J. Phycol.

39, 610616 (2003)

IN VITRO SOMATIC EMBRYOGENESIS AND REGENERATION OF SOMATIC EMBRYOS

FROM PIGMENTED CALLUS OF KAPPAPHYCUS ALVAREZII (DOTY)
DOTY (RHODOPHYTA, GIGARTINALES) 1
C. R. K. Reddy,2 G. Raja Krishna Kumar, A. K. Siddhanta, A. Tewari
Marine Algae and Marine Environment Discipline, Central Salt and Marine Chemicals Research Institute, Bhavnagar 364 002, India

and
K. Eswaran
CSMCRI-Marine Algal Research Station, Mandapam Camp 623 519, Tamil Nadu, India

In vitro somatic embryogenesis and regeneration

of somatic embryos to whole plants through micropropagules was successfully demonstrated from pigmented uniseriate filamentous callus of Kappaphycus
alvarezii (Doty) Doty in axenic cultures. More than
80% of the explants cultured on 1.5% (w/v) agarsolidified Provasoli enriched seawater (PES) medium
showed callus development. The callus induction
rate was consistently higher for laboratory-adapted
plants. The excised callus grew well in subcultures
and maintained its growth for prolonged periods if
transferred to fresh medium in regular intervals.
Some subcultured calli (10%) did undergo transformation and produced densely pigmented spherical or oval-shaped micropropagules (15 mm in diameter) that subsequently developed into young
plantlets in liquid PES medium. The micropropagule
production was further improved through somatic
embryogenesis by a novel method of culturing thin
slices of pigmented callus with naphthaleneacetic
acid (NAA) or a mixture of NAA and 6-benzylaminopurine. Transfer of embryogenic callus along with
tiny somatic embryos to liquid medium and swirling
on orbital shaker facilitated rapid growth and morphogenesis of somatic embryos into micropropagules
that grew into whole plants in subsequent cultivation
in the sea. The daily growth rate of one tissue cultured plant was monitored for seven generations in
field and found to be as high as 1.51.8 times over
farmed plants. The prolific somatic embryogenesis
together with high germination potential of somatic
embryos observed in this study offers a promising
tool for rapid and mass clonal production of seed
stock of Kappaphycus for commercial farming.

Abbreviations: BAP, 6-benzylaminopurine; ESS, Erd

Schreibers seawater; NAA, naphthaleneacetic acid;
PES, Provasoli enriched seawater
The phenomenon of in vitro somatic embryogenesis in tissue culture is common in woody plants, including angiosperms and gymnosperms, and reported
from almost all the plant families (Ammirato 1983,
Tisserat 1985, Jain et al. 1994). The major advantage
of somatic embryogenesis is that it provides a powerful tool for economical clonal propagation for largescale production of plants of commercial interest
(Jain et al. 1994). Somatic embryos usually originate
from induced embryogenic cells within callus tissue
(Sharp et al. 1980). Somatic embryos can also develop
directly from pre-embryonic determined cells that
are programmed for embryonic differentiation. Thus,
somatic embryogenesis could be induced in two distinctly different media, one with auxin for initiation
of embryonic cells in callus tissue and the second
without auxin for subsequent cytodifferentiation of
embryoids. Over the last two decades, an increasing
number of studies have dealt with seaweed tissue culture, following the techniques used in higher plants.
Callus induction and thallus regeneration have been
reported from a wide variety of economically important seaweeds (Polne-Fuller and Gibor 1987, Evans
and Butler 1988, Butler and Evans 1990, Aguirre-Lipperheide et al. 1995).
Although the initial emphasis has been on development of techniques for production of callus and thallus regeneration, their application for in vitro selection and propagation of desired strains (Dawes et al.
1993, Kirihara et al. 1997) and production of secondary metabolites (Lawlor 1990, Maliakal et al. 2001)
has also been investigated. In addition, production of
value added products (Cheney et al. 1987, Liu et al.
1990) from callus cultures has been accomplished in
a few seaweeds. Dawes and Koch (1991) demonstrated use of micropropagule (microcuttings of thallus segments) and callus cultures as a means of maintenance and propagation of select seed stock of Eucheuma
denticulatum (Burnan) Collins and Kappaphycus alvarezii that are farmed on a commercial scale in the Phil-

Key index words: clonal propagation; embedded culture; Kappaphycus; micropropagules; pigmented callus; somatic embryogenesis; somatic embryos; uniseriate branched filamentous callus

1Received
2Author

23 June 2002. Accepted 25 February 2003.

for correspondence: e-mail: salt@csir.res.in.

