ANATOMICAL STUDY OF Sansevieria zeylanica LEAVES AFFECTED BY VEHICULAR EMISSIONS

by

Parangat, John Kelly R. Bayona, Gem L. Misola, Charisse M. Bacunot, Lowie S. Antonio, Nathaniel D.

A special problem submitted to Prof. Liezel M. Magtoto Department of Biology College of Science University of the Philippines Baguio

In partial fulfilment of the requirements of the course in Plant Anatomy

September 14, 2012

ABSTRACT This study deals with the effect of air pollution on different plant structures on the responses of Sansevieria zeylanica to vehicular emissions in three selected sites in Baguio City: UP Drive, UP Campus, and Botanical Garden Nursery whose intensities of vehicular emissions were evaluated by monitoring the vehicular volume along these sites over a 24-hour period. Since only UP Drive is the site where vehicles can pass through, a qualitative comparison is made ranging from light, moderate and heavy. The vehicular volume for UP Drive is 48,063. The responses of S. zeylanica to vehicular emissions were determined using the plant leaves stomatal index and density, stomatal aperture length, guard cell size, and size of epidermal cells. Stomatal index and Stomatal Density were calculated using a Low Power Objective with a magnification of 100x. Stomatal Aperture, Size of Epidermal Cells and Guard Cells were observed under a compound light microscope at High Power Objective with a magnification of 400x. Results showed that samples from Botanical garden have the longest stomatal aperture among the three sites. UP drive and UP campus have aperture length with means that were statistically equal. Botanical garden has the smallest epidermal cell area compared to the two sites. The mean guard cell area of UP Campus was the largest and UP Drive was the smallest. Lastly, UP Drive has the smallest stomatal index. The indexes of Botanical garden and UP Campus were statistically equal. It can be drawn from this study that vehicular emissions decrease the length of stomatal aperture, increase epidermal cell size, decrease guard cell area and stomatal index. Results were analyzed using One-way ANOVA, further supported by SNK, Dunkan Test and Pearsons Correlation test.

INTRODUCTION In todays growing economy, there is a great increase in pollution as the population also increases. One of the major environmental threats that our country is facing today is vehicular emissions. Vehicular emission remains a threat to environmental problem which is expected to increase as the vehicle ownership increases in the country. In response to this problem, there has been attention drawn to the effect of these vehicular emissions to the growth of the plants. There is a growing concern that vehicular emissions generally affect the gas exchange in plants. In this study, the leaves of Sansevieria zeylanica were examined. A leaf epidermis is composed of compactly arranged cells, cuticle and stomata. The leaf may be amphistomatic, epistomatic or most commonly, hypostomatic. The stomata are scattered in the broad dicotyledon leaves while they occur in rows parallel with the long axis of the leaf in the narrow elongated leaves of monocots. The stomata may be located above the surface of the epidermis, on the same level or below it. Stomata are small apertures found in the epidermis of vascular plants, (Esau 1965) specifically they occur on stems, leaves, flowers and fruits but not on aerial roots. They occur on both surfaces of many leaves (amphistomatous) or on only one surface (hypostomatous or epistomatous). Stomata are bounded by guard cells. Stomata, from the Greek word stoma which means mouth provides an essential connection between the internal air spaces of plants and the external atmosphere. These pores are associated with cuticle bordered by pairs of structurally and physiologically specialized guard cells and adjacent epidermal cells termed subsidiary cells. These subsidiary cells, (Jarvis and Mansfield, 1981) form the stomatal complex and facilitate gas movement through the epidermis. In the absence of stomata, most plants will not survive the terrestrial environment since supply of carbon dioxide will be inadequate for photosynthesis, but at the same time the unavoidable loss of water vapor through them creates the danger of dehydration. Therefore, according to Cowan, 1982 and Raschke, 1976 the capability of the stomata to adjust their apertures is very important for the survival of the plants.

According to literatures, at maturity of a leaf, the number of stomata per unit leaf area may or may not be constant. The number of stomata in a certain leaf area may be affected by different environmental factors, one of which is pollution caused by vehicular emissions.

