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UNIVERSITY OF CAPE COAST

SCHOOL OF BIOLOGICAL SCIENCES

EFFECT OF VEHICULAR EMISSIONS ON ALLAMANDA CATHARTICA

GYAN, ERNEST TAWIAH

A DISSERTATION PRESENTED TO THE ENVIRONMENTAL SCIENCE DEPARTMENT, UNIVERSITY OF CAPE COAST, IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF A DEGREE IN B.Sc. (Environmental Science)

JUNE, 2006

DECLARATION

I, Ernest Tawiah Gyan, hereby declare that the work herein presented in this dissertation, “Effect of vehicular emissions on allamanda cathartica”, is the result of my own research except otherwise stated. I further affirm that this work has never been submitted in part or whole in this university or elsewhere for the award of a degree.

_____________________ Date:________________

GYAN, ERNEST TAWIAH

(Student)

_____________________ Date:________________

PROF. H. K. AKOTOYE

(Supervisor and Head of Department)

DEDICATION

This work is dedicated unreservedly and with deep gratitude to my mother and family.

I greatly love you.

TABLE OF CONTENTS

CONTENTS PAGES

Declaration i

Dedication ii

Table of Contents iii

Acknowledgements iv

Abstract v

CHAPTER ONE

Introduction 1

CHAPTER TWO

Materials and Methods 11

CHAPTER THREE

Results 14

CHAPTER FOUR

Discussion and Conclusions 26

References 34

Appendix 1 37

Appendix 2 45

Appendix 3 48

ACKNOWLEDGEMENTS

I am most grateful to Prof. H. K. Akotoye for the fatherly supervision and guidance through the entire course of this project.

I am extremely grateful to my mother, Madam Georgina Sarkodie. How possibly could I have reached this far without your invaluable financial help, emotional assistance and moral support? Thanks. May the Almighty God richly bless you. And may He grant you long life, good health, peace and everlasting prosperity.

My Love, thanks, and prayers for my siblings Ophelia, Linda, Amanda and Maria. Thanks for your prayers and support. You understood me, even when I failed to understand myself.

My profound thanks go to Miss Margaret Sarpong-Nsiah ( Human Biology Department) for her motherly support. In the latter moments of my academic period, when the waves of the sea became strongest against me, you were always on my side. You are a true mother. Thanks Mum.

Thank you Prof. J. J. Fletcher for motivating me. You always listened to my ideas and have always been willing to assist me in any way possible.

Great thanks go to Mr. Emmanuel Agyei Heneku ( UCC Hospital) and family for their love, care and assistance.

I appreciate and thank Mr. T. K. Baidoo (Chief Technician, Dept. of Botany), Mr. Ankrah, Mr. Otoo (Herbarium, UCC), Mr. Agyarkwa (Herbarium, UCC), for their selfless technical assistance.

Last but not least, a few words of appreciation are also in order to my friends Alice Nutsugah, Emmanuel Agyekum, Boniface Aabeyir, Norbert Agana, Pastor Isaiah Jojo Morrison and all the people who supported me to make this work a success. Many thanks and God’s blessings to you all.

ABSTRACT

Allamanda cathartica is an ornamental plant used for various decorations. As a Hypostomatic shrub, it has both stomata and trichomes only on the lower surface of its leaves. Vehicular emissions have effect on it. Emissions from vehicles can cause its stomata to shrink and clog the stomatal pores. It has an abnormality of shrunken stomata. The stomatal frequency of Allamanda cathartica is directly proportional to vehicular emissions. The development and growth of its epidermal cells are initially impeded by low levels of vehicular emissions, but they later on respond with an increase in epidermal cell numbers in the presence of high pollution. Its stomatal size is directly proportional to pollution increases. The stomatal pore size and stomatal index of Allamanda cathartica are directly proportional to increases in pollution resulting from vehicular emissions.

CHAPTER ONE

INTRODUCTION

Vehicular emission mostly occur when a mixture of the lighter liquid hydrocarbons, called petrol, is chiefly used as a fuel for internal-combustion. The petrol is produced by the fractional distillation of petrol oil. Alkanes burn in a plentiful supply of air, or of oxygen alone, to produce carbon dioxide and water only. Methane is the major constituent of natural gas, and octane, C₈H₁₈, is a major component of petrol. This fractional distillation of petrol oil has a natural tendency to make petrol emit lead that is dangerous to both humans and plants. Nitric oxide is an atmospheric pollutant produced by motor cars and released in their exhaust fumes. It is one of the main gases, beside sulphur dioxide, that form acid rain to have adverse effect on soil, monuments, water bodies and plants. Where there are low wind speeds under an inversion, pollutants such as car exhaust fumes are not dispersed and high concentrations can develop-especially around urban centres with high number of vehicular activities (Nicholls, 2004).

Unlike the stratospheric ozone, which is natural, vehicular emission contribute immensely to the formation of ground-level ozone. Ground-level ozone is a dangerous pollutant in smog and is produced by the interaction of hydrocarbons and nitrogen oxides (resulting from domestic-, industrial- and vehicular emissions) under the influence of sunlight. Ozone causes serious crop damage. Often, the first noticeable effects of pollution from the exhaust of cars are aesthetic. These include sunlight and visibility reduction due to tiny particles suspended in air. In effect, photosynthetic plants may not obtain sufficient sunlight to produce carbohydrate. There are also claims that acid rain, resulting from vehicle exhaust, has caused widespread damage to forests in Europe. Thus, vehicle exhaust adversely affect the health of animals and plants and the chemical nature of the atmosphere (Ridge, 1991).

