively in several tropical
countries as an alternative livelihood opportunity. Current carrageenan industry
worldwide utilizes around 140,000 dry tons harvested through aquaculture practices and produces around 50,000 tons carrageenan with a market value over
US$ 600 million (Guiry, 2008). Gelidium and Gracilaria are the two important
agarophytes that form the major part of total agarophytes consumed by world
agar industry. Annually, about 125,000 wet tons of agarophytes are processed and
7,500 tons agar produced with a value of US$132 million (Hanisak, 1998; Chopin
and Bastarache, 2004).
The most emerging and promising component of seaweed-based industry
is the phycosupplement, which is estimated at 1.22 million wet tons and valued
at US$53 million (Hanisak, 1998; Chopin and Bastarache, 2004). Most of the tonnage
309

310

C.R.K. REDDY ET AL.

is used for the manufacturing of soil additives, fertilizers (agrichemicals), and

animal feeds. In fact, the Canadian-based private company Acadian Seaplants
Limited (ASL), the only large commercial seaweed grower outside of Asia, is a
leading exporter of phycosupplement products and accounts for 3040% of total
world market. Further, it is also a world leader in the development of a landbased seawater tank cultivation system involving several seaweed strains. Some of
these cultivated seaweeds with edible application have recently found exclusive
niche market in Japan providing much higher added value for the crop than those
cultured earlier for the purpose of carrageenan. Similarly, the central salt and
Marine Chemicals Research Institute (CSMCRI), India, has developed and patented an integrated technology for production of sap (80% fresh wt) from red
alga Kappaphycus alvarezii along with a residue that yields k-carrageenan
(Eswaran et al., 2005). The sap has turned out to be a promising liquid fertilizer
with all essential plant nutrients. Given that the fertilizer will be required in large
volumes, there could be a demand for producing very large quantities of biomass
in India.
It is evident from the above that global seaweed industry today consumes
large volumes of raw material that are largely met from the intensive farming practices. Therefore, the initial studies, in general, on cultivation have largely focused
on domestication and selection of strains from wild stocks to multiple the production potentials of cultivated species (Santelices, 1992). Further, the outbreak of
diseases like red rot and chytrid blight in Porphyra (Fujita, 1990; Park et al., 2006)
and ice-ice and epiphytic infection in Kappaphycus (Largo et al., 1995a, b;
Vairappan et al., 2008) have also been reported to impact the crop yields of farmed
red algae causing severe economical losses to the farming industry. As in agriculture, the need to produce better strains for cultivation has been the main impetus
behind the biotechnological developments in the red algae. That is, techniques like
tissue culture, mutagenesis, and selection developed in the 1980s and then protoplast fusion in the 1990s because classical breeding methods were of little benefit
in red macroalgal cultivation. The earlier studies pursued to develop the field of
cellular biotechnology of seaweeds have truly succeeded and resulted in establishment of pioneering methods for routine tissue and protoplast culture from a wide
range of seaweeds including red algae (see reviews by Polne-Fuller, 1988; Butler
and Evans, 1990; Garcia-Reina et al., 1991; Reddy et al., 1994; Aguirre-Lipperheide
et al., 1995; Reddy et al., 2008a; Baweja et al., 2009). The advances made in cellular
biotechnology have provided the spurt in the studies on molecular biotechnology
of seaweeds (see reviews by Stevens and Purton, 1997; Minocha, 2003; Walker
et al., 2005; Chan et al., 2006; Reddy et al., 2008b) to realize the benefits offered by
seaweed resources to the fullest extent. The previous reports have dealt with either
cellular or molecular biotechnology of seaweeds and rarely covering both. The
objective of the present report is to review the progress made and discuss the current status of application of cellular and molecular biotechnology techniques
exclusively for red seaweeds.

DEVELOPMENTS IN BIOTECHNOLOGY OF RED ALGAE

311

2. An Overview of Cellular Biotechnology and Their Applications

The cellular biotechnology mainly comprises the tissue, callus, and protoplast culture
and offers vast opportunities in the area of plant biotechnology, and allowed widespread use of cell culture for in vitro genetic manipulation, plant propagation, and
production of commercially valuable metabolites. The progress achieved in respective areas is briefly reviewed in the following sections.

2.1. STATUS OF TISSUE AND CALLUS CULTURE IN RED ALGAE

The commercial farming of seaweeds especially agarophytes and carrageenophytes
traditionally made use of vegetative thalli for their propagation in the sea (Dublin,
2005; Hurtado et al., 2006; Molloy, 2006; Ganesan et al., 2006, 2009). The practice
of selection of seed stock of fastest growing plants of one season as the seed for
the next season in Kappaphycus and Eucheuma led to decline in the crop production (Dawes et al., 1993), which had been attributed to the use of reduced genetic
diversity of seed stock. Consequently, several researchers including Cheney (1999)
argued that new varieties would have to be produced and preserved using tissue
culture and genetic manipulation techniques, which not only increase the crop
production rates but also provide a variety of benefits with consequential implications in overall seaweed biotechnology.
There are 51 species belonging to 26 genera that have been successfully grown
in tissue culture, including virtually every major commercially valuable species
(Table 1). The callus induction rates for red algae varied greatly from species to
species. A number of tissue culture studies dealt with agarophytes and carrageenophytes have demonstrated the production of viable micropropagules with a promising application of micropropagation for mariculture (Dawes and Koch, 1991;
Reddy et al., 2003; Rajakrishna Kumar et al., 2004, 2007). Later, a technique
producing substantially high yields of micropropagules (Fig. 1) from pigmented
filamentous callus has been accomplished for commercially important red alga
Kappaphycus alvarezii (Reddy et al., 2003). Another important and emerging
development in this area of research has been the establishment of in vitro culture
system and photo bioreactors specifically suitable for bioprocess technology for
production of useful secondary metabolites with potential medicinal applications
(e.g., halogenated monoterpenes, agardhilactone) from select multicellular marine
macroalgae under optimized culture conditions (Malaikal et al., 2001; Rorrer and
Cheney, 2004). To date, there are only two published reports where tissue culture
progeny has been transplanted in the sea and studied for growth. Incidentally, both
studies have dealt with farmed Kappaphycus and reported daily growth rates of
55.5% over 85 days cultivation period in one case (Dawes et al., 1993) and 1.51.8
times over the farmed plants propagated through vegetative means in another case
(Reddy et al., 2003).

E. denticulatum
E. uncinatum
Furcellaria fastigata
Gelidium robustum
Gelidiella acerosa
G. vagum
Gigartina exasperata
Gloiopeltis tenax
Gracilaria chilensis
G. corticata
G. papenfussii

Eucheuma denticulatum

NAA, BAP, IAA, K

NAA, BAP, K, Spm
2-4D, BAP, IAA,
Colchicine
PAA, Zea
PAA, IBA, IAA, NAA,
K, 2ipBAP, Zea
PAA, Zea
NR
NR
NR
NAA, BAP, IAA, K
NR
NR
IAA, BAP
IAA, K
NAA, BAP, IAA, K
NR

NAA
PAA, NAA, BAP, K,
2,4,5 T, IAA
IAA, BAP
IAA, BAP
IAA, BAP
NR
K, NAA
IAA, BAP
NR

Agardhiella subulata

Ahnfeltiopsis flabelliformis
Carpopeltis affinis
C. prolifera
Ceramium kondoi
Chondrus crispus
Chondracanthus tenellus
Kappaphycus alvarezii

PGRs

Species

ESS/2
PES
MS
PES
PES
MS
PES
ASP12-NTA
PES
PES
ASP12-NTA

ESS
PES
VS 50, F/2 50,
ASP12-NTA
ESS/2
ESS

ASP12-NTA
ASP12-NTA
ASP12-NTA
MS
SWMD1
ASP12-NTA
PES

mASP12-NTA
f/5

Medium

Table 1. List of red algae for which tissue and callus culture accomplished.

PR
PR
CI
PR
PR
CI
PR
CI
CI
CI & PR
PR

CI & PR
PR

NR
NR
NR
3
16
2
93
15
3
10
NR
60
2

SE
CI
CI & PR

PR
CI & PR
CI & PR
CI
PI
CI & PR
PR

PR
PR

Status

90
100
100

38
96
60
2
NR
18
10

NR
NR

Callus induction (%)

Hurtado and Biter (2007)

Dawes and Koch (1991)
and Dawes et al. (1993)
Hurtado and Cheney (2003)
Polne-Fuller and Gibor (1987)
Gusev et al. (1987)
Polne-Fuller and Gibor (1987)
Rajakrishna Kumar et al. (2004)
Gusev et al. (1987)
Polne-Fuller and Gibor (1987)
Huang and Fujita (1997)
Collantes et al. (2004)
Rajakrishna Kumar et al. (2007)
Polne-Fuller and Gibor (1987)

Cheney et al. (1987)

Bradley and Cheney (1990)
and Huang et al. (1998)
Huang and Fujita (1997)
Huang and Fujita (1997)
Huang and Fujita (1997)
Gusev et al. (1987)
Chen and Taylor (1978) and Chen (1982)
Huang and Fujita (1997)
Polne-Fuller and Gibor (1987), Dawes
and Koch (1991) and Dawes et al. (1993)
Reddy et al. (2003)
Munoz et al. (2006)
Hayashi et al. (2008)

Reference

312
C.R.K. REDDY ET AL.

K, IAA, 2-4D
K, IAA, 2-4D
IAA, BAP
IAA, BAP, 2-4D
NR
NR
IAA, 2-4D, BAP
IAA, BAP
NR
NR
NR
IAA, BAP
IAA, BAP
IAA, BAP
NAA, BAP, IAA, K
IAA, 2-4D, K
K, BAP, 2-4D, NAA
NR
K, BAP, NAA, 2-4D
IAA, BAP
IAA, BAP
Zea, PAA

G. perplexa
G. tenuistipitata
G. textori
G. vermiculophylla
G. verrucosa

NR
NR

NR
NR
NAA, K
NR

Phyllophora nervosa
Porphyra perforata

P. lenceolata
P. nereocystis
P. umbilicalis
P. yezoensis

L. paniculata
L. undulata
Meristotheca papulosa
Ochtodes secundriramea

Laurencia sp.

Gracilariopsis tenuifrons
Grateloupia acuminata
G. doryphora
G. dichotoma
G. filiformis
G. filicina
G. turuturu
G. imbricata
Hypnea musciformis

PGRs

Species

MS
ASPC-1 &
ASP12-NTA
ASP6-F2
ASP12-NTA
La
NR

ASP12-NTA
ASP12-NTA
ASP12-NTA
ASP12-NTA
M&S
ASP6-F2
ASP12-NTA
ASP12-NTA
mPES
ASP12-NTA
Von stosch
ASP12-NTA
ASP12-NTA
ASP12-NTA
PES
ASP12-NTA
PES
PES
mMS
ASP12-NTA
ASP12-NTA
ASP12-NTA

Medium

84
90
80

18
89

2
NR

36
57
90
NR
18
40
NR

NR

100
100
5
NR
4
30
NR
92
100

Callus induction (%)

PR
PR
CI
PR

CI&PR
PR

CI
CI
CI
CI & PR
CI& PR
CI & PR
PR
CI
BF
CI
CI&PR
CI
CI
CI
CI
CI
PR
SR
CI
CI
CI
PR

Status

(continued)

Polne-Fuller and Gibor (1987)

Polne-Fuller and Gibor (1987)
Liu and Kloareg (1992)
Yamazaki et al. (1998) and Hafting (1999)

Yokoya et al. (2004)

Yokoya et al. (2004)
Huang and Fujita (1997)
Yokoya et al. (1999)
Gusev et al. (1987)
Kaczyna and Megnet (1993)
Yokoya (2000)
Huang and Fujita (1997)
Robaina et al. (1990)
Yokoya and Handro (1996)
Yokoya et al. (1993)
Huang and Fujita (1997)
Huang and Fujita (1997)
Huang and Fujita (1997)
Rajakrishna Kumar et al. (2007)
Yokoya et al. (2003)
Garcia-Reina et al. (1988)
Robaina et al. (1992)
Gusev et al. (1987)
Huang and Fujita (1997)
Huang and Fujita (1997)
Malaikal et al. (2001) and
Rorrer and Cheney (2004)
Gusev et al. (1987)
Polne-Fuller and Gibor (1987)

Reference

DEVELOPMENTS IN BIOTECHNOLOGY OF RED ALGAE

313

IAA, BAP
NAA, K
NR

IAA, BAP
K, BAP, NAA, 2-4D
NR
NR

Prionitis crispata
Pterocladia capillacea

Ptilophora subcostata
Rhodymenia pertusa
Smithora naiadum
Solieria filiformis

ASP12-NTA
mPES
NSW, PES,
mNSW, mPES
ASP12-NTA
mMS
ASPC1
NaNO3 +
NaH2PO4

Medium

39
NR
85
90.3

65
12

Callus induction (%)

CI
CI
PR
CI

CI
CI
CI&BS

Status

Huang and Fujita (1997)

Gusev et al. (1987)
Polne-Fuller and Gibor (1987)
Robeldo and Garcia-Reina (1993)

Huang and Fujita (1997)

Liu and Gordon (1987)
Liu et al. (1990)

Reference

ASP6-F2, artificial seawater medium (Fries); ASP-12NTA, artificial seawater medium with nitrlotriacetic acid; PES, provosoli enriched seawater medium; ESS,
erdshcribers seawater; MS, Murashige and Skoog; BAP, benzylaminopurine; 2-4D, 2,4,dichlorophenoxy acetic acid; IAA, indole acetic acid; K, kinetin; NAA,
naphthalene acetic acid; PAA, phenyl acetic acid; 2,4,5 T, IAA, 2,4,5 trichlorophenoxy acetic acid; Zea: zeatin; Spm, spermine; NR, not reported; BF, bud formation;
CI, callus induction; PR, plant regeneration; SE, somatic embryogenesis; AV, adventitve embryogenesis; BS, biochemical study; SD, shoot differentiation.

