Paper 8697

ISSN (Online) 2581-9429
IJARSCT
International Journal of Advanced Research in Science, Communication and Technology (IJARSCT)
Volume 3, Issue 2, March 2023

Impact Factor: 7.301
Effects of Heavy Metals (Nickel & Lead) on Plant

Growth, Protein, Proline, Chlorophyll and Phenol
Content on Sugarcane Saccharum Plants at In-
Vivo Condition
D. Divyashree1 and G. Lavanya2
Research Scholar, PG and Research Department of Microbiology1
Assistant Professor, PG and Research Department of Microbiology2
Kamban College of Arts and Science for Women, Tiruvannamalai, Tamil Nadu, India
Abstract: The tropic and subtropics region Sugarcane is one of the most important cash crops. Crystal
sugar mainly made from Sugarcane. Lower amounts of Nickel caused a conspicuous increase of plant
growth in sugarcane Cultivar 2.0 and 4.0mM of protein and nickel content increased in leaves of
Sugarcane. The most extreme abatement in sugar of 85.1% was seen at 4.0 mM portion of nickel. Younger
leaves of lead treated plants showed greatest impact through yellowing of leaf and decreased leaf region
and bowing of edge of leaf in sugarcane plants. Restraint in the growth of sugarcane plants was begun even
at 1.0 mM portion of Pb. At 4.0 mM portion of Pb 15 folds decrease of sugar content was noticed. Protein
content of this plant showed positive reactions with supply of 1.0 to 4.0 mM centralization of Pb. The
grouping of chlorophyll 'a' and 'b' was additionally diminished with increasing convergence of nickel.
Chlorophyll 'a' was more delicate at all centralization of Ni than chlorophyll 'b'. All out chlorophyll items
in sugarcane plant was invigorated at 1.0 mM of this metal. The convergence of 'a', 'b' and absolute
chlorophyll in leaves of sugarcane plants diminished altogether with increasing levels of lead. Pheophytin
additionally showed same pattern with nickel, aside from pheophytin'b' at 1.0 mM fixation. The plants that
were developed at 2.0 mM dose of nickel gave least action of this catalyst when contrasted with control.
Action of α, β what's more, absolute amylase in plant leaves expanded at expanding concentration of nickel.
Catalase movement was viewed as diminished at abundance portions of lead. Greatest decrease in catalase
action was estimated at 2.0 mM of Pb. Activity of α, β what's more, absolute amylase showed variable
outcomes with various expanding grouping of lead in sugarcane plants. α- amylase had non significant
response at 1.0 and 4.0 mM portion of Pb and inhibitory impact at 2.0 mM convergence of Pb. Action of β-
amylase repressed at 1.0 and 4.0 mM of Pb and stimulated at 2.0 mM fixation.
Keywords: Heavy Metals.
I. INTRODUCTION
The term heavy metals refers to any metallic element that has a relatively high density and is toxic or poisonous even at
low concentration ( Lenntech Water Treatment and Air Purification 2004).Heavy metals include lead (Pb), cadmium
(Cd), nickel (Ni),cobalt (Co), iron (Fe), zinc (Zn), chromium (Cr), iron (Fe),arsenic (As),silver(Ag)and the platinum
group elements. Heavy metals are largely found in dispersed form in rock formations. Industrializations and
urbanization have increased the anthropogenic contribution of heavy metals in biosphere. Heavy metal toxicity in plants
varies with plant species, specific metal, concentration, chemical form and soil composition and pH, as many heavy
metals are considered to be essential for plant growth.
Lead continues to be used widely in many industrial processes and occurs as a contaminant in all environmental
compartments (soils, water, the atmosphere, and living organisms).Trace elements including nickel are present in
different chemical forms because they are associated to diverse organic and inorganic components of the soils such a
Copyright to IJARSCT DOI: 10.48175/568 292

