COMPARATIVE LEAF ANATOMY OF TETRASTIGMA
COMPARATIVE LEAF ANATOMY OF TETRASTIGMA SPECIES (VITACEAE) FROM
GUNUNG MULU NATIONAL PARK, SARAWAK
NUR AISHAH BINTI AMIR (42999)
Bachelor of Science in Honor
(Plant Resources Science and Management)
2016
Comparative leaf anatomy of Tetrastigma species (Vitaceae) from Gunung Mulu
National Park, Sarawak
Nur Aishah binti Amir
This dissertation is submitted in partial fulfilment of requirements for the Degree Bachelor
of Science with Honours in Plant Resource Science and Management
Department of Plant Science and Environmental Ecology
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
2016
Approval sheet
Name of candidate: Nur Aishah binti Amir
Title of thesis: Comparative leaf anatomy of Tetrastigma species (Vitaceae) from Gunung
Mulu National Park, Sarawak
_____________________________________
(Dr. Aida Shafreena binti Ahmad Puad)
Supervisor
Plant Resource Science and Management
Faculty of Resource Science and Management
Universiti Malaysia Sarawak
Date:
_____________________________________
(Dr. Freddy Yeo Kuok San)
Programme coordinator
Plant Resource Science and Management
Faculty of Resource Science and Management
Universiti Malaysia Sarawak
Date:
I
DECLARATION
No portion of the work referred to in this report has been submitted in support of an
application for another degree of qualification of this or any other university or institution of
higher learning.
_______________________________
Nur Aishah binti Amir
Plant Resources Science and Management Programmme
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
Date:
II
ACKNOWLEDGEMENTS
Alhamdulillah, thanks to Allah SWT because I have managed to complete this thesis although
had through many ups and downs in this project until the end.
First and foremost I would like to express my gratitude to my research supervisor, Dr. Aida
Shafreena binti Ahmad Puad for her kind guidance and dedication to me in completing this
project. Thanks also to postgraduate students, Wan Nur Fatiha bt Wan Zakaria and Siti Noor
Aishah for helping me to get the project samples and shared knowledges.
To my mother, this thesis I have dedicated specially for you. Thank you for your endless moral
and financial support. Without you, I might not overcome this hardship and made me who I
am right now. Many thanks also to my siblings for their help and encouragement in my study.
Lastly, many thanks to my fellow systematic friends Zarifah bt. Zainal Abidin, Janice Manyie,
and Noor Hazwan Fahmy that helped me in labwork. Special gratitudes also to Azra Hamzi b.
Hasani, Vievianna anak Charlie, Nurul Fahana bt. Abidin, Clora anak Jilan and other
coursemates for your supportive help thorughout this research.
Thank You.
III
TABLE OF CONTENTS
Approval Sheet…………………………………………………………...………
I
Declaration………………………………………………………………...……..
II
Acknowledgement………………………………………………………...……..
III
Table of Contents…………………………………………………………..…….
IV
List of Abbreviations……………………………………………………......……
VI
List of Tables……………………………………………………………......…...
VII
List of Figures………………………………………………………………..…..
VIII
Abstract………………………………………………………………………..…
IX
1.0: Introduction
1.1: Background………………………………………………………...
1
1.2: Problem statement and objective…………………………………..
2
2.0: Literature Review
2.1: Vitaceae Juss……………………………………………………….. 3
2.2: Tetrastigma (Miq.) Planch…………………………………………. 4
2.3: Leaf anatomical variation………………………………………….. 5
2.4: Gunung Mulu National Park...........................................................
6
3.0: Materials and methods
3.1: Specimen preparation leaf blade and petiole...................................
7
3.2: Sectioning and tissue clearing.........................................................
7
3.3: Double staining…………………………………………………….
7
3.4: Dehydration, mounting and drying………………………………… 9
3.5: Data analysis……………………………………………………….. 9
IV
4.0: Result and discussion
4.1: Leaf anatomical descriptions of Tetrastigma species……………… 10
4.1.1: Tetrastigma pedunculare (Wall) Planch…………………...
11
4.1.2: Tetrastigma rafflesiae (Miq.) Planch……………………… 13
4.1.3: Tetrastigma dichotomum (Blume) Planch………………… 15
4.1.4: Tetrastigma diepenhorstii (Miq.) Latiff…………………...
17
4.1.5: Tetrastigma megacarpum Latiff…………………………… 19
4.2: Leaf midrib…………………………………………………………. 21
4.3: Leaf lamina…………………………………………………………. 21
4.4: Leaf margin………………………………………………………… 24
4.5: Taxonomic implication…………………………………………….. 27
Conclusion and Recommendation……………………………………………….. 29
References………………………………………………………………………... 30
V
LIST OF ABBREVIATION
% = Percentage
µm = micrometre
o
C = Temperature (degree Celcius)
cm3= cubic centimetre
CO2 = Carbon dioxide
ha = hectare
km = kilometre
mm = Millimetre
NP = National park
sq = square
TS = transverse section
UNESCO = United Nations Educational, Scientific and Cultural Organisations
VI
LIST OF TABLES
Page
Table 1
Specimen collected from Gunung Mulu
NP
8
Table 2
Anatomical characteristic of midrib
between Tetrastigma species
22
Table 3
Anatomical features of lamina and leaf
margin between Tetrastigma species
25
VII
LIST OF FIGURES
Page
Figure 1
Presence of vascular bundles in the midrib of T.
pedunculare (medullary and abaxial part)
10
Figure 2
Presence of druses in the adaxial side of the
midrib of T. pedunculare
10
Figure 3
TS of midrib of T. pedunculare
12
Figure 4
TS of lamina of T. pedunculare
12
Figure 5
TS of leaf margin of T. pedunculare
12
Figure 6
TS of midrib of T. rafflesiae
14
Figure 7
TS of lamina of T. rafflesiae
14
Figure 8
TS of midrib of T. rafflesiae
14
Figure 9
TS of midrib of T. dichotomum
16
Figure 10
TS of midrib of T. dichotomum
16
Figure 11
TS of midrib of T. dichotomum
16
Figure 12
TS of midrib of T. diepenhorstii
18
Figure 13
TS of midrib of T. diepenhorstii
18
Figure 14
TS of midrib of T. diepenhorstii
18
Figure 15
TS of midrib of T. megacarpum
20
Figure 16
TS of midrib of T. megacarpum
20
Figure 17
TS of midrib of T. megacarpum
20
Figure 18
Midrib for Tetrastigma species studied under
light microscopy
23
Figure 19
Leaf margin for Tetrastigma species studied
under light microscopy
26
VIII
Comparative leaf anatomy of Tetrastigma species (Vitaceae) from Gunung Mulu National
Park, Sarawak
Nur Aishah binti Amir
Plant Science and Management Resource Programme
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
Tetrastigma is a genus from the Vitaceae family that consist about 122 species worldwide. This species can be
found in subtropical regions of South East Asia and Australia. Study on morphology of Tetrastigma has been
done extensively. However, anatomical study on this genus is still limited. Therefore, the objective of this study
was to investigate the anatomical variations in leaves of Tetrastigma species from Gunung Mulu National Park in
order to see if anatomical characteristics from leaves can be used to identify species. In this study, the specimens
collected from Gunung Mulu National Park, Miri were used to observed anatomical characteristics of the midrib,
lamina, and leaf margin. Sliding microtome was used in leaf sectioning and double-stained in Safranin and Alcian
green. Then, the dehydration was done in an alcohol series. The section mounted in Euparal before left the
section to dry in the oven at 60ºC. The sections were observed under the light microscope and images captured
using a digital camera fitted on a microscope and processed using Motic Image Plus 2.0 software and saved as
JPEG file. The anatomical characteristics that have been observed in this study are vascular bundles, outline of
midrib as well as the presence of the druses and trichomes in theTetrastigma species.
Keywords: Gunung Mulu National Park, leaf anatomy, species identification, Tetrastigma, Vitaceae
ABSTRAK
Tetrastigma adalah genus dari keluarga Vitaceae yang merangkupi 122 spesies dari seluruh dunia. Spesies ini
boleh dijumpai di kawasan subtropical Asia Tenggara dan Australia. Kajian morfologi Tetrastigma telah
dilakukan secara meluas. Walaubagaimanapun, kajian variasi anatomi untuk genus ini agak terhad. Oleh itu,
objektif kajian ini adalah untuk mengkaji variasi anatomi daun Tetrastigma spesies dari Taman Negara Gunung
Mulu bagi melihat sama ada ciri-ciri anatomi ini dapat menyumbang kepada pengenalpastian spesies. Dalam
kajian ini, spesimen telah diambil dari Taman Negara Gunung Mulu, Miri bagi memerhati ciri-ciri anatomi di
bahagian tulang daun, lamina dan tulang tepu. Mesin mikrotom gelungsur telah digunakan untuk memotong
bahagian daun manakal pewarnaan berganda menggunakan Safranin dan Alcian Hijau. Dehidrasi telah
dilakukan mengikut siri alcohol. Bahagian daun dilekap menggunakan Euparal sebelum dikeringkan dalam oven
pada 60ºC. Daun diperhatikan menggunakan mikroskop majmuk dan diproses melalui aplikasi Motic Image 2.0
Plus dan disimpan sebagai fail JPEG. Ciri-ciri anatomi yang telah diperhatikan dalam kajian ini adalah berkas
vascular, garis kasar tulang daun serta kehadiran druses dan trikom dalam species Tetrastigma.
Kata kunci: Taman Negara Gunung Mulu, anatomi daun, identifikasi spesies, Tetrastigma, Vitaceae
IX
1.0 INTRODUCTION
1.1 Background
Tetrastigma (Miq.) Planch is the largest genus in the family Vitaceae (grape family)
and consists of about 122 species (Latiff, 1984). The genus distributed in subtropical
region of South East Asia and Australia (Nais, 2001). Tetrastigma is recognised due to its
4-lobed stigma features in the pistillate flower and is classified under the section Vitis (Yeo
et al., 2012).