PGRs

Species

Table 1. (continued)

314
C.R.K. REDDY ET AL.

DEVELOPMENTS IN BIOTECHNOLOGY OF RED ALGAE

315

Figure 1. Mass production of micropropagules from pigmented filamentous callus of Kappaphycus

alvarezii.

The role of plant growth regulators (PGRs) on callus induction and differentiation in multicellular algae is often reported with divergent views (Bradley, 1991;
Evans and Trewavas, 1991). Nevertheless, indole-3-acetic acid (IAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and kinetin had stimulatory role in callus formation,
growth, and regeneration both in intercalary and apical explants of Gracilaria
tenuistipitata, G. perplexa, and Grateloupia dichotoma (Yokoya and Handro, 1996;
Yokoya et al., 2004). Additions of a combination of auxins and cytokinins to culture medium resulted in induction of callus in Gracilaria verrucosa (Kaczyna and
Megnet, 1993). The implications of PGRs on callus formation have also been
reported to vary with the seaweed and photon flux densities used during explant
culture. The brown color morph of Hypnea musciformis had the highest rate of
callus formation in high photon irradiance with low concentration of IAA, while
green color morphs produced calli with 2,4-D irrespective of concentration and
photon flux densities used. Similarly, IAA:BA (5:1 mg l1) stimulated callus formation in Kappaphycus strain originated from tetraspore germination. Treatment of
explants with colchicin (0.01%) for 14 days in half strength von Stoschs medium
with glycerol (90 mM) further increased the regeneration potential of callus
(Hayashi et al., 2008). A water-soluble extract from Laurencia sp. increased callus

316

C.R.K. REDDY ET AL.

formation on explants of the same species (Robaina et al., 1992). In view of these
conflicting findings, it would be interesting to test the use of some PGR conjugates
that have been recently reported in a number of seaweeds (Stirk et al., 2005).
Cell suspension cultures for red algae have not been so far achieved except
for Porphyra (Chen, 1989). The possible reason could be attributable to their callus
structure and morphology. In most cases, the callus from macroalgae is composed
of uniseriate branched filaments and is rigid (nonfriable). In contrast to friable
callus of higher plants, the filamentous callus regenerate directly into full plants
via microplantlet stage when transferred to agitated liquid cultures. The bioprocess
engineering studies carried out in Agardhiella subulata (Huang et al., 1998),
Ochtodes secundiramea (Malaikal et al., 2001), and Portieria hornemannii
(Barahona and Rorrer, 2003) have utilized the microplantlets and filamentous
clumps obtained from cell and tissue culture for continuous culture system. While
these suspension culture systems may not look like a typical cell culture system of
a land plant, they are functionally analogous to cell suspension cultures of higher
plants and differ in many ways from whole plants. They typically consist of a
mass of tiny shoots, which can be subcultured and grown in photo-bioreactors in
the same fashion as a cell suspension culture.
2.2. TRENDS IN PROTOPLAST RESEARCH OF RED ALGAE
Protoplasts form another important component of cellular biotechnology. Protoplast
isolation, regeneration, and somatic hybridization through protoplast fusion are
of particular interest in seaweed biotechnology and genetic improvement of
cultivars. Protoplasts are living plant cells devoid of rigid cell wall and with their
totipotent potential and homogeneous cell system facilitate the exploration of
several aspects of modern biotechnology. Treatment of cells or tissues with specific
cell wall degrading enzymes under defined conditions results in total removal of
their complex polysaccharide cell wall. Although the first protoplasts produced
from marine macroalgae were obtained using mechanical methods (e.g., Tatewaki
and Nagata, 1970; Enomoto and Hirose, 1972), it was not until the discovery
of suitable enzymes for cell wall digestion for the field to gain momentum in
late 1980s and early 1990s. Soon after, protoplasts had been isolated from over
40 species (Table 2) of different red algae. The two important genera that
undoubtedly received overwhelming attention for development of protoplast
studies are Gracilaria and Porphyra because of their proven commercial importance in food and biotechnology industry.
The totipotency property of algal protoplasts has allowed their consideration
for a variety of applications in both fundamental and applied research. The protoplasts of Porphyra have been used for establishing the select cell lines having resistance to low salinity (Iwabuchi, 1995), higher temperatures (Masuda et al., 1995),
and with altered amino acid and amino acid analog contents (Yamashita and Fujita,
1996). In another study, protoplasts from Porphyra have also been successfully

317

DEVELOPMENTS IN BIOTECHNOLOGY OF RED ALGAE

Table 2. Red algal species for which protoplast production reported.

Species
Acrosorium
polyneurum

Bangia
atropurpurea

Chondrus
crispus

Gelidium
robustum
Gracialria
asiatica

Enzyme composition
Cellulase R-10
Macerozyme
Agarase
Papain
Mannitol
Pretreatment
Papain
Mannitol
MES
Treatment
Agarase
-1,4-Mannanase
Xylanase
Mannitol
MES
pH
Carragenase
Cellulase
Pectolyase
Macerozyme
Sorbitol
Sea Salt
pH
Pretreatment
NaCl
MgCl2
Tris
PMSF
Treatment
Cellulase
Carragenase
Sorbitol
NaCl
MgCl2
PMSF
KCl
Tris
pH

Pretreatment
SW:DW
Mannitol
Treatment
Crude Agarase
Cellulase
Sea snail enzyme
SW:DW

Yield (g1 f wt.)

4%
2%
50 U
2%
0.6 M

Status Reference
PI

Yamaguchi
et al. (1989)

PI

Araki et al. (1994)

BS

Smith and
Bidwell (1989)

5.8 108

PI

Le Gall et al. (1990)

18.5 108

PI

Coury et al. (1993)

PI

Yan and Wang (1993)

5.7 106
2%
0.5 M
20 mM
1U
1U
1U
0.7 M
20 mM
6.0
1%
1%
1%
0.5%
14.5%
2.4%
7.5
450 mM
120 mM
100 mM
0.2 mM
1%
180 U
0.35 M
0.5 M
40 mM
0.2 mM
5 mM
50 mM
6.0

60:40
1M

4%
1%
60:40
(continued)

318

C.R.K. REDDY ET AL.

Table 2. (continued)
Species

G. changii

G. chilensis
G. chorda

G. filicina

G. gigas

G. lemaneiformis

Enzyme composition
Mannitol
CaCl2
pH
Pretreatment
SW:DW
Mannitol
Treatment
Cellulase
Macerozyme
Agarase
MES
pH
NA
Cellulase
Macerozyme
Pectolyase
Abalone acetone
powder
Mannitol
CaCl2
pH
Pretreatment
Papain
NaCl
Mannitol
Tris
pH
Treatment
Bacterial crude
extract
Cellulase
Macerozyme
Top shell powder
MES
pH
Cellulase
Macerozyme
Pectolyase
Abalone acetone
powder
Mannitol
CaCl2
pH
Cellulase R-10
Macarozyme
Agarase
Pectolyase
Mannitol

Yield (g1 f wt.)

Status Reference

58 104

PR

Yeong et al. (2007)

NA
0.220 105

PR
PI

Cheney (1990)
Chou and Lu (1989)

10 106

PI

Yamaguchi
et al. (1989)

0.220 105

PI

Chou and
Lu (1989)

310 105

CW

Cheney et al. (1986)

0.8 M
5 mm
6.26.5
60:40
1.0 M
2%
1%
10 U/ml
50
6.0
NA
3%
3%
0.5%
10%
0.8 M
5 mM
5.86.0
2%
2%
0.7 M
50 mM
7.5

0.5%
0.2%
2.5%
25 mM
6.0
3%
3%
0.5%
10%
0.8 M
5 mM
5.86.0
3%
3%
1%
0.5%
1M

(continued)

319

DEVELOPMENTS IN BIOTECHNOLOGY OF RED ALGAE

Table 2. (continued)
Species

G. lemaneiformis

G. salicornia

G. sordida

G. tenuistipitata

G. tikvahiae

G. verrucosa

G. verrucosa

Enzyme composition
CaCl2
SW:DW
MES
pH
Cellulase
Agarase
Mannitol
pH
Cellulase
Macerozyme
Pectolyase
Abalone powder
Mannitol
CaCl2
pH
Cellulase
Agarase
Mannitol
pH
Cellulase
Macerozyme
Pectolyase
Abalone powder
Mannitol
CaCl2
pH
Cellulase R-10
Macerozyme
Agarase
Pectolyase
Mannitol
CaCl2
SW:DW
MES
pH
Cellulase
Agarase
Mannitol
pH
Pretreatment
CaCl2.2H2O
Mannitol
Tris MES
pH
Treatment
Agarase
CaCl22H2O

5 mM
60:40
50 mM
6.0
2%
0.01%
0.4 M
7.0
3%
3%
0.5%
10%
0.8 M
5 mM
5.86.0
2%
0.01%
0.4 M
7.0
3%
3%
0.5%
10%
0.8 M
5 mM
5.86.0
3%
3%
1%
0.5%
1M
5 mM
60:40
50 mM
6.0
2%
0.01%
0.4 M
7.0

Yield (g1 f wt.)

Status Reference

105 107

PI

Bjork et al. (1990)

0.220 105

PI

Chou and
Lu (1989)

105107

PI

Bjork et al. (1990)

0.220 105

PI

Chou and Lu (1989)

Bjork et al. (1990)

310 105

PR

Cheney et al. (1986)

Cheney (1990)

105107

PI

Bjork et al. (1990)

Araki et al. (1998)

810 106

CW

Mollet et al. (1995)

10 mM
0.4 M
10 mM
6.0
25 U
10 mM
(continued)

320

C.R.K. REDDY ET AL.

Table 2. (continued)
Species

Grateloupia
sparsa

G. filicina

G. turuturu

Halymenia
formosa

Kappaphycus
alvarezii

Enzyme composition
Cellulase
Mannitol
Tris Mes
pH
Cellulase
Macerozyme
Agarase
Papain
Mannitol
Pretreatment
Papain
NaCl
Mannitol
Tris
pH
Treatment
Bacterial extract
Cellulase
Macerozyme R-10
Top shell powder
MES
pH
Pretreatment
Papain
NaCl
Mannitol
Tris
pH
Treatment
Bacterial crude
extract
Cellulase
Macerozyme R-10
Top shell powder
MES
pH
Cellulase
Abalone acetone
powder
Mannitol
pH
Crude cellulase
Pure k carragenase
k & i carragenase
Sorbitol (Os/kg)
Abalone powder
Cellulase

1.25%
0.8 mM
10 mM
6.0
4%
2%
50 U
2%
0.6 M

Yield (g1 f wt.)

