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EXPERIMENT 1

Morpho-Anatomical Studies of Solanum nigrum L. Grown Under Two

Different Environmental Conditions

INTRODUCTION

Management of flyash is a major environmental and economic concern as

a result of the coal-fired power generators all over the world. Flyash consists of

minute glass-like particles, and its deposition on leaves inhibits the normal rate

of transpiration and photosynthesis in the affected plants. Fly ash also affects the

physicochemical characteristics of soil because it is generally basic, rich in

various essential and non essential elements, but poor in both nitrogen and

available phosphorus.

Nearly 15-30% of the total amount of residue generated during burning of

coal in thermal power plants constitute fly ash. Fly ash particles when dumped,

cause a serious problem, to human and animal (Page et al., 1979; Borm, 1997), as

well as to the plants. Fly ash adversely affects plant growth by interfering with

physiological processes, and by influencing pigment concentrations,

bioaccumulations, and cell structures (Darrall, 1989). Photosynthesis,

respiration, stomatal activity, stomatal conductance, transpiration, water uptake,

anatomy of wood and bark, transport process and the overall plant biomass is

adversely affected.

In the present study, the effects of long-term (Seed germination-seed-set

stage) exposures of leaf and whole above ground plant parts of the common

medicinal weed plants, Solanum nigrum to coal-smoke pollutants (fly ash) were

investigated. Estimation of air pollutants from Kasimpur Thermal Power Plant,

Aligarh were examined from 0.5 Km fly ash polluted area) to 15 km away from

source of emission.

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In India out of 90 million tones of fly ash generated annually about

12 million tones is being utilized (Srivastava, 2002). In coming years this figure is

expected to reach 200 million tones by 2012. The Kasimpur Thermal Power

Plants runs on sulphur rich low grade, bituminous type of coal, brought from

different collieries of north India (Table-1), combustion of coal causes emission

of gases which affect plant, animal and human life in the vicinity of the power

plant (Table-2), respective wind velocity throughout the year at the vicinity of

Thermal Power Plant (Table-3.) and the texture of the fly as amended soil (Table-

4).

The Aligarh district (University Campus) is situated in Uttar Pradesh in a

fertile agricultural plain between the Ganga and Yamuna rivers. Geographically

the district falls between 270 29' N and 280 11' latitude and 770 29' E and 780 38'

Longitude about 187 meters above the sea level. Kasimpur the site of thermal

power plant complex, is located in the Morthal Pargana of Koil Tahsil (District) of

Aligarh between 270 59' N and 280 3' N latitude and 780 8' E and 780 93' E

longitude.

This experiment was aimed at finding out the effect of fly ash disposition

on (i) plant biomass (ii) leaf area (iii) Stomatal aperture size (iv) stomatal shape

(v) stomatal indices (vi) size and number of trichomes (vii) septation of

trichomes (viii) shape and size of vessels (ix) shape and size of sieve cells (x)

stomatal conductance (xi) chlorophyll pigment concentrations and (xii)

characteristics of fly ash.

MATERIAL AND METHODS

Plant Sampling and Analysis:

The plant of Solanum nigrum were collected at the same intervals of time

from two sites i.e. Kasimpur area within the radius of 0.5 Km from the source of

fly ash emission, and University Campus 15 Km away from the source which was

considered as control. The plants were cut at the margins of roots and shoots.

Their lengths were measured with the help of meter scale and fresh weights

53

were taken using physical balance. The roots and the shoots were placed

separately in bamboo paper envelops, kept and their dry weighs were

determined.

To study foliar characteristics, replicates of freshly collected and fully

expanded mature leaves of same age and size of S. nigrum were fixed in

formaline-aceto-alcohol (FAA) and stored in 70% Ethanol (Johansen, 1940). For

obtaining epidermal peel, leaf pieces of one cm2 were cut and treated with 30%

HNO3. The peels were washed twice in distilled water, stained with iron alum

and haematoxylin and mounted in Canada balsam for microscopic examination

(Ghouse and Younus, 1972). To determine the number of stomata and the size of

stomatal pore aperture, one cm2 piece were selected. At 10× and 40×

magnifications, necessary photographs were taken.