There are several researches and articles concerning the relationship of the stomata and atmospheric condition. One of these was Alistair M. Hetherington & F. Ian Woodwards article The role of stomata in sensing and driving environmental change. The article explains how the stomata on the surface of the leaves and stalks regulate gases in and out of the plants body. It also showed recent data from diverse fields that establish their central importance to plant physiology, evolution and global ecology. According to the authors, Stomatal morphology, distribution and behaviour respond to a spectrum of signals, from intracellular signalling to global climatic change. Such concerted adaptation results from a web of control systems, reminiscent of a scale-free network, whose untangling requires integrated approaches beyond those currently used. The study Stomatal density and stomatal index as indicators of paleoatmospheric CO 2 concentration was also concerned in the inverse relationship between atmospheric CO2 concentration and stomatal density and/or stomatal index. This study was done by D.L. Royer of the Yale University Department of Geology and Geophysics, New Haven, USA. Some excerpt of the studys abstract said that: According to Duldulao and Gomez, leaf gross morphological changes like as yellowing and browning, deformity in shape, spotting, drying of leaf margins and less hairy features were more experiential in plants from the more polluted site than in the control site. In the study the stomatal size and stomatal index was considered significant in affecting interaction of plant site and growth stage. The two factors, plant site and plant type significantly affects chlorophyll content of the leaves. Epidermal leaf surface features, including stomates, trichomes and chlorophyll content in plants growing along roadsides were altered due to the stresses of vehicular exhaust emission with high traffic density in urban areas. The alterations can be considered as pointers of environmental stresses. The effects of pollution on plants include mottled foliage, burning at leaf tips or margins, twig dieback, stunted growth, premature leaf drop, delayed maturity, abortion or early drop of blossoms, and reduced yield or quality. In general, the visible injury to plants is of three types: (1) collapse of leaf tissue with the development of necrotic patterns, (2) yellowing or other color changes, and (3) alterations in growth or premature loss of foliage. Injury from air pollution can be confused with the symptoms caused by fungi, bacteria, viruses, nematodes,

insects, nutritional deficiencies and toxicities, and the adverse effects of temperature, wind, and water. Plant injury caused by air pollution is most common near large cities, smelters, refineries, electric power plants, airports, highways, incinerators, refuse dumps, pulp and paper mills, and coal-, gas-, or petroleum-burning furnaces. Plant injury also occurs near industries that produce brick, pottery, cement, aluminum, copper, nickel, iron or steel, zinc, acids, ceramics, glass, phosphate fertilizers, paints and stains, rubbers, soaps and detergents, and other chemicals. Damage in isolated areas occurs when pollutants are spread long distances by wind currents.

Factors that govern the extent of damage and the region where air pollution is a problem are (1) type and concentration of pollutants, (2) distance from the source, (3) length of exposure, and (4) meteorological conditions. For some pollutants, damage can occur at levels below Environmental Protection Agency standards.

Other important factors are city size and location, land topography, soil moisture and nutrient supply, maturity of plant tissues, time of year, and species and variety of plants. A soil moisture deficit or extremes of temperature, humidity, and light often alter a plants response to an air pollutant.

Dr. Kent reports that nitrogen dioxide, a byproduct of combustion from car engines or open fires, can slow the growth of plants. Fortunately, rainfall transforms nitrogen dioxide into nitric acid, which adds nitrogen to the soil and actually benefits plants. However, carbon monoxide is less benign. This component of car exhaust is poisonous to humans and will stunt the growth of plants. Some evergreens will drop their leaves completely when exposed to carbon monoxide. Plant responses to air pollution are helpful in the following ways. It establishes the early presence of air-borne contaminants, determines the geographical distribution of the pollutants, and helps estimates the concentration of pollutants. It also provides a passive system for collecting pollutants for chemical analyses later and obtains direct identification of different air pollutants on the basis of plant species and variety affected.

Sansevieria zeylanica commonly known as snake plant or bowstring hemp is a succulent plant that can be grown in high light. This plant can tolerate low humidity, low water and feeding. Plants often form dense clumps from a spreading rhizome or stolons. An attempt was then made in this study to know and identify the plant structures that may serve as an indicator of the levels of carbon dioxide and other pollutants in the atmosphere. Attention was primarily focused on the stomata of Sansevieria zeylanica leaf. Stomata have been shown to affect the cellular respiration of Sansevieria zeylanica. This paper aims to (1) compare the anatomical differences of Sansevieria zeylanica exposed to vehicular emissions with plants from the unpolluted site, (2) note the effect of air pollutants in the anatomy of the test plant, and (3) correlate results from literatures with that of the study.

This study is important because it is used to monitor the ability of Sansevieria zeylanica to adapt to the environment and its capability as a bio-indicator for air pollution. The study will significantly back up the recent studies on increasing air pollution and its anatomical effect on plant. This information gathered can be used to monitor air quality by using anatomical structure as the parameters of air pollution caused by vehicular emissions.

The study focuses on the microscopic epidermal effects of air pollution on different parameters on the plant species. The microscopic epidermal parameters used are size of epidermis, size of guard cells, stomatal aperture, stomatal size and stomatal index. The study is limited to the effects of air pollution in just one plant species Sansevieria zeylanica. It is also well stated in the study that only surface sections were done and no cross sections were made. Also, the age of each plant is only assumed by measuring the height. The researchers were not able to plant cuttings of S. zeylanica and three sites were only selected because of the limited time.

METHODOLOGY

Study Area

Three locations were identified for the collection of the specimen in different places of Baguio City namely University of the Philippines drive (UP Drive) at Governor Pack Road, inside the campus of University of the Philippines Baguio beside Human Kinetics Program (HKP) building and Botanical gardens nursery beside the forest located in Leonard wood road which is the controlled variable.