Status Reference

71077108

CW

Chen and Chiang (1995)

10 106

PI

Yamaguchi
et al. (1989)
Chen and Chiang (1994)

10 106

PI

Yamaguchi
et al. (1989)

2 104

PI

Chou and Lu (1989)

11.2 107

PI

Zablackis et al. (1993)

8.2 103

PR

Salvador and
Serrano (2005)
(continued)

2%
2%
0.7 M
50 mM
7.5

0.5%
0.2%
2.5%
25 mM
6.0
2%
2%
0.7 M
50 mM
7.5

0.5%
0.2%
2.5%
25 mM
6.0
5%
6.7%
1M
6.0
1%
195 U
108 U
1.5
5%
2%

321

DEVELOPMENTS IN BIOTECHNOLOGY OF RED ALGAE

Table 2. (continued)
Species

Laurencia
obtusa

Palmaria
palmata

Palmaria
palmata

Plocamium
cartilagineum

Porphyra.sp

P. angusta

P. crispata

P. dentata
P. lanceolata

P. leucosticta

Enzyme composition
Mannitol
CaCl2
pH
Sea urchin powder
Sorbitol
CaCl2
Phosphate buffer
pH
Cellulase
Abalone powder
PES
Phosphate buffer
Mannitol
CaCl2
pH
Cellulase R-10
Macerozyme R-10
Abalone enzyme
Bovine albumin
Na3C6H5O7.2H2O
Mannitol
pH
Sea urchin powder
Sorbitol
CaCl2
Phosphate buffer
pH
Crude enzyme
Paua gut extract
Aspergillus extract
Cellulase
Abalone powder
Mannitolo
pH
Cellulase
Abalone powder
Mannitol
pH

1.0 M
5 mM
6.06.1
10%
0.8M
3.4 mM
0.1 M
6.0
3%
0.10.2%
60%
40%
0.1 M
5 mM
6.06.2
2%
1%
2%
0.5%
50 mM
0.8 M
5.8
10%
0.8 M
3.4 mM

Abalone powder
Sorbitol
HEPES
pH
Sea snail
Cellulase
Pectolyase
Sorbitol
CaCl2

10%
0.6 M
50 mM
6.0
2%
1.5%
0.5%
0.5 M
0.08%

6.0
187.5 L
37.5 L
125 L
5%
6.7%
1M
6.0
5%
6.7%
1M
6.0

Yield (g1 f wt.)

Status Reference

PI

Balestri et al. (1989)

46 107

PI

Liu and Kloareg (1992)

Nikolaeva
et al. (1999)

5 107

PS

Le Gall et al. (2004)

PI

Balestri et al. (1989)

500/field

PI

Packer (1994)

2 105

PI

Chou and Lu (1989)

2 105

PI

Chou and Lu (1989)

Gall et al. (1993)

PI
PR

Gall et al. (1993)

Polne-Fuller
et al. (1984)

PR

Chen (1987)

(continued)

322

C.R.K. REDDY ET AL.

Table 2. (continued)
Species

Enzyme composition

NaH2PO4
Ca(H2PO4)H2O
pH
P. linearis
Sea snail extract
Cellulase R-10
Sorbitol
pH
P. linearis
Agarase
Mannitol
pH
P. nereocystis
Pretreatment
Papain
Mannitol
MES
pH
Treatment
Abalone powder
Mannitol
MES
pH
P. okamurae
Pretreatment
Proteolyase
Mannitol
HEPES
pH
Treatment
Bacterial extract
Dextran sulfate
HEPES
pH
P. okhaensis
Protease P6
Cellulase
Macerozyme R-10
Agarase
Abalone powder
Dextran sulfate
NaCl
HEPES
pH
P. perforata
Abalone acetone
powder
Sorbitol
HEPES
pH
P. pseudolinearis Pretreatment
Proteolyase
Mannitol
HEPES

Yield (g1 f wt.)

0.01%
0.01%
7.0
2%
3%
0.5 M
7.0
0.2%
0.1 M
6.8

Status Reference

PR

Chen et al. (1988)

Chen (1989)

PR

Chen et al. (1995)

1218 106

PR

Waaland et al. (1990)

5 105
1.5 107

PI

Fujita and
Saito (1990)

23.2 0.24
106

PI

Dippakore
et al. (2005)

PR

Polne-Fuller et al. (1984,

1990)
Saga et al. (1986)

PR

Fujita and
Saito (1990)

10%
0.5 M
50 mM
6.0
2%
0.5 M
50 mM
6.0
5%
0.6 M
50 mM
8.0

0.5%,
50 mM
8.0
1%
2%
2%
50 U
1%
0.5%
1%
25 mM
6.0
10%
0.6M
50 mM
6.0
5%
0.6 M
50 mM

5 1051.5
107

(continued)

DEVELOPMENTS IN BIOTECHNOLOGY OF RED ALGAE

323

Table 2. (continued)
Species

P. seriata

P. suborbiculata

P. suborbiculata

P. tenera

Enzyme composition
pH
Treatment
Bacterial extract
Dextran sulfate
HEPES
pH
Pretreatment
Proteolyase
Mannitol
HEPES
pH
Treatment
Bacterial crude
extract
Dextran sulfate
HEPES
pH
Limpets extract
Cellulase
Glucose
Pretreatment
Proteolyase
Mannitol
HEPES
pH
Treatment
Bacterial crude
extract
Dextran sulfate
HEPES
pH
Pretreatment
Papain
Mannitol
Tris
pH
Treatment
Abalone acetone
powder
Mannitol
CaCl2
NaCl
MES
pH
Pretreatment
Protease
Mannitol
HEPES

Yield (g1 f wt.)

Status Reference

5 1051.5
107

PI

Fujita and
Saito (1990)

CW

Tang (1982)

5 1051.5
107

PI

Fujita and Saito (1990)

2.5 106

PI

Song Ho and Chung

(1988)

5 1051.5
107

PI

Fujita and Saito (1990)

8.0

0.5%,
50 mM
8.0
5%
0.6 M
50 mM
8.0

0.5%,
50 mM
8.0
2%
2.0 M
5%
0.6 M
50 mM
8.0

0.5%,
50 mM
8.0
2.5%
0.7M
50 mM
7.5

0.7 M
5 mM
2%
25 mM
7.5
5%
0.6 M
50 mM

(continued)

324

C.R.K. REDDY ET AL.

Table 2. (continued)
Species

P. tenuipedalis

P. yezoensis

P. yezoensis

Solieira
filiformis

Enzyme composition
pH
Treatment
Bacterial crude
extract
Dextran sulfate
HEPES
pH
Pretreatment
Protease
Mannitol
HEPES
pH
Treatment
Bacterial crude
extract
Dextran sulfate
HEPES
pH
Pretreatment
Papain
Mannitol
MES
Treatment
b-1,4-Mannanase
b-1,4-xylanase
pH
Pretreatment
Protease
Mannitol
HEPES
pH
Treatment
Bacterial extract
Dextran sulfate
HEPES
pH
b-Mannanase,
Mannitol
Cellulysin
Cellulase
Abalone powder
Agarase
Pectolyase
Carragenase
Mannitol
pH

Yield (g1 f wt.)

Status Reference

8.0

0.5%,
50 mM
8.0
51051.5 107 PR

Fujita and Saito (1990)

Saga and Sakai (1984)
Fujita and Migita (1985)

8.6 105

PR

Araki et al. (1987)

Yamaguchi et al. (1989)

5 1051.5
107

PI

Fujita and Saito (1990)

PI

Ootsukaa et al. (2006)

PI

Gomez-Pinchetti and
Garcia Reina (1993)

5%
0.6 M
50 mM
8.0

0.5%,
50 mM
8.0
2%
0.7 M
50 mM
1U
1U
6.0
5%
0.6 M
50 mM
8.0

0.5%,
50 mM
8.0
5U
0.7 M
3%
3%
3%
0.01%
0.25%
30 U
0.4 M
6.0

8.7 105

BS, biochemical study; CW, cell wall formation; PI, protoplast isolation; PR, plant regeneration; GS, genetic
study; NA, not accessible; SW, seawater; DW, distilled water.

DEVELOPMENTS IN BIOTECHNOLOGY OF RED ALGAE

325

tested for their seeding and regeneration in laboratory conditions (Dippakore et al.,
2005). There also exist a number of examples where protoplasts have been used
initially for investigating the basic physiological and biochemical mechanisms. The
inorganic carbon uptake and assimilation mechanisms in Chondrus crispus (Smith
and Bidwell, 1989) and Gracilaria tenuistipitata (Haglund et al., 1992) have been
investigated using photosynthetically active protoplasts. A number of studies have
also confirmed that the physiological state of protoplasts is as same as that of intact
plants (Davison and Polne-Fuller, 1990; Amano and Noda, 1992). Secretion of
carrageenan fragments by cultured protoplasts of Kappaphycus alvarezii var.
tambalang has also been reported (Zablackis et al., 1993).
In the past, most seaweed strain improvement efforts have been confined to
the use of classical breeding techniques (strain selection, mutagenesis, and sexual
hybridization). Such efforts have met with little success in producing commercially
valuable strains in red algae. However, the major barrier in classical breeding techniques, especially sexual hybridization, is obtaining both sexes of both species at a
time. For example, in Kappaphycus and Gelidiella, male plants are rare and in some
species unknown. Further, the red algae appear to lack interspecific interfertility.
In contrast, somatic hybridization by means of protoplast fusion facilitates mixing
of two genomes of different origin, which is otherwise not possible due to sexual
incompatibility. This technique, in contrary to genetic engineering, provides an
important means to transfer the traits particularly polygenetic in nature.
The first report of successful protoplast fusion and fusion product regeneration
has been between two color morphs of red alga Porphyra yezoensis, which is
popularly consumed as human food (Fujita and Migita, 1987). Of the total 21
reports available on the protoplast fusion, 17 are related to commercially important red seaweeds (Table 3) and of which, 11 are of intrageneric fusion and the rest
five are of intergeneric fusions (Uppalapati and Fujita, 2000; Uppalapati et al.,
2000; Reddy et al., 2008b). The genus Porphyra, being an important edible red alga
and also susceptible to many marine pathogens, has been the first target for application of these techniques to the genetic improvement of this alga. Fujita and his
coworkers have extensively carried out interspecific protoplast fusion between
Porphyra yezoensis and P. pseudolinearis and screened the F1 hybrids for resistance
to red rot and chytrid blight. These studies have revealed that some F1 thalli
showed relatively low infection levels to chytrid blight than red rot when compared
with the parent P. yezoensis (Fujita and Uppalapati, 1997). The intergeneric F1
fusion products thalli of P. yezoensis and Bangia atropurpurea showed very low
infection levels to both red rot and chytrid blight in comparison with P. yezoensis
(Fujita, 1993). The heterokaryons regenerated were mostly characterized at biochemical levels. Later studies with intergeneric protoplast fusion between Porphyra
yezoensis and Monostroma nitidum have employed RAPD markers in addition to
biochemical parameters for characterizing the somatic hybrids (Kito et al., 1998).
The methods for screening and selection of heterokaryons are underdeveloped
for seaweeds. The efficient implementation of screening methodologies using
differential fluorescence labeling, and genetic and metabolic complementation can

326

C.R.K. REDDY ET AL.

Table 3. Present status of protoplast fusion and regeneration in different red algae.
Fusion species

Method of fusion

Status

Reference

Enteromorpha Porphra yezoensis

Gracilaria chilensis G. tikvahiae
P. yezoensis P. yezoensis
P. yezoensis P. pseudolinearis
P. yezoensis P. haitanensis
P. yezoensis P. tenera (green)

PEG
PEG
PEG
Electrofusion
PEG
PEG

PF
PD
PD
PD
CF
CF

P. yezoensis P. suborbiculata
P. yezoensis P. vietnamensis
P. tenera P. suborbiculata
P. linearis P. miniata
P. suborbiculata P. tenuipedalis
P. yezoensis Bangia atropurpurea
P. pseudolinearis B. atropurpurea
P. yezoensis Monostroma nitidum
P. yezoensis P. tenuipedalis

Electrofusion
No information
No information
Electrofusion
Electrofusion
No information
No information
PEG
PEG

CF
CF
CF
CF
CF
CF
CF
PD
PD

P. yezoensis Enteromorpha compressa

P. yezoensis M. nitidum

PEG and electrofusion

PEG and electrofusion

PF
PF

Saga et al. (1986)

Cheney (1990)
Fujita and Migita (1987)
Fujita and Saito (1990)
Dai et al. (1993)
Araki and Morishita
(1990)
Mizukami et al. (1995)
Matsumoto et al. (1995)
Matsumoto et al. (1995)
Chen et al. (1995)
Achiha (1995)
Fujita (1993)
Fujita (1993)
Kito et al. (1998)
Uppalapati and Fujita
(2000)
Uppalapati et al. (2000)
Uppalapati et al. (2000)

PF, protoplast fusion; PD, protoplast development; CF, callus development.

greatly accelerate the success rate. Also, the frequency of cybrids from somatic
hybridization can be further enhanced by inactivating the nuclei of either of partner by x-rays, g-rays, and mutagenic treatments.
To circumvent some of the inherent problems associated with protoplasts
like poor survival rates, especially for anatomically more complex red algae,
Cheney (1999) used a technique called sporeprotoplast fusion and reported
greater survival rates of hybrids in a number of red macroalgal genera, including
Porphyra, Gracilaria, and Chondrus.
3. Molecular Biotechnology of Red Algae
The genetic engineering of red seaweeds is aimed at the genetic improvement of
commercially important species for the production of gel forming polysaccharides,
improved nutritional quality and growth rates, the production of fine chemicals
(medicines and pigments), the production of disease-resistant varieties, and for
their potential use in phyto-remediation of aquatic environments. The advances
made in molecular approaches have led to better understanding of the red algal
systematics, phylogeny, and their evolutionary relationships. The red algal population studies using molecular markers have also been briefly discussed in the context
of molecular biotechnology.