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soluble exchangeable bound to carbonate ,Fe , Mn oxides, organic matter and residual (Sudhakaretal. 1992) . Lead is
very important as far as persistence in the environment is concerned.
Heavy metal binding to various functional groups of proteins, primarily SH groups would modify protein conformation
and result in the loss of activity in many enzymes comprising SH groups in their activity centers. Besides, metal
binding to SH-groups of the physiologically active compounds of low molecular weight would also interfere with cell
metabolism (Mildvan1970).Thus binding to SH-groups alone would already give rise to diverse metabolic disturbance.
Heavy metals differ in their affinity for oxygen, nitrogen- and sulfur- containing ligands, and these differences depend
on the physical and chemical properties. Properties of heavy metal ions.
Saccharumofficinarum,
Genus : Saccharum
Species : Saccharumofficinarum
Sugarcane is a large, strong-growing species of grass in the genus saccharum. It originated in SoutheastAsia and is
now cultivated in tropical and subtropical countries worldwide for the production of sugar and other
products.Saccharumofficinarum, a perennial plant, grows in clumps consisting of a number of strong unbranched stems.
A network of rhizomes forms under the soil which sends up secondary shoots near the parent plant. The stems vary in
colour, being green, pinkish, or purple and can reach 5 m(16 ft)in height. They are jointed, nodes being present at the
bases of the alternate leaves. The internodes contain a fibrous white pith immersed in sugary sap. The elongated, linear,
green leaves have thick midribs and saw-toothed edges and grow to a length of about 30 to 60 cm (12 to 24 in) and
width of 5 cm (2.0 in). The terminal inflorescence is a panicleup to 60 cm (24 in) long, a pinkish plume that is broadest
at the base and tapering towards the top. The spikeletsare borne on side branches and are about 3 mm (0.12 in) long and
are concealed in tufts of long, silky hair. The fruits are dry and each one contains a single seed.
Sugarcane harvest typically occurs before the plants flower, as the flowering process causes a reduction in sugar
content. Sugarcane belongs to the genus Saccharum which has numerous species that are grown to obtain sugarcane
which is used to manufacture sugar. Saccharumofficinarum is the plant that is used to produce about 70% of the total
sugar produced worldwide.Sugarcane is a perennial plant that grows in the tropical and sub tropical regions, loves a lot
of sun and water but does not like its roots to be waterlogged. It is native to South Asia. Sugarcane has jointed and stout
stalks that are rich in sugar. It is the most grown crop in the world. Primarily used for sugar production, with about 70 -
80% of the sugar being produced from it and most of the rest being produced from sugar beets. Sugar making from
sugarcane developed in India about 5000 years ago. It spread to Europe in the 1400's. It is propagated by cutting the
stems and planting them this being the most common method of growing sugarcane.
Heavy metals are known to induce free radical formation and a consequent oxidative damage in senescing leaf cells
under light(pandaandpatra2000).The objective of study was to find out the toxicity level of nickel and lead on plant
growth, pigment contents, proteins contents, phenolic contents , flavoinds content and certain antioxidative enzymes
Nutrition Facts
1. Sugarcane contains various essential minerals like calcium, phosphorus, iron, magnesium and potassium.
2. Sugarcane juice is rich in flavonoid and phenolic compounds which are powerful antioxidants that prevent cell
damage by free radicals and reduce risk of cancer and heart disease.
3. It has a low glycemic index between 30-40, about half that of refined sugar. It has no simple sugars. The sugar
is in raw form.
4. Sugarcane juice is easily digestible, provides instant energy and hydrates the body.
5. Sugarcane juice is alkaline, contains soluble fiber, proteins and vitamins A, C and Bcomplex.
II. MATERIALS AND METHODS

2.1 Soil Preparation
In the present study local cultivar of saccharumofficinarum(sugarcane) was selected to study the effect of selected
heavy metals nickel and lead. The soil samples were collected from three different areas and where the agricultural
lands were irrigated with polluted water. The collected soil samples were air-dried, passed through a 2-mm mesh sieve
to remove any roots and stones, mixed together properly, and used for further studies and send to soil analysis

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(AAS).Sample more than present of heavy metals so, decline the plant growth. Soil samples (1kg each) were then
packed in sterile polythene bags and maintained at green house for 30 days.
Control soils were set up without heavy metal and all the set-ups were prepared in 4 replicates.
2.2 Seed Inoculation

Seeds of saccharumofficinarum(sugarcane) were sown. Watering was done at regular intervals. The whole experiment
was conducted in the green house condition. The morphological enzymatic and non enzymatic changes were studied at
30 days interval after initiation of heavy metal stress. Atomic absorption spectrometry was used to analyse the metal
uptake by plants at the respective intervals (PritiBhardwajet.al., 2009)
2.3 Heavy Metal Analysis Of Soil Plant Sample