In this study, leaf samples of Tetrastigma were taken from Gunung Mulu National
Park. Gunung Mulu National Park, the largest national park in Sarawak is situated in Miri,
Sarawak. It is known as a UNESCO World Heritage Site due to its conservation value.
Gunung Mulu National Park covers an area of 544 sq. km gives a breathtaking scenery of
Mother Nature to be experienced and enjoyed. This park is a home to various species flora
and fauna and spectacular attractions including limestone pinnacles and magnificent caves
such as Deer Cave (Hazebroek & Abang Kashim, 2002).
Leaf anatomical variation has proven useful in taxonomy and systematic studies.
Leaves generally consists of two groups of marginal meristems which are the marginal that
give rise to the adaxial and abaxial epidermis of the leaf, while the submarginal generally
give rise to all internal tissue including procambium and mesophyll cells (Cutler et al.,
2008). Observation and descriptions that forms to study the bases of plant anatomical
require an understanding of cellular orientation terminology such as transverse, tangential
and radial in order to describe cell and tissue surfaces in sectioned tissues (Dickinson,
2000). Although the leaf is made up of the same tissue systems it has its own significant
variation that distinguished one species from another.
1
1.2 Problem statement and objective
Leaf anatomical study on Tetrastigma species in Sarawak is poorly studied.
Previous study by Najmaddin et al. (2013) only limited to a few species from Vitaceae of
which only one species studied was from Tetrastigma. The identification of Tetrastigma
species based on sterile materials alone may sometimes results in wrong identification.
Therefore, there is a need for the use of anatomical evidence in addition to leaf
morphological characteristics in the absence of reproductive material for species
identification. Hence, the objective of this study was to investigate the anatomical
differences in leaves of Tetrastigma species in order to see whether anatomical
characteristics in addition to morphological characteristics can be used to identify the
species.
2
2.0 LITERATURE REVIEW
2.1 Vitaceae Juss.
Vitaceae is known as a grape family which consist about 900 species from 15
genera which have the liana growth habit that produces profuse stem-derived adventitious
roots (Chen, 2009; Nikolov et al., 2014). Some of known genera in this family are Vitis,
Cayratia, Tetrastigma and Cissus. This family distributed mostly throughout tropical and
temperate areas in Asia and Australia.
The members of this family are woody climber with vines. The leaves are simple,
digitately to 1-3 pinnately compound, alternate or opposed arrangement, with multicellular,
caducous spherical structures known as “pearl glands” on leaf surface. The flowers are
small, 4-7 merous, and axillary in panicles. Fruits are berry and contain about 1-4 seeds.
Seeds are endoskeleton with dorsal chalazal characteristics that can be extended to
different genera (Zhiduan et al., 2007). Each of the genus in this family has its own distinct
characteristics that could distinguished from one genus to another. For example, genus
Tetrastigma can be identified by its 4-lobed stigma in pistillate flower, while Vitis is
identified by its united petals at the apex and shed as a cap-like calyptra. Other genera such
as Parthenocissus, Yua and Ampelopsis are identified as having 5-merous petals (Zhiduan
et al., 2007)
Vitaceae has its own importance economically and medicinally. Vitis is the most
economically important genus that produced mainly great wines in Europe region such as
Vitis vinifera and V. labrusca through fermentation process. In medicinal uses,
Parthenocissus leaf and roots can be used to treat ailments for human (Chen, 2009).
Another study on leaves extract of Tetrastigma leucastophylum (Densst.) done by Krishna
et al. (2014) stated that the investigation of leaf morphology showed its importance in
ethnomedicinal uses.
3
2.2 Tetrastigma Miq. Planch
Tetrastigma is known to be the largest genus in Vitaceae which comprised about
122 species (Latiff, 1984). This genus is mostly distributed in sub-tropic Asian and
expanding to Australia as well (Chen et al., 2011).
Due to its 4-lobed stigma characters in the pistillate flower, Tetrastigma gained its
genus name which derived from Latin word, tetra meaning four (Latiff, 1991). For the leaf
morphological features of Tetrastigma, they are commonly described as simple type, rarely
mixed with 2 or 3-foliate leaves (Ren & Wen, 2007). Tetrastigma species are climbers with
large vines growing by its tendrils. It has striate and terete stems with very prominent
lenticels. Its inflorescence is pedunculate with cymose structure in which peduncle length
differs in different sex. The plants are dioecious, and the fruits are pulpy berries type (Nais,
2001). Some species of Tetrastigma can be identified by the type of leaf. For example T.
scortechinii has simple leaves but other species such as T. cruciatum, T. dubium and T.
curtisii are predominantly trifoliate leaves (Latiff, 1984).
In the phylogenetic analysis, this genus is well-supported clade with close affinity
to Cayratia and Cysphostemma of which both Tetrastigma and Cysphostemma are
monophyletic while Cayratia is paraphyletic (Trias-Blasi et al., 2012).
Tetrastigma is known as the host plant to Rafflesia species, the biggest flower in the
world. According to Mat-Salleh (1991), it is remained unknown on how Rafflesia seeds
could germinate and grow inside the vine of Tetrastigma. The seeds of Rafflesia may
disperse to host through dispersal agent such as squirrel, ants, pigs, and elephants (Adam et
al., 2013). The seeds will germinate and forms new plant on its host. The characteristic of
Tetrastigma to reproduce vegetatively by lateral runners enables Rafflesia to spread by
'tagging-along' with the newly established host (Nais, 2001).
4
2.3 Leaf anatomical studies
Leaf is the primary organ of plant that holds an important role in the basic
physiological function in order for plant to live. Leaves are function to manufacture food
materials through photosynthesis process, transport of assimilated material and also
transpiration process (Cutler et al., 2008).
All cell composition in the leaf structure is initiated to develop and grow. Generally,
leaf anatomy consists of epidermis cell with stomata, mesophyll cell and vascular tissue
with their own specialized functions (Cutler et al., 2008). For example, stomata forms and
provide with specialized opening of guard cell for transpiration process, while bulliform
cells to function in rolling and unrolling of leaves due to the loss or uptake of water
(Dickison, 2000).
Leaf anatomical study also helps in species identification in order to compare with
other species in taxonomic classification. Although the leaf is made up of the same tissue
systems, the arrangements of leaf tissue in each species are different due to the physical
and environmental factor that contributes to the evolution of leaf structure in anatomy and
morphology such as water availability, light intensity, ecological niche and herbivores
(Cutler et al., 2008)
Previous study of comparative leaf anatomy of selected species in Vitaceae was
done by Najmaddin et al. (2013) showed that anatomical characteristics can be used to
discriminate species. Four selected species which are Tetrastigma cruciatum Craib &
Gagnep., Pterisanthes miquelii Planch., Cissus hastate Miq. and Nothonocissus spicifera
showed different anatomical features on transverse section in leaf petiole and midrib. In
transverse section of petiole, P. miquelii, C. hastata and N. spicifera showed subcircular
with concave adaxial surface, while T. cruciatum shows a rounded abaxial and rounded
lateral projection in abaxial surface. For midrib transverse section, P. miquelii shows
5
simple and unicellular glandular trichomes, while C. hastata has “V” shape on abaxial
surface and N. spicifera has “U” shape occasionally (Najmaddin et al., 2013).
2.4 Gunung Mulu National Park
Gunung Mulu National Park is located in Miri with the size of about 544 sq. km. Its
unique features with high biodiversity gained its recognition as UNESCO World Heritage
Place (Hazebroek & Abang Kashim, 2002).
Gunung Mulu, 2,376 m above sea level is the highest mountain in the park with
massive sandstone features. It also acts as the home to one of the longest network of caves
in the world. The Sarawak Chamber is the largest underground chamber in the world.. Deer
Cave is the biggest cave passage in the world and Clear Water Cave is the longest cave in
South East Asia and can be found in this chamber. (Hazebroek & Abang Kashim, 2002).
Gunung Mulu National Park covers an evergreen tropical forest that exhibit more
than 3500 species of floras and also being habitat for wildlife animals. This forest gives the
most luxuriant of all plant communities and probably has the greatest formation due to the
changes and continuation of forest types in specific zone and altitude. Alluvial forest,
dipterocarp forest, heath forest and forest over limestone are the types of forest found in
this park (Proctor et al., 1983). In dipterocarp forest, it occurs on the lower slopes up to
800 m where most of the big trees belonging to the family Dipterocarpaceae such as
“kapur bukit” (Dryobalanops beccarii). Heath forest or kerangas forest are mainly
composed of medium sized and small trees within even canopy and few emergents. Young
trees, palms specifically rattan and pitcher plants forms a moderately dense understorey.
Plant life in alluvial forest is strongly influenced by soil development where they occurred
on frequently flooded, recent river sediments such as Ficus species (Hazebroek & Abang
Kashim, 2002).
6
3.0 MATERIALS AND METHODS
3.1 Leaf sample
In this study, mature leaf samples obtained from Gunung Mulu National Park were
fixed in a mixture of alcohol and glacial acetic acid in a ratio of 3:1 (Table 1). Three
replicates were sectioned for each species studied. The leaves were sectioned transversely
through the middle region of midrib, lamina, and leaf margin.
3.2 Sectioning and tissue clearing
Leaf samples were washed by running it with tap water for 20 minutes to clean
from acetic acid and sectioned on the sliding microtome. The specimen held firmly in a
universal clamp with polystyrene supporter and the position of specimen was set up in
upright position. The microtome was set up to section about 20-30 μm thick or following
the suitability of the specimen and were cut into 15-20 sections of leaf tissues enough to
prepare for 3 slides for each section. The sections were transferred into a petri dish using a
wet brush. Tissue clearing was done in the fume cupboard. All tissues were bleached in
Clorox solution for 1-3 minutes before staining process.
3.3 Double staining
In this process, Safranin and Alcian green solution were used as staining agents.