DEVELOPMENTS IN BIOTECHNOLOGY OF RED ALGAE

327

3.1. STATUS OF GENETIC ENGINEERING

The genetic engineering studies carried out in macroalgae so far have mainly
dealt with commercially valuable red and brown seaweeds only. The rate and
efficiency of gene expression delimits the progress of studies in seaweeds when
compared with unicellular algae (Fukuda et al., 2008). Table 4 shows the status
of genetic engineering studies accomplished for red algal taxa. The early studies
have demonstrated the transient expression of the b-glucoridinase reporter gene
(uidA) fused to the CaMV-35S promoter in a number of macroalgal taxa using
various gene transfer methods including biolistic particle bombardment, vector mediated, and electroporation (Kurtzman and Cheney, 1991; Kubler et al.,
1994; Kuang et al., 1998). In another study, Gan et al. (2003) reported the transient expression of reporter gene lacZ fused to the SV40 promoter in Gracilaria
changii. The subsequent studies attempted to use homologous promoters for
foreign gene expression in Porphyra protoplasts using a portion of 18S rDNA
in a vector pQD-GUS, and compared the expression of GUS protein with that
of the parent pBS-GUS vector. The resultant transformants showed increased
GUS activity with pQD-GUS compared with those of pBS-GUS. Following the
Agrobacterium mediated gene transfer, Bernasconi et al. (2004) reported transformation of Porphyra yezoensis expressing bacterial nitroreductase gene (nfs1)
with abilities to detoxify trinitrotoluene (TNT). The attempts were also made
for efficient transient expression of gene construct with GAPDH promoter of
P. yezoensis by particle bombardment (Fukuda et al., 2008). Although progress
has been made in the genetic engineering of red macroalgae, it is not well developed as that for kelps as evidenced by recent reports by Qin et al. (2005). Some
of the reasons for this may have to do with the lack of appropriate promoters
and other factors described in Fukuda et al. (2008). The continuing research in
genetic engineering will lead to the development of transgenic red algae with
desired and improved qualities.
Table 4. Red algal species for which genetic engineering accomplished.
Species

Mode of gene transfer

Status of expression Reference

Chondrus crispus
Gracilaria changii
Kappaphycus alvarezii
Porphyra leucosticta
P. miniata
P. tenuipedalis
P. yezoensis
P. yezoensis
P. yezoensis
P. yezoensis
P. yezoensis
P. yezoensis

Vector mediated
Biolistic particle
Biolistic particle
Electroporation
Electroporation
Electroporation
Electroporation
Vector mediated
Electroporation
Vector mediated
Vector mediated
Electroporation

Stable
Transient
Transient
Transient
Transient
Transient
Transient
Stable
Transient
Stable
Transient
Transient

Collen et al. (2006)

Gan et al. (2003)
Kurtzman and Cheney (1991)
Lin et al. (2001)
Kubler et al. (1994)
Achiha (1995)
Kuang et al. (1998)
Cheney (1999)
He et al. (2001)
Cheney (2001)
Liu et al. (2003)
Mizukami et al. (2004)

328

C.R.K. REDDY ET AL.

3.2. SEQUENCE ANALYZED MARKERS FOR RED SEAWEED

SYSTEMATICS
The red algae with complex alternation of life cycles and great morphological plasticity have necessitated the investigations into their genetic constituents.
Furthermore, the mating patterns, gene flow among the individuals during sexual
reproduction, and genetic structure of populations are also not well understood for
this diverse group of plants. In this context, classical approaches like study of reproductive anatomy (Gargiulo et al., 1992), chemistry (Bird et al., 1987), cytogenetics,
and karyology (Plastino and Oliveira, 1988; Bird et al., 1990; Godin et al., 1993) have
been employed to understand the taxonomical and evolutionary hierarchy of different red algal taxa. The application of molecular tools and techniques has advanced
the resolution of taxonomy and evolutionary concepts about the red seaweeds.
The often used molecular markers for studying the systematics and evolutionary
relationship of red seaweeds are shown in Table 5. The molecular approaches that
were used to study the systematics of algae include the DNA fingerprinting (Rice
and Bird, 1990; Wattier et al., 1997) and gene sequence data (Destombe and Douglas,
1991; Bird et al., 1994; Goff et al., 1994). The studies on phylogenetic relatedness of
seaweeds have progressed with the generation of sequence data from the conserved
organelle markers of nuclear encoded internal transcribe region (Bhattacharya et al.,
1990; Goff et al., 1994; Bellorin et al., 2002), plastid-encoded rbcL gene and rubisco
spacer gene (Wang et al., 2000; Clerck et al., 2005), mitochondrial cox2, cox3 gene,
and cox spacer region (Zuccarello et al., 1999; Saunders, 2005; Robba et al., 2006;
Yang et al., 2008). The application of multiple markers provided additional opportunity to distinguish the differentiations at genus or species level in Porphyra based
on plastid-encoded psaA, psbA gene, and phycoerythrin gene (Yang and Boo, 2004).
Recently, partial amplication of intergenic spacer sequence has been reported
in P. haitensis (Li et al., 2009). The intergenic spacer sequences with high evolution rates than internal transcribed spacer sequences can be employed with greater
efficiency for hierarchal distinctness of even closely related species.

3.3. DEVELOPMENTS IN MOLECULAR GENETIC MARKERS

The genetic markers have been developed for the identification of plant variants, the
analysis of phylogenetic and genetic diversity, hybrid confirmation, genome mapping, and gene tagging for marker-assisted selection studies. The discriminatory
markers were developed using restriction fragment length polymorphism (RFLP)
for Porphyra (Stiller and Waaland, 1993; Teasdale et al., 2002; Niwa et al., 2006)
and others also for the screening of population of Gracilaria (Candia et al., 1999).
The advancement in the genetic studies was possible only after the development
of PCR-based genetic markers due to their simplicity and high reproducibility.
Of the PCR-based markers, RAPD markers are quite successful enough to classify even at species level and used in Gracilaria (Gonzlez et al., 1996; Lim et al.,

DEVELOPMENTS IN BIOTECHNOLOGY OF RED ALGAE

329

Table 5. Phylogenetic markers for red algae.

Gene

Primer sequence

Reference

cox 1

5-TCAACAAATCATAAAGATATTGG-3
5-ACTTCTGGATGTCCAAAAAAYCA-3
5-CCT GTN TTA GCA GGW GCT ATT
ACA ATG C-3
5-ACA GTA TAC ATA TGA TGN GCT CAA AC-3
5-GTACCWTCTTTDRGRRKDAAATGTGATGC-3
5-GGATCTACWAGATGRAAWGGATGTC-3
5-dTGTACACACCGCCCGTCGC-3
5-dATATGCTTAARTTCAGCGGGT-3
5-GGTGAATTCCATACGCTAAA ATG-3
5-GCAGCTGTAACATTCATGTA-3
5-CCCTTTATGCGATGGAAAGA-3
5-GCAGCAGTTACATTCATATA-3
5-TAATAACGAGAACCTTCTGG-3
5-TARTAACGAGARCCYTCWGG-3
5-TGTGGACCTCTACAAACAGC-3
5-CCCCATAGTTCCCAAT-3
5-TATACTTCTACAGACACAGCTGA-3
5-ATTTCACACAGGAAACAGCTATGACATGTCAAA
TAAT GGTAGTCCCCA-3
A Fwd. 5-GA(G/T)TGTCC(T/G)GG(A/T)CATTTTGG-3
D Fwd. 5-TACAATGC (T/C)GA(T/C)TT(T/C)GA(T/C)GG-3
F Revs. 5-C(T/A)CG(T/A)CC(T/A)CCCAT(T/A)GC(A/G)TGG-3
GRevs.5-TG(A/G)AA(I/C/T)GTITT(I/C/T)AG(I/T)GTCAT(C/T)TG-3
psaA130F 5-AACWACWACTTGGATTTGGAA-3
psaA180F 5-GATAGTCAWACHAGTTCWTTAGA-3
psaA1600R 5-GCATGAATATGRTGWACCAT-3
psaA1760R 5-CCTCTWCCWGGWCCATCRCAWGG-3
psbA-F 5-ATGACTGCTACTTTAGAAAGACG-3
psbA500F 5-CTCTGATGGWATGCCWYTAGG-3
psbA600R 5-CCAAATACACCAGCAACACC-3
psbA-R2 5-TCATGCATWACTTCCATACCTA-3
psbA-R1 5-GCTAAATCTARWGGGAAGTTGTG-3

Saunders
(2005)
Geraldino
et al. (2006)

cox 1

cox2-3
spacer
Nuclear
ITS
rbcl
rbcl
rbcS
Rubisco
spacer
Rubisco
spacer
RPB1

psaA

psbA

Zuccarello
et al. (1999)
Bellorin
et al. (2002)
Wang et al.
(2000)
Wang et al.
(2000)
Lee et al.
(2001)
Maggs et al.
(1992)
Zuccarello
et al. (1999)
Stiller and
Hall (1997)

Yoon et al.
(2002)

Yoon et al.
(2002)

2001), Gelidium (Patwary et al., 1993; Bouza et al., 2006), Porphyra (Dutcher and
Kapraun, 1994; Huh et al., 2006). Even the sex specific RAPD markers have been
investigated for Gracilaria gracilis and G. Changii (Martinez et al., 1999; Sim et al.,
2007). Precision of genetic marker studies have further advanced following the
development of AFLP markers, which offered greater sensitivity and reproducibility when compared with previously developed DNA markers. The first report
of use of AFLP marker was by Donaldson et al. (1998) while studying the population of Chondrus crispus. The subsequent studies carried out on population genetics of Porphyra (Iitsuka et al., 2002; Niwa et al., 2006) and Chondrus (Donaldson
et al., 2000) have also employed the AFLP markers. Further refinement in the
development of genetic marker studies were made by codominant ISSR markers in

330

C.R.K. REDDY ET AL.

Gracilaria lemaneiformis, Chondrus crispus (Xue et al., 2006; Wanga et al., 2007).
In addition to random genetic markers, cleaved analyzed polymorphic sequence
(CAPS) of four gene loci, beta-tubulin gene (-tubulin), elongation factor-1 gene
(EF-1), type II DNA topoisomerase gene (TOP2), and the c-subunit of the vacuolar-type H -ATPase gene (V-ATPase) for studying the genetic diversity within
related species of P. yezoensis (Park et al., 2007).

3.4. GENOMIC ORGANIZATION OF RED SEAWEED

The last few years have witnessed considerable progress in the field of algal
genomics. Recently, complete genome sequences from the unicellular red alga
Cyanidioschyzon merolae have been published (Matsuzaki et al., 2004). In
addition, there are several cDNA sequencing projects underway for numerous algal species. This wealth of genomic data is serving as a powerful means
for the development and application of recombinant techniques for these species. Expressed sequence tag (EST) approach inferred the data about coding
sequences from the whole genome. The ESTs approach has been used for the
first time in red alga Gracilaria gracilis to identify the genes encoding for
carbohydrate metabolism (Lluisma and Ragan, 1997). The progress in EST studies
led to the construction of cDNA libraries for several species of red algae including
Porphyra haitanensis, P. yezoensis, Griffithsia okiensis, Gracilaria changii, G.
gracilis, Chondrus crispus, and Galdieria sulphuraria (Table 6). These cDNA
libraries referred to coding genes related to metabolic pathways (Weber et al.,
2004), photosynthesis, nucleic acid synthesis, repair and processing, cellular
maintenance, stress responses (Lluisma and Ragan, 1997). This technique has
been further utilized to understand the control of complex tri-phasic life cycle
with the identification of the phase determining genes (Liu et al., 1994; Ren
et al., 2006; Kakinuma et al., 2006; Colln et al., 2006, 2007). Several genomes
have been completed for red macroalgae, including plastid genomes for a species of Gracilaria and Porphyra and mitochondrial genome for Chondrus and

Table 6. Expressed sequence tags data of red seaweeds.

Species

No. of ESTs

Reference

Porphyra haitanensis
Griffithsia okiensis
Gracilaria changii
Chondrus crispus
Porphyra yezoensis
Galdieria sulphuraria
Porphyra yezoensis
Gracilaria gracilis

5,318
1,104
8,088
4,054
1,152
5,270
81
200

Xiaolei et al. (2007)

Lee et al. (2007)
Teo et al. (2007)
Collen et al. (2006)
Kakinuma et al. (2006)
Weber et al. (2004)
Lee et al. (2000)
Lluisma and Ragan (1997)

DEVELOPMENTS IN BIOTECHNOLOGY OF RED ALGAE

331

Table 7. Genome organization of red seaweeds.