Botanical Garden

UP Campus UP DRIVE

The three areas are receiving different intensities of air pollutants and exhaust particles from smoke produced by vehicles passing in the said areas. The sites will be rated in terms of the

vehicular volume passing in it, the one with the greatest vehicular volume will be regarded with high air pollutants from vehicular emissions followed by moderate and then low.

Monitoring the vehicular Flow The density of vehicles passing through the roads of University of the Philippines Drive (UP drive) was known from a recent thesis last 2012 which was quantified by number of vehicles passing in each sites in a 24-hour cycle (Salvador, 2011). The highly polluted site which is the UP Drive has 48, 063 vehicles/24-hour cycle.

Test specimen

Kingdom: Plantae Phylum: Magnoliophyta Class: Liliopsida Order: Asparagales Family: Asparagaceae Genus: Sansevieria Species: Sansevieria zeylanica

Sansevieria zeylanica is a monocot plant, succulent herb without stem having thick fibrous leaves transversely banded in light and dark green crossbands. Its leaves are concave in the middle which can grow up to 60 cm. Its common names are devils tongue and tiger plant (Madulid, as cited by Lallana, 2011).

Sampling Method Three replicates of Sansevieria zeylanica leaves ranging from 24 32 inches, assuming that the plants are of similar ages were selected randomly from each site. It was immediately put to a polyethylene bag and was returned to lab for preparing and cutting sections as soon as possible to prevent high rate of dehydration. (Duldulao, 2008) In each plant, surface sectioning

in the Adaxial and Abaxial region of the leaf were done and 10 samples were measured in each sections for statistical analysis. A total of 60 samples were measured in each site for more reliable results.

Test Protocol and Parameters

Fine sections of leaves were taken from the surface and were put to clean water to prevent dehydration. Sections were fixed with FAA, dehydrated with 40% ethanol for about 3060 seconds and were rinsed with 50%ethanol. Specimens were then mounted with Canada Balsam. Prepared sections were examined under the microscope for observing its stomatal index, guard cell area, size of stomatal aperture and size of epidermal cells. Stomatal type and epidermal cells were also identified. The abaxial and adaxial parts of the leaf were used as a separate component.

Stomatal Index

The

stomatal

index

was

computed

using

the

formula

using Low Power Objective under the magnification of 100x with an eyepiece of 10x.

Stomatal Density

As adapted from Wuytack, the stomatal density was calculated by the number of stomata per 1mm2 under a magnification of 100x, a Low Power Objective and an eyepiece of 10x.

Guard Cells, Epidermal Size, Size of Stomatal Aperture

Other parameters such as length of stomatal aperture, area of guard cell, and epidermal size were measured by calibrated ocular micrometer using High Power Objective with an eyepiece of 10x.

Data Analysis

The stomatal index, stomatal density, guard cell area, size of stomatal aperture and size of epidermal cells of leaves of Sansevieria zeylanica were compared through one-way Anova using SPSS v. 20 further supported by SNK, Dunkan Test and Pearsons Correlation test.

RESULTS AND DISCUSSION Vehicular Volume in the Three Study Sites There were three sites where the specimens were collected--UP Drive , UP Campus and Botanical Garden. The UP Drive was set as the polluted site and the Botanical Garden was the non-polluted site. UP drive was one of the busiest roads in Baguio City. It was being used by vehicles starting from motorcycles to Public Utility Vehicles like jeepneys and buses. Dark smokes in the UP drive were always seen because the road was an inclined so the vehicles are required to change gear and release black smokes from their engines. The UP Campus was set as the semi-polluted site because vehicles were passing by the UP Campus but not as much as in the UP Drive but not lesser than in the Botanical Garden. The Botanical Garden was the least polluted in the site and it was more far from the main road. The vehicular volume was set to heavy, medium or light. The most polluted site was set as Heavy. The intermediate site was set as medium and the least polluted site was listed as light.

Data Analysis The researchers used One-way Anova to analyse the Aperture length, ordinary epidermal size (Length, width and area), Guard cell size (Length, width and area) and stomatal index that were gathered from the specimens of the three different sites. The software IBM SPSS Statistics version 20 was used in the analysis. This analysis involved a sample, a sampling distribution and a population therefore certain parametric assumptions were required to ensure the compatibility of the components.

These assumptions were: a) the data were independent from each other, b) the data were normally distributed and c) the observations in the different groups have nearly equal variances. The first assumption of independence was met in this study because the samples were randomly chosen from the different sites and so were the areas of the leaf where the surface sectioning was done. Almost all of the data gathered in the study were also qualitative and were actual measurements of the parameters being studied. The subjects were also measured only once. The next assumption was that the data came from a normal population or that data were normal. Kolmogorov-Smirnov goodness-of-fit test was used to test this assumption.

