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UNIVERSITI PUTRA MALAYSIA
OPTIMIZATION OF RNA EXTRACTION AND GENERATION OF EXPRESSED SEQUENCE TAGS FROM SARGASSUM BINDERI
(SONDER) J. AGARDH
TONY WONG KOK MIN.
FBSB 2005 15
OPTIMIZATION OF RNA EXTRACTION AND GENERATION OF EXPRESSED SEQUENCE TAGS FROM
Sargassum binderi (SONDER) J. AGARDH
BY
TONY WONG KOK MIN
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirements for the Degree of Master of
Science
July 2005
Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master Science
OPTIMIZATION OF RNA EXTRACTION AND GENERATION OF EXPRESSED SEQUENCE TAGS FROM
Sargassum binderi (SONDER) J. AGARDH
BY
TONY WONG KOK MIN
July 2005
Chairman : Ho Chai Ling, PhD
: Biotechnology and Biomolecular Sciences Faculty
Brown seaweeds are macroalgae in the marine habitat. They are widely
used as food, sources for cosmetics, pharmaceuticals, soap, agar, textile,
and alginate. Sargassum binderi is one of the most abundant brown
seaweeds in Malaysia. Previous studies have shown that S. binderi contains
high quality of alginate compared to commercially used seaweeds such as
Laminaria hyberborea, Macrocystis pyrifera and Ascophyllum nodosum.
Expressed sequence tag (EST) approach is a powerful tool in providing
genetic information of an organism, especially for S. benderi in which little
genetic information is available. This study presents the first attempt in
generating ESTs from S. binden. RNA extraction from seaweeds was the
main challenge in this study, as the RNA yield was low and the
polysaccharide contamination was difficult to be eliminated. A total of ten
different RNA extraction methods (including five modifications) have been
carried out to obtain sufficient RNA of high quality to construct a
representative cDNA library for ESTs generation. The optimized S. binderi-
specific CTAB RNA extraction method developed in this study was able to
produce high yield of RNA with minimum polysaccharide contamination.
Sufficient amount of mRNA was obtained to construct a primary cDNA library
with a titer of 9.2 x l o5 pfulml. A total of 2051 ESTs were generated and
analyzed from the amplified cDNA library of S. binden (with a titer of 1.31 x
l o 9 pfulml). The ESTs were putatively identified by comparison to the non-
redundant peptide database in NCBI. Approximately 82% of the ESTs were
assigned as unknown and novel sequences that are potentially important for
new gene discovery, whereas the 18% ESTs with significant matches to the
database were classified into various putative functional groups, including
protein synthesis, energy, protein destination and storage, metabolism, cell
structureldivision, diseaseldefense, signal transduction, transcription, and
miscellaneous. The EST information generated from this study may
contribute towards better understanding of the biochemistry and molecular
biology of S. binder; and other brown seaweeds in the future.
Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains
OPTlMASl PNGEKSTRAKAN RNA DAN PENJANAAN TAG JUJUKAN TEREKSPRES DARIPADA Sargassum binderi (SONDER) J . AGARDH
Oleh
TONY WONG KOK MIN
Julai 2005
Pengerusi : Ho Chai Ling, PhD
Fakulti : Bioteknologi dan Sains Biomolekul
Rumpai laut perang adalah makroalga di laut. la digunakan secara meluas
di dalam industri makanan, kosmetik, farmasi, sabun, agar, tekstil and
alginate. Sargassum binderi merupakan salah satu rumpai laut perang yang
boleh didapati dengan banyaknya di Malaysia. Hasil penyelidikan awal
menunjukkan bahawa S. binderi mengandungi alginate berkualiti tinggi
berbanding dengan rumpai laut perang lain yang digunakan di dalam industri
seperti Laminaria hyberborea, Macrocystis pyrifera dan Ascophyllum
nodosum. Pendekatan tag jujukan terekspres (expressed sequence tag -
EST) amat berguna untuk memperolehi maklumat genetik sesuatu
organisma, terutamanya untuk S. binderi yang mempunyai maklumat genetik
yang terhad. Kajian ini merupakan penyelidikan terulung untuk memperolehi
EST daripada S. binden. Pengekstrakan RNA merupakan suatu cabaran
utama dalam pengajian ini, di mana kuantiti RNA yang diperolehi adalah
rendah dan dicemari polisakarida yang susah dipisa hkan. Sejumlah sepuluh
kaedah pengekstrakan RNA (termasuk lima pengubahsuaian) 'ielah
rnU!g#&&$N SWLfAN ABWL S4Ub WWIw WfFU W Y *
dijalankan untuk mendapat RNA yang cukup dan berkualiti demi pembinaan
perpustakaan cDNA untuk penjanaan EST. Kaedah pengekstrakan RNA
yang telah diubahsuaikan khas untuk S. binderi, dapat menghasilkan RNA
yang berkuantiti tinggi dengan pencemaran polisakarida yang minimum.