Various countries have set standards in legislation in the form of concentration levels that are believed to be low enough to protect humans and plants. However, the nature of the problem requires the implementation of internal environmental treaties, and to this end 49 countries agreed in March 1985 on a United Nations Convention to protect the ozone layer and damage to plants. This “Montreal Protocol”, which was renegotiated in 1990 and 1992, called for the phase-out of certain chlorocarbons and fluorocarbons by the end of the Century. In addition, several international protocols have been aimed specifically at reducing the incidence of acid rain that adversely affect the outer layers of plants and the nutrients they get from the soil. Vehicular emission cause Trans-boundary Air Pollution (TAP). Concern over trans-boundary air pollution, including acid rain, in Europe has led to the United Nations Economic Commission for Europe (UNECE) developing air quality guidelines, called Critical Loads and Levels, which represent thresholds below which it is believed that damage will not occur to different ecological systems. Critical Loads are based on the amount of acidity that an ecosystem can tolerate being deposited indefinitely. Critical Levels are concentrations of ozone, sulphur dioxide, and nitrogen oxides over different averaging times and applicable to different categories of vegetation. Massive cuts are being made in emission in Europe in order to move towards these thresholds. In contrast, in many developing countries, pollutant concentrations are rising very rapidly due to increased industrialization and motor traffic. Concern in such places is primarily with impacts on human health and plants in cities. The World Health Organization has published air quality guidelines designed to protect health and plant survival (Bell, 2004).

The effect of vehicular emission on plants needs to be ascertained due to plant benefits to humans. Plants are a seemingly endless source of benefits to humans. The over 250,000 species of angiosperms, in their roots, stems, leaves, and even flowers, provide food for almost all the animal world. Firstly, many of the flowering food plants are herbs, which are relatively small, soft-stemmed plants. Most of the herbs are annuals, but they are also found as biennials and perennials. Secondly, many medicines are derived from plants. For example, quinine comes from the bark of the cinchona; digitalis comes form the foxglove. Thirdly, although synthetics have made great in roads in the manufacture of fabrics, natural fibres such as flax (form the family Linaceae) and cotton are still commonly used for clothing, drapes, upholstery, etc. Fourthly, the decorative uses of plants range from vast landscaping designs, involving great tracts of trees, flowers, and shrubs, to a simple flower arrangement in a vase. Fifthly, wood is used in furniture making and construction, and use of wood as an alternative source of energy has seen a resurgence in recent decades in the United States in response to unstable oil prices. It is the only source of energy in many countries ( a situation that has led to widespread deforestation, which in turn has precipitated devastating erosion and flooding). Coal itself is a plant product-albeit from ancient trees and more primitive tracheophytes. Lastly, thousands of industrial chemicals are derived from the plant world- plastics, turpentine, tannin, and rubber, to name just a few. Although it is now generally produced synthetically, rubber was originally made from the latex of the rubber trees, as in the family of Apocynaceae (Fried, 1990).

Allamanda cathartica belong to the family of Apocynaceae. Most plants of the Apocynaceae family are sensitive to vehicular emissions, making them good indicators of the effect of vehicular emission on plants. Members of this family are mainly climbers or shrubs. A few however are trees and herbs. There is the presence of latex. The leaves are simple and are either opposite or whorl in arrangement. The flowers are bisexual, regular and hypogynous in cymose type of inflorescence. The flowers are usually funnel shaped, also referred to as Salverform. Although the floral parts are pentamerous, the carpels are bicarpilate. Aestivation of the corolla is contorted or twisted. The fruit type is a follicle or it may be a berry or a drupe. Members of this family are useful plants as they have both medicinal and socio-economic importance. Examples are Thevetia sp., Vinca sp., Plumeria sp., Alstonia sp., Allamanda cathartica, Allamanda schotti and Allamanda nerifolia (Anon, 2002; Metcalfe and Chalk, 1983).

Anything that affect the leaf epidermal structure or function of a plant will inevitably affect the normal functioning of that plant as a result of the unique anatomy and physiology of the epidermis. Hence, the effect of vehicular emission can be seen from an alteration in one or more of epidermal structures and functions, to which they affect.

Cells, the fundamental units of life, are associated in various ways with each other, forming coherent masses, or tissues. Moreover, the principal tissues of vascular plants are grouped together into larger units on the basis of their continuity throughout the plant body. These larger units, known as tissue systems, are readily recognized, often with the unaided eye. There are three tissue systems, and their presence in root, stem, and leaf reveals both the basic similarity of the plant organs and the continuity of the plant body. The three tissue systems are (1) the dermal tissue system, (2) the vascular tissue system, and (3) the ground (or fundamental) tissue system. The tissue systems are initiated during the development of the embryo, where their precursors are represented by the primary meristem- protoderm, procambium, and ground meristem, respectively.

The dermal tissue, of which we are concerned, is represented by the epidermis (Raven et al., 1992).

The epidermis constitutes the dermal tissue system of leaves, floral parts, fruits, and seeds and of stems and roots until they undergo considerable secondary growth (Mauseth, 1988).

The epidermal tissue system is derived from the dermatogen of the apical meristem and forms the epidermis (epi, upon; derma, skin) or outermost skin layer, which extends over the entire surface of the plant (Dutta, 2002).

While most cells are not continuous, epidermal cells form a continuous layer on the surface of the plant body in the primary state. They show various special characteristics related to their superficial position. The main mass of epidermal cells, the epidermal cells proper, vary in shape but are often tabular (Esau, 1977). For all, the epidermis is part of the leaf structure.

In large plants such as trees, leaves are the only structures which contain chloroplasts; the stem (or trunk) and branches serve only as a leaf support system (although young stems may be green). Leaves are therefore, in general, the chief photosynthetic organs (Ridge, 1991).