Table 1. One-Sample Kolmogorov-Smirnov Test For Normality Parameter Aperture length Epidermal Width Epidermal Length Epidermal Area Guard Cell Width Guard Cell Length Guard Cell Area Stomatal Density Stomatal Index Significance Value .008 .000 .071 .187 .000 .000 .098 .792 .856

If the significance value was greater than .05, the data set was normal and not normal if it was less than 0.05. Based in table 1 above, the significance value of the guard cell area, epidermal area, epidermal length, stomatal index and stomatal density were greater than 0.05 therefore these parameters are normal (Appendix, Table 1). The aperture length, epidermal width, guard cell width and guard cell length are non-normal data. These parameters did not

meet the assumption of normality of Anova. However, Anova can still be used on these observations because biological data are usually non-normal data. The final assumption of Anova is that the data are homoscedastic. The researchers used Levens test to validate this homogeneity of the variances. Table 2. Test of Homogeneity of Variances Parameter Aperture length Epidermal Width Epidermal Length Epidermal Area Guard Cell Width Guard Cell Length Guard Cell Area Stomatal Density Stomatal Index Significance Value .084 .587 .151 .660 .067 .000 .239 .811 .441

If the significance value was greater than .05, the data set was not normal and normal if it was less than 0.05. Table 2 shows that only the data on the guard cell length have equal variances (Appendix, Table 2). All of the other parameters were not homoscedastic. If the assumptions of Anova were not met, the level of significance of the test and the sensitivity of the F statistic to real departures from the null hypothesis would be affected and as a result, the results validity might also be affected. Anova was still used and to ensure the accuracy and validity of the test, the data were also analysed using the nonparametric tests Kruskal-Wallis Analysis of Ranks and Duncan analysis. The null hypothesis in this analysis was that the aperture length, epidermal wall width, epidermal wall length, guard cell width, guard cell length, epidermal area, guard cell area, stomatal density and the stomatal index of the Sansevieria zeylanica species from the three different places with varying air conditions were equal. The alternative hypothesis was that there

were significant differences in these parameters between the specimens from the three different sizes.

Table 3. ANOVA Parameter Aperture length Epidermal Width Epidermal Length Epidermal Area Guard Cell Width Guard Cell Length Guard Cell Area Stomatal Density Stomatal Index Significance Value .000 .003 .000 .001 .000 .000 .000 .079 .001

Table 3 above showed the result of the analysis of variance. The significance values of the parameters of the aperture length, epidermal width, epidermal length, epidermal area, Guard cell width, guard cell length and guard cell of the specimens were less than 0.05(Appendix, Table 3). This means that these parameters in the three different sites have significant difference. The significance value of the stomatal density was greater than 0.05, therefore the plants from the three different sites have no significant difference in their stomatal density. Since some of the assumptions of Anova are not met, the post hoc tests Kruskal-Wallis Analysis of Ranks and Duncan analysis were used to ensure the validity of the Anova.

STOMATAL APERTURE LENGTH Table 4. Aperture Length Location UP Drive UP Campus Botanical Garden Mean Aperture Length (m) 29.5208 30.3333 34.7292

The results of the data analysis showed that the aperture length of the specimens from UP Drive and UP campus were the same and were smaller than the aperture length of the specimens from Botanical Garden (Appendix Table 4). The Botanical garden specimens have the longest aperture with a length of 34.72 micrometers. The aperture lengths of the specimens of UP Drive and UP campus which were 29.52 and 30.33 micrometers have no significant difference. Thus the level of air quality is directly proportional to the size of the stomatal aperture which is as the level of air quality increase, the larger the stomatal aperture and as the level of air quality decrease, the smaller the stomatal aperture is. According to Robinson, et. al. air pollutants such as SO 2 and O3 in high concentrations can usually cause stomatal closure. At low concentration, stomatal conductance is often increased. There are two mechanisms underlying the example, the need to suppress transpiration may take interference with stomatal control have recently been precedence over the intake of CO2 for photosynthesis identified, one involving O3 and the other CO2. The study of Omasa and Oneo shows that stomatal aperture has a significant difference when air pollutants is present. The stomatal aperture is inversely proportional to the level of air pollutants which is as stomatal aperture decreases as the level of air pollutants increases. Their study focused on the digital image processing technique of capturing images of the stomata as it is adapting to the artificial environment that was created by the researchers. In the second half of their study, Omasa and Oneo examined the responses to SO2 of neighboring stomata in a small leaf region of an intact growing plant. These stomata showed almost uniform and constant k, until about 20 min after the start of the exposure, and then a wide

variety of stomatal movements began; the largest value in k, was about twice as large as the smallest value at 45 min and became about three times as large at 90 min. Water-soaking and wilting began to appear in the subsidiary cells at about 55 min, when k, was a local maximum value, and then all the stomata began to close. This phenomenon was assumed to be caused by increased water loss from the subsidiary cell due to SO2, which affects the membrane and osmotic pressure, with a difference resulting in the turgor between the guard cell and the subsidiary cell (Heath, 1980 as cited by Omasa and Oneo).