610

SOMATIC EMBRYOGENESIS IN KAPPAPHYCUS

ippines. Nevertheless, there is no information available on mass production of micropropagules clonally

from seaweed callus tissues.
Kappaphycus alvarezii (Doty) Doty ( Eucheuma striatum (Schmitz) Doty) is a major commercial source of
-carrageenan and is presently being grown in our experimental farm at Mandapam (Tamil Nadu, India).
Recently, its cultivation on a commercial scale has also
commenced in Indian waters. This study was initiated
to develop in vitro cell culture techniques to establish
productive strains with high yields of quality phycocolloid either through somaclonal variation or protoplast fusion (somatic hybridization) for commercial
farming of this alga on the Indian coast. Here we report on tissue culture of K. alvarezii, with in vitro somatic embryogenesis and mass clonal production of
propagules through somatic embryos.

materials and methods

Culture development. Kappaphycus alvarezii was collected from
our experimental farm at Mandapam (9 16 N, 798 E), Tamil
Nadu and brought to the Bhavnagar (Gujarat) laboratory under cool conditions (2223 C). After arrival, the branches were
trimmed to about 10 cm and cleaned thoroughly in filtered seawater (Whatman GF/C, Maidstone, UK) with a soft brush. The
branches were used for initiation of unialgal cultures and for
acclimatization to laboratory conditions by growing in Provasoli
enriched seawater (PES) media (Provasoli 1968), modified f/2
(Waaland and Watson 1980), and modified Erd Schreibers seawater (ESS) (Suto 1959). To eliminate diatom growth, GeO 2
(10 mgL1) was added to all culture media during first 2 weeks
of culture. During the acclimatization period, algae were continuously aerated and maintained at 22  1 C under cool
white fluorescent tube lights at 35 mol photonsm2s1 with a
12:12-h light:dark cycle.
Preparation of axenic material. Apical thallus fragments of approximately 5 cm in length were selected for tissue culture.
Axenic explants were established by following surface sterilization methods (Polne-Fuller and Gibor 1984, Huang and Fujita
1997a). The select explants were manually cleaned thoroughly
with a brush in filtered seawater under a stereo zoom microscope (Olympus SZH10, Tokyo, Japan) and in 0.5% liquid detergent (Charmy green, Lion Co. Ltd., Tokyo, Japan) in sterile
seawater for 10 min. Explants were then treated with 2% povidone iodine (available iodine 0.5% w/v) in sterile seawater for
3 min to eliminate surface microbes. Further, sterilization was
continued in sterile PES medium containing 3% filter-sterilized
(Millex-GV 0.22 m, Millipore S.A., Molsheim, France) broadspectrum antibiotic mixture (Polne-Fuller and Gibor 1984) for
2 days. During antibiotic treatment, the cultures were maintained as static cultures under similar conditions as described
for acclimatization. The sterility of the treated fragments was
confirmed by testing on Zobell agar plates for about 3 weeks in
a bacterial incubator.
Explant culture. After antibiotic treatment, the fragments
were thoroughly cleaned with sterile seawater and excised into
4- to 5-mm length explants. Each explant was then wiped gently
with sterile filter papers (Whatman no. 1, Maidstone, UK) to remove moisture and any mucilaginous exuded from cut ends before the inoculation on explant culture medium. The explants
were inoculated into Petri dishes (15 explants in each) with 20
mL of 1.5% (w/v) bacto-agar-solidified PES medium and cultured as described above except with light reduced to 5 mol
photonsm2s1. After 2 weeks, callus induction rate was calculated by counting the number of explants with callus. To ensure sustainable callus growth, the explants bearing callus were
transferred regularly to fresh culture medium at 30-day inter-

