ACKNOWLEDGEMENT

It gives me great pleasure in writing this acknowledgement as a token of gratitude

to people, who have always been supportive and helpful throughout the training
work. I bow my head to the almighty for blessing god showered on me, which
alone helped me to complete this project.
I would like to express my deep sense of gratitude and hearty thanks to Dr.
Sanjay Biyani (Director, Academics), Biyani Girls College, Jaipur, for providing
me permission, support and help at required time during my entire work.
I take great opportunity to thank Dr. Priyanka Dadhupanthi Associate
Professor and Head, Department of biotechnology, Biyani Girls College, Jaipur,
for his excellent guidance.
I have boundless pleasure in expressing profound sense of reverence and
gratitude to my esteemed advisor Dr. Sunita Rao (Assistant Professor, Biyani
Girls College) for prudent and didactic guidance, patronage, keen interest and
acuminous suggestion in planning and execution of this research work and
preparation of this manuscript. Her analytical quick perusal of the manuscript and
constant inspiration in completion of this elusive task is not only praise worthy but
also unforgettable.
I express my cordial thanks to Dr. Sunita Rao for her support throughout
my training programme.
My family has been an emotional anchor; my parents have supported me in
all my trials and tribulations and encouraged me to reach my goals.
I express my cordial thanks to my devoted and caring friends for timely
support.

SAKSHI PANDEY

1 |3 7
 DECLARATION
I hereby declare that the project entitled “Effect of zinc metal on pea (Pisum Sativum)
plant” presented in this report is a record of experimental work carried out by me. The
work embodied in it has not been submitted for the award of any other degree or diploma.

Date:

Place: Jaipur ` (SAKSHI PANDEY)

2 |3 7
CONTENTS PAGE

1. INTRODUCTION 4-13
2. LITERATURE REVIEW 14-16
3. MATERIALS AND METHODOLOGY 17-25
4. RESULT AND DISCUSSION 26-332
5. CONCLUSION 33
6. SUMMARY 34
7. REFERENCES 35-37

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 Chapter 1

INTRODUCTION

HEAVY METALS AND THEIR TOXICITY IN PLANTS

A heavy metal is toxic when relatively it is dense metal or metalloid that is noted for its noted for
its potential toxicity, especially in environmental contexts. Heavy metal toxicity means excess of
required concentration or it is unwanted which were found naturally on the earth , and become
concentration as a result of human caused activity, enter in plant, animals and human tissues via
inhalation , diet and manual handlings , and can bind to and interfere with the functioning of vital
cellular components.

Heavy metals include - the transition-metal elements essential to plant nutrition.

 Iron (Fe)
 Zinc (Zn)
 Manganese (Mn).
 Copper (Cu)
 Nickel (Ni)
 Molybdenum (Mo)
 Cobalt (Co) [Which is required for the nitrogen fixation in leguminous crops.]

The non-essential elements

 Chromium (Cr)
 Cadmium (Cd)
 Mercury (Hg)
 Lead (Pb).

Effect of heavy metal

The heavy metal available for plant uptake are those present as soluble components in the
soil solution or those solubilized by root exudates. Plants require certain heavy metals for
their growth and upkeep, excessive amount of these metals can become toxic to plants and
ability of plants to accumulate essential metals equally enables them to acquire other non-

4 |3 7
 essential metals. The toxic effect lead to a decrease in plant growth which finally results in
the death of plants.

All these elements are toxic to crop plants at high tissue concentrations. Nevertheless, Mn
toxicity can reduce crop yields on acidic soils, and Mn and Fe toxicities occur on
waterlogged or flooded soils. Toxicities can also arise in soils enriched in specific heavy
metals by the weathering of the underlying rocks or anthropogenic activities.
hyperaccumulate heavy metals are also providing insight into the ability of plants to exclude
and tolerate heavy metals in their tissues.

PLANT SELECTED: Pisum sativum

REASONS BEHIND IT’S SELCTION:

 Pea plant is easy to grow in limited area.

 It shows chlorosis at higher concentration of Zn metal.
 In pea plant the Zn treatment depicts the reduction in root, steam and leaves growth.