mRNA yang cukup telah digunakan untuk membina perpustakaan cDNA
utama dengan titer 9.2 x l o 5 pfulml. Sejumlah 2051 EST telah dijana dan
dianalisis daripada perpustakaan cDNA yang telah diamplifikasi (titer 1.31 x
lo9 pfulml). EST dikenalpasti melalui perbandingan dengan pangkalan data
peptida yang tidak berulang di NCBI. Sebanyak 82% EST telah digolongkan
dalam kategori 'tidak diketahui' and 'kategori baru', yang berpotensi dan
berkepentingan dalam penemuan gen-gen baru. Di samping itu, sebanyak
18% EST yang lain telah digolong dalam pelbagai kumpulan berdasarkan
kepada fungsi putative EST tersebut, iaitu sintesis protin, tenaga, destinasi
dan penyimpanan protin, metabolisms, strukturlpembahagian sell
penyakitlpertahanan, pemindahan isyarat, transkripsi dan serbaneka.
Maklumat EST yang terkumpul dalam kajian ini, akan menyumbang ke arah
pemahaman yang lebih mendalam mengenai biokimia dan biologi molecular
S. bindefi dan rumpai laut perang di masa akan datang.
ACKNOWLEDGEMENTS
Million thanks to Dr. Ho Chai Ling, my main supervisor, for giving me a good
opportunity to pursue my studies in the field of molecular biology. Her
knowledge, guidance, openness, enthusiasm, and inspiration will always
serve to me as an example of the perfect supervisor. Not to forget Assoc.
Prof. Dr. Raha Abdul Rahim and Prof. Dr. Phang Siew Moi, my co-
supervisors, for their guidance and advice throughout my study.
I would like to extend my gratitude to my lab mates: Dr. Sudha, Yeen Yee,
Seddon, Dang, Swee Sen and many others for their kind advice, assistance
and support. Special thanks to Mr. Lee Weng Wah and Mr. Lee Yang Ping
for assisting me in sequence analysis; members at the Algae Laboratory,
University of Malaya, for their knowledge in seaweeds identification.
I would like to acknowledge the Malaysia Toray Science Foundation (MTSF)
and the fundamental Research grant, the Ministry of Education, Malaysia, for
providing financial support in this research project and Graduate Research
Assistance fellowship.
Lastly, not to forget my family for their faith, love and emotional support. For
that, I dedicate this dissertation to them.
I certify that an Examination Committee met on 2oth J U I ~ 2005 to conduct the final examination of Tony Wong Kok Min on his Master of Science thesis entitled "Optimization of RNA Extraction and Generation of Expressed Sequence Tags from Sargassum binder; (Sonder) J . Agardh" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows:
Gulam Rusul Rahmat Ali, PhD Professor Faculty of Food Science and Technology Universiti Putra Malaysia (Chairman)
Maziah Mahmood, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner)
Norihan Mohd Salleh, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner)
Rofina Yasmin Othman, PhD Associate Professor Institute of Biological Sciences Universiti Malaya (External Examiner)
School of Graduate Studies Universiti Putra Malaysia
Date: 2 5 OCT 2005
This thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Master of Science degree. The members of the Supervisory Committee are as follows:
Ho Chai Ling, PhD Lecturer Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman)
Raha Abdul Rahim, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member)
Phang Siew Moi, PhD Professor Institute of Biological Sciences Universiti Malaya (Member)
AlNl IDERIS, PhD Professor1 Dean School of Graduate Studies Universiti Putra Malaysia
Date: 17 NOV 2005
. . . Vll l
DECRALATION
I hereby declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.