Species

Genome

Size (bp)

Reference

Galdieria sulphuraria
Gracilaria tenuistipitata
Cyanidioschyzon merolae
Cyanidioschyzon merolae
Cyanidioschyzon merolae
Porphyra yezoensis
Chondrus crispus
Porphyra purpurea
Porphyra purpurea

Nuclear
Plastid
Nuclear
Plastid
Mitochondria
Plastid
Mitochondria
Mitochondria
Plastid

8,000,000
183,883
16,520,305
149,987
32,211
191,952
25,836
36,753
191,028

Barbier et al. (2005)

Hagopian et al. (2004)
Matsuzaki et al. (2004)
Matsuzaki et al. (2004)
Matsuzaki et al. (2004)
Reith and Munholland (1995)
Burger et al. (1999)
Burger et al. (1999)
Reith and Munholland (1995)

Porphyra (Table 7). However, the efforts are underway to complete the nuclear
genome of Porphyra umbilicalis by the USs Dept. of Energy Joint Genome
Institute (JGI).
4. Conclusions
It is evident from the foregoing account that there is considerable progress made in both
cellular and molecular biotechnology for red algae. The success achieved in cellular
biotechnology will undoubtedly be useful in the studies of genetic transformation and
bioprocess engineering of red seaweeds. The transformed cell lines can now be grown
with greater confidence and success. Also, the progress made in the development of
a variety of molecular markers will prove useful in elucidating the phylogenetic and
evolutionary relationship among the species while aiding the algal systematics. The
ongoing genomic projects will certainly provide a large pool of genetic elements such
as promoters, which can be effectively used for transgene expression as well as inventory of genes that could be used as possible targets for genetic engineering programs
aimed at manipulation of metabolic pathways in the near future.
5. Acknowledgments
We are grateful to a reviewer for his valuable suggestions and comments on
the first draft of this manuscript. Both the editors (Prof. Joseph Seckbach
and Prof. David Chapman) of this volume are also thanked for their keen
interest in our contribution. The seaweed biotechnology research carried
out at CSMCRI is largely from the funding support received from the
Council of Scientific and Industrial Research (NWP 018) and Department of
Science and Technology, New Delhi, and is gratefully acknowledged. One
of the coauthors (VG) thankfully acknowledges the DST, New Delhi, for
fellowship (JRF).

332

C.R.K. REDDY ET AL.

6. References
Achiha, H. (1995) Crossbreeding of Porphyra tenuipedalis by electroporation. Kaiyo Monthly 27:
671677.
Aguirre-Lipperheide, M., Estrada-Rodriguez, F.J. and Evans, L.V. (1995) Facts, problems and needs in
seaweed tissue culture: an appraisal. J. Phycol. 31: 677688.
Amano, H. and Noda, H. (1992) Proteins of protoplasts from several seaweeds. Nippon Suisan
Gakkaishi 58(2): 291299.
Araki, T., Aoki, T. and Kitamikado, M. (1987) Preparation and regeneration of protoplasts from wild
type of Porphyra yezoensis and green variant of P. tenera. Nippon Suisan Gakkaishi 53: 16231627.
Araki, T. and Morishita, T. (1990) Fusion of protoplasts from wild type Porphyra yezoensis and green
type P. tenera thalli (Rhodophyta). Nippon Suisan Gakkaishi 56: 1161.
Araki, T., Hayakawa, M., Tamaru, Y., Yoshimatsu, K. and Morishita, T. (1994) Isolation and regeneration of haploid protoplasts from Bangia atropurpurea (Rhodophyta) with marine bacterial
enzymes. J. Phycol. 30: 10401046.
Araki, T., Inoue, N. and Morishita, T. (1998) Purification and characterization of b-1,3-xylanase from
a marine bacterium, Alcaligenes sp. XY-234. J. Gen. Appl. Microbiol. 44: 269274.
Balestri, E., Della Pieta, F. and Cinelli, F. (1989) Production of protoplasts from two red marine algae:
Laurencia obtusa (Hudson) Lamourax and Plocamium cartilagineum (L.) Dixon, from Mediterranean Sea, In: S. Miyachi, I. Karube and T. Ishida (eds.) Current Topics in Marine Biotechnology.
The Japanese Society for Marine Biotechnology, Tokyo, pp. 375378.
Barahona, L.F. and Rorrer, G.L. (2003) Isolation of halogenated monoterpenes from bioreactor-cultured microplantlets of the macrophytic red algae Ochtodes secundiramea and Portieria hornemannii. J. Nat. Prod. 66: 743751.
Barbier, G., Oesterhelt, C., Larson, M.D., Halgren, R.G., Wilkerson, C., Garavito, R.M., Benning,
C. and Weber, A.P.M. (2005) Comparative genomics of two closely related unicellular thermoacidophilic red algae, Galdieria sulphuraria and Cyanidioschyzon merolae, reveals the molecular
basis of the metabolic flexibility of Galdieria sulphuraria and significant differences in carbohydrate metabolism of both algae. Plant Physiol. 137: 460474.
Baweja, P., Sahoo, D., Jimnez, P.G. and Robaina, R.R. (2009) Seaweed tissue culture as applied to
biotechnology: problems, achievements and prospects. Phycol. Res. 57: 4558.
Bellorin, A.M., Oliveira, M.C. and Oliveira E.C. (2002) Phylogeny and systematic of the marine algal
family Gracilariaceae (Gracilariales, Rhodophyta) based on small subunit rDNA and its ITS
sequences of Atlantic and Pacific species. J. Phycol. 38: 551563.
Bernasconi, P., Cruz-Uribe, T., Rorrer, G., Bruce, N. and Cheney, D. (2004) Development of a TNTdetoxifying strain of the seaweed Porphyra yezoensis through genetic engineering. J. Phycol.
40(Suppl. 1): 31.
Bhattacharya, D., Elwood, H.J., Goff, L.J. and Sogin, M.L. (1990) Phylogeny of Gracilaria lemaneiformis (Rhodophyta) based on sequence analysis of its small subunit ribosomal RNA coding
region. J. Phycol. 26: 181186.
Bird, C.J., Helleur, R.J., Hayes, E.R. and McLachlan, J. (1987) Analytical pyrolysis as a taxonomic
tool in Gracilaria (Rhodophyta, Gigartinales). Hydrobiologia 151/152: 207211.
Bird, C.J., Nelson, W.A., Rice, E.L., Ryan, K.G. and Villemur, R. (1990) A critical comparison of
Gracilaria chilensis and G. sordid (Rhodophyta, Gracilariales). J. Appl. Phycol. 2: 375382.
Bird, C.J., Ragan, M.A., Critchley, A.T., Rice, E.L. and Gutell, R.R. (1994) Molecular relationships
among the Gracilariaceae (Rhodophyta): further observations on some undetermined species.
Eur. J. Phycol. 29: 195202.
Bjork, M., Ekmanm, P., Wallin, A. and Pedersen, M. (1990) Effect of growth rate and other factors on
protoplast yield from four species of Gracilaria (Rhodophyta). Bot. Mar. 33: 433439.
Bouza, N., Caujap-Castells, J., Gonzlez-Prez, M.A. and Sosa, P.A. (2006) Genetic structure of
natural populations in the red algae Gelidium canariense (Gelidiales, Rhodophyta) investigated by
random amplified polymorphic DNA (RAPD) markers. J. Phycol. 42: 304311.

DEVELOPMENTS IN BIOTECHNOLOGY OF RED ALGAE

333

Bradley, P.M. and Cheney, D.P. (1990) Some effects of plant growth regulators on tissue cultures of the
red alga Agardhiella subulata (Gigartinales, Rhodophyta). Hydrobiologia 204/205: 353360.
Bradley, P.M. (1991) Plant hormones do have a role in controlling growth and development of algae.
J. Phycol. 27: 317321.
Burger, G., Saint-Louis, D., Gray, M.W. and Lang, B.F. (1999) Complete sequence of the mitochondrial DNA of the red algae Porphyra purpurea: cyanobacterial introns and shared ancestry of red
and green algae. Plant Cell 11: 16751694.
Butler, D.M. and Evans, L.V. (1990) Cell and tissue culture of macroalgae, In: Akatsuka I (ed.)
Introduction to Applied Phycology. SPB Academic Publishing, The Hague, The Netherlands,
pp. 629645.
Candia, A., Gonzlez, M.A., Montoya, R., Gmez, P. and Nelson, W. (1999) Comparison of ITS
RFLP patterns of Gracilaria (Rhodophyceae, Gracilariales) populations from Chile and New
Zealand and an examination of interfertility of Chilean morphotypes. J. Appl. Phycol. 11:
185193.
Chan, C.Y., Ho, C.L. and Phang, S.M. (2006) Trends in seaweed research. Trends Plant Sci. 11:
165166.
Chen, L.C.-M. and Taylor, A.R.A. (1978) Medullary tissue culture of the red alga Chondrus crispus.
Can. J. Bot. 56: 883886.
Chen, L.C.-M. (1982) Callus-like formations from Irish moss. Biol. Bull. Nat. Taiwan Nor. Uni. 17: 6367.
Chen, L.C.-M. (1987) Protoplasts morphogenesis of Porphyra leucosticte in culture. Bot. Mar.
30: 399403.
Chen, L.C.-M., Hong, M.F. and Craigie, J.S. (1988) Protoplasts development from Porphyra linearis
an edible marine red alga, In: K.I. Puite, J.J.M. Dons, H.J. Huizing, A.J. Kool, M. Koornneef, and F.A. Krens (eds.) Progress in Plant Protoplasts Research, Vol. 7. Kluwer, Dordrecht, The
Netherlands, pp. 123124.
Chen, L.C.-M. (1989) Cell suspension culture from Porphyra linearis (Rhodophyta) a multicellular
marine red alga. J. Appl. Phycol. 1: 153159.
Chen, L.C.M., McCracken, I.R. and Xie, Z.K. (1995) Electrofusion of protoplasts of two species of
Porphyra (Rhodophyta). Bot. Mar. 38: 335338.
Chen, Y.C. and Chiang, Y.M. (1994) Development of protoplasts from Grateloupia sparsa and
G . filicina (Halymeniaceae, Rhodophyta). Bot. Mar. 37: 361366.
Chen, Y.C. and Chiang, Y.M. (1995) Ultra structure of cell wall regeneration from isolated protoplasts
of Grateolopia sparsa (Halymeniaceae, Rhodophyta). Bot. Mar. 38: 393399.
Cheney, D.P., Mar, E., Saga, N. and van der Meer, J. (1986) Protoplasts isolation and cell division in
the agar producing seaweed Gracialria (Rhodophyta). J. Phycol. 22: 238243.
Cheney, D.P., Luistro, A.H. and Bradley, P.M. (1987) Carrageenan analysis of tissue culture and whole
plants of Agardhiella subulata. Hydrobiologia 151/152: 161166.
Cheney, D.P. (1990) Genetic improvement of seaweeds through protoplasts fusion, In: C. Yarish, C.
Penniman, and P. Van Patten (eds.) Economically Important Marine Plants of the Atlantic: Their
Biology and Cultivation. Univ. Conn. Sea Grant Program, Storrs, pp. 1525.
Cheney, D.P. (1999) Strain improvement of seaweeds through genetic manipulation: current status,
World Aquaculture, 30: 5556.
Cheney, D.P., Metz, B. and Stiller, J. (2001) Agrobacterium-mediated genetic transformation in the
macroscopic marine red alga Porphyra yezoensis. J. Phycol. 37 (Suppl): 11.
Chopin, T. and Bastarache, S. (2004) Mariculture in Canada: finfish, shellfish and seaweed. World
Aquacult. 35: 3741.
Chou, H.N. and Lu, H.K. (1989) Protoplasts from seaweeds: isolation, culture and fusion, In: S. Miyachi, I. Karube, and Y. Ishida (eds.) Current Topics in Marine Biotechnology. The Japanese Society
for Marine Biotechnology, Tokyo, pp. 227230.
Clerck, O.D., Gavio, B., Fredericq, S., Brbara, I. and Coppejans, E. (2005) Systematics of Grateloupia filicina (Halymeniaceae, Rhodophyta), based on rbcl sequence analyses and morphological
evidence, including the reinstatement of g. Minima and the description of G. capensis sp. Nov. J.
Phycol. 41: 391410.

334

C.R.K. REDDY ET AL.

