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.