All the soil samples and plant samples were air dried, sieved to (<2mm) and thoroughly mixed, soil analysis was done
for analyzing the heavy metals like nickel and lead .
2.4 Analytical Techniques

Fresh plant samples were used for the estimation of proline, proteins, chlorophyll and phenolic contents was done with
spectrometric assay.
2.5 Biochemical Analysis

Determination of proline content
Free proline content was estimated by following the method of Bates et al.(1973).Fresh 0.5gm root and shoot samples
were homogenized in 5ml of 3% sulposalicyclic acid using a mortar and pestle.About 2ml of extract was taken in test
tube and to it 2ml of glacial acetic acid and 2ml ofNinhydrin reagent were added.The reaction mixture was boiled in a
water bath at 100C for 30 mintues.After cooling the reaction mixture, 4ml of toluene was added .After thorough mixing
,the chormophore containing toluene was separated and absorbance of redcolour developed was read at 520nm against
toluene black on UV-visible spectrophotometer(Chemito,UV-2600).The proline concentration was determined using
calibration curve and expressed as mg proline per g fresh weight of tissue.
Estimation of protein content

Protein content was determined by the method of lowery.0.5g of plant sample (shoot)was homogenized in 10ml of
20% Trichloroacetic acid(TCA).The homogenate was centrifuged in 10minutes for 3000rpm.The supernant was
discharged and the pellet was re-extracted with 5ml of 0.1NaoH.1ml of the extract was taken in a test tube.After that
solution was mixed well and kept in dark for 10 minutes.Later 0.5ml of folinphenol reagent was added and the mixture
was kept in dark for 30 minutes. The sample was read at 660nm in the UV-spectrophotometer.
Total phenol content

0.5g of fresh sample was incubated homogenized 3.0ml 0f KPO4 buffer using mortar and pestle. The extract was
centrifuged at 2000rpm for 10minutes.The supernatant was transferred to a tube.0.5ml of extract and add 0.1ml of folin
incubate at room temperature for 15minutes.After add 2.5ml of saturated Naco3,read at 520nm against Guaiol standard
on UV-visible spectrophotometer.
Determination of Total chlorophyll content

The material was processed in the fresh state immediately after collection .After fine chopping portions weighing 0.5g
were measured off on an analytic balance. The measured –off material was then homogenized in a homogenizer with
the addition of 10ml of 80%acetone.A primary acetone extract containing all chloroplast pigments was obtained in this
way. The extract was centrifuged at 2500rpm for 5minutes.Since the concentration of pigments was in most cases too
great for reading to be performed on a spectrophotometer, the obtained extract was diluted by adding 9ml of 80%
acetone per ml of extract. The extract produced in this way was subjected to read at 663nm UV-visible
spectrophotometer.

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III. RESULTS AND DISCUSSION
Heavy metal PB&NI analysis of soil
The concentration of heavy metals Lead& Nickel were analysed in soil samples (Control ,A&C).The results clearly
indicates that Lead& Nickel were increase accumulated in experimental soil when compared to contro. .In the soil
sample B more lead is accumulated, so there was no seed germination. Lead is known to inhibit seed germination of
(Morzuk and funicclli1982).Inhibition of germination may result from the interference of lead with important enzyme
(Baker1972).
Lead effect on plants
Lead (Pb) is one of the most abundant toxic elements in the soil. It exerts adverse effect or morphology, growth and
photosynthetic process of plant.
Nickel effect on plants
Nickel concentration is increasing in certain areas by human activities such as mining works, emission of smelters
burning of coal and oil sewage (Gimeno-Garciaet.al,1996).
Heavy metal PB&NI analysis of plant( saccharumofficinarum)
Concentration of heavy metals Lead & Nickel were analysed in plant samples grown in (Control, A&C).The results
clearly indicates that the above said heavy metals were increasely accumulated in experimental plants when compared
to control (Fig 2).
Figure 2: Heavy metals (Pb&Ni) analysis of soil test
0.25
Co
nc
Soil 0.2
test analysis
en 0.2
tra
tio0.15
0.13
n
Pb
of
0.1 0.08 Ni
pb
&n 0.06
i 0.05 0.04
0.03 0.03
0.02
0
A B C D
SOIL TEST
Figure 3: Heavymetals (Pb&Ni) analysis of plant test
co 0.6
nc 0.51 plant test analysis
en 0.5
0.42
tra
0.4 0.36
tio
0.3
n 0.3
of 0.22 0.22 pb
pb 0.2 ni
&n
0.1
0
Z1 Z2 Z3
plant test