Next, the sections were cleaned from Clorox solution by washed three times using distilled
water before staining. A few drops of Safranin solution dropped in the petri dish and left
for 10 minutes. After that, the solution were washed with distilled water then replaced with
a few drops of Alcian green solution and left it for another 5 minutes.
7
Table 1: Specimen collection from Gunung Mulu National Park, Miri
Species
T. dichotoum (Blume) Planch.
Collection number
Tetra56
Elevation
Along trail to Camp 3 (1261 m)
Collector's names
W.N.Fatihah, S.N.Aishah
T. dichotoum (Blume) Planch.
Tetra52
Camp 3 (1416 m)
W.N.Fatihah, S.N.Aishah
T. dichotoum (Blume) Planch.
Tetra48
Summit trail Camp 4 (2297 m)
W.N.Fatihah, S.N.Aishah
T. diepenhorstii (Miq.) Latiff
Tetra62
Along traiil to HQ (88 m)
W.N.Fatihah, S.N.Aishah
T. diepenhorstii (Miq.) Latiff
Tetra57
From Camp 3 to HQ (742 m)
W.N.Fatihah, S.N.Aishah
T. diepenhorstii (Miq.) Latiff
Tetra0020
Camp 1 (120 m)
W.N.Fatihah, S.N.Aishah
T. megacarpum Latiff
Tetra9
Camp 3 (Ca.1300 m)
W.N.Fatihah, S.N.Aishah
T. megacarpum Latiff
Tetra23
Kenyalang loop (26 m)
W.N.Fatihah, S.N.Aishah
T. megacarpum Latiff
Tetra44
Night-walk loop (79 m)
W.N.Fatihah, S.N.Aishah
T. pedunculare (Wall) Planch.
Tetra6
Along trail to HQ (22 m)
W.N.Fatihah, S.N.Aishah
T. pedunculare (Wall) Planch.
Tetra39
Botany loop (47 m)
W.N.Fatihah, S.N.Aishah
T. pedunculare (Wall) Planch.
Tetra63
Alongside road to Royal Mulu (67 m)
W.N.Fatihah, S.N.Aishah
T. rafflesiae (Miq.) Planch.
Tetra30
Kenyalang loop (46 m)
W.N.Fatihah, S.N.Aishah
T. rafflesiae (Miq.) Planch.
Tetra46
Night-walk loop (51 m)
W.N.Fatihah, S.N.Aishah
T. rafflesiae (Miq.) Planch.
Tetra59
Along trail to HQ (38 m)
W.N.Fatihah, S.N.Aishah
8
3.4 Dehydration, mounting and drying
Dehydration were done in series of ascending alcohol concentration of 50%, 75%
(with additional of HCl solution), 95% and 100% alcohol solution. This process was done
in a fume cupboard.
After dehydration process, mounting process was done by transferred sections from
petri dish on to slide by using a small and soft paintbrush that was wetted with 100%
alcohol. Each slide was consisting of 3-4 sections. A drop of Euparal was put on the section
and cover slip was put on the slide slowly. Bubbles were avoided in the sections by
pressing the cover slip slowly.
For drying process, the slide (with temporary labels) was dried in the oven with
temperature of 40o C to 60o C for about two weeks. Finally, the slide was labelled with
permanent label on the right side of the slide.
3.5 Data analysis
Lamina, leaf margin and midrib transversely sectioned were observed under the
light microscope. Photomicrographs of sections were taken using compound microscope
with a digital camera attached to it. Images were processed using Motic Image Plus 2.0
software
and
saved
as
9
JPEG
file.
4.0 RESULTS AND DISCUSSION
4.1 Leaf anatomical descriptions of Tetrastigma species
Leaf anatomical variations of the midrib, lamina and leaf margin of five Tetrastigma
species are shown in Figure 1-15. Figure 1-3 show the transverse sections of T.
pedunculare (Wall) Planch. while Figure 4-6 show the transverse sections of T. rafflesia
(Miq.) Planch. Figure 7-9 show the transverse section of T. dichotomum (Blume) Planch
and Figure 10-12 show the transverse section of T. diepenhorstii (Miq.) Planch while
Figure 13-15 shows the transverse section of T. megacarpum Latiff. Midrib of Tetrastigma
species in this study can be divided into two parts: medullary and abaxial side (Figure 1).
Presence of druses also can be observed in Tetrastigma species studied (Figure 2).
medullary
abaxial
Figure 1: Presence of vascular bundles in the midrib of T. pedunculare (medullary and
abaxial part)
Druses
Figure 2: Presence of druses in the adaxial side of the midrib of T. pedunculare
10
4.1.1 Tetrastigma pedunculare (Wall) Planch
Midrib: The outline for adaxial is convex while the outline for abaxial is wide “U” shape
with prominent and truncated bottom. Vascular bundle is discontinuous closed system with
“O” shape that consist of outer vascular bundles and medullary bundles. Outer vascular
bundle consists of discontinuous of several vascular bundle arranged in ¾ circle shape.
Medullary vascular bundle consists of three vascular bundle at the adaxial side. Secretory
canal, druses and collenchyma are present in the midrib (Figure 3).
Lamina and leaf margin: Tetrastigma pedunculare shows one layer of epidermis covered
with very thin cuticle on adaxial surface. Mesophyll palisade consist of two layers beneath
epidermal layer. Trichome presents on the abaxial surface. Druses are present and
randomly scattered in the lamina (Figure 4). The outline of leaf margin shows recurve
downward with rounded and tapering end (Figure 5).
11
co
sc
Figure 3: TS of midrib of T. pedunculare. Scale = 100 µm
ep
dr
mp
tr
Figure 4: TS of lamina of T. pedunculare. Scale = 100 µm
ct
Figure 5: TS of leaf margin of T. pedunculare. Scale = 100 µm
Note: co: collenchyma; sc: secretory canal; mp: mesophyll palisade; ct: cuticle; ep:
epidermis; dr: druses; tr: trichome
12
4.1.2 Tetrastigma rafflesiae (Miq.) Planch
Midrib: The outline for adaxial is convex while the abaxial outline shows wide “U” shape
with prominent and rounded convexity. Vascular bundle is closed system with “O” shape
that consists of outer vascular bundles and medullary bundles. Outer vascular bundle
consists of discontinouous of several vascular bundle arranged in ¾ circle shape.
Medullary vascular bundle consists of single vascular bundle at the adaxial side. Druses
and collenchyma are present (Figure 6).
Lamina and leaf margin: Tetrastigma rafflesia shows uniseriate, one layer of epidermis
covered with very thin and smooth cuticle on adaxial surface. Mesophyll palisade consist
of two layers. Collenchyma and druses are present (Figure 7). The outline of leaf margin
shows slightly recurved downwards with rounded end (Figure 8).
13
dr
co
Figure 6: TS of midrib of T. rafflesiae. Scale = 100 µm
mp
dr
ep
Figure 7: TS of lamina of T. rafflesiae. Scale = 100 µm
ct
ep
Figure 8: TS of leaf margin of T. rafflesiae. Scale = 100 µm
Note: co: collenchyma; mp: mesophyll palisade; ct: cuticle; ep: epidermis; dr: druses
14
4.1.3 Tetrastigma dichotomum (Blume) Planch
Midrib: The outline for adaxial is convex while the abaxial is “U” shape with prominent
and rounded convexity. Vascular pattern closed system consist of outer and medullary
vascular bundle. Outer vascular bundle consist of discontinuous of several vascular bundle
arranged in ¾ circle shape. Medullary vascular bundle consists of single vascular bundle at
the adaxial side. Druses are present in abundance (Figure 9).
Lamina and leaf margin: Tetrastigma dichotomum show a uniseriate, one layer of
epidermis covered with thin and smooth cuticle on adaxial surface. Mesophyll palisade
consist of two layers. Druses are also present on lamina section (Figure 10). The outline of
leaf margin is slightly bend downward with a tapered end (Figure 11).
15
co
sc
dr
Figure 9: TS of midrib of T. dichotomum. Scale = 100 µm
ct
mp
ep
dr
Figure 10: TS of lamina of T. dichotomum. Scale = 100 µm
ct
Figure 11: TS of leaf margin of T. dichotomum. Scale = 100 µm
Note: co: collenchyma; sc: secretory canal; mp: mesophyll palisade; ct: cuticle; ep:
epidermis; dr: druses
16
4.1.4 Tetrastigma diepenhorstii (Miq.) Latiff
Midrib: The outline for adaxial is convex while the abaxial outline shows “U” shape with
prominent and rounded convexity. Vascular bundle closed system consist of outer vascular
bundle and medullary bundles. Outer vascular bundle consists of discontinuous of several
vascular bundle arranged in ¾ circle shape. Medullary vascular bundle consists of single
vascular bundle at the adaxial side. Druses, collenchyma and secretory cells are present
(Figure 12).
Lamina and leaf margin: Tetrastigma diepenhorstii show a uniseriate, one layer of
epidermis covered with thin and smooth cuticle on adaxial surface. Mesophyll palisade
consist only one layer. Druses are arranged in the mesophyll palisade (Figure 13). The leaf
margin is straight and recurved downwards with rounded end (Figure 14).
17
co
dr
Figure 12: TS of midrib of T. diepenhorstii. Scale = 100 µm
ct
dr
ep
mp
Figure 13: TS of lamina of T. diepenhorstii. Scale = 100 µm
Figure 14: TS of leaf margin of T. diepenhorstii. Scale = 100 µm
Note: co: collenchyma; sc: secretory canal; mp: mesophyll palisade; ct: cuticle; ep:
epidermis; dr: druses
18
4.1.5 Tetrastigma megacarpum Latiff
Midrib: The outline for adaxial is convex while the abaxial outline shows “U” shape with
prominent and irregular convexity. Vascular bundle closed system consits of outer vascular
bundles and medullary bundles. Outer vascular bundle consits of discontinuous of few
vascular bundle arranged in ½ circle shape. Medullary vascular bundle consists of single
vascular bundle at the adaxial side. Druses are present (Figure 15).