TONY WONG KOK MIN
TABLE OF CONTENTS
Page
ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL DECLARATION LlST OF TABLES LlST OF FIGURES LlST OF ABBREVIATIONS
CHAPTER
INTRODUCTION
LITERATURE REVIEW 2.1 Seaweed
2.1.1 Classification 2.1.2 Morphology 2.1.3 The importance of seaweed
2.2 Phaeophyta 2.2.1 Sargassum
2.3 The applications of brown seaweed 2.3.1 The industrial values of alginate 2.3.2 Human's favourite nutritional food 2.3.3 Dried seaweed as animal food 2.3.4 Biosorption of heavy metals
2.4 Molecular study of phaeophyta 2.4.1 RNA extraction from seaweeds 2.4.2 Molecular taxonomic studies of Sargassum
SPP. 2.4.3 The expressed sequence tags (EST) approach
and its applications to seaweed research 2.4.4 Gene classes of brown seaweed
MATERIALS AND METHODS 3.1 Sample collection and preparation 3.2 RNA extraction
3.2.1 Method 1: CTAB method (Apt et a/. 1995) 3.2.2 Method 2: LiCI-SDS method (Kim et a/. 1997) 3.2.3 Method 3: CsCl method (GliSin eta/. 1974) 3.2.4 Method 4: RNeasy Plant Mini Kit (Qiagen) 3.2.5 Method 5: ~ppendorf@ Perfect RNA Mini kit
3.3 Optimization of CTAB method 3.3.1 Method 1 . I : Modified CTAB method 1 3.3.2 Method 1.2: Modified CTAB method 2 3.3.3 Method 1.3: Modified CTAB method 3 3.3.4 Method 1.4: Modified CTAB method 4
I I
iv v i vii ix xii ...
X l l l
xv
3.3.5 Method 1.5: Modified CTAB method 5 Quantification of total RNA 3.4.1 Spectrophotometer 3.4.2 Formaldehyde-agarose gel electrophoresis mRNA isolation Quantification of mRNA cDNA library construction and EST generation 3.7.1 The first strand cDNA synthesis 3.7.2 The second strand cDNA synthesis 3.7.3 Blunting the cDNA termini 3.7.4 Ligation of the EcoR I adapter 3.7.5 Phosphorylation of the EcoR I ends 3.7.6 Digestion with Xho I 3.7.7 Size fractionation by agarose gel
electrophoresis 3.7.8 Ligation of DNA to the Uni-ZAP XR vector 3.7.9 Preparation of the host bacteria 3.7.1 0 The DNA packaging 3.7.1 1 Plating and titering 3.7.12 Amplification of the Uni-ZAP XR library 3.7.1 3 Single clone in vivo excision 3.7. I4 Plasmid isolation 3.7.15 Mass excision 3.7.16 Large-scale plasmid DNA extraction Confirmation of cDNA inserts 3.8.1 Polymerase chain reaction Sequence analyses
RESULTS 4.1 Optimization of RNA extraction 4.2 mRNA isolation 4.3 cDNA library construction 4.4 Analysis of sequences 4.5 Generation and identification of ESTs
DISCUSSION 5.1 RNA extraction optimization 5.2 Optimization of the CTAB method for RNA extraction
from S. binden 5.3 mRNA isolation 5.4 Construction of cDNA library 5.5 Sequence analyses of ESTs
CONCLUSION 6.1 Conclusion 6.2 Recommendation for further studies
REFERENCES APPENDICES BIODATA OF THE AUTHOR
LIST OF TABLES
Table
2.1 Summary of the three macroalgae divisions and their significant characteristics (adapted from Harold and Michael 1978; David 2002).
2.2 The applications of alginate in food (reproduced from Chapman and Chapman 1980).
Page
5
2.3 The applications of alginate in industries (reproduced from 13 Chapman and Chapman 1980).
2.4 Production of seaweed for food in Japan (reproduced from McHugh 1991).
2.5 Comparison of fucoxanthin and iodine content between brown seaweed (Ascophyllum spp.) and grass (reproduced from Chapman and Chapman 1980).