EPIDERMAL SIZE AND AREA Table 5. Epidermal Width Location Botanical Garden UP Campus UP Drive Mean Epidermal Width (m) 18.5000 19.1667 20.7083

The result of SNK analysis showed that the plant from UP drive has the widest epidermal width which has a width of 20.71 micrometers (Appendix, Table 5). It also showed that the epidermal width of the specimens from Botanical garden and UP campus were 18.50 micrometers and 19.167 micrometers respectively. The Duncan analysis also showed the same result, that the epidermal width of specimens from Botanical garden and UP campus are the same and are smaller than the Epidermal width of specimens from UP drive. Table 6. Epidermal Length Location Botanical Garden UP Drive UP Campus Mean Epidermal Length (m) 67.6250 71.3333 80.5833

The Epidermal cells of the Sansevieria specimens fom UP campus have the longest epidermal with a mean size of 80.58 micrometers based on the SNK test. Duncan analysis has the same result with SNK, which was, the epidermal of the specimens from Botanical garden and UP Drive which have lengths of 67.63 m and 71.33 m respectively, were equal but were shorter compared to the epidermal length of specimens from UP campus (Appendix, Table 6). Table 7. Epidermal Area Location Botanical Garden UP Drive UP Campus Epidermal Area (m)2 1269.0625 1480.7292 1540.9375

Both of the SNK and Duncan test showed that the specimens from UP campus have the highest epidermal area while those from Botanical garden have the lowest epidermal area. The area of the specimens from UP, UP drive and Botanical garden were 1540.94 m2, 1480.73 m2 and 1269.06 m2 respectively (Appendix, Table 7). The epidermic cells generally have a decreased size in the leaves exposed to the pollutants(GOSTON,2009). In the paper of Meerabai, the pigeon pea plants growing in the vicinity of a silicon industry decreased in size, Average size of the epidermal cell decreases as pollutants increases. Various authors underlined the reduction of plant growth, as a consequence of pollution stress (Gupta and Iqba, 2005; Maruthi Sridhar et al., 2005, 2007; Gostin, 2009). In this paper, plants from UP Campus and UP Drive have almost the same epidermal area size, 1540.94 m2 And 1480.73 m2 respectively, while replicates from Botanical Nursery have an epidermal are size (are) of 1269.06 m2.

GUARD CELL SIZE AND AREA Table 8. Guard Cell width Location UP Drive Botanical Garden UP Campus Guard Cell Width (m) 8.5000 11.1458 11.3542

The SNK and Duncan analysis showed the same result in the guard cell width of the three different sites (Appendix, Table 8). UP and botanical garden specimens have widest guard cells with sizes of 11.35 m and 11.14 m respectively. The table also showed that the UP Drive specimens have the smallest width of 8.5 m. Table 9. Guard Cell Length Location Botanical Garden UP Campus UP Drive Guard Cell Length (m) 37.1667 39.3583 40.3958

Both of the non-parametric analysis showed that the specimens from Botanical garden have the shortest epidermal cell (Appendix, Table 9). The guard cell length from the site was 37.17 m. The specimens from UP drive and UP campus have no significant difference in their guard cell length but are longer than the Botanical Garden specimens. Table 10. Guard Cell Area Location UP Drive Botanical Garden UP Campus Guard Cell Area (m)2 343.4896 414.3490 447.7135

The post hoc tests showed the same results in the Guard Cell Area (Appendix, Table 10). Table 10 above showed that the guard cells from UP drive have the smallest area of 343.49 m2 while the specimens from UP campus have the highest guard cell area of 447.71 m2. In other words, the guard cell area from UP Drive is lower than the specimens from botanical and the guard cell area of Sansevieria from botanical garden is lower than the area from UP campus. Being one a highly populated and industrialized area in the country, City of Baguio has a serious problem on air pollution. Since air pollution is one of the major problem in many heavily populated and industrialized countries (Kambezidis et al. 1996). Vehicular emissions, one cause of air pollution have direct or indirect effect on the metabolism of the roadside plants( Viskari et al., 2000). Opening of stomata ideally achieves an acceptable compromise between the plants need to acquire carbon dioxide from the atmosphere for photosynthesis and water loss by transpiration (Harrison, 2001). Based on the results of the statistical tests done, the guard cell areas from the three different site: Boatanical garden, UP campus and UP drive have a significant differences. Carbon monoxide, oxides of nitrogen and sulfur, different particulate matters, lead and other substances are the different pollutants that are released to the atmosphere as a result of incomplete combustion in the automobile engines. Along with the study, the effect of these emissions on the length and width of the guard cells were studied. In the previous studies, it was shown that the guard cell size is generally affected by pollutants. In correlation with the past study of Irina Neta Gostin about air pollution effects to some Fabaceae species, plants exposed to vehicular emissions tend to have a smaller guard cell size. As for the result of the study, Specimens from UP drive being the most polluted site among the three experimental site, have the smallest size. While specimens collected from the botanical garden (control variable) has the largest stomatal guard cell size.