The epidermis of a plant serves various functions which vehicular emission may adversely alter: (1) The primary function of the epidermis is protection of the internal tissues against mechanical injury, excessive heat or cold, fluctuations of temperature, attacks of parasitic fungi and bacteria, and against the leaching effect of rain. This is possible due to the presence of cuticle, hairs, tannin, gum, etc. (2) Prevention of excessive evaporation of water from the internal tissues by the development of thick cuticles, wax and other deposition, cutinized hairs, scales, multiple epidermis, etc, is another important function of the epidermis. (3) Strong cuticles and cutinized hairs, particularly a dense coating of hairs, protect the plant against intense illumination and excessive radiation of heat. (4) The epidermis also has to protect the plant against attacks by herbivorous animals. This is done with the help of sharp and stiff hairs ( as in some Cucurbits), a dense coating of hairs (as in Gnaphalium) stinging hairs (as in Nettles), glandular hairs as in Boerhavia, silica particles (as in many grasses, e.g. lemon grass, Equisetum, etc) and raphides (as in many Aroids). (5) The epidermis also acts as a storehouse of water, as in desert plants. (6) The epidermis sometimes has some minor functions like photosynthesis, secretion, etc (Dutta, 2002). (7) A more positive, less defensive role of the epidermis is in reproduction, at least in angiosperms. And (8) secretion function, as the epidermis can be extremely active in it. It may contain cells (which by their absorption or loss of water can cause leaves to open when moisture is available and close when it is not.

Considering this diversity of function, it is somewhat surprising that there seem to be only four basic types of mature epidermal cells: Ordinary epidermal cells, guard cells, trichomes, and root hairs.

Ordinary epidermal cells are basically the cells that lie between the more specialized cells of the epidermis, and they are typically the most numerous and cover the greatest proportion of the plant body ( Montenegro, 1974). Tall or columnar epidermal cells are rare, being mentioned in only 18 families by Metcalfe and Chalk (1979), such as Chrysobalanaceae, Ericaceae, and Thymelaeaceae. An almost universal feature of ordinary epidermal cells is that they are firmly attached to each other along their sides and attached less firmly to the tissues beneath. Ordinary epidermal cells are usually unremarkably cytological. Chloroplasts are rare (Martin and Juniper, 1970), except in aquatic angiosperms, such as Myriophyllum, Ranunculus, Potamogeton, and Sagittaria (Linsbauer, 1930) and in ferns (Meyer, 1962; Wylie, 1948).

Smoke or fumes from vehicles exhaust can block a plant’s stomata to minimize photosynthetic activities of that plant. The epidermis contains holes (stomatal pores) whose size can be increased or decreased by the swelling of the adjacent guard cells. Stomata are found on virtually all green parts of a plant, especially the leaves and stems. On the leaves, they are typically more abundant on the abaxial surface, with the upper surface having fewer or even none. The daxial surfaces of leaves typically have about 100 stomata per mm², and in the leaves of many deciduous trees the density can be ten times as high (Ting, 1982). Densities as great as 2230 per mm² occur in Miconia pycnoneura (Howard, 1969), whereas in Macropanax, Schefflera, and Ozoroa the entire epidermis consist of virtually only guard cells and immediately neighbouring cells (Solereder, 1908). In the floating leaves of water lily (Nymphaea), abaxial stomata would be submerged and useless; only the adaxial surface has stomata. Similarly, the aquatic angiosperms, in which the entire plant is submerged and incapable of transpiration, have been reported by Solereder (1908) to lack stomata totally: Ceratophyllaceae, Nymphaeaceae, Podostemonaceae, and some Ranunculaceae. In Callitriche heterophylla, some leaves are submerged and have few stomata; other leaves are emergent and have numerous stomata (Deschamp and Cooke, 1985). Very low densities occur in certain cloud forest plants: 22 stomata per mm² in Peperomia emarginella (Howard, 1969). However, it is worth noting that Allamamda cathartica has stomata restricted only to the lower epidermis.

Two basic types of guard cells occur: dumbbell-shaped ones in the grass family and Cyperaceae (sedges), and crescent-shaped ones in the rest (Ziegler et al., 1974). The functional unit is, therefore, not just the guard cells but the stomatal complex: the guard cells together with the adjacent epidermal cells. If these adjacent cells are distinct in size, shape, or cell contents, then they are termed the subsidiary cells.

Stomata may either be found on both upper and lower epidermis or, may be restricted to only the lower or upper epidermis. If the stomata is present in both upper and lower epidermis, it is called Amphistomatic. On the other hand, if the stomata is present in only the lower epidermis, it is called Hypostomatic. And finally, if the stomata is found only on the upper surface, it is called Epistomatic.

The epidermal cell wall may either be sinously walled (i.e. thick walls), irregularly walled or straightly walled.

There are five most common types of stomatal complexes that are generally said to be the morphological type of stomata: anomocytic, paracytic, diacytic, actinocytic and anisocytic (Anon, 2002).

The anomocytic type includes those epidermises in which there are no obvious subsidiary cells. The guard cells appear to be embedded in ordinary epidermal cells. Thus, the stoma is surrounded by ordinary epidermal cells (Metcalfe and Chalk, 1983).

The paracytic type is easily recognizable: each guard cell is accompanied by one or more subsidiary cells that are aligned parallel with it. Thus, the stoma is surrounded by 2 subsidiary cells. These occur in the Convolvulaceae, Luguminosae, Magnoliaceae, and Rubiaceae, among others. This was formerly known as the rubiaceous type.