4.1 The comparisons of various RNA extraction methods.
4.2 Comparisons of the modified CTAB methods.
S. binderi ESTs statistics.
LIST OF FIGURES
Figure
2.1 Pictures showing the drawing of Sargassum plant (a), branch with air-filled vesicles and receptacle (b) and perennial primary shoot and holdfast (c) (reproduced from Sean 1997).
A summary of cDNA cloning and expressed sequence tag (EST) sequencing (adapted from Rudd 2003).
Formaldehyde agarose gel electrophoresis of the total RNA extracted using method 1.4. The two distinct bands showed the 18s and 28s ribosomal RNA.
Page
9
Electrophoretical analysis of DNA inserts from random 58 selected plaques. The DNA inserts ranged from 500 to 2,000 bp by comparing to the A-Pst I marker (200 ng).
Distribution of sequences quality. From 2,304 sequences, 2,051 (89%) were readable sequences with length more than 100 bp, 11 8 (5%) were readable sequences with length less than 100 bp, and 135 (6%) were non-readable sequences.
The size distribution of ESTs. It showed that majority of the ESTs ranged from 801 to 900 bp.
ESTs with homology to sequences from various organisms. Most of the ESTs showed significant homology to plant species (33.7 %).
The frequency of ESTs in the TUCs. Most of the TUCs (89%) contained less than 5 ESTs and were assumed to be expressed at low to intermediate level. Only 11% of the TUCs fall into categories with frequencies of more than 5 ESTs.
The top five highly expressed putative functional ESTs from 63 S. binder;.
Classification of the non-redundant ESTs. This pie chart shows that 56% of the ESTs are unknown sequences, followed by 26% are novel sequences, and 18% sequences with putative functions.
... Xll l
Putative functional groups of the non-redundant ESTs according to their proposed biological roles or biochemical functions
xiv r"
LIST OF ABBREVIATIONS
BLAST
CTAB
dCTP
DEPC
dGTP
DNA
DNase
dNTPs
DTT
dTTP
EDTA
EtBr
beta
lambda
microgram
microliter
degree centigrade
percentage
Basic Local Alignment Search Tool
base pairs
complementary DNA
chloride
cesium chloride
hexacetyltrimethyl ammonium bromide
2'-deoxy-adenosine-5'-triphosphate
2'-deoxy-cytidine-5'-triphosphate
diethyl pyrocarbonate
2'-deoxy-guanosine-5'-triphosphate
deoxyribonucleic acid
deoxyribonuclease
deoxynucleotides
dithiothreitol
thymidine-5'-tryphosphate
ethylenediaminetetraacetic acid
ethidium bromide
gram
HCI
kb
I
LB
LiCl
M
MgS04
min
ml
mM
mRNA
NaCl
NaOAc
NaOH
NCBl
n g
OD
PCR
hydrochloric acid
kilo base pairs
liter
Luria-bertani
lithium chloride
molar
magnesium sulfate
minute
milliliter
millimolar
messenger RNA
sodium chloride
sodium acetate
sodium hydroxide
National Center for Biotechnology Information
nanogram
optical density
polymerase chain reaction
~ f u plaque forming units
RNA ribonucleic acid
RNase A ribonuclease A
rPm revolution per minute
SDS sodium dodecyl sulphate
volume per volume
weight per volume
CHAPTER 1
INTRODUCTION
Seaweeds are macroalgae in the marine habitat. They can be used as food,
sources for cosmetics, pharmaceuticals, soap, agar and textile. There are
mainly three groups of seaweed: Phaeophyta (brown algae), Rhodophyta
(red algae) and Chlorophyta (green algae) (Graham and Wilcox 2000), and
each of them has its own important commercial values. The brown
seaweeds are producer of alginate (Ertesvag and Valla 1998). Alginate is
widely used in industry due to its viscosity and water binding capacity, and
its ability to form therrnostable gels with divalent cations (Ertesvag et al.
1998). Sargassum binderi is one of the most abundant brown seaweeds in
Malaysia. Previous studies have shown that S. binderi contains high quality
of alginate compared to commercially used seaweeds such as Laminaria
hyberborea, Macrocystis pyrifera and Ascophyllum nodosum.