1.1 GENERAL INTRODUCTION OF Pisum sativum L (PEA)

The pea is most commonly the small spherical seed or the seed-pod of the pod fruit Pisum
sativum. Each pod contains several peas, which can be green or yellow. Pea pods are
botanically fruit, since they contain seeds and developed from the ovary of a (pea) flower. The
name is also used to describe other edible seeds from the Fabaceae such as the pigeon
pea (Cajanus cajan), the cowpea (Vigna unguiculata), and the seeds from several species
of Lathyrus.

P. sativum is an annual plant, with a life cycle of one year. It is a cool-season crop grown in
many parts of the world; planting can take place from winter to early summer depending on
location. The average pea weighs between 0.1 and 0.36 grams. The immature peas (and in snow
peas the tender pod as well) are used as a vegetable, fresh, frozen or canned; varieties of the
species typically called field peas are grown to produce dry peas like the split pea shelled from
the matured pod. These are the basis of pease porridge and pea soup, staples of medieval cuisine;
in Europe, consuming fresh immature green peas was an innovation of Early Modern cuisine.

5 |3 7
The wild pea is restricted to the Mediterranean basin and the Near East. The earliest
archaeological finds of peas date from the late neolithic era of current Greece, Syria, Turkey and
Jordan. In Egypt, early finds date from ca. 4800–4400 BC in the Nile delta area, and
from ca. 3800–3600 BC in Upper Egypt. The pea was also present in Georgia in the 5th
millennium BC. Farther east, the finds are younger. Peas were present
in Afghanistan ca. 2000 BC, in Harappa, Pakistan, and in northwest India in 2250–1750 BC. In
the second half of the 2nd millennium BC, this pulse crop appears in the Ganges Basin and
southern India.

1.2 BIOLOGICAL CLASSIFICATION

Kingdom: Plantae

Division : Angiosperms

Subdivision : Eudicots

Class: Rosids

Order: Fabales

Family: Fabaceae

Genus: Pisum

Species: P. sativum

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1.2 Uses

Peas are cultivated for the fresh green seeds, tender green pods, dried seeds and foliage (Duke,
1981). Green peas are eaten cooked as a vegetable, and are marketed fresh, canned, or frozen
while ripe dried peas are used whole, split, or made into flour (Davies et al., 1985). In some parts
of the world, dried peas are consumed split as dahl, roasted, parched or boiled. "Green peas are
the number one processed vegetable specifically in UK and USA. Green foliage of garden pea is
also used as vegetable in parts of Asia and Africa. Leaves are used as a pot herb in Burma and
parts of Africa" (Kay, 1979). Some cultivars are grown for their tender green pods, which are
eaten cooked or raw. "Oil from ripened seed has antisex hormonic effects; produces sterility and
antagonizes effect of male hormone" (Duke, 1981). "Based on protein digestibility of peas in
broilers, it is reported that the natural protein of peas and faba bean is almost entirely digested in
the small intestine and the impaired performance in literature was attributed to an increased
secretion of endogenous protein" (Huisman and Van der Poil, 1994). Pea is being used in a
growing snack market. One snack item is prepared by soaking the peas overnight and frying
them in palm oil or coating them with other food items such as rice flour before frying for the
purpose of imparting different flavors. Another product is prepared by finely grinding the peas
and extruding them under pressure to create different shapes. The different shapes are then fried,
seasoned and packaged (Jambunathan et al., 1994). "Cultivars such as 'Alaska' 'Super Alaska,'
'Supergreen,' and 'Alaska Wilt Resistant have long been the standard type of canning pea.
Wrinkled-seeded garden peas are sweeter than smooth seeded types. Marrowfats is a large
flattened pea type that is popular for making snack items and are also used for reconstitution and
canning in the UK. Canning peas are divided into two classes: Alaska and sweet. Of the sweet
type is 'Surprise' (most important) and 'Resistant Surprise' (gaining in popularity because of its
disease resistance). Some midsummer varieties are: 'Pride,' 'Early Perfection,' 'Ace,' and
'Wasatch'. Late cultivars, which ripen about 14 days after 'Alaska,' are: 'Perfection' and its
various strains, as 'Dark-green Perfection,' 'Bridget,' 'Superior,' 'New Era,' ëPugetí and
'Shoshone.' New cultivars, too numerous to mention, have been entered into production in the US
and other countries. Canning cultivars usually have a tough skin that holds its shape during
canning. For home garden use, 'Little Marvel,' 'Laxton Progress,' 'World's Record,' 'Wando' holds
quality well in hot weather" (Duke, 1981).