611

vals. After 2 months of explant culture, callus outgrowth was excised from the explant and subcultured separately on fresh medium under similar conditions except with light intensity
increased to 35 mol photonsm2s1.
Effect of agar, light, and plant growth regulators on callus induction. To obtain optimal and consistent callus induction rates
and growth in explants, some of the culture media supplements such as agar, growth regulators, and photon flux densities were standardized. All culture conditions and media used
for this purpose were the same as adopted for explant culture.
Fifty explants were used for each observation. To investigate
the optimal photon flux densities, explants were cultured at 5,
30, and 70 mol photonsm2s1, for agar at 0.8%, 1.0%, 1.5%,
2.0%, and 3.0%, and for growth regulators (naphthaleneacetic
acid [NAA] and 6-benzylaminopurine [BAP]) at 0.1 and 1.0
mgL1 each or in combination. In all cases, callus induction
rate in explants was determined after 2 weeks of explant culture and expressed as mean of total explants.
Induction of embryogenic callus and somatic embryogenesis in pigmented filamentous callus. For the induction of embryogenic callus, 60-day-old subcultured callus clumps were thinly sliced and
cultured as blocked embedded in 0.4% bacto-agar or 0.6% agarose (low gelling temperature, type VII, Sigma, St. Louis, MO,
USA) solidified PES medium. The sliced blocks ( n  15) were
transferred to Petri dishes with 20 mL of culture medium with
low gelling agar and cultured under similar conditions used for
subcultured callus. While implanting the callus slices into agar,
care was exercised to avoid exposure to heat and to rapidly cool
the agar after transplantation of callus slices. The subculture of
embedded filamentous callus was carried out regularly at 40- to
45-day interval. The effect of NAA and BAP on somatic embryogenesis of pigmented callus was also determined.
Micropropagule culture. The agar containing embryogenic
callus with tiny somatic embryos was cut into blocks with a scalpel and transferred to conical flasks with 50 mL of liquid PES
medium. The flasks were then placed on a portable rotary
shaker (New Brunswick Scientific, Edison, New Brunswick, NJ,
USA) and swirled at 100 rpm for about 1 month and later transferred to aerated flasks (nonaxenic) and grown until the germlings attained lengths of 35 cm, which is the minimum size for
field transplantation. During micropropagule culture, the media was replenished at weekly intervals. The culture conditions
used for micropropagules were the same as described for acclimatization.
Field cultivation of regenerated plants from somatic embryos. The
viability of regenerated plants in field cultivation was tested at
CSMCRIs field station at Mandapam. Ten laboratory-grown
germlings, 3 months old and 35 cm in length, were transported in a cooler (2223 C) for field cultivation. Individual
plants were weighed and tied to floating long lines. To minimize grazing and detachment of germlings from the cultivation
rope, the germlings were grown in closed transparent polythene bags with perforations. The growth rate of tissue-cultured germlings was monitored and compared with farmed
plants (control) over seven generations from September 1999
to May 2001. The cultivation period for the first five generations was 90 days each and for the sixth and seventh was 60 days
each. The growth of individual plants was measured at 30-day
interval during the cultivation period as increase in fresh biomass, and their daily growth rates were calculated following the
formula of Dawes et al. (1993).

results
Culture development and axenic culture. During acclimatization, bleaching and degeneration of the distal
ends of the cuttings were observed in all tested media.
However, the fronds grown in PES medium recovered
rapidly in 1 month and showed growth in apical tips
and regenerated new branches. Thus, PES medium
was chosen for subsequent tissue culture experimen-