Ten mature leaves from five plants at both the sites were randomly

selected to calculate leaf area, for this purpose an outline of the shape of each leaf

was drawn on rice papers and the area occupied was measured with the help of

planimeter. The Stomal Index (I) was calculated by the formula: the number of

stomata (S) and epidermal cell (E) in a unit area (Salisbury, 1972).

100×+

=IS

SI

The Stomatal conductance were measured using LI-62,00 portable

photosynthesis system (LI-COR; Lincoln, Nebraska, USA) in the morning Sunny

day.

The length of trichome was also measured with micrometer scale and

their septation and numbers were counted under microscope.

Macerations were prepared using Ghouse and Yunus, (1972) technique,

temporary slides were prepared and stained with safranin and mounted in

glycerin to gathered data on sieve cells and vessels cells.

54

Estimation of Chlorophyll:

For estimation of chlorophyll, one g fresh leaves of S. nigrum collected from

both the sites were crushed in mortar and pestle containing 50 ml of 80%

acetone and filtered through Whatman (No.1) filter paper. The filtrate was

transferred to 100 ml volumetric flask and the volume was made upto the mark

with 80% acetone. The transmittance was read at 480, 510, 645 and 663 nm on

UV-1700 Pharma Spec spectrophotometer (Shimadzu, Japan). The amount of

chlorophyll a, b and Carotenoids was determined as mg/g of fresh leaf according

to the formula given by MacLachlan and Zalik (1963).

Physico-chemical Characteristics of Fly Ash:

The physical and chemical properties of the fly ash, obtained from

Kasimpur Thermal Power Plant, were analyzed by different methods. The

texture of fly ash in relation to particle size was determined by hydrometer

method (Allen et al., 1974). The electrical conductivity of the fly ash was

measured by conductivity meter (Elico., Co Ltd. Hyderabad, India). The pH was

measured with the help of pH meter after obtaining an extract from fly ash and

water suspension in the ratio of 1:1 (w/v). Total organic carbon, total nitrogen,

and total phosphorus were analyzed by Degtjareff method (Walkey and Black,

1934); Microkjeldahl method (Nelson and Sommers, 1972) and Molybdenum

blue method (Allen et al., 1974), respectively. The total metal elements were

determined by mixed acid digestion using conc. HNO3, H2SO4 and HClO4 with the

help of atomic absorption spectrophotometer (SensAA., GBC Scientific

equipment, Australia) (Allen et al., 1974). The equipment was calibrated at the

beginning and end of each testing session by injecting various volumes of

standard solutions by the analyst.

The data obtained were analyzed using student’s‘t’ test and the software

used were Spss-17. The graphs were plaughted using the software Sigma Plot-11.

55

OBSERVATIONS

Fly ash particles are very fine and thus tend to remain air-borne for a long

period. Fly ash dust under certain conditions of humidity, stick to the leaves and

promote chemical as well as physical injuries, and small necrotic dark

brown/black spots appear on the leaves of plant (plate-1 C and D).

Shoot and Root Length:

The plants of Solanum nigrum, at the control site were taller than those at

polluted site i.e. Kasimpur Thermal Power Plant. The reduction in the shoot

length (19.86%) was comparatively less than in the root length (46%). The

length of the shoot at control site was 60.4 cm (± 4.3) and at polluted site 48.4 cm

(± 4.3). On the other hand the length of the root at control site was 30.00 cm (±

5.3) and at polluted site 16.2 cm (± 2.8). Difference in the length of the shoot and

the root could be observed (Plate-1 A and B; Fig- 1 A and B).

Fresh and Dry Weights of Shoots and Roots:

Fresh and dry weights of plant grown at control site were higher than

those of polluted site. The reductions in both fresh and dry weights of shoots

were (13.6%) and (35.2%) and of roots were (24.3%) and (29.1%), respectively.