In the diacytic type (formerly the caryophyllaceous type), there are two large subsidiary cells that completely surround the guard cells and are aligned perpendicularly or at right angles to the long axis. These are common in the Acanthaceae, Carryophyllaceae, and others.

In the actinocytic type, the guard cells are surrounded by many subsidiary cells aligned radially around them.

In the anisocytic type (formerly Cruciferous type), the stomatal complexes can be somewhat difficult to recognize. They consist of three unequally sized subsidiary cells, which may not be very distinct from ordinary epidermal cells. Two (2) of the cells are ordinary epidermal cells but one (1) of them is a subsidiary cell. This type can be found in Cruciferae, Solanaceae, and others.

The term trichome (or hairs) is applied to an artificial grouping of all cells that project markedly out of the plane of the epidermis (Theobald et al., 1979). The functions of trichomes are wonderfully diverse. The glandular trichomes can secrete water, salt, nectar, mucilage, terpenes, adhesives, digestive enzymes and irritants that sting. Other trichomes absorb water and salts (Benzing and Pridgeon, 1983). The nonglandular trichomes can, along with the cuticle and waxes, protect against excessive sunlight.

Other unusual epidermal cells, apart from the four basic types, are Lithocysts, Silica cells, Cork cells and Bulliform cells (Metcalfe and Chalk, 1979)

The leaf epidermal structure may be viewed from two (2) perspectives, vis-à-vis, the surface view and the transverse section view.

In this project, Allamanda cathartica leaves were selected for investigations with the ultimate aim of gaining some basic insight into the effect of vehicular emission on plants.

The specific aims and objectives however, are:

· To determine the stomata size

· To find out the stomata frequency

· To study the type of epidermal cell wall

· To determine the stomatal index and;

· To determine if there are any stomatal abnormalities or not.

CHAPTER TWO

MATERIALS AND METHODS

The materials used and the methods followed in this study are outlined below.

2.1 Materials

2.1.1 Heavily Polluted Plant Materials

Foliar materials of Allamanda cathartica were collected fresh from three replicates located in a suspected heavily polluted zone (“UCC Eastgate-Science” Road) in Cape Coast on long 1⁰07 and 1⁰15W, lat 5⁰00 and 15’N.

2.1.2 Moderately Polluted Plant Materials

Foliar materials of Allamanda cathartica were collected fresh from three replicates located in a suspected moderately polluted zone (UCC Grounds and Gardens) in Cape Coast on long 1⁰07 and 1⁰15W, lat 5⁰00 and 15’N.

2.1.3 Non-Polluted Plant Material ( Experimental Control)

Foliar materials of Allamanda cathartica were collected fresh from three replicates located in a suspected non-polluted zone ( vicinity of the Faculty of Education and the Faculty of Social Sciences) in Cape Coast on long 1⁰07 and 1⁰15W, lat 5⁰00 and 15’N.

2.1.4 Other Materials

Light microscope, Photomicroscope, Slides, Cover slips, Telecounter, Stage micrometer, Eyepiece micrometer, Glycerol and Black polyethene bags

2.2 Methods

Critical observation was made on the “UCC Eastgate-Science” Road to discover the period of time in which heavy vehicular traffic occur. This period of time was noted down.

Three replicates of the Allamanda cathartica were randomly selected from the UCC “Eastgate-Science” Road during periods of heavy traffic. A picture of the this ornamental plant was taken with the help of a camera. Foliar materials were collected fresh from these three replicates and were carefully placed in three separate polyethene bags. The polyethene bags were tightly closed to prevent the withering of the foliar materials through evaporation, and were subsequently carried to the laboratory.

Through careful observatory skills, the light microscope was calibrated with the aid of a stage micrometer and an eyepiece micrometer.

The collected foliar materials were carefully washed with copious amount of water to rid them from any dirt. Three leaves were randomly selected from each of the three replicates. Epidermal peels from each leaf were stripped off by the “Hold and Tear” method, thoroughly washed with water and temporarily mounted in 50% glycerol on a clean slide. The slides were covered with the Cover slips and then mounted on the light microscope.

The surface views of the prepared slides of all the samples were examined under the calibrated light microscope. The type of epidermal cell wall and the distribution of mature stomata on the leaf surface were observed and noted down. Following, the morphological type of stomata was carefully observed and recorded. Nevertheless, the stomatal frequency and the number of epidermal cells were determined by the aid of a telecounter, and the results noted down. This was followed by the determination of the stomatal length, stomatal breadth and the pore size. The results were then recorded. The stomatal measurements were the averages of three field of views or observations of the three randomly selected leafs per each of the three collected replicates, whose sum were 100 or more; viewed with a 40x objective and 10x eyepiece. Pictures of the upper epidermis (Plate 1), lower epidermis (Plate 2) and any observed peculiar features were taken with the help of a camera and a photomicroscope. Finally, any features of abnormalities (Plate 3) were noted down.

The above procedure was repeated for foliar materials collected from the UCC Grounds and Gardens and, the vicinity of the Faculty of Education and the Faculty of Social Sciences. All observations and measurements made were carefully noted down.

CHAPTER THREE

RESULTS

Following the methods outlined in the previous chapter, the following results are presented.

3.1 FIELD OBSERVATION

Habit of the Allamanda cathartica: Straggling shrubs.

Period of heavy vehicular traffic: 7: 30 a.m. – 12:30 p.m.

3.2 EPIDERMAL STRUCTURE AND STOMATAL FEATURES

Epidermal Wall Pattern: Irregularly walled.

Trichomes Presence and Nature: Trichomes were found only on the lower epidermis. The Trichomes were Multicellular type of Nonglandular trichomes (Peltate hairs).