612

C. R. K. REDDY ET AL.

tation, and the regenerated fronds were used as

source material for further tissue culture. Treatment
of K. alvarezii fragments with 0.5% detergent for 10
min, 2% iodine for 3 min, and followed by incubation
in 3% broad-spectrum antibiotic mixture for 2 days
resulted in 90%95% of cultured explants free of bacteria. Exposures to higher concentrations for prolonged periods caused bleaching of explants, and exposure to lower concentrations with shorter durations
were found to be ineffective and gave explants with inadequate sterility.
Callus induction and somatic embryogenesis. Callus induction as filamentous outgrowths was observed from
the cut surface of the explant during the first 2 weeks
after initiation of explant culture. New cells developed from both medullary and cortical regions of the
cut end of the explants, which, through subsequent
cell divisions, gave rise to pigmented uniseriatebranched filaments. In 2 months, the callus growth
became prominent and spread all over the cut surface, forming a bright whitish caplike structure (Fig.
1a) at both cut ends of explant, if they were placed
horizontal to agar surface. The callus induction rate
in explants of laboratory-acclimatized plants was
found to be
80%. The callus growth did not continue in explants that were obtained from plants in
the early stages of acclimatization. Most of the explants cultured for callus induction during the first 2
months of the acclimatization period showed bleaching with callus after the second week onward in cultures.
Although callus formation at the cut ends of explant was common, occasionally induction from all
over the surface of the explant (Fig. 1b) and from the
intact apical tips (Fig. 1c) was also observed when the
explants were from the apical region. Except for the
apical cells, cells of filamentous callus were elongated
(width 1015 m and length 25200 m) and highly
vacuolated with discoid chloroplasts spread all over
the cell. The apical cells were short and oval in shape.
The initial growth of callus was uniseriate branched
filamentous type (Fig. 1d), followed by formation of
disorganized cell mass. Upon excision from the explant, all subcultured calli formed clumps with prolific filamentous outgrowths (Fig. 1e) in 2 months.
These clumps of filaments were subcultured and
could be maintained as filamentous clumps for over 2
years in our laboratory by regularly transferring them
to fresh medium at bimonthly intervals. Moreover,
some cultured calli penetrated the agar bed. The prolonged culture of some excised callus (10%) for
more than 2 months without further subculture on
fresh agar plates resulted in development of densely
pigmented spherical or oval-shaped micropropagules
of 15 mm in diameter (Fig. 1f) on the callus. The micropropagules were deep reddish brown in color and
firm with dense tissue. The density of micropropagules, however, was found to be higher on the periphery of the callus, presumably because of its more
intimate contact with the nutrient medium. Because

some filaments from subcultured calli found penetrated in the agar, an attempt was made to implant
and culture thin sections of pigmented callus tissues
inside agar or agarose bed (Fig. 1g). In 1 month, all
implanted blocks in the agar medium showed luxuriant growth of filamentous callus outgrowths with
dense cell contents and pigmentation on all sides of
transplanted callus tissues (Fig. 1h). After 2 months,
the transplanted regenerated callus developed into
embryonic callus with groups of spherical cells at the
apical region. In addition, there were reddish brown
globular pigment bodies distributed all over the surface, imparting brownish colored spots on the agar
plate. The embryonic filaments from inserted blocks
eventually produced dark reddish brown-pigmented
microcolonies of globular or oval-shaped somatic embryos (Fig. 1, i and j ).
The somatic embryos usually occurred as tiny buds
on the surface of elongated cells near apical regions
of the filaments. These buds originated from a single
cell, which was small in size (10 m) and characterized by dense cytoplasmic contents and pigmentation.
Addition of 0.11.0 mgL1 NAA or a mixture of 0.1
mgL1 each NAA and BAP to the culture medium enhanced callus growth, embryogenesis, and differentiation of embryos into micropropagules compared with
the controls (data not shown). Transfer of such embryogenic callus blocks to liquid cultures on a rotary
shaker facilitated morphogenesis of somatic embryos
into micropropagules after about 40 days in culture
(Fig. 1k). The seedlings (Fig. 1, l and m) obtained
from the somatic embryos grew rapidly in polythene
bags in situ, forming new plants similar to farmed
plants. The polythene bags (Fig. 1n) prevented grazing and minimized settlement of epiphytic growth. All
10 regenerated plants survived with higher growth
rates than parental samples with one plant (Fig. 1o)
having a daily growth rate of 8.11%. That plant was
further monitored and found to have 1.51.8 times
the daily growth rate of the existing farm plants over
seven generations (Fig. 2).
Effect of agar, light, and plant growth regulators on callus induction. Apparently, agar concentration in the
culture medium did not affect the callus induction in
explants except at lower concentration (0.8%), where
bud development was dominant (64%) over callus
formation. The callus growth in such explants was always suppressed and only bud growth progressed.
Agar concentrations above 1.5% always showed callus
formation and restricted bud development in explants. However, the callus induction rate for 3% agar
was declined (64%) compared with 1.5% agar (82%).
Similarly, the explants grown under different photon
flux densities did not show significant differences in
callus induction rate, but their continued exposure to
higher light intensities (70 mol photonsm2s1)
caused bleaching at initial stages of callus growth. Additions of plant growth regulators such as NAA and
BAP to explant culture medium showed no increase
in callus induction rate or callus growth. The callus in-