The fresh weight of shoot at control site was 39.6 g (± 2.4) and the root was 5.9 g

(± .54), while dry weight of the shoot at control site was 14.2 g (± .86) and of the

root was 2.6 g (± .32). The fresh and the dry weights of the shoots and the roots

at polluted site were 34.2 g (± 2.3), 9.2 g (± 1.5) and 4.4 g (± .45), 1.8 g (± .32)

respectively. Difference in the fresh and the dry weights of both the sites could

be observed (Fig- 1 C, D, E and F).

Photosynthetic Pigments:

The decline in amount of pigments in the leaves followed a similar trend.

There was higher reduction in the contents of Chl-b than Chl-a and carotenoid

pigments at polluted site. The reductions in Chl-a (35.9%), Chl-b (58.6%) and

carotenoid (23.2%) were found in the plants of polluted site.

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The pigment concentration at control site were, Chl-a 0.298 (± 0.2), Chl-b

0.382 (± .02) and carotenoid 0.297 mg/g of leaf (± .03). On the other hand the

concentrations were 0.191 (±.01), 0.158 (± .004) and 0.228 mg/g of leaf (± .01),

respectively in the plants of polluted sites. The observation could be followed by

(Fig-2 A, B and C).

Stomatal Features:

The type of stomata found in S. nigrum was 100% anomocytic, in which

the epidermal cells around the guard cells were not distinguishedable from other

epidermal cells, that is subsidiary cells resembled with the general epidermal

cells (Plate –3 A and B).

The stomatal aperture in the stomatal apparatus was larger at polluted

site when compared with the plants at control site. Variations in the width of

aperture also showed variation.

The length and width of stomatal apparatus increased by 34.1 % and

27.2 % respectively at polluted site, when compared with control. Similar trend

of increase in the length of stomatal pore (34.2%) and width of stomatal pore

(13.1%) were found. The length and width of stomatal apparatus were found to

be 36.0 µm (± .81) and 26.4 µm (± .49) in the plants at polluted site. Similarly the

length and width of stomatal pore were 16.5 µm (± .46) and 4.9 µm (± .42) were

at polluted site. The length of stomatal apparatus 23.7 µm (± .58) and width of

stomatal apparatus 19.2 µm (± .23), while the length of stomatal pore 10.8 µm

(± .69) and their width 4.2 µm (± .82) were noticed at control site. (Fig-3 A, B, C

and D; Plate-3 C and D).

In comparison to control, stomatal conductance, stomatal index and leaf

area were found reduced when compared with that of polluted site. The

reductions were much higher in leaf area (36.0%) of polluted site, when average

reduction in stomatal conductance (17.8%) and stomatal index (16.5%) were

noticed. The stomatal conductance at control site was 0.84 µmol m-2 s-1 (± 0.56)

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and stomatal index 31.0 (± 1.78). The leaf area was larger at control site 43.4 cm2

(± 3.59) when compared with the polluted site 27.8 cm2 (± 2.48). On the other

hand the stomatal conductance of polluted site was 0.69 µmol m-2 s-1 (± 0.51) and

stomatal index 25.9 (± 2.26). (Fig-4 A, B and C; Plate-3 A and B).

Trichomes:

The numbers of trichomes, average length of trichome, and septation in

trichome were higher at polluted site than control site. The increase in trichome

length was 31.7% and septation 18.1%, number of trichomes was 28.2% at

polluted site. The length of trichome was 36.2 µm (± 1.40), septation 2.0 µm

(± .12) and the number of trichome 6.6 was (± .92) in the plants at control site.

On the other hand the plants at polluted site had higher trichome length 53.0 µm

(± 2.90), their septations 2.5 µm (± .06) and the number of trichomes were

9.2 (± .86). (Fig-5 A, B and C; Plate-4 A, B C and D).

Size of the Vessels and Sieve Cells:

The sizes of vessel elements and sieve tube elements were found

increased in the plants at polluted site than those of the control site. The width of

vessel element width was found to be increased prominently than its length at

the polluted site. The increased in vessel elements length and width were 1.2%

and 6.2% at polluted site. The length and width of sieve tube elements 18.5%

and 16.6% respectively. The length of vessel elements at polluted site was

83.7 µm (± 6.36) and width 19.3 µm (± .49). On the other hand the length and the

width of vessel elements at control site were 82.7 µm (± 2.59) and 18.1 µm

(± 1.09) respectively. The length and width of sieve tube elements were

201.7 µm (± 6.58) and 36.0 µm (± 2.10) respectively, whereas at control site

these were 164.3 µm (± 7.65) and 30.0 µm (± .99) respectively. (Fig-6 A, B, C and

D; Plate-5 A, B, C and D; Plate-6 A, B C and D).