Lamina and leaf margin: Tetrastigma megacarpum show a uniseriate, one layer of
epidermis covered with thin and smooth cuticle on adaxial surface. Mesophyll palisade
consist only one layer. Druses are present (Figure 16). The outline for leaf margin is
recurved downward with rounded end (Figure 17).
19
co
dr
Figure 15: TS of midrib of T. megacarpum. Scale = 100 µm
ct
ep
mp
dr
Figure 16: TS of lamina of T. megacarpum. Scale = 100 µm
ct
mp
ep
dr
Figure 17: TS of leaf margin of T. megacarpum. Scale = 100 µm
Note: co: collenchyma; mp: mesophyll palisade; ct: cuticle; ep: epidermis; dr: druses
20
4.2 Leaf midrib
The transverse section of the midrib of Tetrastigma species studied present several
characteristics and shapes as summarized in Table 2 and Figure 18. The outline of the
midrib of Tetrastigma varies among each species studied. Generally, Tetrastigma species
have convex shape on the adaxial side. However some Tetrastigma species have similar
outline that is having prominent and rounded convexity on the abaxial such as shown in
Tetrastigma rafflesiae (Fig.18b), T. dichotomum (Fig.18c), and T. diepenhorstii (Fig.18d).
Tetrastigma pedunculare has an arc shape on the abaxial (Fig.18a), and T. megacarpum is
prominent with an irregular projection on abaxial side (Fig.18e). Druses are found in
abundance only in T. dichotomum. In general, druses are randomly scattered in the midrib
in all Tetrastigma species studied.
4.3 Leaf lamina
All Tetrastigma species studied show a uniseriate epidermis covered by thin cuticle
on the adaxial surface (Fig.4, 7, 10, 13 and 16). Trichomes only presents on the abaxial
surface of T. pedunculare (Fig. 4).
The layers of mesophyll palisades varies in the Tetrastigma species studied.
Tetrastigma pedunculare (Fig.4), T. rafflesiae (Fig.7) and T. dichotomum (Fig.10) consist
two layers of mesophyll palisade, while both T. diepenhorstii (Fig.13) and T. megacarpum
(Fig.16) consist one layer of mesophyll palisade.
Druses are common features that found in all Tetrastigma species studied. In
general, druses are randomly scattered in the lamina for all Tetrastigma species observed.
However in T. diepenhorstii, druses are arranged only in the mesophyll palisade. The
lamina characteristics are summarized in Table 3.
21
Table 2: Anatomical characteristics of midrib between Tetrastigma species
T. pedunculare
T. rafflesiae
T. dichotomum
T. diepenhorstii
T. megacarpum
Midrib shape
Outline of midrib
Adaxial
Convex
Convex
Convex
Convex
Convex
Abaxial
Wide “U” - prominent
and truncate
Wide “U” - prominent
and rounded
“U” shape - prominent
and rounded
“U” shape - prominent
and rounded
“U” shape - prominent
and irregular
Midrib vascularization
pattern
Closed system,
outer vascular bundle is
¾ circle shape,
medullary consist of
three vascular bundle
Closed system, outer
vascular bundle is ¾
circle shape, medullary
consist of single
vascular bundle
Closed system, outer
vascular bundle is ¾
circle shape, medullary
consist of single
vascular bundle
Closed system, outer
vascular bundle is ¾
circle shape, medullary
consist of single
vascular bundle
Closed system, outer
vascular bundle is ½
circle shape, medullary
consist of single
vascular bundle
Presence of druses
Yes
Yes
Most abundant
Yes
Yes
22
(a)
(b)
(c)
(d)
(e)
Figure 18: TS of midrib for Tetrastigma species studied under light microscopy. (a)T.
pedunculare (b)T. rafflesiae (c)T. dichotomum (d)T. diepenhorstii (e)T. megacarpum. Scale:
a, b, c, d and e = 100 µm
23
4.4 Leaf margin
All Tetrastigma species studied have various shapes of margin (Fig.19). However,
it is not very distinctive. Leaf margin of T. pedunculare recurved downwards with rounded
and tapering end. (Fig.19a). Both T. rafflesiae (Fig.19b) and T. dichotomum (Fig.19c)
margin are straight and slightly bend downwards with rounded and tapering end. As for T.
diepenhorstii, it is straight with rounded end of margin (Fig.19d). Leaf margin of T.
megacarpum is recurved downwards with rounded end (Fig.19e). The features of leaf
margin of Tetrastigma species studied are summarized in Table 3.
24
Table 3: Anatomical features of lamina and leaf margin between Tetrastigma species
T. pedunculare
Presence of trichome
Present on abaxial
surface
Presence of druses
Randomly scattered
T. rafflesiae
T. dichotomum
T. diepenhorstii
T. megacarpum
-
-
-
-
Randomly scattered Randomly scattered
Arranged in the
mesophyll pelisade
Randomly scattered
No. layer of
epidermal cell
1
1
1
1
1
No. layer of
mesophyll pelisade
2
2
2
1
1
Shape of leaf margin
Recurve downwards
with rounded and
tapering end
Slightly recurved
downwards with
rounded end
Slightly bending
downwards with
tapering end
25
Straight then recurved Recurved downwards
downward with
with rounded end
rounded end
(a)
(b)
(c)
(d)
(e)
Figure 19: TS of leaf margin for Tetrastigma species studied under light microscopy. (a)T.
pedunculare (b)T. rafflesiae (c)T. dichotomum (d)T. diepenhorstii (e)T. megacarpum. Scale:
a, b, c, d and e = 100 µm
26
4.2 Taxonomic Implication
Morphological characteristics are very important for species identification.
However, the morphological characteristics are limited and fewer of them support analysis.
Therefore, anatomical characteristics can help in adding more characters for species
identification and analysis. Several studies by Najmaddin et al. (2011; 2013), Talip &
Cutler (2009) and Wosch et al. (2015) have proven anatomical characteristics as useful in
taxonomic purposes.
In the present study, not many diagnostic characteristics are observed. Transverse
section of the midrib does show some diagnostic characteristics especially on the outline of
the midrib and the vascular bundles as shown in Figure 17. From the previous study by
Najmadin et al. (2013), four species under Vitaceae (Tetrastigma cruciatum, Pterisanthes
miquelii, Cissus hastata and Nothocissus spicifera) showed good diagnostic characters by
possessed druses and rhapides. However in this study, rhapides are not observed. This
concludes that druses are common feature found in Vitaceae family as to compare both
previous and present study.
This study has shown the presence of trichomes can differentiate among species in
this species. As an example, the presence of trichomes only present in the leaf lamina of T.
pedunculare that can differentiate this species from other four Tetrastigma species that
have been observed. From previous study by Najmaddin et al., (2011), the presence of
trichome can be observed in the lamina of Vitis vinefera. It shows that the presence of
trichome is quite common in members of Vitaceae family.
27
Therefore, some important characteristics are observed in this study. Generally,
collenchyma and secretory cell are all present in the midrib in all Tetrastigma species
studied. The midrib vascularization pattern also showed similar feature in the medullary
vascular bundle except for T. pedunculare that consist of three vascular bundles. The
outline of the midrib of this study and previous study by Najmaddin et al. (2011) showed
similarity of an arc shape on the adaxial side.
28
5.0 CONCLUSION AND RECOMMENDATION
In conclusion, anatomical characteristics of Tetrastigma species in this study such
as outline of midrib, vascular bundle pattern and presence of trichome is proven to help in
species identification to a certain extent. The presence of druses is also a common
characteristic for Tetrastigma species.
A more comprehensive study on the leaf anatomy of Tetrastigma species is
suggested as there are limited sources that would provide conclusive evidence of good
anatomical characteristics. Skill level in sectioning should be improved in order to obtain a
good slide for observation to identify characteristics in this species.
29
REFERENCES
Adam, J.H., Mohamed, R., Juhari, M.A.A, Nik Ariff, N.N.F, & Wan, K.L. (2013). Rafflesia
sharifa-hapsahiae (Rafflesiaceae), a new species from Peninsular Malaysia.
Turkish Journal of Botany, 37, 1038-1044.
Chen, I. (2009). History of Vitaceae inferred from morphology based on phylogeny and the
fossil record of seeds. Published dissertation for the degree Doctor of Philosophy.
University of Florida Unites States of America.
Chen, P., Chen I., & Wen J. (2011). The first phylogenetic analysis of Tetrastigma (Miq.)
Planch, the host of Rafflesiaceae. Taxon, 60(2), 499-512.
Cutler, D.F., Botha, T., & Stevenson, D.W. (2008). Plant anatomy: An applied approach.
MA, USA: Blackwell Publishing.
Dickison, W. (2000). Integrative Plant Anatomy. CA, USA: Hardcourt Academic Press.
Hazebroek, H. P. & Abang Kashim b. Abang Morshidi. (2002). A Guide to Gunung Mulu
National Park. Sabah, Malaysia: Natural History Publications (Borneo) Sdn. Bhd.
Krishna, T.P.A., Ajeesh Krishna, T.P., Chithra, N.D., Deepa, P.E., Darsana, U., Sreelekha,
K.P., Juliet, S., Naish, S.N., Ravindran, R., Kumar, K.G.A., & Ghosh, S. (2014).
Acaricidal
activity of petroleum ether extract of leaves of Tetrastigma
leucostaphylum (densst.)
Alston against Rhipicephalus (Boophilus) annulatus.
The Scientific World Journal, 1-6.
Latiff, A. (1984). Studies in Malesian Vitaceae VII. The genus Tetrastigma in the Malay
Peninsula. Garden Bulletin Singapore, 36(2), 213-216.
Latiff, A. (1991). Studies in Malesian Vitaceae X. Two new species of Tetrastigma from
Borneo. Blumea, 35, 559-564.
Mat Salleh, K. (1991). Rafflesia magnificent flower of Sabah. Kota Kinabalu, Malaysia:
Borneo Publishing Company.