SOMATIC EMBRYOGENESIS IN KAPPAPHYCUS

613

Fig. 1. Callus induction, subculture, somatic embryogenesis, and field cultivation of Kappaphycus alvarezii. (a) Profuse growth of
filamentous callus on explants after 60 days. Scale bar, 1 mm. (b) Explant with callus development all over the surface. Scale bar, 1
mm. (c) Explant with apical callus. Scale bar, 1 mm. (d) Uniseriate filamentous callus growth. Scale bar, 50 m. (e) Subculture of excised callus 50 days old. Scale bar, 500 m. (f) Subcultured callus with oval- or spherical-shaped pigmented micropropagules after 60
days in culture. Scale bar, 1.5 mm. (g) Growth of pigmented callus from implanted slices in agar plate (0.4% agar). Scale bar, 2 cm.
(h) A single block of filamentous callus growth inside the soft gel. Scale bar, 1 mm. (i) Formation of somatic embryos in embryogenic
callus. Scale bar, 40 m. (j) Magnified view of globular and oval-shaped somatic embryos on filamentous callus. Scale bar, 40 m. (k)
Germination of micropropagules in liquid PES medium. Scale bar, 6 mm. (l) Young plantlets of Kappaphycus with multiple shoots.
Scale bar, 1 cm. (m) Fully grown laboratory plants in aerated culture after 90 days. Scale bar, 3 cm. (n) Field-grown tissue-cultured regenerated plants from micropropagules by long line method after 50 days. Scale bar, 30 cm. (o) Comparison of parent plant (L) and
regenerated plant (R) after 50 days. Scale bar, 10 cm.

614

C. R. K. REDDY ET AL.

Fig. 2. Comparison of growth of control (farmed plant, ) with that of tissue-cultured Kappaphycus () in field cultivation. Vertical bars, SD (n  10).

duction rate in both control and hormone-treated explants ranged between 67% and 84%.
discussion
Our results demonstrate a novel method for clonal
propagation using somatic embryos from uniseriate
pigmented filamentous callus of the red seaweed K.
alvarezii. Culture methods allowed routine preparation of axenic explants, subculture of excised callus,
and embedded callus culture for somatic embryogenesis and culture of micropropagules. Callus or calluslike growth has been reported from several species of
red algae (Aguirre-Lipperheide et al. 1995, Huang
and Fujita 1997b). The origin of callus and its morphology described for seaweeds varied greatly with
species (Yokoya et al. 1993). Callus, in general, is described as mass of a disorganized growth of cells from
the differentiated tissue as a result of wounding (Yeoman 1987). Though callus development in K. alvarezii
mainly occurred at the cut ends, it also formed directly from intact surfaces and from apical tips of the
explants (nonwound) as the case of Solieria filiformis
(Ktzing) Gabrielson (Robledo and Garcia-Reina
1993), Meristotheca papulosa J. Agardh (Huang and

Fujita 1997a), and Gracilariopsis tenuifrons (Bird et Oliveira) Fredricq et Hommersand (Yokoya 2000).
In red algae, production of filamentous callus is
common, whereas compact cell masses are rarely reported (Robaina et al. 1992, Kaczyna and Megnet
1993, Huang and Fujita 1997b). In K. alvarezii, callus
formation was first observed at the cut surface of the
explant as filamentous type, originating from both
medullary and cortical regions of the explant. In contrast, callus in E. uncinatum and K. alvarezii developed
from the cortical tissue (Polne-Fuller and Gibor 1987)
and in E. denticulatum and K. alvarezii from medullary
tissue (Dawes and Koch 1991). The differences in origin of callus on explants were attributed to the concentration of solidifying agent (agar) used for explant
culture medium (Dawes and Koch 1991). Although
agar content did not affect callus induction in K. alvarezii, a lower concentration (0.8%) favored bud development. In species of Laurencia and Grateloupia, bud
formation was dependent on physical characteristics
(osmolality and solidity) of the culture medium
(Robaina et al. 1990). Apart from physical properties
of the explant culture medium and the nature of explant, the water or moisture content of the culture
medium also seems to play an important role in deter-