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Physico-Chemical Characteristics of Fly Ash:

Fly ash samples were collected from Kasimpur Thermal Power Plant area

and analyzed for different parameters. Data of Table (5) were analyzed through

atomic absorption spectrophotometeric technique available in the Department of

Botany, Aligarh Muslim University, Aligarh.

DISCUSSION

The aim of the experiment was to find out the effects of fly ash deposition

on the growth of the plant of Solanum nigrum, and certain morphological and

anatomical features such as, leaf area, photosynthetic pigment, stomatal index,

stomatal conductance, stomatal aperture, number and length of trichomes, size

of vessel elements and sieve tube elements. The data revealed that the growth

parameters (lengths of the shoots and the roots; fresh and dry weight of the

shoots and the roots; leaf area; photosynthetic pigments; stomatal index and

stomatal conductance) decreased whereas the length and the width of stomata;

length and width of stoma; length of trichomes, number of trichomes and their

septation; length and width of vessel elements, and length and width of sieve

tube elements increased at Kasimpur Thermal Power Plant (the polluted site)

when compared with the non-polluted site (control), the campus of Aligarh

Muslim University.

Particles of fly ash are fine and tend to remain suspended in air for a long

period of time. Fly ash dust, under certain conditions of humidity, sticks to the

leaves or fruits and promotes chemical as well as physical injuries. Stunted

growth, low photosynthetic pigment content, reduced leaf area, decrease in

biomass, low stomatal conductivity and stomatal index in the plants grown at fly

ash polluted site were noticed, which might be attributed to the toxic effects of

specific fly ash constituents such as B, As, Se, Mo, Al and Cd. These elements are

readily absorbed by the plants and are accumulated in the plant tissues. Plants

have various intracellular sites and/or metabolism, hence may readily suffer

from the additive and synergistic effects of different toxic metal presents in the

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fly ash. An increase in available Ca and Mg in the absence of an increase K may

antagonizes plant K uptake and eventually causes K difiency symptoms. The

impact of fly ash on plants has also been investigated in selected fly ash disposal

sites with minimal natural vegetation. It is evident that the fly ash had dual

effects on various plants i.e. promotion and inhibition of growth depending upon

the dose. Some toxic compounds, namely dibenzofuran and dienzo-p-dioxine and

metals namely Ni, As, Cd, Pb, Se, Zn, Ca, etc were reported to occur in fly ash, that

were thought to be responsible in poor growth of many vegetable plants

including aquatic species. It is generally observed that most of the plant growing

at fly ash disposal sites exhibited suppressed growth which might be due to

deficiency of nitrogen in the fly ash rich soils (Lazar et al 2008 and Singh and

Kolay, 2009). Decrease in length and width of Solanum nigrum at polluted site,

under the stress of fly ash, might be attributed to the toxic effects of the heavy

metals and other derivatives formed as a result of interference of metallic

elements.

The declination in growth parameters i.e., length and weigh of S. nigrum

might also be due to deposition of fly ash in higher amount on the above ground

parts. Both the high alkaline pH and the excess levels of soluble elements

released from fly ash might induce hazardous effects on plant root and the

rhizosphere. Increased soil pH might result in loss of applied as well as

indigenous soil nitrogen. The high pH in fly ash may be hazardous towards major

groups microbes that play an important role in nitrogen fixation a loss in length

and weight of S. nigrum might also be due to change in soil pH.