STOMATAL DENSITY Table 11. Stomatal Density Location UP Drive Botanical Garden UP Campus Stomatal Density 10.1667 11.5000 13.5000

The results of the post hoc analysis on the stomatal density are the same with the Anova (Appendix, Table 11). There was no significant difference between the stomatal densities of the leaves from the three different sites. The Duncan analysis however showed a different analysis. According to the Duncan test, there was a difference in the stomatal density of the specimens from Botanical garden and from UP campus When plants are exposed to air pollutants, a physiological and anatomical change takes place and may exhibit visible damage to its part. As plants are immobile and more sensitive in terms of physiological reaction to the most prevalent air pollutants than humans and animals, they better reflect local conditions (Nali and Lorenzini 2007). For these reasons, plants are the most common used bio-indicators in air quality biomonitoring studies. As cited in Wuytack, more specifically, its morphological and anatomical parameters are used, such as specific leaf area and stomatal density which have been proven to be useful as indicators of air quality (Balasooriya et al.,2009) Grasses typically have lower stomatal densities than deciduous trees. The size and shape of stomata also vary with different plant species and environmental conditions. For example, grasses have guard cells that resemble slender dumbbells whereas trees and shrubs have guard cells that resemble kidney beans. (Swarthout, 2010). Results show that a low mean of 11.72222222 is found in the species of Sansevieria considering the three sites. To optimize stomatal closure efficiency, stomatal density increases and stomatal pore surface decreases due to increasing levels of air pollution. (Balasooriya et al. 2009; Elagoz et al. 2006; Verma and Singh 2006)

While studying the stomatal density (Wuytack, 2010), their results showed that the stomatal density in Antwerp city, the highly polluted area was higher than in Zoersel, the less polluted area. Their study confirmed that stomatal density increases due to increasing levels of air pollution. The exchange of CO2 and water vapour between leaf and atmosphere is principally controlled by stomatal density and their mean aperture. (Lake and Woodward 200) stomatal densities change cin responae to changing atmospheric levels of co2 and pollutants. Places with high amounts of atmospheric pollutants tend to have increased number of leaf stomata, while lower amounts of atmospheric pollutants promotes a decreased number of stomata.(Kouwenberg et al,2003) The modification of the frequency and sizes of stomata as a response to the environmental stress is an important manner of controlling the absorption of pollutants by plants(Gostin,2009). Stomatal characteristics are often used for bio monitoring of air quality and the majority of the results on the response of the stomatal characteristics to air pollution are unanimous (Balasooriya et al, 2009) to optimize stomatal closure efficiency, stomatal density increases and stomatal pore surface decreases due to increasing levels of air pollution. This adaptation could decrease the amount of poisonous gases getting into leaf tissues and thus protect the plant against pollution. In this experiment, the plants from the UP Campus have the highest stomatal density of 13.5 while plants from the UP Drive have the lowest value with a mean of 10.16666667. This doesnt parallel most related literatures. However, the increase of SI is not a common feature of plant species exposed to air pollution. Verma et al. (2006) find a significant decrease of stomatal density and stomatal index in Ipomea pes-tigridis grown under various degrees of environmental stresses (coal-smoke pollutants). A reduction of stomata is also found in response to elevated CO 2 concentrations, frequently present in city centres (Williams et al. 1986). The reduction in stomatal densities and their pore size may be important for controlling absorption of pollutants (Verma et al. 2006), but will limit photosynthesis at the same time.

STOMATAL INDEX Table 12. Stomatal Index Location UP Drive Botanical Garden UP Campus Stomatal Index .6983333 1.2566667 1.3155500

The two non-parametric showed the same result on the specimens stomatal index (Appendix 12). The specimens from UP campus and botanical garden have the same stomatal index. Their stomatal indexes were higher compared to the stomatal index of the specimens from UP drive. Nowadays, there have been observed increased in number of industries and automobile vehicles which continuously add toxic gases and other substances to the environment. These toxic or pollutants have long term effects on plants by influencing CO 2 contents, light intensity, temperature and precipitation. (Jahan, 1992)

As seen in Table 12 of appendix, UP campus and botanical garden have the higher mean of stomatal index than stomatal index of the specimens from UP Drive. It also showed that there is a significant difference between the stomatal index of leaves from UP Drive and stomatal indices of leaves from UP Campus and botanical garden. Also, implicitly stated, stomatal index was inversely proportional to the site with high pollution. This suggests that pollution might have cause a decrease in stomatal index of the plant because it might have damage the stomata and leaf epidermal cells (Salvador, 2011). The shift in the stomatal index can be attributed to high amounts of carbon dioxide and sulfur dioxide emitted by automobiles. (Tanner et al., as cited by Salvador, 2011) Also, long term exposure to elevated sulfur dioxide levels triggers a phenotypic response of reduced number of stomata compared to the number of epidermal cells in order to minimize the inhibiting effects of SO2 on photosynthesis. The presence of particles which was brought from pollution in the

stomata can be seen caused by increase in temperature of foliage (Rai and Kuretshtha as cited by Salvador, 2011). Correlations Pearsons Correlation Analysis has shown that the stomatal index was positively correlated with the aperture length and guard cell area (see Appendix, Table 13). Moreover, the stomatal index and stomatal density were significantly correlated with each other.