SOMATIC EMBRYOGENESIS IN KAPPAPHYCUS

mining the nature of outgrowths (callus or bud),

which develop from the cut end of the explants.
In seaweeds, the rate of callus induction and
growth is minimal. To obtain consistent and increased percentage of callus induction and callus
growth, plant growth regulators such as auxins and cytokinins are applied individually or in various combinations to explant culture media (Bradley and
Cheney 1990, Dawes and Koch 1991, Kaczyna and
Megnet 1993, Huang and Fujita 1997b). In this study,
we obtained callus induction rates as high as 80% in
K. alvarezii using ordinary solidified PES medium
without any other culture additives. Supplementation
of NAA or BAP individually or in combination (1:1)
to explant culture medium did not result in increases
in either the callus induction rate or callus growth.
The high callus induction rate observed in this study
could be partly due to use of newly grown thallus fragments that were developed during the acclimatization
period in the laboratory conditions. On some occasions, 100% callus induction was possible when laboratory-grown plants were used. This suggests the importance of adapting plants to laboratory conditions
before initiation of tissue culture experimentation.
The success of subculture of excised callus on solid
medium independent of explants has been reported
for a few red seaweeds (Liu and Gordon 1987, PolneFuller and Gibor 1987, Huang and Fujita 1997a). The
cessation of callus growth upon excision has been reported in Grateloupia doryphora (Montagne) Howe
(Robaina et al. 1992) and Gracilaria verrucosa (Huds.)
Papenfuss (Kaczyna and Megnet 1993). Consequently,
subculturing of callus is preferred in liquid cultures
because it not only supports growth but also induces
morphogenesis in the callus. Conversely, the excised
callus of K. alvarezii responded positively to our subculture and has been grown successfully for more
than 2 years in our laboratory by periodically subculturing on fresh medium. The success in subculturing
also allowed the production of micropropagules and
somatic embryos. Implantation of thin slices of callus
into agar enhanced the process of formation of embryogenic callus followed by somatic embryogenesis.
Interestingly, NAA and BAP stimulated cell division
and somatic embryogenesis in implanted tissues compared with untreated callus. The regeneration of somatic embryos into micropropagules was high, with
most forming young plantlets in aerated flasks. Thus,
mass production of micropropagules through embryogenesis of callus culture is possible for macroalgae. Further, seedlings grew rapidly in the field in
polythene bags, forming plants that were morphologically identical to the farmed ones. One plant had a
growth rate of 1.51.8 times over field plants.
In conclusion, the profuse somatic embryogenesis
together with the high regeneration rate of somatic
embryos described in this study could serve as a potential tool for mass clonal propagation of desired
seed stock of both Eucheuma and Kappaphycus for commercial farming, besides forming an ideal starting ma-

615

terial for both fundamental and applied research in

macroalgae. Further, the tissue culture techniques, including an embedded culture method, provide an
ideal option for maintenance and storage of germplasm in live conditions for prolonged periods.
We thank Dr. Pushpito K. Ghosh, Director, CSMCRI, for kind
help and encouragement. We also thank Prof. Clinton J. Dawes,
Department of Biology, University of South Florida, USA and
two anonymous reviewers for critically reviewing this manuscript. Financial support from Department of Biotechnology
(Sanction No. BT/PR 1721/AAQ/03/89/ 99), New Delhi and
Council of Scientific and Industrial Research (CSIR), New Delhi
is gratefully acknowledged. One of the authors (G. R. K. K.) also
thanks CSIR for the award of Senior Research Fellowship.
Aguirre-Lipperheide, M., Estrada-Rodriguez, F. J. & Evans, L. V.
1995. Facts, problems and needs in seaweed tissue culture: an
appraisal. J. Phycol. 31:67788.
Ammirato, P. V. 1983. Embryogenesis. In Evans, D. A., Sharp, W. R.,
Ammirato, P. V. & Yamada, Y. [Eds.] Handbook of Plant Cell Culture, Vol. I: Techniques for Propagation and Breeding. Macmillan
Publishing, New York, pp. 82123.
Bradley, P. M. & Cheney, D. P. 1990. Some effects of plant growth
regulators on tissue cultures of the marine alga Agardhiella
subulata (Gigartinales, Rhodophyta). Hydrobiologia 204/205:
35360.
Butler, D. M. & 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.
62945.
Cheney, D. P., Luistro, A. H. & Bradley, P. M. 1987. Carrageenan
analysis of tissue cultures and whole plants of Agardhiella subulata. Hydrobiologia 151/152:1616.
Dawes, C. J. & 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. 6:214.
Dawes, C. J., Trono, G. C. & Lluisma, A. O. 1993. Clonal propagation of Eucheuma denticulatum and Kappaphycus alvarezii for
Philippines seaweed farms. Hydrobiologia 260/261:37983.
Evans, L. V. & Butler, D. M. 1988. Seaweed biotechnologycurrent
status and future prospects. In Rogers, L. J. & Gallon, J. R.
[Eds.] Biochemistry of the Algae and Cyanobacteria. Clarendon
Press, Oxford, pp. 33550.
Huang, W. & Fujita, Y. 1997a. Callus induction and thallus regeneration of the red alga Meristotheca papulosa (Rhodophyta, Gigartinales). Bot. Mar. 40:5561.
Huang, W. & Fujita, Y. 1997b. Callus induction and thallus regeneration in some species of red algae. Phycol. Res. 45:10511.
Jain, S. M., Gupta, P. K. & Newton, R. J. 1994. Somatic Embryogenesis
in Woody Plants. Vol. 3. Gymnosperms. Kluwer Academic, Dordrecht, The Netherlands, 388 pp.
Kaczyna, F. & Megnet, R. 1993. The effects of glycerol and plant
growth regulators on Gracilaria verrucosa (Gigartinales, Rhodophyceae). Hydrobiologia 268:5764.
Kirihara, S., Fujikawa, Y. & Notoya, M. 1997. Axenic tissue culture
of Sargassum confuscum C. Agardh (Phaeophyta) as source of
seeds for artificial marine forests. J. Mar. Biotechnol. 5:1426.
Lawlor, H. J. 1990. Tissue culture of brown seaweeds. Aust. J. Biotechnol. 4:2604.
Liu, X. W. & Gordon, M. E. 1987. Tissue and cell culture of New
Zealand Pterocladia and Porphyra species. Hydrobiologia 151/152:
14754.
Liu, X. W., Rochas, C. & Kloareg, B. 1990. Callus culture of Pterocladia capillacea (Rhodophyta) and analysis of cell wall polysaccharides. J. Appl. Phycol. 2:297303.
Maliakal, S., Cheney, D. P. & Rorrer, G. L. 2001. Halogenated
monoterpene production in regenerated plantlet cultures of
Ochtodes secundiramea (Rhodophyta, Cryptonemiales). J. Phycol.
37:10109.