There are reports of toxic effects of specific fly ash constituents in plants,

especially B, As, Se, Mo, V, Al and Cd which are considered to be extremely

hazardous to plants if accumulated in the plant tissues (Townsend and Hodgson

1973; Hodgson and Buckley, 1975; Adriano et al., 1980; El-Mogazi et al., 1988;

Inouhe et al., 1994; Singh et al., 1997; Rai et al., 2000). It is also possible that the

toxic constituents of fly ash might have interfered in the metabolic reactions of

S. nigrum that cause suppression in growth.

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Reduction in plant growth of S. nigrum might be attributed to higher

concentration of some metals, such as Cu, Fe, Zn and Ni. As greater availability of

metals results in lowering of pH of fly-ash leading to detrimental effects on

plants (Tripathi et al., 2000). Plants have various intracellular sites and/or

metabolisms that are sensitive to metal ions and, hence, may readily suffer from

the additive and synergistic effects of different toxic metals (Woolhouse, 1983)

present in fly-ash. In turn, plants that have a detoxifying or immobilising

mechanism against specific heavy metals could be the possible candidates for

reclamation of fly-ash polluted soils. Root is the first organ that comes in contact

with the toxic metals in soils and most of the toxic metals can be deposited in the

root tissues. Metal deposition in the root may restrict movement of the toxic

metals to the leaves and other shoot organs (Mishra and Shukla 1986; Inouhe

et al., 1994). Therefore, the different tolerance characteristics of roots could be

an important factor limiting the overall plant growth responses to the fly-ash

constituents.

Potassium deficiency in plants grown on lagoon ash was found to be

mainly caused by the high Ca content of the ash. An increase in available Ca and

Mg in the absence of an increase in available K may antagonise plant K uptake,

and eventually cause K deficiency as has been observed by Plank and Martens

1974. One of the reasons in planth growth reduction of S. nigrum might be the

hinderance in K uptake due to antagonistic effect of Ca and Mg. Although

nutrients available in fly-ash might be beneficial to the plants through soil

application or foliar dusting (Mishra and Shukla, 1986), but regression lines for

tomato yield against foliar boron showed accumulation of around 350 mg of B or

more per gram dry weight of leaf that proved injurious to growth and yield. This

might be true for S. nigrum which showed decrease in plant growth.

Because fly-ash lacks nitrogen, its application, especially at higher

concentrations, results in severe deficiency of nitrogen in soil which is reflected

in plant tissue. Defeciency of nitrogen is an important factor responsible for the

suppressed growth and yield. Reduction in growth of S. nigrum, defiency of

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nitrogen seems to be the major constrain. Aluminium and manganese toxicity in

fly-ash, on the other hand, exhibited different degrees of responses from the

various indicator plants employed, ranging from complete tolerance (Atriplex),

through partial tolerance (spinach) to great sensitivity (barley). Aluminium-

induced root abnormalities were not evident with ash-barley, but were due no

doubt to the high ash calcium (Hewitt, 1948). In S. nigrum there might be some

some role of Aluminium toxicity that was expressed as reduction in plant growth.

Leguminous vegetation, such as Cassia siamea and Pisum sativum, were found to

accumulate Zn, Cu, Ni and Fe at various doses of fly-ash application (25, 50, 75

and 100% fly-ash in soil; Tripathi et al., 2000). Flyash has been utilised to boost

the productivity of a few agricultural crops and leguminous trees (Kumar et al.,

2001; Rai et al., 2002; Tripathi et al., 2002). Cassia siamea has been found to have

antioxidants and a metal detoxification potential when grown on fly-ash and fly-

ash amended with press mud (Kumar et al., 2002). In S. nigrum, contrary to the

leguminous plant an overall reduction in plant growth was noticed.

Any alternative in the soil affects its physical and chemical characteristics.

Addition of fly ash into soil due to aerial deposition is an alteration that results in

the change of physiochemical characteristics of the soil. The important aspect of

fly ash amendment is the change in texture of the soil. Because of a definite size

of ash particles, the soil texture characteristics are shifted from sandy-loam to

loamy-sand type. Increase in cation exchange capacity, pH, and water holding

capacity have been found due to fly ash deposition. Depending upon the size of

ash particles, porosity of soil might increase or decrease. Higher concentration of

carbonates and bicarbonates in the soil due to fly ash addition (Siddiqui et al.,

2004). One of the reason that could be attributed to plant growth reduction is the

change in soil physic-chemical characteristics.