Therefore, it will be interesting to understand the biological functions of S.
binderi through functional genomic studies. In this study, expressed
sequence tag (EST) approach is chosen for the initial molecular studies on
S. binden, as very little genomic information is available. ESTs are partial
sequences of cDNAs that can be used to characterize gene expression in
organisms or tissues. These sequences or tags have been proven useful in
many applications, such as recovery of full-length cDNA or genomic clones,
discovery of novel genes, recognition of exons, delineation of protein
families, development of genetic maps, identification of organism- or tissue-
specific genes, and investigation of unknown function (Lluisma and Ragan
1997). This study presents the first attempt in generating ESTs from S.
binderi.
However, extraction of nucleic acids from seaweeds has always been
problematic. During the nucleic acid extraction process, secondary
metabolites and polysaccharides are always released after the disruption of
cell (Kim et a/. 1997). Besides, well established RNA extraction methods for
higher plants may not work well in seaweeds, in which the seaweed
polysaccharides may have different properties compared to higher plants.
Therefore, optimization of RNA extraction from S. binderi is a necessary
procedure in order to obtain high yield of RNA with minimum contamination.
The objectives of this study are to develop a RNA extraction method for S.
binderi to obtain high yield of pure RNA, to construct a cDNA library from S.
binderi, and to generate and analyze 1,000 ESTs from this seaweed for
further functional genomic studies.
CHAPTER 2
LITERATURE REVIEW
Seaweed
Seaweeds are macroalgae or macroscopic members of the divisions
Chlorophyta, Phaeophyta, and Rhodophyta living in the sea. They are plants
visible to the naked eye, generally growing attached to solid substrate
between and below the tide marks and remain stationary throughout life
(Chapman 1979; Dawson 1956). Seaweeds rarely grow in the free floating
state. However, there are sizable quantities of the brown alga Sargassum
living in the free floating state in the Sargasso Sea northeast of the
Caribbean and in the Gulf of Thailand (Dawson 1956).
The brown and red varieties are the more important seaweeds for
commercial purposes. Seaweeds are phytosynthetic and able to
manufacture sugar from water and carbon dioxide. Seaweeds contain
chlorophyll, but this is clearly evident only in the green seaweeds. The colour
may vary considerably according to environmental factors, such as the
supply of nutrients, intensity of light, and time of the year. Red seaweeds
may sometimes appear yellow, green or purple, and brown seaweeds may
appear yellow, orange or greenish-black (Dawson 1956).
2.1 . I Classification
Although seaweeds are divided into red, brown and green seaweeds, they
can not be differentiated by their colours. The classification is dependent on
the presence of pigments, plastid organization and the properties of their cell
wall. Table 2.1 shows the significant characteristics of seaweed in different
divisions.
2.1.2 Morphology
Seaweeds are very plantlike in appearance, having root, stem, and leaf
analogs in the form of anchoring holdfast, stipes, and blades (Linda and Lee
2000). A multicellular seaweed plant body is usually called thallus (Chapman
1979). A thallus may be a simple filament, a wide, broad sheet, a frond with
flat or cylindrical divisions, or a clumped or close crust on rock, stone, shell
or debris. Reproduction is by single cells produced over wide areas of the
surface, in restricted patches, or on special branches (Alan 1977). The
reproduction systems of seaweed may include vegetative propagation,
asexual reproduction and sexual reproduction (Chapman 1979).
Table 2.1. Summary of the three macroalgae divisions and their significant characteristics (adapted from Harold and Michael 1978; David 2002)
Division Phaeophyta Rhodophyta C hlorophyta
Common Brown algae Red algae Green algae name
Pigments and Chlorophyll a, c; p- Chlorophyll a, (d Chlorophyll a, b; plastid carotene, fucoxanthin in some a-, p-, and y- organization and several other florideophycidae); carotenes, in xanthophylls; 2-6 R- and C- several photosynthetic thylakoidslstack. phycocyanin, xanthophylls; 2-5 species allophycocyanin; thylakoidslstack.
R- and B- phycoertthrin. a-, p-carotene, several xanthophylls; thylakoids single, not associated.
Stored food Laminaran @-I ,3- glucopyranoside, predominantly); mannitol.
Cell wall
Examples
Floridean starch Starch (amylase (glycogen-like) and amylopectin)
(oil in some).