Nature of Leaves ( Stomatal Distribution on Leaf Surface): Hypostomatic

Morphological Type of Stomata: Paracytic

Type of Guard Cell: Crescent-Shaped

Stomatal Abnormality: Shrunken Stomata

Table 3.1 Comparison of mean stomatal frequencies of Allamanda cathartica in 3 zones

Replicates	T₁	T₂	T₃
1	56.111	37.667	30.444
2	56.667	38.000	28.667
3	57.111	38.222	29.333
Means	56.630	37.963	29.481

Key: T₁ = Treatment means of stomatal frequencies of Allamanda cathartica found in a Highly Polluted zone.

T₂ = Treatment means of stomatal frequencies of Allamanda cathartica found in a Moderately Polluted zone.

T₃= Treatment means of stomatal frequencies of Allamanda cathartica found in a Non-Polluted zone.

Table 3.2 Comparison of mean epidermal cell numbers of Allamanda cathartica in 3 zones

Replicates	T₁	T₂	T₃
1	125.667	131.000	165.778
2	127.444	119.889	162.111
3	129.444	120.333	169.222
Means	127.518	123.741	165.703

Key: T₁ = Treatment means of epidermal cell numbers of Allamanda cathartica found in a Highly Polluted zone.

T₂= Treatment means of epidermal cell numbers of Allamanda cathartica found in a Moderately Polluted zone.

T₃ = Treatment means of epidermal cell numbers of Allamanda cathartica found in a Non-Polluted zone.

Table 3.3 Comparison of mean stomatal lengths of Allamanda cathartica in 3 zones

Replicates	T₁/µm	T₂/µm	T₃/µm
1	36.888	30.668	25.112
2	40.444	28.444	25.112
3	37.332	28.888	24.444
Means	38.221	29.333	24.889

Key: T₁ = Treatment means of stomatal lengths of Allamanda cathartica found in a Highly Polluted zone.

T₂= Treatment means of stomatal lengths of Allamanda cathartica found in a Moderately Polluted zone.

T₃= Treatment means of stomatal lengths of Allamanda cathartica found in a Non-Polluted zone.

Table 3.4 Comparison of mean stomatal breadth of Allamanda cathartica in 3 zones

Replicates	T₁/µm	T₂/µm	T₃/µm
1	21.332	14.888	14.044
2	19.112	16.444	14.668
3	19.776	14.888	13.332
Means	20.073	15.407	14.015

Key: T₁ = Treatment means of stomatal breadth of Allamanda cathartica found in a Highly Polluted zone.

T₂ = Treatment means of stomatal breadth of Allamanda cathartica found in a Moderately Polluted zone.

T₃= Treatment means of stomatal breadth of Allamanda cathartica found in a Non-Polluted zone.

Table 3.5 Comparison of mean stomatal size of Allamanda cathartica in 3 zones

Replicates	T₁/µm	T₂/µm	T₃/µm
1	36.888 X 21.332	30.668 X 14.888	25.112 X 14.044
2	40.444 X 19.112	28.444 X 16.444	25.112 X 14.668
3	37.332 X 19.776	28.888 X 14.888	24.444 X 13.332
Means	38.221 X 20.073	29.333 X 15.407	24.889 X 14.015

Key: T₁ = Treatment means of stomatal size of Allamanda cathartica found in a Highly Polluted zone.

T₂ = Treatment means of stomatal size of Allamanda cathartica found in a Moderately Polluted zone.

T₃= Treatment means of stomatal size of Allamanda cathartica found in a Non-Polluted zone.

Table 3.6 Comparison of mean stomatal pore breadth of Allamanda cathartica in 3 zones

Replicates	T₁/µm	T₂/µm	T₃/µm
1	3.244	2.444	2.532
2	3.600	2.888	2.800
3	2.400	2.224	2.800
Means	3.081	2.519	2.711

Key: T₁ = Treatment means of stomatal pore breadth of Allamanda cathartica found in a Highly Polluted zone.

T₂ = Treatment means of stomatal pore breadth of Allamanda cathartica found in a Moderately Polluted zone.

T₃ = Treatment means of stomatal pore breadth of Allamanda cathartica found in a Non-Polluted zone.

Table 3.7 Comparison of mean stomatal pore size of Allamanda cathartica in 3 zones

Replicates	T₁/µm	T₂/µm	T₃/µm
1	20.000 X 3.244	20.000 X 2.444	20.000 X 2.532
2	20.000 X 3.600	20.000 X 2.888	20.000 X 2.800
3	20.000 X 2.400	20.000 X 2.224	20.000 X 2.800
Means	20.000 X 3.081	20.000 X 2.519	20.000 X 2.711

Key: T₁ = Treatment means of stomatal pore size of Allamanda cathartica found in a Highly Polluted zone.

T₂ = Treatment means of stomatal pore size of Allamanda cathartica found in a Moderately Polluted zone.

T₃ = Treatment means of stomatal pore size of Allamanda cathartica found in a Non-Polluted zone.

Table 3.8 Comparison of mean stomatal index of Allamanda cathartica in 3 zones

Replicates	T₁/%	T₂/%	T₃/%
1	30.868	22.332	15.515
2	30.779	24.068	15.026
3	30.613	24.106	14.773
Means	30.753	23.502	15.105

Key: T₁ = Treatment means of stomatal index of Allamanda cathartica found in a Highly Polluted zone.

T₂ = Treatment means of stomatal index of Allamanda cathartica found in a Moderately Polluted zone.

T₃ = Treatment means of stomatal index of Allamanda cathartica found in a Non-Polluted zone.