616

C. R. K. REDDY ET AL.

Polne-Fuller, M. & Gibor, A. 1984. Developmental studies in Porphyra. I. Blade differentiation in Porphyra perforata as expressed
by morphology, enzymatic digestion and protoplast regeneration. J. Phycol. 20:60916.
Polne-Fuller, M. & Gibor, A. 1987. Calluses and callus growth in
seaweeds: induction and culture. Hydrobiologia 151/152:1318.
Provasoli, L. 1968. Media and prospects for the cultivation of marine algae. In Watanabe, A. & Hattore, A. [Eds.] Culture and
Collection of Algae. Jap. Soc. Plant. Physiol. Tokyo. pp. 6375.
Robaina, R. R., Garcia-Reina, G. & Luque, A. 1990. The effects of
the physical characteristics of the culture medium on the development of red seaweeds in tissue culture. Hydrobiologia 204/
205:13742.
Robaina, R. R., Garcia-Jimenez, P. & Luque, A. 1992. The growth
pattern and structure of callus from the red alga Laurencia sp.
(Rhodophyta, Cermiales) compared to shoot regeneration.
Bot. Mar. 35:26772.
Robledo, D. R. & Garcia-Reina, G. 1993. Apical callus formation in
Solieria filiformis (Gigartinales, Rhodophyta) cultured in tanks.
Hydrobiologia 260/261:4016.

Sharp, W. R., Sondahl, M. R., Caldas, L. S., & Maraffa, S. B. 1980.

The physiology of in vitro asexual embryogenesis. Hortic. Rev. 2:
268310.
Suto, S. 1959. Skeletonema no tame no jinkou baiyoueki. SuisanZouskoku. 7:1719 [in Japanese].
Tisserat, B. 1985. Embryogenesis, organogenesis and plant regeneration. In Dixon, R. A. [Ed.] Plant Cell Culture: A Practical Approach. IRL Press, Oxford, pp. 79105.
Waaland, S. A. & Watson, B. A. 1980. Isolation of a cell fusion hormone from Griffithsia pacifica Kylin, a red alga. Planta (Berl.)
149:4937.
Yeoman, M. M. 1987. Bypassing the plant. Ann. Bot. 60:15774.
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. 48:13342.
Yokoya, N. S., Guimaraes, M. P. B. S. & Handro, W. 1993. Development of callus like structures and plant regeneration in thallus
segments of Grateloupia filiformis Ktzing (Rhodophyta). Hydrobiologia 260/261:40713.