Collantes, G., Melo, C. and Candia, A. (2004) Micropropagation by explants of Gracilaria chilensis.
J. Appl. Phycol. 16: 203213.
Collen, J., Roeder, V., Rousvoal, S., Collin, O., Kloareg, B. and Boyen, C. (2006) An expressed sequence
tag analysis of thallus and regenerating protoplasts of Chondrus crispus (Gigartinales, Rhodophyceae). J. Phycol. 42: 104112.
Colln, J., Marsollier, G.I., Lger, J.J. and Boyen, C. (2007) Response of the transcriptome of
the intertidal red seaweed Chondrus crispus to controlled and natural stresses. New Phytol.
176: 4555.
Coury, D.A., Naganuma, T., Polne-Fuller, M. and Gibor, A. (1993) Protoplasts of Gelidium robustum
(Rhodophyta). Hydrobiologia 260/261: 421427.
Dai, J., Zhang, Q. and Bao, Z. (1993) Genetic, breeding and seedling raising experiments with Porphyra protoplasts. Aquaculture 111: 139145.
Davison, I.R. and Polne-Fuller, M. (1990) Photosynthesis in protoplasts of Macrocystis pyrifera
(Phaeophytae). J. Phycol. 26: 384387.
Dawes, C.J. and Koch, E.W. (1991) Branch, micropropagules and tissue culture of the red algae
Eucheuma denticulatum and Kappaphycus alvarezii farmed in the Philippines. J. Appl. Phycol.
6: 2124.
Dawes, C.J., Trono, G.C. and Lluisma, A.O. (1993) Clonal propagation of Eucheuma denticulatum and
Kappaphycus alvarezii for Philippine seaweed farms. Hydrobiologia 260/261: 379383.
Destombe, C. and Douglas, S.E. (1991) Rubisco spacer sequence divergence in the rhodophyte alga
Gracilaria verrucosa and closely related species. Curr. Genet. 19: 395398.
Dippakore, S., Reddy, C.R.K. and Jha, B. (2005) Production and seeding of protoplasts of Porphyra
okhaensis (Bangiales, Rhodophyta) in laboratory culture. J. Appl. Phycol. 17: 331337.
Donaldson, S.L., Chopin, T. and Saunders, G.W. (1998) Amplified fragment length polymorphism
(AFLP) as a source of genetic markers for red algae. J. Appl. Phycol. 10: 365370.
Donaldson, S.L., Chopin, T. and Saunders, G.W. (2000) An assessment of the AFLP method for
investigating population structure in the red alga Chondrus crispus Stackhouse (Gigartinales, Florideophyceae). J. Appl. Phycol. 12: 2535.
Dublin, E.P. (2005) Simplified handbook on Eucheuma seaweed GUSO farming. Degussa Texturant Systems France SASU, In: A.T. Critchely, M. Ohno and D.B. Largo (eds.) World Seaweed
Resources (in DVD format). ETI Information Services Ltd, Berkshire, U.K. pp. 41.
Dutcher, J.A. and Kapraun, D.F. (1994) Random amplified polymorphic DNA (RAPD) identification of genetic variation in three species of Porphyra (Bangiales, Rhodophyta). J. Appl. Phycol.
6: 267273.
Enomoto, K. and Hirose, H. (1972) Culture studies on artificially induced aplanospores in the marine alga
Boergesenia forbesii (Harvey) Feldman (Chlorophyceae, Siphonocladales). Phycologia 11: 119122.
Eswaran, K., Ghosh, P.K., Siddhanta, A.K., Patolia, J.S., Periyasamy, C., Mehta, A.S., Mody, K.H.,
Ramavat, B.K., Prasad, K., Rajyaguru, M.R., Kulandaivel, S., Reddy, C.R.K., Pandya, J.B. and
Tiwari, A. (2005) Integrated method for production of carrageenan and liquid fertilizer from
fresh seaweeds, US Patent No. 6,893,479, May 2005.
Evans, L.V. and Trewavas, A. (1991) Is algal development controlled by plant growth substances?
J. Phycol. 27: 322326.
Fujita, Y. and Migita, S. (1985) Isolation and culture of protoplasts from seaweeds. Bull. Fac. Fish
Nagasaki Univ. 57: 3945.
Fujita, Y. and Migita, S. (1987) Fusion of protoplasts from thalli of two different color types in Porphyra yezoensis Ueda and development of fusion products. Jpn. J. Phycol. 35: 201208.
Fujita, Y. (1990) Diseases of cultivated Porphyra in Japan, In: Akatsuka, I. (ed.) Introduction to Applied
Phycology. SPB Academic Publishing, The Hague, The Netherlands, pp. 177190
Fujita, Y. and Saito, M. (1990) Protoplasts isolation and fusion in Porphyra (Bangiales, Rhodophyta).
Hydrobiologia 204/205: 161166.
Fujita, Y. (1993) Crossbreeding of green and red algae by cell fusion. Kaiyo Monthly 25: 690695.
Fujia, Y. and Uppalapati, S.R. (1997) Genetic improvement of Porphyra through cell culture techniques: present status and future prospects. Nat. His. Res. 3: 7181.

DEVELOPMENTS IN BIOTECHNOLOGY OF RED ALGAE

335

Fukuda, S., Mikamia, K., Ujib, T., Parka, E., Ohbac, T., Asadac, K., Kitadea, Y., Endoa, H., Katoc,
I. and Saga, N. (2008) Factors influencing efficiency of transient gene expression in the red macrophyte Porphyra yezoensis. Plant Sci. 174: 329339.
Gall, E., Chiang, Y.M. and Kloareg, B. (1993) Isolation and regeneration of protoplasts from Porphyra crispate and Porphyra dentate. Eur. J. Phycol. 28: 277283.
Gan, Y.S., Qin, S., Othman, R.Y., Yu, D.Z. and Phang, S.M. (2003) Transient gene expression
of LacZ in partical bombared Gracilaria Changii (Gracilariales, Rhodophyta). J. Appl. Phycol.
15: 345349.
Ganesan, M., Thiruppathai, S. and Jha, B. (2006) Mariculture of Hypnea musciformis (Wulfen) Lamouroux in South east coast of India. Aquaculture 256: 201211.
Ganesan, M., Thiruppathai, S., Eswaran, K., Reddy C.R.K. and Jha, B. (2009) Cultivation of Gelidiella acerosa in the open sea on the southeastern coast of India. Mar. Ecol. Prog. Seri. DOI:
10.3354/meps07891.
Garcia-Reina, G.L., Romero, R.R. and Luque, A. (1988) Regeneration of Thalliclones from Laurencia
sp. (Rhodophyta), In: M.S.S. Paris, F. Mavituna, and J.M. Novais (eds.) Plant Cell Biotechnology.
Springer, New York, pp. 8186.
Garcia-Reina, G.L., Gomez-Pinchetti, J.L., Robeldo, D.R. and Sosa, P. (1991) Actual potential and
speculative applications of seaweed cellular biotechnology: some specific comments on Gelidium.
Hydrobiologia 221: 181194.
Gargiulo, G.M., De Masi, F. and Tripodi, G. (1992) Morphology, reproduction and taxonomy of the
Mediterranean species of Gracilaria (Gracilariales, Rhodophyta). Phycologia 31: 5380.
Geraldino, P.J.L., Yang, E.C. and Boo, S.M. (2006) Morphology and molecular phylogeny of Hypnea
flexicaulis (Gigartinales, Rhodophyta) from Korea. Algae 21: 417423.
Godin, J., Destombe, C. and Maggs, C.A. (1993) Unusual chromosome number of Gracilaria verrucosa
(Gracilariales, Rhodophyta) in the Cape Gris-Nez area, northern France. Phycologia 32: 291294.
Goff, L.J., Moon, D.A. and Coleman, A.W. (1994) Molecular delineation of species and species relationships in the red algal agarophytes Gracilariopsis and Gracilaria (Gracilariales). J. Phycol.
30: 521537.
Gomez-Pinchetti, J.L. and Garcia Reina, G. (1993) Enzymes from marine phycophages that degrade
cell walls of seaweeds. Mar. Biol. 116: 553558.
Gonzlez, M., Montoya, R., Candia, A., Gmez, P. and Cisternas, M. (1996) Organellar DNA restriction fragment length polymorphism (RFLP) and nuclear random amplified polymorphic DNA
(RAPD) analyses of morphotypes of Gracilaria (Gracilariales, Rhodophyta) from Chile. Hydrobiologia 326327: 229234.
Guiry, M.D. (2008) Seaweed site. World-wide electronic publication, National University of Ireland,
Galway. http://www.seaweed.ie/. Accessed 20 Nov. 2008.
Guiry, M.D. and Guiry, G.M. (2009) AlgaeBase. World-wide electronic publication, National University of Ireland, Galway. http://www.algaebase.org; searched on 20 June 2009.
Gusev, M.V., Tambiev, A.H., Kirikora, N.N., Shelyastina, N.N. and Aslanyan, R.R. (1987) Callus
formation in seven species of agarophyte marine algae. Mar Biol 95:593597.
Hafting, J.T. (1999) A novel technique for propagation of Porphyra yezoensis Ueda blades in suspension cultures via monospores. J. Appl. Phycol. 11: 361367.
Haglund, K., Bjork, M., Ramazanov, Z., Garcia-Reina, G. and Pederson, M. (1992) Role of carbonic
anhydrase in photosynthesis and inorganic carbon assimilation in the red alga Gracilaria tenuistipitata. Planta 187: 275281.
Hagopian, J.C., Reis, M., Kitajima, J.P., Bhattacharya, D. and de Oliveira, M.C. (2004) Comparitive
analysis of the complete plastid genome sequence of the red alga Gracilaria tenuistipitata var.
Liui provides insight into the evolution of rhodoplasts and their relationship to other plastids.
J. Mol. Evol. 59: 464477.
Hanisak, M.D. (1998) Seaweed cultivation global trends. World Aquac. 29: 1821.
Hayashi, L., Yokoya, N.S., Kikuchi, D.M. and Oliveira, E.C. (2008) Callus induction and
micropropagation improved by colchicine and phytoregulators in Kappaphycus alvarezii
(Rhodophyta, Solieriaceae). J. Appl. Phycol. 20(5): 653659.

336

C.R.K. REDDY ET AL.

He, P., Yao, Q., Chen, Q., Guo, M., Xiong, A., Wu, W. and Ma, J. (2001) Transfering and expression
of glucose oxidase gene-Gluc in Porphyra yezoensis. J. Phycol. 37: 23.
Huang, W. and Fujita, Y. (1997) Callus induction and thallus regeneration in some species of red algae.
Phycol. Res. 45: 105111.
Huang, Y.M., Maliakal, S., Cheney, D. and Rorrer, G. (1998) Comparison of development, photosynthesis and growth of filament clump and regenerated microplantlet cultures of Agardhiella
subulata (Rhodophyta, Gigartinales). J. Phycol. 34: 893901.
Huh, M.K., Lee, B.K. and Lee, H.Y. (2006) Genetic diversity and phylogenetic relationships in five
Porphyra species revealed by RAPD analysis. Protistology 4: 245250.
Hurtado, A.Q. and Cheney, D.P. (2003) Propagule production of Eucheuma denticulatum (Burman)
Collins et Harvey by tissue culture. Bot. Mar. 46: 338341.
Hurtado, Q., Lhonneur, G.B. and Critchley, A.T. (2006) Kappaphycus cottonii farming, In: A.T.
Critchely, M. Ohno and D.B. Largo (eds.) World Seaweed Resources (in DVD format). ETI Information Services Ltd, Berkshire, U.K.
Hurtado, A.Q. and Biter, A.B. (2007) Plantlet regeneration of Kappaphycus alvarezii var. adik-adik by
tissue culture. J. Appl. Phycol. 19: 783786.
Iitsuka, O., Nakamura, K., Ozaki, A., Okamoto, N. and Saga, N. (2002) Genetic information of three
pure lines of Porphyra yezoensis (Bangiales, Rhodophyta) obtained by AFLP analysis. Fisheries
Sci. 68: 11131117.
Iwabuchi, M. (1995) Color mutations in regenerated thalli of isolated protoplasts from Porphyra
yezoensis. Bull. Fukuoka Fisheries Marine Technol. Res. Center (Jpn.) 4: 5758.
Kaczyna, F. and Megnet, R. (1993) The effects of glycerol and plant growth regulators on Gracilaria
verrucosa (Gigartinales, Rhodophyceae). Hydrobiologia 268: 5764.
Kakinuma, M., Kaneko, I., Coury, D., Suzuki, T. and Amano, H. (2006) Isolation and identification
of gametogenesis-related genes in Porphyra yezoensis (Rhodophyta) were identified using subtracted cDNA libraries. J. Appl. Phycol. 18, 489496.
Kito, H., Kunimoto, M., Kamanishi, Y. and Mizukami, Y. (1998) Protoplasts fusion between Monostroma nitidum and Porphyra yezoensis and subsequent growth of hybrid plants. J. Appl. Phycol.
10: 1521.
Kuang, M., Wang, S.J., Li, Y., Shen, D.L. and Tseng, C.K. (1998) Transient expression of exogenous
GUS gene in Porphyra yezoensis (Rhodophyta). Chin. J. Oceanol. Limnol. 16: 5661.
Kubler, J.E., Minocha, S.C. and Mathieson, A.C. (1994) Transient expression of the GUS reporter
gene in protoplasts of Porphyra miniata (Rhodophyta). J. Mar. Biotechnol. 1: 165169.
Kurtzman, A.L. and Cheney, D.P. (1991) Direct gene transfer and transient gene expression in a
marine red alga using the biolistic method. J. Phycol. 27(Suppl.): 42 pp.
Largo, D.B., Fukami, K. and Nishijima, T. (1995a) Occasional pathogenic bacteria promoting iceice
disease in the carrageenan-producing red algae Kappaphycus alvarezii and Eucheuma denticulatum (Solieriaceae, Gigartinales, Rhodophyta). J. Appl. Phycol. 7: 545554.
Largo, D.B., Fukami, K., Nishijima, T. and Ohno, M. (1995b) Laboratory-induced development of
the iceice disease of the farmed red algae Kappaphycus alvarezii and Eucheuma denticulatum
(Solieriaceae, Gigartinales, Rhodophyta). J. Appl. Phycol. 7: 539543.
Le Gall, Y., Braud, J.P. and Kloareg, B. (1990) Protoplast production in Chondrus crispus gametophytes (Gigartinales, Rhodophyta). Pl. Cell Rep. 8: 582585.
Le Gall, L., Rusig, A.M. and Cosson, J. (2004) Organisation of the microtubular cytoskeleton in protoplasts from Palmaria palmate (Palmariales, Rhodophyta). Bot. Mar. 47: 231237.
Lee, E.K., Seo, S.B., Kim, T.H., Sung, S.K., An, G., Lee, C.H. and Kim, Y.J. (2000) Analysis of
expressed sequence tags of Porphyra yezoensis. Mol. Cell. 10(3): 338342.
Lee, H., Lee, H.K., An, G. and Lee, Y.K. (2007) Analysis of expressed sequence tags from the red alga
Griffithsia okiensis. J. Microbiol. 45: 541546.
Lee, S.R., Oak, J.H., Suh, Y. and Lee, I.K. (2001) Phylogenetic utility of rbcS sequences: an example
from Antihamnion and related genera (Ceramiaceae, Rhodophyta). J. Phycol. 37: 10831090.
Li, Y., Shen, S., He, L., Xu, P. and Lu, S. (2009) Sequence analysis of rDNA intergenic spacer (IGS) of
Porphyra haitanensis. J. Appl. Phycol. DOI: 10.1007/s10811-009-9441-x.