Table 3.9 Comparison of Least Significant Ranges for the Stomatal Frequencies

	T₁	T₂	T₃
T₁	_	SIG	SIG
T₂	SIG	_	SIG
T₃	SIG	SIG	_

KEY: SIG = Significant difference at 5% level at Duncan’s Multiple Range Test (DMRT)

T_1,T₂and T_{3 =}Treatment mean in highly polluted zone, moderately polluted zone and non-polluted zone respectively.

Table 3.10 Comparison of Least Significant Ranges for the Epidermal Cell Numbers

	T₁	T₂	T₃
T₁	_	NS	SIG
T₂	NS	_	SIG
T₃	SIG	SIG	_

KEY: SIG = Significant difference at 5% level at Duncan’s Multiple Range Test (DMRT)

NS = No Significant difference at 5% level at Duncan’s Multiple Range Test (DMRT)

T_1,T₂and T_{3 =}Treatment mean in highly polluted zone, moderately polluted zone and non-polluted zone respectively.

Table 3.11 Comparison of Least Significant Ranges for the Stomatal Length

	T₁	T₂	T₃
T₁	_	SIG	SIG
T₂	SIG	_	SIG
T₃	SIG	SIG	_

KEY: SIG = Significant difference at 5% level at Duncan’s Multiple Range Test (DMRT)

NS = No Significant difference at 5% level at Duncan’s Multiple Range Test (DMRT)

T_1,T₂and T_{3 =}Treatment mean in highly polluted zone, moderately polluted zone and non-polluted zone respectively.

Table 3.12 Comparison of Least Significant Ranges for the Stomatal Breadth

	T₁	T₂	T₃
T₁	_	SIG	SIG
T₂	SIG	_	NS
T₃	SIG	NS	_

KEY: SIG = Significant difference at 5% level at Duncan’s Multiple Range Test (DMRT)

NS = No Significant difference at 5% level at Duncan’s Multiple Range Test (DMRT)

T_1,T₂and T_{3 =}Treatment mean in highly polluted zone, moderately polluted zone and non-polluted zone respectively.

Table 3.13 Comparison of Least Significant Ranges for the Stomatal Index

	T₁	T₂	T₃
T₁	_	SIG	SIG
T₂	SIG	_	SIG
T₃	SIG	SIG	_

KEY: SIG = Significant difference at 5% level at Duncan’s Multiple Range Test (DMRT)

NS = No Significant difference at 5% level at Duncan’s Multiple Range Test (DMRT)

T_1,T₂and T_{3 =}Treatment mean in highly polluted zone, moderately polluted zone and non-polluted zone respectively.

X400

Plate 1: Upper Epidermis of Allamanda cathartica Showing Absence of Stomata and Trichomes.

X400

Plate 2: Lower Epidermis of Allamanda cathartica Located in a Non-Polluted Area and Showing Open Stomata with Little or No Vehicular Particles Clogging The Stomatal Pores.

X400

Plate 3: Lower Epidermis of Allamanda cathartica from a Highly Polluted Area Showing Shrunken Stomatal Abnormality and Stomatal Pores That Are Heavily Clogged with Vehicular Particles.

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Plate 4: Lower Epidermis of Allamanda cathartica Showing A Trichome And Heavily Polluted Stomata

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Plate 5: Close View of the Lower Epidermis of Allamanda cathartica Showing Irregular Epidermal Walls, Paracytic Stomata and Crescent-Shaped Guard Cells.

X 0.14

Plate 6: Allamanda cathartica in a Highly Polluted Area of “UCC Eastgate_Science” Road

CHAPTER FOUR

DISCUSSION AND CONCLUSIONS

The effective functioning of the epidermis is crucial to the survival of a plant.
This should have stimulated great interest in epidermal studies. Despite the morphological and physiological importance of the epidermis information on stomatal structure, distribution and frequency in relationship to vehicular emissions is lacking or incomplete for many ornamental plants, such as Allamanda cathartica. Unlike the family Apocynaceae in which little or no work on epidermal structures have been made, studies are gradually been carried out in many families such as the family Zingiberaceae, Boraginaceae and Convolculaceae. For example, Nyauame and Gill (1990) cited that earlier contributions to phytodermology of the family Zingiberaceae were by Barthelat and Tomlinson, beginning as early as 1893. He further reported that gradual studies in this family following these earlier works, were made by Stebbins & Khush, El-Gazzar & Hamza in the 1960s and 1970s. Nevertheless, it was cited by Nyawuame and Gill that importance of morphological types of mature stoma and the determination of their ontogenic pathway have been stressed by various authors such as Bessis & Guyot, Gill, Martin & Juniper, Metcalfe, Priestley, Ramayya & Rao and Stace. However, none of these works were reported to have been carried out in the family Apocynaceae.

In this study, the Habit of Allamanda cathartica was found to be a straggling shrub. The period of heavy vehicular traffic at the time of research was from 7:30 a.m. to 12:30 p.m. This was observed because most students and lecturers in the University of Cape Coast and its environs (in which the research work was carried out) have lecture schedules beginning at 7:30 a.m. Nevertheless, most breaks from academic works occur between the hours of 11:30 a.m. and 12:30 p.m. for both lecturers and students. However, minimal vehicular traffic was observed for the other periods because different lecture schedules have different closure periods. The epidermal wall was found to be irregularly walled, as also found in Aniseia martinicensis of the family Convolvulaceae.