Nais, J. (2001). Rafflesia of the world. Sabah, Malaysia: Sabah Parks & National History
Publications (Borneo) Sdn. Bhd.
Najmaddin, C., Hussin, K. & Maideen, H. (2011). Comparative study on anatomy and
palynology of the three variety of Vitis vinefera varity (family Vitaceae). African
Journal of Biotechnology, 10(74), 16866-16874.
Najmaddin, C., Hussin, K. & Maideen, H. (2013). Comparative leaf anatomy of selected
species in Vitaceae and Leeaceae. American Journal of Applied Science, 10(4),
414-417.
Nikolov, L.A., Tomlinson, P.B., Manickam, S., Endress, P.K., Kramer, E.M. & Daris, C.C.
(2014). Holoparasitic Rafflesiaceae possess the most reduced endophytes and yet
give rise to the world's largest flowers. Annals of Botany, 114, 233-242.
30
Proctor, J., Anderson, J.M., Chai, P. & Vallack, H.W. (1983). Ecological studies in four
contrasting lowland rain forests in Gunung Mulu National Park, Sarawak: Forest
environment, structure and floristics. The Journal of Ecology, 7(1), 237-260.
Ren, H. & Wen. J. (2007). Tetrastigma. Flora of China, 12, 195-208.
Talip, N. & Cutler, D.F. (2009). Leaf anatomical and micromorphological characters of
Malaysian Parashorea (Dipterocarpaceae). Journal of Tropical Science, 21(2), 156157.
Trias-Blasi, A., Parnell, J.A.N. & Hodkinson, T.R. (2012). Multi-gene region phylogenetic
analysis of the grape family (Vitaceae). BioOne Biology, Ecology and
Environmental Science, 37(4), 941-950.
Wosch, L., Imig, D.C., Cervi, A.C., Moura, B.B., Budel, J.M., & Santos, C.A.M. (2015).
Comparative study of Passiflora taxa leaves: I. A morpho-anatomic profile. Revista
Brasileira de Farmacognosia, 25, 328-343.
Yeo, C.K., Ang, W.F., & Lok, A.F.S.L. (2012). Tetrastigma Planch. (Vitaceae) of
Singapore. Nature in Singapore, 5, 263-270.
Zhiduan, C., Ren, H. & Wen, J. (2007). Vitaceae. Flora of China, 12, 173-222.
31
GUNUNG MULU NATIONAL PARK, SARAWAK
NUR AISHAH BINTI AMIR (42999)
Bachelor of Science in Honor
(Plant Resources Science and Management)
2016
Comparative leaf anatomy of Tetrastigma species (Vitaceae) from Gunung Mulu
National Park, Sarawak
Nur Aishah binti Amir
This dissertation is submitted in partial fulfilment of requirements for the Degree Bachelor
of Science with Honours in Plant Resource Science and Management
Department of Plant Science and Environmental Ecology
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
2016
Approval sheet
Name of candidate: Nur Aishah binti Amir
Title of thesis: Comparative leaf anatomy of Tetrastigma species (Vitaceae) from Gunung
Mulu National Park, Sarawak
_____________________________________
(Dr. Aida Shafreena binti Ahmad Puad)
Supervisor
Plant Resource Science and Management
Faculty of Resource Science and Management
Universiti Malaysia Sarawak
Date:
_____________________________________
(Dr. Freddy Yeo Kuok San)
Programme coordinator
Plant Resource Science and Management
Faculty of Resource Science and Management
Universiti Malaysia Sarawak
Date:
I
DECLARATION
No portion of the work referred to in this report has been submitted in support of an
application for another degree of qualification of this or any other university or institution of
higher learning.
_______________________________
Nur Aishah binti Amir
Plant Resources Science and Management Programmme
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
Date:
II
ACKNOWLEDGEMENTS
Alhamdulillah, thanks to Allah SWT because I have managed to complete this thesis although
had through many ups and downs in this project until the end.
First and foremost I would like to express my gratitude to my research supervisor, Dr. Aida
Shafreena binti Ahmad Puad for her kind guidance and dedication to me in completing this
project. Thanks also to postgraduate students, Wan Nur Fatiha bt Wan Zakaria and Siti Noor
Aishah for helping me to get the project samples and shared knowledges.
To my mother, this thesis I have dedicated specially for you. Thank you for your endless moral
and financial support. Without you, I might not overcome this hardship and made me who I
am right now. Many thanks also to my siblings for their help and encouragement in my study.
Lastly, many thanks to my fellow systematic friends Zarifah bt. Zainal Abidin, Janice Manyie,
and Noor Hazwan Fahmy that helped me in labwork. Special gratitudes also to Azra Hamzi b.
Hasani, Vievianna anak Charlie, Nurul Fahana bt. Abidin, Clora anak Jilan and other
coursemates for your supportive help thorughout this research.
Thank You.
III
TABLE OF CONTENTS
Approval Sheet…………………………………………………………...………
I
Declaration………………………………………………………………...……..
II
Acknowledgement………………………………………………………...……..
III
Table of Contents…………………………………………………………..…….
IV
List of Abbreviations……………………………………………………......……
VI
List of Tables……………………………………………………………......…...
VII
List of Figures………………………………………………………………..…..
VIII
Abstract………………………………………………………………………..…
IX
1.0: Introduction
1.1: Background………………………………………………………...
1
1.2: Problem statement and objective…………………………………..
2
2.0: Literature Review
2.1: Vitaceae Juss……………………………………………………….. 3
2.2: Tetrastigma (Miq.) Planch…………………………………………. 4
2.3: Leaf anatomical variation………………………………………….. 5
2.4: Gunung Mulu National Park...........................................................
6
3.0: Materials and methods
3.1: Specimen preparation leaf blade and petiole...................................
7
3.2: Sectioning and tissue clearing.........................................................
7
3.3: Double staining…………………………………………………….
7
3.4: Dehydration, mounting and drying………………………………… 9
3.5: Data analysis……………………………………………………….. 9
IV
4.0: Result and discussion
4.1: Leaf anatomical descriptions of Tetrastigma species……………… 10
4.1.1: Tetrastigma pedunculare (Wall) Planch…………………...
11
4.1.2: Tetrastigma rafflesiae (Miq.) Planch……………………… 13
4.1.3: Tetrastigma dichotomum (Blume) Planch………………… 15
4.1.4: Tetrastigma diepenhorstii (Miq.) Latiff…………………...
17
4.1.5: Tetrastigma megacarpum Latiff…………………………… 19
4.2: Leaf midrib…………………………………………………………. 21
4.3: Leaf lamina…………………………………………………………. 21
4.4: Leaf margin………………………………………………………… 24
4.5: Taxonomic implication…………………………………………….. 27
Conclusion and Recommendation……………………………………………….. 29
References………………………………………………………………………... 30
V
LIST OF ABBREVIATION
% = Percentage
µm = micrometre
o
C = Temperature (degree Celcius)
cm3= cubic centimetre
CO2 = Carbon dioxide
ha = hectare
km = kilometre
mm = Millimetre
NP = National park
sq = square
TS = transverse section
UNESCO = United Nations Educational, Scientific and Cultural Organisations
VI
LIST OF TABLES
Page
Table 1
Specimen collected from Gunung Mulu
NP
8
Table 2
Anatomical characteristic of midrib
between Tetrastigma species
22
Table 3
Anatomical features of lamina and leaf
margin between Tetrastigma species
25
VII
LIST OF FIGURES
Page
Figure 1
Presence of vascular bundles in the midrib of T.
pedunculare (medullary and abaxial part)
10
Figure 2
Presence of druses in the adaxial side of the
midrib of T. pedunculare
10
Figure 3
TS of midrib of T. pedunculare
12
Figure 4
TS of lamina of T. pedunculare
12
Figure 5
TS of leaf margin of T. pedunculare
12
Figure 6
TS of midrib of T. rafflesiae
14
Figure 7
TS of lamina of T. rafflesiae
14
Figure 8
TS of midrib of T. rafflesiae
14
Figure 9
TS of midrib of T. dichotomum
16
Figure 10
TS of midrib of T. dichotomum
16
Figure 11
TS of midrib of T. dichotomum
16
Figure 12
TS of midrib of T. diepenhorstii
18
Figure 13
TS of midrib of T. diepenhorstii
18
Figure 14
TS of midrib of T. diepenhorstii
18
Figure 15
TS of midrib of T. megacarpum
20
Figure 16
TS of midrib of T. megacarpum
20
Figure 17
TS of midrib of T. megacarpum
20
Figure 18
Midrib for Tetrastigma species studied under
light microscopy
23
Figure 19
Leaf margin for Tetrastigma species studied
under light microscopy
26
VIII
Comparative leaf anatomy of Tetrastigma species (Vitaceae) from Gunung Mulu National
Park, Sarawak
Nur Aishah binti Amir
Plant Science and Management Resource Programme
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
Tetrastigma is a genus from the Vitaceae family that consist about 122 species worldwide. This species can be
found in subtropical regions of South East Asia and Australia. Study on morphology of Tetrastigma has been
done extensively. However, anatomical study on this genus is still limited. Therefore, the objective of this study
was to investigate the anatomical variations in leaves of Tetrastigma species from Gunung Mulu National Park in
order to see if anatomical characteristics from leaves can be used to identify species. In this study, the specimens
collected from Gunung Mulu National Park, Miri were used to observed anatomical characteristics of the midrib,
lamina, and leaf margin. Sliding microtome was used in leaf sectioning and double-stained in Safranin and Alcian
green. Then, the dehydration was done in an alcohol series. The section mounted in Euparal before left the
section to dry in the oven at 60ºC. The sections were observed under the light microscope and images captured
using a digital camera fitted on a microscope and processed using Motic Image Plus 2.0 software and saved as
JPEG file. The anatomical characteristics that have been observed in this study are vascular bundles, outline of
midrib as well as the presence of the druses and trichomes in theTetrastigma species.