7 |3 7
a. Traditional Medicinal Uses

"Seeds are thought to cause dysentery when eaten raw. In Spain, flour is considered emollient
and resolvent, applied as a cataplasm. It has been reported that seeds contain trypsin and
chymotrypsin which could be used for contraceptive, ecbolic. fungistatic and spermicide"(Duke,
1981). Smart (1990) reported that there are no significant amounts of toxicity or anti-metabolites
in peas.

1.3. Botanical Characteristics:

a. Taxonomy, Morphology and Floral Biology

Duke (1981) reported that garden peas are treated as P. sativum ssp. hortense Asch. & Graebn.,
field peas as P. sativum ssp. arvense (L.) Poir., and edible podded peas as P. sativum ssp.
macrocarpon; early dwarf pea as P. sativum var humile. Later, Smart (1990), based on studies
undertaken by Ben-Zeíev and Zohary (1973), and Polhill and van der Maesen et al., (1985)
reported that pea comprises only two species, viz; Pisum sativum and P. fulvum Sibeth. & Smith.
"It is a self pollinated annual herb, bushy or climbing, glabrous, usually glaucous; stems weak,
round, and slender, 30-150 cm long; leaves alternate, pinnate with 1-3 pairs of leaflets and a
terminal branched tendril leaflets ovate or elliptic, 1.5-6 cm long" (Duke, 1981). The leaf type
could be conventional, semi-leafless and leafless (Davies et al., 1985). Leaf size in most cases
increases up to the first node bearing the first flower. Stipules are large, leaf like and up to 10 cm
long. The inflorescence of pea is a raceme arising from the axil of the leaf. Corolla white, or
pink, or purple; pods swollen or compressed, short-stalked, straight or curved, 4-15 cm long, 1.5-
2.5 cm wide, 2-10 seeded, 2-valved, dehiscent on both sutures (Gritton, 1980; Duke, 1981). The
node at which the first flower emerges is characteristic of a given variety; in temperate regions
the number of nodes at which the first flower emerges is reported to vary from 4 in the earliest to
about 25 in late maturing types under field conditions (Gritton, 1980). Flowers borne on the
same peduncle produce pods that mature at different times, the youngest being at the tip. On a
whole plant basis, flowering is sequential and upwards from node to node. Seeds are globose or
angled, smooth or wrinkled, exalbuminous, whitish, gray, green, or brownish; 100 seeds can
weigh from 10 to 36 grams; germination cryptocotylar (Duke, 1981).

8 |3 7
b. Ecology

Peas require a cool, relatively humid climate and are grown at higher altitudes in tropics with
temperatures from 7 to 30°C (Duke, 1981; Davies, 1985) and production is concentrated between
the Tropics of Cancer and 50° N (Davies, 1985). As a winter annual, pea tolerates frost to -2°C
in the seedling stage, although top growth may be affected at -6°C. Winter hardly peas can
withstand -10°C, and with snow cover protection, tolerance can be increased to -40°C
(Slinkard et al., 1994). As described in Slinkard et al. (1994), the optimum temperature levels for
the vegetative and reproductive periods of peas were reported to be 21 and 16°C, and 16 and
10°C (day and night), respectively. "Temperatures above 27°C shorten the growing period and
adversely affect pollination. A hot spell is more damaging to peas than a light frost. Peas can be
grown successfully during midsummer and early fall in those areas having relatively low
temperatures and a good rainfall, or where irrigation is practiced. For very early crops, a sandy
loam is preferred; for large yields where earliness is not a factor, a well-drained clay loam or silt
loam is preferred" (Duke, 1981).