CONCLUSION The study was done to compare the effects of air pollution in the stomatal aperture length, epidermal size (length, width and area), guard cell size (length, width and area), stomatal index and stomatal density of the Sansevieria zeylanica in three different locations with varying air conditions. These parameters vary in the different sites of the study. Botanical garden was the least air-polluted site, UP campus was the moderately polluted site and UP Drive was the most polluted site. The results revealed that Botanical garden has the longest stomatal aperture among the three sites. UP drive and UP campus have aperture length with means that were statistically equal. Botanical garden has also the smallest epidermal wall area compared to the two sites. UP campus and UP drive have their epidermal wall areas statistically equal. The mean guard cell area of UP Campus was the largest and UP Drive was the smallest. Lastly, UP Drive has the smallest stomatal index. The indexes of Botanical garden and UP Campus were statistically equal. Objectives were met. It can be drawn from this study that vehicular emissions decrease the length of stomatal aperture, increase epidermal cell size, decrease guard cell area and stomatal index. Results were analyzed using One-way ANOVA, further supported by SNK, Dunkan Test and Pearsons Correlation test.

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APPENDIX DATA ANALYSIS Table 1. One-Sample Kolmogorov-Smirnov Test Apertu Epider Epider Epider Guard GuardC Guard Stomat Stoma

reLeng malWid malLen malAr th N Norm al Para meter sa,b Mea n Std. Dev iatio n Abs Most Extre me Differ ences olut e Posi tive Neg ativ e Kolmogoro v-Smirnov Z Asymp. Sig. (2tailed) a. Test distribution is Normal. b. Calculated from data. .008 .000 .071 .187 1.671 2.928 1.293 1.089 -.119 -.126 -.096 -.043 .125 .218 .081 .081 .125 .218 .096 .081 3.7571 1 180 31.527 8 th 180 19.458 3 gth 180 73.1806 ea 180

Cellwi ellLengt CellAr alDensi talInde dth 180 h 180 38.9736 ea 180 401.85 07 ty 18 x 18

1430.2 10.333 431 3

11.722 1.0901 2 833

3.7022 15.6110 425.83 2.0494 1 0 264 9

3.46959

84.635 67

2.6302 .36551 7 610

.152

.155

.091

.153

.143

.133

.099

.091

.133

.135

-.152

-.155

-.074

-.153

-.143

2.040

2.082

1.227

.650

.606

.000

.000

.098

.792

.856

Table 2. Test of Homogeneity of Variances Levene Statistic ApertureLength EpidermalWidth EpidermalLength EpidermalArea GuardCellwidth GuardCellLength GuardCellArea StomatalDensity StomatalIndex 2.516 .534 1.912 .416 2.752 10.190 1.443 .212 .865 df1 2 2 2 2 2 2 2 2 2 df2 177 177 177 177 177 177 177 15 15 Sig. .084 .587 .151 .660 .067 .000 .239 .811 .441

Table 3. ANOVA Sum of Squares Between Groups ApertureLength Within Groups Total Between EpidermalWidth Groups Within Groups Total Between EpidermalLength Groups Within Groups Total 942.205 1584.531 2526.736 153.958 2299.479 2453.437 5344.653 38278.229 43622.882 2 177 179 2 177 179 2 177 179 2672.326 216.261 12.357 .000 76.979 12.991 5.925 .003 df Mean Square 471.102 8.952 52.624 .000 F Sig.

Between EpidermalArea Groups Within Groups Total Between GuardCellwidth Groups Within Groups Total Between GuardCellLength Groups Within Groups Total Between GuardCellArea Groups Within Groups Total Between StomatalDensity Groups Within Groups Total Between Groups StomatalIndex Within Groups Total

2446876.736 30011807.943 32458684.679 303.802 448.073 751.875 326.147 1828.666 2154.812 339937.599 942274.600 1282212.198 33.778 83.833 117.611 1.392 .879 2.271

2 1223438.368 177 179 2 177 179 2 177 179 2 177 179 2 15 17 2 15 17 .696 .059 16.889 5.589 169968.799 5323.585 163.073 10.331 151.901 2.531 169558.237