DEVELOPMENTS IN BIOTECHNOLOGY OF RED ALGAE

337

Lim, P.E., Thong, K.L. and Phang, S.M. (2001) Molecular differentiation of two morphological variants of Gracilaria salicornia. J. Appl. Phycol. 13: 335342.
Lin, C.M., Larsen, J., Yarish, C. and Chen, T. (2001) A novel gene transfer in Porphyra. J. Phycol.
37(Suppl.): 2930.
Liu, H.Q., Yu, W.G., Dai. J,X., Gong, Q.H., Yang, K.F. and Zang, Y. (2003) Increasing the transient
expression of GUS gene in Porphyra yezoensis by 18S rDNA targeted homologous recombination. J. Appl. Phycol. 15: 371377.
Liu, Q.Y., Meer, J.P. and Reith, M.E. (1994) Isolation and characterization of phase-specific complementary DNAs from sporophytes and gametophytes of Porphyra purpurea (Rhodophyta) using
subtracted complementary DNA libraries. J. Phycol. 30: 513520.
Liu, X.W. and Gordon, M.E. (1987) Tissue and cell culture of New Zealand Pterocladia and Porphyra
species. Twelfth international seaweed symposium. Hydrobiologia 151/152: 147154.
Liu, X.W., Rochas, C. and Kloareg, B. (1990) Callus culture of Pterocaldia capillacea (Rhodophyta)
and analysis of cell wall polysaccharides. J. Appl. Phycol. 2: 297303.
Liu, X.W. and Kloareg, B. (1992) Explant axenisation for tissue culture in marine macro algae. Chin.
J. Oceanol. Limnol. 10: 268277.
Lluisma, A.O. and Ragan, M.A. (1997) Expressed sequence tags (ESTs) from the marine red alga
Gracilaria gracilis. J. Appl. Phycol. 9: 287293.
Maggs, C.A., Douglas, S.E., Fenety, J. and Bird, J.C. (1992) A molecular and morphological analysis of
the Gymnogongrus devoniensis (Rhodophyta) complex in the north Atlantic. J. Phycol. 28: 214232.
Malaikal, S., Cheney, D. and Rorrer, G.L. (2001) Halogenated monoterpenes production in regenerated
plantlet cultures of Ochtodes secundiramea (Rhodophyta, Cryptonemiales). J. Phycol. 37: 10101019.
Martinez, E.A., Destombe, C., Quillet, M.C. and Valero M. (1999) Identification of random amplified
polymorphic DNA (RAPD) markers highly linked to sex determination in the red alga Gracilaria
gracilis. Mol Ecol 8: 15331538.
Masuda, K., Mizuta, A. and Tanida, K. (1995) Selective breeding of Porphyra using isolated thallus
protoplasts. Kaiyo Monthly 27(11): 655660.
Matsuzaki, M., Misumi, O., Shin-I, T., Maruyama, S., Takahara, M., Miyagishima, S.Y., Mori, T.,
Nishida, K., Yagisawa, F., Nishida, K., Yoshida, Y., Nishimura, Y., Nakao, S., Kobayashi, T.,
Momoyama, Y., Higashiyama, T., Minoda, A., Sano, M., Nomoto, H., Oishi, K., Hayashi, H.,
Ohta, F., Nishizaka, S., Haga, S., Miura, S., Morishita, T., Kabeya, Y., Terasawa, K., Suzuki, Y.,
Ishii, Y., Asakawa, S., Takano, H., Ohta, N., Kuroiwa, H., Tanaka, K., Shimizu, N., Sugano, S.,
Sato, N., Nozaki, H., Ogasawara, N., Kohara, Y. and Kuroiwa, T. (2004) Genome sequence of the
ultrasmall unicellular red alga Cyanidioschyzon merolae 10D. Nature 428: 653657.
McHugh, D.J. (2003) A guide to the seaweed industry. FAO Fisheries Technical Paper 441: 1105.
Minocha, S.C. (2003) Genetic engineering of seaweeds: current status and perspectives, In: A.R.O.
Chapaman, R.J. Anderson, V.J. Vreeland and I.R. Davidson (eds.) Proceedings of the 17th International Seaweed Symposium. Oxford Univ. Press, New York, pp. 1926.
Mizukami, Y., Okauchi, M., Kito, H., Ishimoto, S., Ishida, T. and Fuseya, M. (1995) Culture and
development of electrically fused protoplasts from red marine algae, Porphyra yezoensis and P.
suborbiculata. Aquaculture 132: 361367.
Mizukami, Y., Hado, M., Kito, H., Kunimoto, M. and Murase, N. (2004) Reporter gene introduction
and transient expression in protoplasts of Porphyra yezoensis. J. Appl. Phycol. 16: 2329.
Mollet, J.C., Verdus, M.C., Kling, R. and Morvan, H. (1995) Improved protoplast yield and cell wall
regeneration in Gracilaria verrucosa (Huds.) Papenfuss (Gracilariales, Rhodophyta). J. Exp. Bot.
46: 239247.
Molloy, F.J. (2006) Seaweed resources of Nambia, In: A.T. Critchely, M. Ohno and D.B. Largo (eds.)
World Seaweed Resources (in DVD format). ETI Information Services Ltd, Berkshire, U.K.
Munoz, J., Armando, C., Lopez, C., Patino, R. and Robledo, D. (2006) Use of plant growth regulators
in micropropagation of Kappaphycus alvarezii (Doty) in airlift bioreactors). J. Appl. Phycol.
18: 209218.
Nikolaeva, E.V., Usov, A.I., Sinitsyn, A.P. and Tambiev, A.H. (1999) Degradation of agarrophytic red
algal cell wall components by new crude enzyme preparations. J. Appl. Phycol. 11: 385389.

338

C.R.K. REDDY ET AL.

Niwa, K., Kikuchi, N., Iwabuchi. M. and Aruga, Y. (2006) Morphological and AFLP variation
of Porphyra yezoensis Ueda form, narawaensis Miura (Bangiales, Rhodophyta). Phycol. Res.
52: 180190.
Ootsukaa, S., Sagaa, N., Suzukib, K., Inoue, A. and Ojimab, T. (2006) Isolation and cloning of an
endo--1,4-mannanase from Pacific abalone Haliotis discus hannai. J. Biot. 125: 269280.
Packer, M.A. (1994) Protoplast isolation from single cells and small tissue fragments of wild Porphyra
fronds (Rhodophyta). Bot. Mar. 37: 101108.
Park, C.S., Kakinuma, M. and Amano, H. (2006) Forecasting infections of the red rot disease on Porphyra yezoensis Ueda (Rhodophyta) cultivation farms. J. Appl. Phycol. 18: 295299.
Park, E.J., Fukuda, S., Endo, H. and Saga, N. (2007) Genetic polymorphism within Porphyra yezoensis
(Bangiales, Rhodophyta) and related species from Japan and Korea detected by cleaved amplified
polymorphic sequence analysis. Eur. J. Phycol. 42: 2940.
Patwary, M.U., MacKay, R.M. and Meer, J.P. (1993) Revealing genetic markers in Gelidium vagum
(Rhodophyta) through the random amplified polymorphic DNA (RAPD) technique. J. Phycol.
29: 216222.
Plastino, E.M. and Oliveira, E.C. (1988) Sterility barriers among species of Gracilaria (Rhodophyta,
Gigartinales) from the So Paulo littoral, Brazil. Br. Phycol. J. 23: 26771.
Polne-Fuller, M., Biniaminov, M. and Gibor, A. (1984) Vegitative propagation of Porphyra perforata.
Proc. Int. Seaweed Symp. 11: 308313.
Polne-Fuller, M. and Gibor, A. (1987) Calluses and callus-like growth in seaweeds: induction and
culture. Hydrobiologia 151/152: 131138.
Polne-Fuller, M. (1988) The past, present and future of tissue culture and biotechnology of seaweeds,
In: Stadler, T., Mollion, J., Verdus, M.C., Karamanos, Y., Morvan, C.D. (eds.) Algal Biotechnology. Elsevier, London, pp. 731.
Polne-Fuller, M., Rogerson, A., Amano, H. and Gibor. A. (1990) Digestion of seaweeds by the marine
amoeba Trichosphaerium. Hydrobiologia 204/205: 409413.
Qin, S., Jiang, P. and Tseng, C.K. (2005) Transforming kelp into a marine bioreactor. Trends Biotechnol. 23: 264268.
Rajakrishna Kumar, G., Reddy, C.R.K., Ganesan, M., Tiruppathi, S., Dipakkore, S., Eswaran, K.,
Subba Rao, P.V. and Jha, B. (2004) Tissue culture and regeneration of thallus from callus of
Gelidiella acerosa (Gelidiales, Rhodophyta). Phycologia 43: 596602.
Rajakrishna Kumar, G., Reddy, C.R.K. and Jha, B. (2007) Callus induction and thallus regeneration from
the callus of phycocolloid yielding seaweeds from the Indian coast. J. Appl. Phycol. 19: 1525.
Reddy, C.R.K., Fujita, Y. and Bajaj, Y.P.S. (1994) Somatic hybridisation in algae, In: Y.P.S. Bajaj
(ed.) Biotechnology in Agriculture and Forestry 27. Somatic Hybridisation in Crop Improvement I.
Springer, Heidelberg, pp. 483502.
Reddy, C.R.K., Krishnakumar Kumar, G., Eswaran, K., Siddhanta, A.K. and Tewari, A. (2003) In
vitro somatic embryogenesis and regeneration of somatic embryos from pigmented callus of Kappaphycus alvarezii (Gigartinales, Rhodophyta). J. Phycol. 39: 610616.
Reddy, C.R.K., Jha, B., Fujita, Y. and Ohno, M. (2008a) Seaweed micropropagation techniques and
their potentials: an overview. J. Appl. Phycol. 20: 609617.
Reddy, C.R.K., Gupta, M.K., Mantri, V.A. and Jha, B. (2008b) Seaweed protoplasts: status, biotechnological perspectives and needs. J. Appl. Phycol. 20: 619632.
Reith, M. and Munholland, J. (1995) Complete nucleotide sequence of the Porphyra purpurea chloroplast genome. Plant Mol. Bio. Repo. 13: 333335.
Ren, X., Zhang, X. and Sui, Z. (2006) Identification of phase relative genes in tetrasporophytes and
female gametophytes of Gracilaria/Gracilariopsis lemaneiformis (Gracilariales, Rhodophyta).
Electron. J. Biotechnol. 9: 127132.
Rice, E.L. and Bird, C.J. (1990) Relationships among geographically distant populations of Gracilaria
verrucosa (Gracilariales, Rhodophyta) and related species. Phycologia 29: 501510.
Robaina, R.R., Garcia, J.P., Garcia-Reina, G. and Luque, A. (1990) Morphogenetic effect of glycerol
on tissue cultures of the red seaweed Grateloupia doryphora. J. Appl. Phycol. 2: 137143.