Keywords: Gunung Mulu National Park, leaf anatomy, species identification, Tetrastigma, Vitaceae
ABSTRAK
Tetrastigma adalah genus dari keluarga Vitaceae yang merangkupi 122 spesies dari seluruh dunia. Spesies ini
boleh dijumpai di kawasan subtropical Asia Tenggara dan Australia. Kajian morfologi Tetrastigma telah
dilakukan secara meluas. Walaubagaimanapun, kajian variasi anatomi untuk genus ini agak terhad. Oleh itu,
objektif kajian ini adalah untuk mengkaji variasi anatomi daun Tetrastigma spesies dari Taman Negara Gunung
Mulu bagi melihat sama ada ciri-ciri anatomi ini dapat menyumbang kepada pengenalpastian spesies. Dalam
kajian ini, spesimen telah diambil dari Taman Negara Gunung Mulu, Miri bagi memerhati ciri-ciri anatomi di
bahagian tulang daun, lamina dan tulang tepu. Mesin mikrotom gelungsur telah digunakan untuk memotong
bahagian daun manakal pewarnaan berganda menggunakan Safranin dan Alcian Hijau. Dehidrasi telah
dilakukan mengikut siri alcohol. Bahagian daun dilekap menggunakan Euparal sebelum dikeringkan dalam oven
pada 60ºC. Daun diperhatikan menggunakan mikroskop majmuk dan diproses melalui aplikasi Motic Image 2.0
Plus dan disimpan sebagai fail JPEG. Ciri-ciri anatomi yang telah diperhatikan dalam kajian ini adalah berkas
vascular, garis kasar tulang daun serta kehadiran druses dan trikom dalam species Tetrastigma.
Kata kunci: Taman Negara Gunung Mulu, anatomi daun, identifikasi spesies, Tetrastigma, Vitaceae
IX
1.0 INTRODUCTION
1.1 Background
Tetrastigma (Miq.) Planch is the largest genus in the family Vitaceae (grape family)
and consists of about 122 species (Latiff, 1984). The genus distributed in subtropical
region of South East Asia and Australia (Nais, 2001). Tetrastigma is recognised due to its
4-lobed stigma features in the pistillate flower and is classified under the section Vitis (Yeo
et al., 2012).
In this study, leaf samples of Tetrastigma were taken from Gunung Mulu National
Park. Gunung Mulu National Park, the largest national park in Sarawak is situated in Miri,
Sarawak. It is known as a UNESCO World Heritage Site due to its conservation value.
Gunung Mulu National Park covers an area of 544 sq. km gives a breathtaking scenery of
Mother Nature to be experienced and enjoyed. This park is a home to various species flora
and fauna and spectacular attractions including limestone pinnacles and magnificent caves
such as Deer Cave (Hazebroek & Abang Kashim, 2002).
Leaf anatomical variation has proven useful in taxonomy and systematic studies.
Leaves generally consists of two groups of marginal meristems which are the marginal that
give rise to the adaxial and abaxial epidermis of the leaf, while the submarginal generally
give rise to all internal tissue including procambium and mesophyll cells (Cutler et al.,
2008). Observation and descriptions that forms to study the bases of plant anatomical
require an understanding of cellular orientation terminology such as transverse, tangential
and radial in order to describe cell and tissue surfaces in sectioned tissues (Dickinson,
2000). Although the leaf is made up of the same tissue systems it has its own significant
variation that distinguished one species from another.
1
1.2 Problem statement and objective
Leaf anatomical study on Tetrastigma species in Sarawak is poorly studied.
Previous study by Najmaddin et al. (2013) only limited to a few species from Vitaceae of
which only one species studied was from Tetrastigma. The identification of Tetrastigma
species based on sterile materials alone may sometimes results in wrong identification.
Therefore, there is a need for the use of anatomical evidence in addition to leaf
morphological characteristics in the absence of reproductive material for species
identification. Hence, the objective of this study was to investigate the anatomical
differences in leaves of Tetrastigma species in order to see whether anatomical
characteristics in addition to morphological characteristics can be used to identify the
species.
2
2.0 LITERATURE REVIEW
2.1 Vitaceae Juss.
Vitaceae is known as a grape family which consist about 900 species from 15
genera which have the liana growth habit that produces profuse stem-derived adventitious
roots (Chen, 2009; Nikolov et al., 2014). Some of known genera in this family are Vitis,
Cayratia, Tetrastigma and Cissus. This family distributed mostly throughout tropical and
temperate areas in Asia and Australia.
The members of this family are woody climber with vines. The leaves are simple,
digitately to 1-3 pinnately compound, alternate or opposed arrangement, with multicellular,
caducous spherical structures known as “pearl glands” on leaf surface. The flowers are
small, 4-7 merous, and axillary in panicles. Fruits are berry and contain about 1-4 seeds.
Seeds are endoskeleton with dorsal chalazal characteristics that can be extended to
different genera (Zhiduan et al., 2007). Each of the genus in this family has its own distinct
characteristics that could distinguished from one genus to another. For example, genus
Tetrastigma can be identified by its 4-lobed stigma in pistillate flower, while Vitis is
identified by its united petals at the apex and shed as a cap-like calyptra. Other genera such
as Parthenocissus, Yua and Ampelopsis are identified as having 5-merous petals (Zhiduan
et al., 2007)
Vitaceae has its own importance economically and medicinally. Vitis is the most
economically important genus that produced mainly great wines in Europe region such as
Vitis vinifera and V. labrusca through fermentation process. In medicinal uses,
Parthenocissus leaf and roots can be used to treat ailments for human (Chen, 2009).
Another study on leaves extract of Tetrastigma leucastophylum (Densst.) done by Krishna
et al. (2014) stated that the investigation of leaf morphology showed its importance in
ethnomedicinal uses.
3
2.2 Tetrastigma Miq. Planch
Tetrastigma is known to be the largest genus in Vitaceae which comprised about
122 species (Latiff, 1984). This genus is mostly distributed in sub-tropic Asian and
expanding to Australia as well (Chen et al., 2011).
Due to its 4-lobed stigma characters in the pistillate flower, Tetrastigma gained its
genus name which derived from Latin word, tetra meaning four (Latiff, 1991). For the leaf
morphological features of Tetrastigma, they are commonly described as simple type, rarely
mixed with 2 or 3-foliate leaves (Ren & Wen, 2007). Tetrastigma species are climbers with
large vines growing by its tendrils. It has striate and terete stems with very prominent
lenticels. Its inflorescence is pedunculate with cymose structure in which peduncle length
differs in different sex. The plants are dioecious, and the fruits are pulpy berries type (Nais,
2001). Some species of Tetrastigma can be identified by the type of leaf. For example T.
scortechinii has simple leaves but other species such as T. cruciatum, T. dubium and T.
curtisii are predominantly trifoliate leaves (Latiff, 1984).
In the phylogenetic analysis, this genus is well-supported clade with close affinity
to Cayratia and Cysphostemma of which both Tetrastigma and Cysphostemma are
monophyletic while Cayratia is paraphyletic (Trias-Blasi et al., 2012).
Tetrastigma is known as the host plant to Rafflesia species, the biggest flower in the
world. According to Mat-Salleh (1991), it is remained unknown on how Rafflesia seeds
could germinate and grow inside the vine of Tetrastigma. The seeds of Rafflesia may
disperse to host through dispersal agent such as squirrel, ants, pigs, and elephants (Adam et
al., 2013). The seeds will germinate and forms new plant on its host. The characteristic of
Tetrastigma to reproduce vegetatively by lateral runners enables Rafflesia to spread by
'tagging-along' with the newly established host (Nais, 2001).
4
2.3 Leaf anatomical studies
Leaf is the primary organ of plant that holds an important role in the basic
physiological function in order for plant to live. Leaves are function to manufacture food
materials through photosynthesis process, transport of assimilated material and also
transpiration process (Cutler et al., 2008).
All cell composition in the leaf structure is initiated to develop and grow. Generally,
leaf anatomy consists of epidermis cell with stomata, mesophyll cell and vascular tissue
with their own specialized functions (Cutler et al., 2008). For example, stomata forms and
provide with specialized opening of guard cell for transpiration process, while bulliform
cells to function in rolling and unrolling of leaves due to the loss or uptake of water
(Dickison, 2000).
Leaf anatomical study also helps in species identification in order to compare with
other species in taxonomic classification. Although the leaf is made up of the same tissue
systems, the arrangements of leaf tissue in each species are different due to the physical
and environmental factor that contributes to the evolution of leaf structure in anatomy and
morphology such as water availability, light intensity, ecological niche and herbivores
(Cutler et al., 2008)
Previous study of comparative leaf anatomy of selected species in Vitaceae was
done by Najmaddin et al. (2013) showed that anatomical characteristics can be used to
discriminate species. Four selected species which are Tetrastigma cruciatum Craib &
Gagnep., Pterisanthes miquelii Planch., Cissus hastate Miq. and Nothonocissus spicifera
showed different anatomical features on transverse section in leaf petiole and midrib. In
transverse section of petiole, P. miquelii, C. hastata and N. spicifera showed subcircular
with concave adaxial surface, while T. cruciatum shows a rounded abaxial and rounded
lateral projection in abaxial surface. For midrib transverse section, P. miquelii shows
5
simple and unicellular glandular trichomes, while C. hastata has “V” shape on abaxial
surface and N. spicifera has “U” shape occasionally (Najmaddin et al., 2013).
2.4 Gunung Mulu National Park
Gunung Mulu National Park is located in Miri with the size of about 544 sq. km. Its
unique features with high biodiversity gained its recognition as UNESCO World Heritage
Place (Hazebroek & Abang Kashim, 2002).
Gunung Mulu, 2,376 m above sea level is the highest mountain in the park with
massive sandstone features. It also acts as the home to one of the longest network of caves
in the world. The Sarawak Chamber is the largest underground chamber in the world.. Deer
Cave is the biggest cave passage in the world and Clear Water Cave is the longest cave in
South East Asia and can be found in this chamber. (Hazebroek & Abang Kashim, 2002).