As a Hypostomatic plant, Allamanda cathartica has both stomata and trichomes found only on the lower surface of the leaf. Although it is a necessity for this ornamental plant to possess the ability of gaseous exchange, it must also be able to prevent impacts from extreme sunlight. Hence, restricting stomata to the lower surface of the leaf is an adaptation to maximize gaseous exchange while minimizing the adverse impact of sunlight from withering the plant, through excessive transpiration. The trichomes observed were Multicellular type of Nonglandular trichomes. This is a feature that protect against excessive sunlight. AS they die and dehydrate, their walls become more refractile and scatter light. Such a structure is also a deterrent against insects, because the hairs can tangle the feet or impale the insect; being dead and empty, the trichomes are of little nutritive value to the insect. They are called Peltate hairs as all the branch cells interconnect along their sides to form a disk or shield-shaped structure with stalks (Plate 4).

The morphological type of stomata was seen as Paracytic type because each stoma was surrounded by two(2) specialized epidermal cells ( called Subsidiary Cells) which were oriented parallel to the long axis of the stoma. This morphological type of mature stomata was found to be similar to that of Aframomum melegueta, Aframomum sceptrum (both of which belong to the family Zingiberaceae) and most of the species found in the family Convolvulaceae, such as Ipomoea obscura, Jacquemontia tamnifolia, Lepistemon owariense and Merremia aegyptica (Nyawuame & Gill, 1990).

Unlike the dumbbell-shaped type of guard cells found in the grass family and Cyperaceae (sedges), the guard cells of Allamanda cathartica were observed as Crescent-shaped type, as found in the other families apart from the aforementioned families above (Ziegler et al., 1974).

There were features of abnormal stomata (Plate 3). These were shrunken stomata. This could have been caused by certain constituents of the emissions from vehicular exhausts that might have impeded the growth and development of some of the stomata.

The stomata were present only in the abaxial surfaces of all the replicates. This is consistent with the findings of Ting (1982) that stomata on the leaves are more abundant on the abaxial surface, with the upper surface having fewer or even none.

From the results, stomatal frequency is directly proportional to vehicular emissions. Thus, the stomatal frequencies of replicates in the highly polluted areas were highest whiles those in the non-polluted areas were least, with replicates in the moderately polluted areas showing moderate stomatal frequencies (with respect to the other two zones). For example, the average stomatal frequencies of replicates in the highly polluted areas, moderately polluted areas and the non-polluted areas were respectively 56.630, 37.963 and 29.481. This implies that the stomatal growth and development of Allamanda cathartica are sensitive to vehicular emissions.

The epidermal cell numbers of the studied replicates showed a gradual decrease from the non-polluted zones to the moderately polluted zones. For example, the mean epidermal cell numbers of the various replicates in the non-polluted zone was 165.703. But, this number decreased to 123.741 with gradual pollution presence, as observed from the moderately polluted site. However, the epidermal cells of all the replicates begun to show an increase with higher pollution, as observed by an increase in the average epidermal cell numbers of 123.741 in the moderately polluted zone to an average of 127.518 in the highly polluted zone. It can be inferred that the epidermal cells in the non-polluted zone were higher because in the absence of pollution they were carrying out their normal functions, as indicated by Dutta (2002). Thus, with the introduction of moderate pollution, there was an inhibitory effect on the sensitive Allamanda cathartica replicates. This effect could have impeded the normal growth and development of the epidermal cells. The gradual increase in the epidermal cells with higher pollution levels could be attributed to the fact that the replicates gained some form of resistance against the vehicular emissions’ effect. This made them to increase their numbers that initially had a greater decrease with moderate pollution. In all the studied areas, the epidermal cell numbers were greater than all the other studied cells. This is in agreement with the findings of Montenegro (1974).

Both stomatal lengths and stomatal breadths showed directly proportional increases with pollution increases. Thus, both the stomatal lengths and stomatal breadths of replicates in the moderately polluted areas were slightly higher than those of the non-polluted zones. For example, the mean stomatal lengths of the moderately polluted zone and the non-polluted zone were respectively 29.333µm and 24.889µm whiles the mean stomatal breadths of replicates in the moderately polluted and non-polluted zones were respectively 15.407µm and 14.015µm. Nevertheless, there was a greater increase in the stomatal lengths and stomatal breadths in the replicates located in the highly polluted zone. For instance, an average stomatal length of 38.221µm in replicates of the highly polluted zone was far greater than the average stomatal lengths of 29.333µm and 24.889µm in the moderately polluted zone and the non-polluted zone respectively. A similar trend was seen in the stomatal breadth as the average stomatal breadth of 20.073µm in the highly polluted zone was far greater than those of the moderately polluted zone (15.407µm) and the non-polluted zone (14.015µm). Thus, stomatal size is directly proportional to vehicular emissions’ effect. This could be attributed to the fact that Allamanda cathartica, being sensitive, has evolved a genetic trait of responding to vehicular emissions’ effect by increasing its stomatal size to maximize the rate of gaseous exchange within its polluted environment. For all things being equal, stomatal size in a way is directly related to the stomatal pore.

From the deductions above, it follows that stomatal pore size should be directly proportionally related to vehicular emissions’ effect. This is true in the sense that the average stomatal pore breadths in the non-polluted zone, moderately polluted zone and the highly polluted zone were 2.711µm, 2.519µm and 3.081µm respectively. This is an attribution that a bigger stomatal pore size is more effective in either carrying out gaseous exchange processes or pollutant expulsion than smaller stomatal pore size.