Plant biomass is one of the important parameter that determines overall

plant growth and performance. An increase or decrease in plant biomass could

be correlated with two plant traits viz., stomatal index and leaf area. These three

parameters, stomatal index, leaf area and biomass were used to provide

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quantitative assessment of the plants response towards increased fly ash

deposition.

From the data it was revealed that leaf area of S. nigrum grown at polluted

site was smaller than that grown at control site. Leaf area is an evidence of a

phenotypic response in relation to photosynthesis potential and growth; the

larger will be the leaf the greater will be the growth rate (Grime, 1979; Raven et

al 1999; Gianoli and Gonzalez-Teuber, 2005; Maseda and Fernandez, 2006; Xu

and Zhou, 2008). The extent of leaf damage from fly ash pollutant is normally

related to the amount of stomatal intake. Significantly fewer and smaller leaves

evoke a direct effect on leaf growth. Reduction in leaf size may be occur as a

natural adaptation because the smaller the leaf, the less the absorption of

noxious gasses, as Gridhar (1981) noted in Impatiens balsamina growing near a

thermal power plant.

From this study it was found that coal smoke pollution invariably reduced

the foliar growth in Solanum nigrum. Earlier findings on other species are in

accordance with these results (Pandey and Pandey, 1996; Nighat et al., 1999,

2000). The extent of leaf damage from fly ash pollutants in normally related to

the amount of stomatal intake. Significantly fewer and smaller leaves have been

thought to be affecting the plant growth. Reduction in leaf size may occur as a

natural adaptation because the smaller the leaf, the lesser will be the absorption

of particulates and noxious gases altering the growth and metabolism of the

plants growing near the thermal power plant complex.

Reduction in chlorophyll contents, as observed in the present study has

been frequently observed in the plants growing under fly ash pollution stress

(Nighat et al., 1999; Banergee et al., 2003). Sensitivity of chlorophyll a or b varies

from species to species. Greater sensitivity of Chl-b, as observed in this study, has

also been reported by various workers (Ajay and Subrahmanyum, 1996; Azam,

2009). The reduction in chlorophyll concentration in the polluted leaves could be

due to chloroplast damage (Pandey et al., 1991), inhibition of chlorophyll

biosynthesis (Esmat, 1990) or enhanced chlorophyll degradation. Chlorophyll a

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is presumed to be degraded to phaeophytin, whereas chlorophyll b molecule

loses its phytol group (Rao and Le Blanc, 1996.)

The number and size of stomata in S. nigrum grown at polluted site was

found to be decreased as is evident from the results. Stomata have been

described as the necessary evil (Sutcliffe, 1974); are essential for carbon-dioxide

acquisition but at the cost of water loss (Beerling et al., 1993). The development

of stomata is considered a critical stage in the evolution of advanced land plants

(Hetherington and Woodward, 2003). In S. nigrum probably fly ash particles

caused hindrance in uptake of Co2 and release of O2 during photosynthesis and

vice-versa during respiration. In addition to this reduction in size and number of

stomata also palyed an important role in altering physiology of affected plants.

Stomatal diffusion resistance, and hence conductance, is directly related to the

size and spacing of stomata on the leaf surface i.e. a trade-off between size and

number of stomata (Jones, 1992; Beerling et al., 1993; Wang et al., 2007).

Compared with stomatal length (eg. Guard cell length or stomatal aperture

length), Stomatal index is relatively plastic (Richardson et al., 2001) and

potentially adaptive to environmental change (Carpenter and Smith, 1975;

Woodward, 1987; Poulos et al., 2007; Lake and woodward, 2008; Sekiya and

Yano, 2008).

Decrease in stomatal conductance under fly ash pollution stress is a

common phenomenon (Kellomaki and Wang, 1997). The reduction may be

attributed to lowered photosynthetic rate and high intercellular Co2,

concentration (Ali et al., 1999 a, b) under the condition of stress. Reduction in

leaf number, leaf size, stomatal frequency and stomatal aperture might have

reduced the rate of photosynthesis that were replaced in reduction in growth of

S. nigrum.