Cellulose, alginic Cellulose, xylans, Cellulose in many acid, and sulfated several sulfated @-I ,4- mucopolysaccharides polysaccharides glucopyranoside), (fucoidan). (galactans) hydroxyproline
calcification in glycosides; some. xylans and
mannans; or wall absent; calcified in some.
Fucus, Laminaria. Sargassum
Gracilaria, Porphyra , Chondms
Enteromorpha, Ulva, Codium
2.1.3 The importance of seaweed
Seaweeds have been harvested for many centuries for numerous uses.
Since the time of Romans it has been used as a fertilizer, especially by
farming communities living close to sea (Alan 1977).
At least 107 genera and 493 species of seaweeds have been recorded as
being economically utilized worldwide (Tseng 1981). Three genera and three
species of Chlorophyta, 10 genera and 13 species of Phaeophyta and four
genera and six species of Rhodophyta are commercially cultivated in the
Asia-Pacific region (Trono 1986). Seaweeds are mainly harvested for the
phycocolloids (agar, carragreenan and alginic acid) (Phang 1984). The
worldwide production of phycocolloids (first value is dry tonnage of raw
material with the weight of phycocolloid in bracket): alginate 500,000
(18,000), carrageenan 200,000 (15,000), agar 180,000 (7,000) (Alan and
Masao 1993). In Japan, the overall production of alginate is about 1,000-
1,500 tons per year in 1994 (Subhuti 2002).
Agar is imported and marketed in Malaysia in four main forms: agar strips,
bacteriological agar (powder), agar desserts (jellies) and flavoured powder
mixes. Malaysia imported 172 tonnes of agar strips worth RM 6.55 millions in
1988, suggesting that a large domestic market exists to support the
production and processing of seaweed in the country. However, competition
from other countries in the region may be a serious constraint for the
development of Malaysian seaweed processing and agar production (Jahara
and Phang 1989).
2.2 Phaeophyta
The phaeophyta (brown algae) is the most complex forms found among the
algae. There are a lot of differences compared to other algae (Table I ) . The
plant body of phaeophyta ranges from a millimeter in size or so to about 70
meters in length. They may be small, branched, attached, in filamentous
forms, or have large plant bodies with certain portion similar to those found
in higher plants. They are found most often firmly attached to various
substrates, often with elaborate holdfast systems. In addition to these
structures, which resemble roots, some forms have stem-like and leaf-like
appendages. However, they are lack of vascular tissues of higher plants
(Trainor 1978).
The colour of the brown algae (although colours may in fact vary from dark
brown or golden brown to olive green), is due to an accessory carotenoid
pigment, fucoxanthin, which masks the other pigments (Table 1) (Boney
1966). All the brown algae, with the exception of the Fucales, have an
alternation of sporophyte and gametophyte generations (Dawson 1956). The
cell wall matrix of marine algae typically consists of acidic polysaccharides.
In the Phaeophyta, the major polysaccharide in the cell wall matrix is
alginate (Hagen and Larsen 1997).
2.2.1 Sargassum
Genus Sargassum is under the division of Phaeophyta, order Fucales, and
family Sargassaceae (Silva 1962). Sargassum is a large genus with more
than 150 species described, occurring in tropical, subtropical, and temperate
zones of both hemispheres (Harold and Michael 1978). They are commonly
called gulf weed and mostly are from tropical countries (Trainor 1978). S.
binder; grow near to the seashore, especially in Malaysia, Philippines, India,
and Australia (Misra 1966).
The Sargassum plant ( Figure 2.1) is a flat, expanded sheet of cellular tissue
(the thallus) that narrows towards the base, so forming the "stipe" or stalk,
which is attached to the plant's habitat by the disc-shaped "holdfast". The
thallus cells are in crowded rows with thick jelly-like cell walls, which give the
plant flexibility and a degree of protection against the violent wave action.
The plant is slippery, helping it to remain moist when uncovered and to
prevent different specimens from chafing against and damaging each other
when being swirled around by the incoming sea. The sliminess also helps
the plant to maintain its elasticity, so that it can stretch and withstand the
sucking and pulling of the sea movements and it is through exuding slime
from its surface that it rids itself of calcium carbonate.
The thallus and its branches each have a conspicuous mid rib, on either side
which are numerous oval-shaped air-filled bladders. Their purpose is to help
buoy the plant when it is submerged, raising its extremities towards the
water-surface and the light.