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Figure 4: Effect of increasing (Pb&Ni) concentration of proline content
Co 0.45
0.4
nc 0.4 PROLINE CONTENT
en 0.35
tra 0.3
0.3
tio
n 0.25
0.2
of 0.2
pr 0.15
oli
0.1
ne
0.05
0
sample(a) sample© control
plant sample
Figure 5: Effect of increasing (Pb&Ni)concentration of protein
Co 0.9 0.8
nc 0.8 PROTEIN CONTENT
en 0.7
tra 0.6
0.6
tio
n 0.5
0.4
of 0.4
pr
0.3
ot
ein0.2
0.1
0
plant sample
Figure 5: Effect of increasing (Pb&Ni) concentration of phenol content
Co 0.2
nc 0.18
PHENOL CONTENT
ent 0.16
rat 0.14
ion 0.12
of
0.1
ph 0.18
0.08
en
ol 0.06 0.12
0.04
0.06
0.02
0
plant sample

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Figure 6: Effect of increasing (Pb&Ni) concentration of chlorophyll content
Co
nc
0.16
en 0.14
tra 0.14 CHLOROPHYLL CONTENT
tio 0.12
n 0.1
of 0.1
chl 0.08
or 0.06
0.06
op
hyl 0.04
l
0.02
0
plant sample
IV. BIOCHEMICAL ANALYSIS

Proline content
In the present investigation, metal stress resulted into a sharp decrease in proline content. However,the metals stress-
reduced in proline accumulation (Fig 3).The amount of proline present in the between samples(A&C) were observed as
0.2mg &0.3mg.The proline concentration is very low ,when compared with control.
Protein content
The amount of protein present in the between samples (A&C) were observed as 0.6mg, 0.4mg respectively .The protein
concentration is very low, when compared with control. Control showed higher protein content than sample (Fig 4).
However, a continous decrease in protein content with increase in metal stress was observed. Heavymetals caused
significant decrease in growth and protein content. Protein content decreased in metals exposed plants at metals
concentration observed by Abdul Ghani(2011)in wheat.
Chlorophyll content
The chlorophyll content was higher in control sample. It was decreased with increase in metal stress(Fig 5).Excess of
nickel in soil causes various physiological alterations and diverse toxicity symptoms such as chlorsis&necrosis in
different plant species(Zornozaetal.d1999)
Phenol content
The total phenol content was gradually decreased with progressing metal stress. However contrasting results were
observed in phenol level in comparing with control(Fig 6). In metal stress plants reduction in phenol content was
observed by ( yingli yang(2011)in wheat.
V. CONCLUSION
In the present investigation, it is concluded that lead and nickel concentration.It was observed that produced toxic
impact on germination and growth of saccharumofficinarum (Sugarcane) plants as compared to control .Seedling
growth is considered as an indicator of metals stress on plant vigor. This shows that vegetable crops have the ability to
uptake the heavy metals through their roots and transport them to the edible portion of the plant that are consumed by
people or fed to animals. Then increased concentration in human food chain over a long time can provoke detectable
damage to health effect.
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ISSN (Online) 2581-9429

Volume 3, Issue 2, March 2023

Effects of Heavy Metals (Nickel & Lead) on Plant

Keywords: Heavy Metals.

Copyright to IJARSCT DOI: 10.48175/568 292

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II. MATERIALS AND METHODS

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2.2 Seed Inoculation

2.3 Heavy Metal Analysis Of Soil Plant Sample

2.4 Analytical Techniques

2.5 Biochemical Analysis

Estimation of protein content

Total phenol content

Determination of Total chlorophyll content

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IV. BIOCHEMICAL ANALYSIS

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Volume 3, Issue 2, March 2023

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