1.4. Crop Culture

a. Field Cultivation

Dry peas serve as rotational crops in the Palouse area in a state of eastern Washington and
Northern Idaho. It is considered as an alternative to the cereal grain production and provides the
basis to break disease cycles in winter wheat, improve soil fertility status and improve weed
control. "Peas reduced the fertilizer requirements of maize by 20-32 kg/ha in India compared
with wheat or fallow, respectively; in France it was estimated that about 50 kg/ha of N are
returned to the soil by peas" (Davies et al., 1985). Pea growing seasons vary from 80-100 days in
semi-arid regions and up to 150 days in humid and temperate areas (Davies et al., 1985).

Peas are propagated only from seed. "At higher temperatures germination is rapid, but seedlings
may die from various pathogens in the soil. As temperature rises during growing season, yield
drops off rapidly. In New York, yields are highest when seeds are planted during first 2 weeks of
April; for each 2-week delay in planting, yield of shelled peas decreased about 400 kg/ha"
(Duke, 1981). Thorough preparation of soil is very important, especially when the seed is
broadcast or planted with a grain drill, as no subsequent cultivation is given thereafter. In the

9 |3 7
Palouse region of eastern Washington and northern Idaho peas are sown between March 25th at
lower elevations to May 10th at higher elevations when the soil temperature is above 4°C; for
planting winter peas, September 15th to the 30th has been recommended (Muehlbauer et al.,
1983). Recommended plant densities vary tremendously depending on soil type, cultivar, seed
size, and biotic factors, particularly diseases. In the Palouse area of Washington state, USA,
spring peas are sown at the rate of 140-195 kg ha-1 or 89-108 seeds m-2, while fall sown peas are
planted at the rate of 85-135 kg ha-1 or 71-104 seeds m-2 (Muehlbauer et al., 1983).

Peas are relatively unresponsive to fertilizers, particularly nitrogen; additions are necessary when
nodulation is poor or fails completely. When the seeds are treated with the Rhizobium, care must
be taken in the choice of fungicide seed treatments to prevent potential toxicity (Muehlbauer et
al., 1983). The amount of nitrogen fixed through symbiosis with Rhizobia is reported to vary
from 71 kg N per hectare in Alabama to 119 kg N per hectare in Wisconsin (Mahler et al., 1988).
The N concentration in pea leaf tissue is reported to range from 1.8 to 2.3% (Mahler et al., 1988).

b. Harvesting

Peas grown for home use or for fresh market are picked by hand before the seeds are fully
matured and still in the pod and are used for immediate consumption. In some cases, gardeners
and commercial growers make two or three pickings, depending on maturity, while other
growers make only one picking, in which the vines are pulled and all pods are removed. "Peas
for processing are harvested with machines of various types. Sometimes vines are cut with a
mowing machine, windrowed and loaded onto trucks with a hay loader. Pea harvesters that mow
the peas and load directly onto trucks are common in major pea growing areas. Pea vines are
hauled to a vining station, where pods are separated from vines, after which seeds are separated
from pods" (Duke, 1981). Current methods of processing pea harvest involve the use of "viners"
that harvest the pods and remove the peas from the pods in one operation in the field. The peas
are then transported to the processing plant where they are quickly processed. Dry peas are
harvested when the pods are completely dry and can be threshed directly in the field by a
combine. For dry peas timely harvest is important for maintaining quality and is usually done
when the seed moisture content is less than 13%. Both premature harvesting and harvesting too
late reduce the quality of the dry pea crop. If the pea crop is over mature, harvesting early in the

10 |3 7
morning or during the evening when relative humidity is low will minimize shattering and seed
breakage (Muehlbauer et al., 1983).