From the equation of stomatal index,

Stomatal Index = Stomatal Number

_________________________________ X 100%,

Stomatal Number + Epidermal Cell Number

it follows that stomatal frequency is directly related to stomatal index. Hence, if stomatal frequency is directly proportional to vehicular emissions, then all things being equal, stomatal index should also be directly proportional to vehicular emissions’ effect. This deduction is true as the average stomatal index in the non-polluted zone, moderately polluted zone and the highly polluted zone were observed to be 15.105%, 23.502% and 30.753% respectively. The replicates in polluted zone need greater stomatal densities than those in comparably lesser polluted zones. This support the findings that plants challenged by unfavourable environmental conditions (for example, vehicular emissions) need greater stomatal densities for effective functions, as noted by Dutta (2002), than those leastly challenged by unfavourable environmental conditions (Ting, 1982; Howard, 1969; Solereder, 1908; Deschamp and Cooke, 1985).

From Plate 1-Picture of the upper epidermis of Allamanda cathartica- it can be observed that there are no stomata and trichomes found. Plate 2, revealing the lower epidermis in the non-polluted site, show that there is little or no sooth (or any other dark particles from vehicular emissions) found to be blocking the stomatal pore. This is because the site is almost pollution free. However, little patches of darkness can be observed as no area in Cape Coast is totally pollution free. This result from the fact that vehicular emissions are trans-boundary air pollutants and can still reach any site in Cape Coast. Plate 3 show a picture of the lower epidermis from the highly polluted area. It is easily observed that almost all the stomatal pores are heavily clogged with sooth and other particles from vehicle exhausts. This greatly affects gaseous exchange processes. Plate 4 shows a picture of a trichome and stomata on the lower surface of a leaf of Allamanda cathartica located in a highly polluted area. The trichome is severely darkened by particles from vehicular emissions. This may interfere with the normal functioning of the trichome.

From the One-Way Analysis of Variance of the stomatal frequency, a statistical significance level (critical value) of 0.050 is greater than the probability value of 0.000. This implies that there were significant differences between the treatment means in the 3 zones. This is supported by the fact that the calculated F-Ratio of 1529.610 is far greater than either the tabulated F-Ratio of 5.140 (at 5% significance level) or 10.920 (at 1% significance level). Although the individual means of the highly polluted zone, moderately polluted zone and the non-polluted zone were 56.630, 37.963 and 29.481 respectively, we are 95% sure that the true means lie within a respective intervals of 55.102-58.158, 36.435-39.491 and 27.953-31.009, based on pooled standard deviation of 0.615 ( as shown in the One-Way Analysis of Variance).

Thus, the Duncan’s Multiple Range Test (DMRT) showed that there were significant differences between the stomatal frequencies found in replicates in all three (3) zones when compared with each other. Thus, the use of the Duncan’s Multiple Range Test (DMRT) revealed that there was no significant difference between the epidermal cell numbers found in the highly polluted area and the moderately polluted area. But, there was a significant difference between the epidermal cell numbers found in the highly polluted area and the non-polluted area. Nevertheless, there was a significant difference between the number of epidermal cells found between the moderately polluted area and the non-polluted area (Benony & Howard, 2000).

There was a significant difference found between the stomatal breadths of replicates located in the highly polluted area and the moderately polluted areas. Moreover, there was a significant difference found between replicates located in both the moderately polluted area and the non-polluted area revealed that there was no significant difference. The stomatal breadths critical value of 0.05 was greater than the probability value of 0.000. This was supported by the result that the calculated F-Ratio of 35.510 was larger than the tabulated F-Ratio value of either 5.140 (at 5% significance level) or 10.920 at 1% significance level). Although the average treatment means in the highly polluted area, moderately polluted area and the non-polluted area were respectively 20.073µm, 15.407µm and 14.015µm respectively, we are 95% confident that the true means or the individual 95% confidence interval range for mean based on pooled standard deviation of 0.9220 will respectively be 2.049µm-4.114µm, 1.486 µm-3.551 µm and 1.678 µm-3.743 µm.

In conclusion, Allamanda cathartical is an ornamental plant that is sensitive to vehicular emissions. The type of stomatal abnormality found was shrunken stomata. As a Hypostomatic shrub, it has both stomata and trichomes only on lower surface of its leaves. Vehicular emissions have effect on it. It can cause its stomata to shrink and clog the stomatal pores. Its stomatal frequency is directly proportional to vehicular emissions. The development and growth of its epidermal cells are initially impeded by vehicular emissions, but they respond with an increase in epidermal cell numbers in the presence of high pollution. Its stomatal size is directly proportional to pollution increases. The stomatal pore size and stomatal index of Allamanda cathartica are directly proportional to increases in pollution resulting from vehicular emissions.

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APPENDIX 1

Table of Stomatal Frequency of Leaf 1 of Allamanda cathartica in the 3 zones

MEANS	Moderately Polluted Zone			Highly Polluted Zone			Non-Polluted Zone
	A	B	C	A	B	C	A	B	C
	35.000	48.000	35.000	63.000	37.000	61.000	28.000	32.000	24.000
	31.000	47.000	39.000	61.000	67.000	53.000	31.000	35.000	30.000
	38.000	40.000	43.000	52.000	68.000	53.000	29.000	25.000	27.000
	34.667	45.000	39.000	58.667	57.333	55.667	29.333	30.667	27.000

KEY: A, B and C represent replicates 1, 2 and 3 respectively.

Table of Stomatal Frequency of Leaf 2 of Allamanda cathartica in the 3 zones

MEANS	Moderately Polluted Zone			Highly Polluted Zone			Non-Polluted Zone
	A	B	C	A	B	C	A	B	C
	36.000	38.000	39.000	52.000	40.000	61.000	33.000	31.000	40.000
	40.000	29.000	36.000	53.000	74.000	56.000	37.000	19.000	27.000
	51.000	43.000	38.000	51.000	67.000	53.000	29.000	28.000	29.000
	42.333	36.667	37.667	55.333	60.333	56.667	33.000	26.000	32.000