Gunung Mulu National Park covers an evergreen tropical forest that exhibit more
than 3500 species of floras and also being habitat for wildlife animals. This forest gives the
most luxuriant of all plant communities and probably has the greatest formation due to the
changes and continuation of forest types in specific zone and altitude. Alluvial forest,
dipterocarp forest, heath forest and forest over limestone are the types of forest found in
this park (Proctor et al., 1983). In dipterocarp forest, it occurs on the lower slopes up to
800 m where most of the big trees belonging to the family Dipterocarpaceae such as
“kapur bukit” (Dryobalanops beccarii). Heath forest or kerangas forest are mainly
composed of medium sized and small trees within even canopy and few emergents. Young
trees, palms specifically rattan and pitcher plants forms a moderately dense understorey.
Plant life in alluvial forest is strongly influenced by soil development where they occurred
on frequently flooded, recent river sediments such as Ficus species (Hazebroek & Abang
Kashim, 2002).
6
3.0 MATERIALS AND METHODS
3.1 Leaf sample
In this study, mature leaf samples obtained from Gunung Mulu National Park were
fixed in a mixture of alcohol and glacial acetic acid in a ratio of 3:1 (Table 1). Three
replicates were sectioned for each species studied. The leaves were sectioned transversely
through the middle region of midrib, lamina, and leaf margin.
3.2 Sectioning and tissue clearing
Leaf samples were washed by running it with tap water for 20 minutes to clean
from acetic acid and sectioned on the sliding microtome. The specimen held firmly in a
universal clamp with polystyrene supporter and the position of specimen was set up in
upright position. The microtome was set up to section about 20-30 μm thick or following
the suitability of the specimen and were cut into 15-20 sections of leaf tissues enough to
prepare for 3 slides for each section. The sections were transferred into a petri dish using a
wet brush. Tissue clearing was done in the fume cupboard. All tissues were bleached in
Clorox solution for 1-3 minutes before staining process.
3.3 Double staining
In this process, Safranin and Alcian green solution were used as staining agents.
Next, the sections were cleaned from Clorox solution by washed three times using distilled
water before staining. A few drops of Safranin solution dropped in the petri dish and left
for 10 minutes. After that, the solution were washed with distilled water then replaced with
a few drops of Alcian green solution and left it for another 5 minutes.
7
Table 1: Specimen collection from Gunung Mulu National Park, Miri
Species
T. dichotoum (Blume) Planch.
Collection number
Tetra56
Elevation
Along trail to Camp 3 (1261 m)
Collector's names
W.N.Fatihah, S.N.Aishah
T. dichotoum (Blume) Planch.
Tetra52
Camp 3 (1416 m)
W.N.Fatihah, S.N.Aishah
T. dichotoum (Blume) Planch.
Tetra48
Summit trail Camp 4 (2297 m)
W.N.Fatihah, S.N.Aishah
T. diepenhorstii (Miq.) Latiff
Tetra62
Along traiil to HQ (88 m)
W.N.Fatihah, S.N.Aishah
T. diepenhorstii (Miq.) Latiff
Tetra57
From Camp 3 to HQ (742 m)
W.N.Fatihah, S.N.Aishah
T. diepenhorstii (Miq.) Latiff
Tetra0020
Camp 1 (120 m)
W.N.Fatihah, S.N.Aishah
T. megacarpum Latiff
Tetra9
Camp 3 (Ca.1300 m)
W.N.Fatihah, S.N.Aishah
T. megacarpum Latiff
Tetra23
Kenyalang loop (26 m)
W.N.Fatihah, S.N.Aishah
T. megacarpum Latiff
Tetra44
Night-walk loop (79 m)
W.N.Fatihah, S.N.Aishah
T. pedunculare (Wall) Planch.
Tetra6
Along trail to HQ (22 m)
W.N.Fatihah, S.N.Aishah
T. pedunculare (Wall) Planch.
Tetra39
Botany loop (47 m)
W.N.Fatihah, S.N.Aishah
T. pedunculare (Wall) Planch.
Tetra63
Alongside road to Royal Mulu (67 m)
W.N.Fatihah, S.N.Aishah
T. rafflesiae (Miq.) Planch.
Tetra30
Kenyalang loop (46 m)
W.N.Fatihah, S.N.Aishah
T. rafflesiae (Miq.) Planch.
Tetra46
Night-walk loop (51 m)
W.N.Fatihah, S.N.Aishah
T. rafflesiae (Miq.) Planch.
Tetra59
Along trail to HQ (38 m)
W.N.Fatihah, S.N.Aishah
8
3.4 Dehydration, mounting and drying
Dehydration were done in series of ascending alcohol concentration of 50%, 75%
(with additional of HCl solution), 95% and 100% alcohol solution. This process was done
in a fume cupboard.
After dehydration process, mounting process was done by transferred sections from
petri dish on to slide by using a small and soft paintbrush that was wetted with 100%
alcohol. Each slide was consisting of 3-4 sections. A drop of Euparal was put on the section
and cover slip was put on the slide slowly. Bubbles were avoided in the sections by
pressing the cover slip slowly.
For drying process, the slide (with temporary labels) was dried in the oven with
temperature of 40o C to 60o C for about two weeks. Finally, the slide was labelled with
permanent label on the right side of the slide.
3.5 Data analysis
Lamina, leaf margin and midrib transversely sectioned were observed under the
light microscope. Photomicrographs of sections were taken using compound microscope
with a digital camera attached to it. Images were processed using Motic Image Plus 2.0
software
and
saved
as
9
JPEG
file.
4.0 RESULTS AND DISCUSSION
4.1 Leaf anatomical descriptions of Tetrastigma species
Leaf anatomical variations of the midrib, lamina and leaf margin of five Tetrastigma
species are shown in Figure 1-15. Figure 1-3 show the transverse sections of T.
pedunculare (Wall) Planch. while Figure 4-6 show the transverse sections of T. rafflesia
(Miq.) Planch. Figure 7-9 show the transverse section of T. dichotomum (Blume) Planch
and Figure 10-12 show the transverse section of T. diepenhorstii (Miq.) Planch while
Figure 13-15 shows the transverse section of T. megacarpum Latiff. Midrib of Tetrastigma
species in this study can be divided into two parts: medullary and abaxial side (Figure 1).
Presence of druses also can be observed in Tetrastigma species studied (Figure 2).
medullary
abaxial
Figure 1: Presence of vascular bundles in the midrib of T. pedunculare (medullary and
abaxial part)
Druses
Figure 2: Presence of druses in the adaxial side of the midrib of T. pedunculare
10
4.1.1 Tetrastigma pedunculare (Wall) Planch
Midrib: The outline for adaxial is convex while the outline for abaxial is wide “U” shape
with prominent and truncated bottom. Vascular bundle is discontinuous closed system with
“O” shape that consist of outer vascular bundles and medullary bundles. Outer vascular
bundle consists of discontinuous of several vascular bundle arranged in ¾ circle shape.
Medullary vascular bundle consists of three vascular bundle at the adaxial side. Secretory
canal, druses and collenchyma are present in the midrib (Figure 3).
Lamina and leaf margin: Tetrastigma pedunculare shows one layer of epidermis covered
with very thin cuticle on adaxial surface. Mesophyll palisade consist of two layers beneath
epidermal layer. Trichome presents on the abaxial surface. Druses are present and
randomly scattered in the lamina (Figure 4). The outline of leaf margin shows recurve
downward with rounded and tapering end (Figure 5).
11
co
sc
Figure 3: TS of midrib of T. pedunculare. Scale = 100 µm
ep
dr
mp
tr
Figure 4: TS of lamina of T. pedunculare. Scale = 100 µm
ct
Figure 5: TS of leaf margin of T. pedunculare. Scale = 100 µm
Note: co: collenchyma; sc: secretory canal; mp: mesophyll palisade; ct: cuticle; ep:
epidermis; dr: druses; tr: trichome
12
4.1.2 Tetrastigma rafflesiae (Miq.) Planch
Midrib: The outline for adaxial is convex while the abaxial outline shows wide “U” shape
with prominent and rounded convexity. Vascular bundle is closed system with “O” shape
that consists of outer vascular bundles and medullary bundles. Outer vascular bundle
consists of discontinouous of several vascular bundle arranged in ¾ circle shape.
Medullary vascular bundle consists of single vascular bundle at the adaxial side. Druses
and collenchyma are present (Figure 6).
Lamina and leaf margin: Tetrastigma rafflesia shows uniseriate, one layer of epidermis
covered with very thin and smooth cuticle on adaxial surface. Mesophyll palisade consist
of two layers. Collenchyma and druses are present (Figure 7). The outline of leaf margin
shows slightly recurved downwards with rounded end (Figure 8).
13
dr
co
Figure 6: TS of midrib of T. rafflesiae. Scale = 100 µm
mp
dr
ep
Figure 7: TS of lamina of T. rafflesiae. Scale = 100 µm
ct
ep
Figure 8: TS of leaf margin of T. rafflesiae. Scale = 100 µm
Note: co: collenchyma; mp: mesophyll palisade; ct: cuticle; ep: epidermis; dr: druses
14
4.1.3 Tetrastigma dichotomum (Blume) Planch
Midrib: The outline for adaxial is convex while the abaxial is “U” shape with prominent
and rounded convexity. Vascular pattern closed system consist of outer and medullary
vascular bundle. Outer vascular bundle consist of discontinuous of several vascular bundle
arranged in ¾ circle shape. Medullary vascular bundle consists of single vascular bundle at
the adaxial side. Druses are present in abundance (Figure 9).
Lamina and leaf margin: Tetrastigma dichotomum show a uniseriate, one layer of
epidermis covered with thin and smooth cuticle on adaxial surface. Mesophyll palisade
consist of two layers. Druses are also present on lamina section (Figure 10). The outline of
leaf margin is slightly bend downward with a tapered end (Figure 11).