7.215

.001

60.005

.000

15.784

.000

31.928

.000

3.022

.079

11.881

.001

Table 4. Aperture Length Location N Subset for alpha = 0.05 1 UP Drive Student-Newman-Keulsa UP Botanical Sig. UP Drive Duncana UP Botanical Sig. Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 60.000. 60 60 60 .139 60 60 60 .139 29.5208 30.3333 34.7292 1.000 29.5208 30.3333 34.7292 1.000 2

Table 5. Epidermal Width Location N Subset for alpha = 0.05 1 Botanical Student-Newman-Keulsa UP UP Drive Sig. Botanical Duncana UP UP Drive Sig. Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 60.000. 60 60 60 .312 60 60 60 .312 18.5000 19.1667 20.7083 1.000 18.5000 19.1667 20.7083 1.000 2

Table 6. Epidermal Length Location N Subset for alpha = 0.05 1 Botanical Student-Newman-Keulsa UP Drive UP Sig. Botanical Duncana UP Drive UP Sig. Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 60.000. 60 60 60 .169 60 60 60 .169 67.6250 71.3333 80.5833 1.000 67.6250 71.3333 80.5833 1.000 2

Table 7. Epidermal Cell Area Location N Subset for alpha = 0.05 1 Botanical Student-Newman-Keulsa UP Drive UP Sig. Botanical Duncana UP Drive UP Sig. Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 60.000. 60 60 60 1.000 60 60 60 1.000 1269.0625 1480.7292 1540.9375 .424 1269.0625 1480.7292 1540.9375 .424 2

Table 8. GuardCellwidth Location N Subset for alpha = 0.05 1 UP Drive Student-Newman-Keulsa Botanical UP Sig. UP Drive Duncana Botanical UP Sig. Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 60.000. 60 60 60 1.000 60 60 60 1.000 8.5000 11.1458 11.3542 .474 8.5000 11.1458 11.3542 .474 2

Table 9. GuardCellLength Location N Subset for alpha = 0.05 1 Botanical Student-Newman-Keulsa UP UP Drive Sig. Botanical Duncana UP UP Drive Sig. Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 60.000. 60 60 60 1.000 60 60 60 1.000 37.1667 39.3583 40.3958 .079 37.1667 39.3583 40.3958 .079 2

Table 10. GuardCellArea Location N Subset for alpha = 0.05 1 UP Drive Student-Newman-Keulsa Botanical UP Sig. UP Drive Duncana Botanical UP Sig. 60 60 60 1.000 1.000 60 60 60 1.000 343.4896 414.3490 447.7135 1.000 1.000 343.4896 414.3490 447.7135 1.000 2 3

Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 60.000.

Table 11. StomatalDensity Location N Subset for alpha = 0.05 1 UP Drive Student-Newman-Keulsa Botanical UP Sig. UP Drive Duncana Botanical UP Sig. Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 6.000. 6 6 6 .344 6 6 6 10.1667 11.5000 13.5000 .067 10.1667 11.5000 11.5000 13.5000 .163 2

Table 12. StomatalIndex Location N Subset for alpha = 0.05 1 UP Drive Student-Newman-Keulsa Botanical UP Sig. UP Drive Duncana Botanical UP Sig. Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 6.000. 6 6 6 1.000 6 6 6 1.000 .6983333 1.2566667 1.3155500 .679 .6983333 1.2566667 1.3155500 .679 2

Table 13. Correlations ApertureLe EpidermalWall GuardCell ngth Pearson Correlat 1 ApertureLengt ion h Sig. (2tailed) N Pearson Correlat -.086 EpidermalWall ion Area Sig. (2tailed) N .249 180 180 .603 180 .967 18 .774 18 1 -.039 -.011 .073 180 .249 180 .053 180 .020 18 .244 18 -.086 .145 .543* .290 Area Area StomatalIn StomatalDe dex nsity

Pearson Correlat .145 GuardCellArea ion Sig. (2tailed) N Pearson Correlat .543* StomatalIndex ion Sig. (2tailed) N Pearson Correlat .290 StomatalDensit ion y Sig. (2tailed) N .244 18 .774 18 .104 18 .000 18 18 .073 .396 .798** 1 .020 18 .967 18 .026 18 18 .000 18 -.011 .523* 1 .798** .053 180 .603 180 180 .026 18 .104 18 -.039 1 .523* .396

*. Correlation is significant at the 0.05 level (2-tailed). **. Correlation is significant at the 0.01 level (2-tailed).

COMPUTATION FOR THE CALIBRATION CONSTANT Tabe 14. Computation of the calibration constant for LPO LPO Trial 1 Stage Ocular 10 10 Trial 2 5 5 Trial 3 10 10

Average: 1 x 10= 1 cc

Tabe 15. Computation of the calibration constant for HPO HPO Trial 1 Stage Ocular 5 20 Trial 2 10 40 Trial 3 15 60 Average: .25 x 10= 2.5 cc