DEVELOPMENTS IN BIOTECHNOLOGY OF RED ALGAE

339

Robaina, R.R., Garcia, J.P. and Luque, A. (1992) The growth pattern and structure of callus from the
red alga Laurencia sp. (Rhodophyta, Ceramiales) compared to shoot regeneration. Bot. Mar.
35: 267272.
Robba, L., Russel, S.J., Barker, G.L. and Bordie, J. (2006) Assessing the use of the mitochondrial cox1
marker for use in DNA barcoding of red algae (Rhodophyta). Am. J. Bot. 93: 11011108.
Robeldo, D.R. and Garcia-Reina, G. (1993) Apical callus formation in Solieria filiformis (Gigartinales,
Rhodophyta) cultured in tanks. Hydrobiologia 260/261: 401406.
Rorrer, G.L. and Cheney, D.P. (2004) Bioprocess engineering of cell and tissue cultures for marine
seaweeds. Aquacult. Eng. 32: 1141.
Saga, N. and Sakai, Y. (1984) Isolation of protoplast from Laminaria and Porphyra. Nippon Suisan
Gakkaishi 50: 1085.
Saga, N., Polne-Fuller, M. and Gibor, A. (1986) Protoplasts from seaweeds: production and fusion.
Beih. Nova Hedweg. 83: 3743.
Salvador, R.C. and Serrano, A.E. (2005) Isolation of protoplasts from tissue fragments of Philippine
cultivars of Kappaphycus alvarezii (Solieriaceae, Rhodophyta). J. Appl. Phycol. 17: 1522.
Santelices, B. (1992) Strain selection of clonal seaweeds, In: F.E. Round and D.J. Chapman (eds.)
Progress in Phycological Research, Vol. 8. Biopress, Bristol, pp. 86115.
Saunders, G.W. (2005) Applying DNA barcoding to red macroalgae: a preliminary appraisal holds
promise for future applications. Philos. Trans. Roy. Soc. Lond. B. Biol. Sci. 360: 18791888.
Siddhanta, A.K., Meena, R., Prasad, G., Chhatbar, M.U., Mehta, G.K., Oza, M.D., Kumar, S. and
Prasad, K. (2009) Development of carbohydrate polymer based new hydrogel materials derived
from seaweed polysaccharides, In: F. Columbus (ed.) Carbohydrate Polymers: Development, Properties and Applications [In press; April 11, 2009]; Nova Science Publishers, Inc.; 400 Oser Avenue,
Suite 1600; Hauppauge, NY 11788.
Sim, M.C., Lim, P.E., Gan, S.Y. and Phang, S.M. (2007) Identification of random amplified polymorphic DNA (RAPD) marker for differentiating male from female and sporophytic thalli of
Gracilaria changii (Rhodophyta). J. Appl. Phycol. 19: 763769.
Smith, R.G. and Bidwell, R.G.S. (1989) Inorganic carbon uptake by photosynthetically active protoplasts of the red macroalga Chondrus crispus. Mar. Biol. 102: 14.
Song Ho, S. and Chung, G.H. (1988) Isolation and purification of protoplasts from Porphyra tenera
thalli. Aquaculture 1: 103108.
Stevens, D.R. and Purton, S. (1997) Genetic engineering of eukarygtic algae: progress and prospects.
J. Phycol. 33: 713722.
Stiller, J.W. and Waaland, J.R. (1993) Molecular analysis reveals cryptic diversity in Porphyra (Rhodophyta). J. Phycol. 29: 506517.
Stiller, J.W. and Hall, B.D. (1997) The origin of red algae: Implications for plastid evolution. PNAS
94: 45204525.
Stirk, W.A., Gold, J.D., Novak, O., Strnad, M. and Van Staden, J. (2005) Changes in endogenous cytokinins
during germination and seedling establishment of Tagetes minuta L. Plant Growth Regul. 47: 17.
Tang, Y. (1982) Isolation and cultivation of the vegetative cells and protoplasts of Porphyra suborbiculata Kjellm. J. Shandong Coll. Oceanol. 12: 3750.
Tatewaki, M. and Nagata, K. (1970) Surviving protoplasts in vitro and their development in Bryopsis.
J. Phycol. 6: 401403.
Teasdale, B., West, A., Taylor, H. and Klein, A. (2002) A simple restriction fragment length polymorphism (RFLP) assay to discriminate common Porphyra (Bangiophyceae, Rhodophyta) taxa from
the Northwest Atlantic. J. Appl. Phycol. 14: 293298.
Teo, S.S., Ho, C.L., Teoh, S., Lee, W.W., Tee, J.M., Rahim, R.A. and Phang, S,M. (2007) Analyses of
expressed sequence tags from an agarophyte, Gracilaria changii (Gracilariales, Rhodophyta). Eur.
J. Phycol. 42: 4146.
Uppalapati, S.R., Morita, T. and Fujita, Y. (2000) A method of high-frequency hetero-fusion of
Porphyra yezoensis protoplasts with Enteromorpha compress or Monostroma nitidum protoplasts.
Bull. Fac. Fish. Nagasaki Uni. 81: 4954.

340

C.R.K. REDDY ET AL.

Uppalapati, S.R. and Fujita, Y. (2000) Red rot resistance in interspecific protoplast fusion product progeny of Porphyra yezoensis and P. tenuipedalis (Bangiales, Rhodophyta). Phycol. Res. 48: 281289.
Vairappan, S.C., Chung, C.S., Hurtado, A.Q., Soya, F.E., Lhonneur, G.B. and Critchley, A. (2008)
Distribution and symptoms of epiphyte infection in major carrageenophyte-producing farms.
J. Appl. Phycol. 20: 477483.
Waaland, J.R., Dickson, L.G. and Watson, B.A. (1990) Protoplasts isolation and regeneration in the
marine red alga Porphyra nereocystis. Planta 181: 522528.
Walker, T.L., Collet, C. and Purton, S. (2005) Algal transgenics in the genomic era. J. Phycol. 41(6):
10771093.
Wang, H.W., Kawaguchi, S., Horiguchi, T. and Masuda, M. (2000) Reinstatement of Grateloupia catenata (Rhodophyta, Halymeniaceae) on the basis of morphology and rbcL sequences. Phycologia 39: 228237.
Wanga, X., Zhaoa, F., Hub, Z., Critchley, A.T., Morrelld, S.L. and Duan, D. (2007) Inter-simple
sequence repeat (ISSR) analysis of genetic variation of Chondrus crispus populations from North
Atlantic. Aqua Bot. 88: 154159.
Wattier, R., Dallas, J.F., Destombe, C., Saumitou-Laprade, P. and Valero, M. (1997) Single locus microsatellites in Gracilariales (Rhodophyta): high level of genetic variability within Gracilaria gracilis and conservation in related species. J. Phycol. 33: 868880.
Weber, A.P., Oesterhelt, C., Gross, W., Brutigam, A., Imboden, L.A., Krassovskaya, I., Linka, N.,
Truchina, J., Schneidereit, J., Voll, H., Voll, L.M., Zimmermann, M., Jamai, A., Riekhof, W.R.,
Yu, B., Garavito, R.M. and Benning, C. (2004) EST-analysis of the thermo-acidophilic red
microalga Galdieria sulphuraria reveals potential for lipid A biosynthesis and unveils the pathway
of carbon export from rhodoplasts. Plant Mol. Biol. 55(1): 1732.
Xiaolei, F., Yongjun, F., Songnian, H. and Guangce, W. (2007) Generation and analysis of 5318
expressed sequence tags from the filamentous sporophyte of Porphyra haitanensis (Rhodophyta).
J. Phycol. 43: 12871294.
Xue, S., Xuecheng, Z., Yunxiang, M., Zhenghong, S. and Song, Q. (2006) Identification of
phase and sex-related ISSR markers of red alga Gracilaria lemaneiformis. J. Oce. Uni. China
5: 8284.
Yamaguchi, K., Araki, T., Aoki, T., Tseng, C. and Kitamikado, M. (1989) Algal cell wall degrading
enzymes from viscera of marine animals. Nippon Suisan Gakkaishi 55: 105110.
Yamashita, Y. and Fujita, Y. (1996) The effects of amino acids on Porphyra protoplast development.
Abstracts for the Autumn Meeting of the Japanese Society of Fisheries, pp. 72.
Yamazaki, A., Nakanishi, K. and Saga, N. (1998) Axenic tissue culture and morphogenesis in Porphyra yezoensis (Bangiales, Rhodophyta). J. Phycol. 34: 10821087.
Yan, X.H. and Wang, S.J. (1993) Regeneration of whole plants from Gracilaria asiatica Chang et Xia
protoplasts (Gracilariaceae, Rhodophyta). Hydrobiologia 260/261: 429436.
Yang, E.C. and Boo, S.M. (2004) Evidence for two independent lineages of Griffithsia (Ceramiaceae,
Rhodophyta) based on plastid proteincoding psaA, psbA, and rbcL gene sequences. Mol.
Phylogenet. Evol. 31: 680688.
Yang, E.C. and Boo, S.M. (2006) A red alga-specific phycoerythrin gene for biodiversity surveys of
callithamnioid red algae. Mol. Ecol. Notes 6: 533535.
Yang, E.C., Kim, M.S., Geraldinon, P.J.L., Sahoo, D., Shin, J.A. and Boo, S.M. (2008) Mitochondrial
cox1 and plastid rbcL genes of Gracilaria vermiculophylla (Gracilariaceae, Rhodophyta). J. Appl.
Phycol. 20: 161168.
Yeong, H.Y., Khalid, N. and Phang, S.M. (2007) Protoplast isolation and regeneration from Gracilaria
changii (Gracilariales, Rhodophyta). J. Appl. Phycol. 20: 641651.
Yokoya, N.S., Guimaraes, M.P.B.S. and Handro, W. (1993) Development of callus like structures
and plant regeneration in thallus segments of Grateloupia filiformis Kutzing (Rhodophyta).
Hydrobiologia 260/261: 407413.
Yokoya, N.S. and Handro,W. (1996) Effects of auxins and cytokinins on tissue culture of Grateloupia
dichotoma (Gigartinales, Rhodophyta). Hydrobiologia 326/327: 393400.

DEVELOPMENTS IN BIOTECHNOLOGY OF RED ALGAE

341

Yokoya, N.S., Kakita, H., Obika, H. and Kitamura, T. (1999) Effects of environmental factors and
plant growth regulators on growth of the red alga Gracilaria vermiculophylla from Shikoku
Island, Japan. Hydrobiologia 398/399: 339347.
Yokoya, N.S. (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, N.S., Plastino, M. and Artel, R. (2003) Physiological responses and pigment characterization of two colour strains of the carrageenophyte Hypnea musciformis (Rhodophyta), In: A.R.O.
Chapman, R.J. Anderson, J. Valerie, and I.R. Vreeland Davison (eds.) Proceedings of the 17th
International Seaweed Symposium. Oxford Uniersity Press, Oxford, UK, pp. 425433.
Yokoya, N.S., West, J.A. and Luchi, A.E. (2004) Effects of plant growth regulators on callus formation, growth and regeneration in axenic tissue culture of Gracilaria tenuistipitata and Gracilaria
perplexa (Gracilariales, Rhodophyta). Phycol. Res. 52: 244254.
Yoon, H.S., Hackett, J.D. and Bhattacharya, D. (2002) A single origin of the peridinin- and
fucoxanthin containing plastids in dinoflagellates through tertiary endosymbiosis. PNAS 99:
1172411729.
Zablackis, E., Vreeland, V. and Kloareg, B. (1993) Isolation of protoplasts from Kappaphycus alvarezii
var. tambalang (Rhodophyta) and secretion of carrageenan fragments by cultured cells. J. Exp.
Bot. 44: 15151522.
Zuccarello, G.C., Burger, G., West, J.A. and King, R.J. (1999) A mitochondrial marker for red algal
intraspecific relationships. Mol. Ecol. 8: 14431447.