15
co
sc
dr
Figure 9: TS of midrib of T. dichotomum. Scale = 100 µm
ct
mp
ep
dr
Figure 10: TS of lamina of T. dichotomum. Scale = 100 µm
ct
Figure 11: TS of leaf margin of T. dichotomum. Scale = 100 µm
Note: co: collenchyma; sc: secretory canal; mp: mesophyll palisade; ct: cuticle; ep:
epidermis; dr: druses
16
4.1.4 Tetrastigma diepenhorstii (Miq.) Latiff
Midrib: The outline for adaxial is convex while the abaxial outline shows “U” shape with
prominent and rounded convexity. Vascular bundle closed system consist of outer vascular
bundle and medullary bundles. Outer vascular bundle consists of discontinuous of several
vascular bundle arranged in ¾ circle shape. Medullary vascular bundle consists of single
vascular bundle at the adaxial side. Druses, collenchyma and secretory cells are present
(Figure 12).
Lamina and leaf margin: Tetrastigma diepenhorstii show a uniseriate, one layer of
epidermis covered with thin and smooth cuticle on adaxial surface. Mesophyll palisade
consist only one layer. Druses are arranged in the mesophyll palisade (Figure 13). The leaf
margin is straight and recurved downwards with rounded end (Figure 14).
17
co
dr
Figure 12: TS of midrib of T. diepenhorstii. Scale = 100 µm
ct
dr
ep
mp
Figure 13: TS of lamina of T. diepenhorstii. Scale = 100 µm
Figure 14: TS of leaf margin of T. diepenhorstii. Scale = 100 µm
Note: co: collenchyma; sc: secretory canal; mp: mesophyll palisade; ct: cuticle; ep:
epidermis; dr: druses
18
4.1.5 Tetrastigma megacarpum Latiff
Midrib: The outline for adaxial is convex while the abaxial outline shows “U” shape with
prominent and irregular convexity. Vascular bundle closed system consits of outer vascular
bundles and medullary bundles. Outer vascular bundle consits of discontinuous of few
vascular bundle arranged in ½ circle shape. Medullary vascular bundle consists of single
vascular bundle at the adaxial side. Druses are present (Figure 15).
Lamina and leaf margin: Tetrastigma megacarpum show a uniseriate, one layer of
epidermis covered with thin and smooth cuticle on adaxial surface. Mesophyll palisade
consist only one layer. Druses are present (Figure 16). The outline for leaf margin is
recurved downward with rounded end (Figure 17).
19
co
dr
Figure 15: TS of midrib of T. megacarpum. Scale = 100 µm
ct
ep
mp
dr
Figure 16: TS of lamina of T. megacarpum. Scale = 100 µm
ct
mp
ep
dr
Figure 17: TS of leaf margin of T. megacarpum. Scale = 100 µm
Note: co: collenchyma; mp: mesophyll palisade; ct: cuticle; ep: epidermis; dr: druses
20
4.2 Leaf midrib
The transverse section of the midrib of Tetrastigma species studied present several
characteristics and shapes as summarized in Table 2 and Figure 18. The outline of the
midrib of Tetrastigma varies among each species studied. Generally, Tetrastigma species
have convex shape on the adaxial side. However some Tetrastigma species have similar
outline that is having prominent and rounded convexity on the abaxial such as shown in
Tetrastigma rafflesiae (Fig.18b), T. dichotomum (Fig.18c), and T. diepenhorstii (Fig.18d).
Tetrastigma pedunculare has an arc shape on the abaxial (Fig.18a), and T. megacarpum is
prominent with an irregular projection on abaxial side (Fig.18e). Druses are found in
abundance only in T. dichotomum. In general, druses are randomly scattered in the midrib
in all Tetrastigma species studied.
4.3 Leaf lamina
All Tetrastigma species studied show a uniseriate epidermis covered by thin cuticle
on the adaxial surface (Fig.4, 7, 10, 13 and 16). Trichomes only presents on the abaxial
surface of T. pedunculare (Fig. 4).
The layers of mesophyll palisades varies in the Tetrastigma species studied.
Tetrastigma pedunculare (Fig.4), T. rafflesiae (Fig.7) and T. dichotomum (Fig.10) consist
two layers of mesophyll palisade, while both T. diepenhorstii (Fig.13) and T. megacarpum
(Fig.16) consist one layer of mesophyll palisade.
Druses are common features that found in all Tetrastigma species studied. In
general, druses are randomly scattered in the lamina for all Tetrastigma species observed.
However in T. diepenhorstii, druses are arranged only in the mesophyll palisade. The
lamina characteristics are summarized in Table 3.
21
Table 2: Anatomical characteristics of midrib between Tetrastigma species
T. pedunculare
T. rafflesiae
T. dichotomum
T. diepenhorstii
T. megacarpum
Midrib shape
Outline of midrib
Adaxial
Convex
Convex
Convex
Convex
Convex
Abaxial
Wide “U” - prominent
and truncate
Wide “U” - prominent
and rounded
“U” shape - prominent
and rounded
“U” shape - prominent
and rounded
“U” shape - prominent
and irregular
Midrib vascularization
pattern
Closed system,
outer vascular bundle is
¾ circle shape,
medullary consist of
three vascular bundle
Closed system, outer
vascular bundle is ¾
circle shape, medullary
consist of single
vascular bundle
Closed system, outer
vascular bundle is ¾
circle shape, medullary
consist of single
vascular bundle
Closed system, outer
vascular bundle is ¾
circle shape, medullary
consist of single
vascular bundle
Closed system, outer
vascular bundle is ½
circle shape, medullary
consist of single
vascular bundle
Presence of druses
Yes
Yes
Most abundant
Yes
Yes
22
(a)
(b)
(c)
(d)
(e)
Figure 18: TS of midrib for Tetrastigma species studied under light microscopy. (a)T.
pedunculare (b)T. rafflesiae (c)T. dichotomum (d)T. diepenhorstii (e)T. megacarpum. Scale:
a, b, c, d and e = 100 µm
23
4.4 Leaf margin
All Tetrastigma species studied have various shapes of margin (Fig.19). However,
it is not very distinctive. Leaf margin of T. pedunculare recurved downwards with rounded
and tapering end. (Fig.19a). Both T. rafflesiae (Fig.19b) and T. dichotomum (Fig.19c)
margin are straight and slightly bend downwards with rounded and tapering end. As for T.
diepenhorstii, it is straight with rounded end of margin (Fig.19d). Leaf margin of T.
megacarpum is recurved downwards with rounded end (Fig.19e). The features of leaf
margin of Tetrastigma species studied are summarized in Table 3.
24
Table 3: Anatomical features of lamina and leaf margin between Tetrastigma species
T. pedunculare
Presence of trichome
Present on abaxial
surface
Presence of druses
Randomly scattered
T. rafflesiae
T. dichotomum
T. diepenhorstii
T. megacarpum
-
-
-
-
Randomly scattered Randomly scattered
Arranged in the
mesophyll pelisade
Randomly scattered
No. layer of
epidermal cell
1
1
1
1
1
No. layer of
mesophyll pelisade
2
2
2
1
1
Shape of leaf margin
Recurve downwards
with rounded and
tapering end
Slightly recurved
downwards with
rounded end
Slightly bending
downwards with
tapering end
25
Straight then recurved Recurved downwards
downward with
with rounded end
rounded end
(a)
(b)
(c)
(d)
(e)
Figure 19: TS of leaf margin for Tetrastigma species studied under light microscopy. (a)T.
pedunculare (b)T. rafflesiae (c)T. dichotomum (d)T. diepenhorstii (e)T. megacarpum. Scale:
a, b, c, d and e = 100 µm
26
4.2 Taxonomic Implication
Morphological characteristics are very important for species identification.
However, the morphological characteristics are limited and fewer of them support analysis.
Therefore, anatomical characteristics can help in adding more characters for species
identification and analysis. Several studies by Najmaddin et al. (2011; 2013), Talip &
Cutler (2009) and Wosch et al. (2015) have proven anatomical characteristics as useful in
taxonomic purposes.
In the present study, not many diagnostic characteristics are observed. Transverse
section of the midrib does show some diagnostic characteristics especially on the outline of
the midrib and the vascular bundles as shown in Figure 17. From the previous study by
Najmadin et al. (2013), four species under Vitaceae (Tetrastigma cruciatum, Pterisanthes
miquelii, Cissus hastata and Nothocissus spicifera) showed good diagnostic characters by
possessed druses and rhapides. However in this study, rhapides are not observed. This
concludes that druses are common feature found in Vitaceae family as to compare both
previous and present study.
This study has shown the presence of trichomes can differentiate among species in
this species. As an example, the presence of trichomes only present in the leaf lamina of T.
pedunculare that can differentiate this species from other four Tetrastigma species that
have been observed. From previous study by Najmaddin et al., (2011), the presence of
trichome can be observed in the lamina of Vitis vinefera. It shows that the presence of
trichome is quite common in members of Vitaceae family.
27
Therefore, some important characteristics are observed in this study. Generally,
collenchyma and secretory cell are all present in the midrib in all Tetrastigma species
studied. The midrib vascularization pattern also showed similar feature in the medullary
vascular bundle except for T. pedunculare that consist of three vascular bundles. The
outline of the midrib of this study and previous study by Najmaddin et al. (2011) showed
similarity of an arc shape on the adaxial side.
28
5.0 CONCLUSION AND RECOMMENDATION
In conclusion, anatomical characteristics of Tetrastigma species in this study such
as outline of midrib, vascular bundle pattern and presence of trichome is proven to help in
species identification to a certain extent. The presence of druses is also a common
characteristic for Tetrastigma species.
A more comprehensive study on the leaf anatomy of Tetrastigma species is
suggested as there are limited sources that would provide conclusive evidence of good
anatomical characteristics. Skill level in sectioning should be improved in order to obtain a
good slide for observation to identify characteristics in this species.
29
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