1.5. Yields and Economics

Pea is among the four important cultivated legumes next to soybean, groundnut, and beans
(Hulse, 1994). Total world dry pea production rose from 8.127 million metric tons in 1979-81 to
14.529 million metric tons in 1994 while acreage varied from 7.488 to 8.060 million hectares for
the same years (FAO, 1994). The highest productivity for pea was reported in France at 5088 kg
per hectare in 1994, about eight times more than the African average yield. In 1994, USA total
acreage was 54, 000 hectares with an average yield of 2587 kg per hectare (FAO, 1994).
Important production areas of the world include France, Russia, Ukraine, Denmark and United
Kingdom in Europe; China and India in Asia; Canada and USA in North America; Chile in
South America; Ethiopia in Africa, and Australia (FAO, 1994). "Throughout temperate regions
both green and dried peas are an important garden and field crop. In the United States, ca.
550,000 MT are produced commercially for food annually, and ca. 200,000 MT of field peas for
feed" (Duke, 1981).

Pea is the predominant export crop in world trade and represents about 40% of the total trade in
pulses (Oram and Agcaoili, 1994). The major exporting countries, excluding the European
Economic Commission (EEC), are Australia, Canada and the USA (Oram and Agcaoili, 1994).
Most of the peas from USA were exported to India, Haiti, Peru, and the Philippines in 1995 and
had a total value of US$ 24,210,499 (Directory of US Suppliers & Industry Information, 1996).

1.6. Biotic Factors

Peas are adversely affected by: Ascochyla pisi, Cladosporium pisicola (leaf spot or
scab), Erysiphe polygoni (powdery mildew), Fusarium oxysporum (wilt), Peronospora
pisi (downy mildew), Phythium sp. (pre emergence damping-off), Botrytis cinerea (grey
mold), Aphanomyces euteiches (common root rot), Thielaviopsis basicola (black root rot),
and Sclerotina sclerotiorum (sclerotina white mold). Pea Early Browning Virus (PEBV), Pea
Enation Mosaic virus (PEMV), Pea Mosaic Virus (PMV), Pea top yellows (PTY), Pea seed-
borne Mosaic Virus (PSbMV) and Pea Streak Virus (PSV) constitute diseases caused by viruses,

11 |3 7
while the most important bacterial disease is caused by Pseudomonas pisi (bacterial blight)
(Muehlbauer et al., 1983; Davies et al., 1985; van Emden et al., 1988).

Insect pests include Aphis cracivora (Groundnut aphid), Acyrthosiphon pisum (Pea
aphid), Kakothrips robustus (Pea thrips), Bruchis pisorum (Pea seed beetle), Callosobruchus
chinensis (Adzuki bean seed beetle), Apion sp. (Seed weevil), Sitona lineatus (Bean
weevil), Contarina pisi (Pea midge), Helicoverpa armigera (African bollworm), Diachrysia
obliqua (Pod borer), Agriotis sp. (Cut worms), Cydia nigricana (Pea moth), Phytomuza
horticola (Leaf minor), Heliothis Zea (American bollworm), Etiella Zinckenella (Lima bean pod
borer), Ophiomyia phaseoli (Bean fly), Delia platura (Bean seed fly), Tetranychus sp. (Spider
mites), Pratylenchus penetrants (Root lesion nematodes), Ditylenchus dipsaci (Stem
nematode), Heterodera goettingiana (Pea cyst nematode), and Meloidogyne javanica (Root knot
nematode) (van Emden et al., 1988; Muehlbauer et al., 1983).

Phytoremediation

Phytoremediation (‘phyto’ means plant) is a generic term for the group of technologies that use
plants for remediating soils, sludges, sediments and water contaminated with organic and
inorganic contaminants. Phytoremediation can be defined as “the efficient use of plants to
remove, detoxify or immobilise environmental contaminants in a growth matrix (soil, water or
sediments) through the natural biological, chemical or physical activities and processes of the
plants”. Plants are unique organisms equipped with remarkable metabolic and absorption
capabilities, as well as transport systems that can take up nutrients or contaminants selectively
from the growth matrix, soil or water. Phytoremediation involves growing plants in a
contaminated matrix, for a required growth period, to remove contaminants from the matrix, or
facilitate immobilisation (binding/containment) or degradation (detoxification) of the pollutants.
The plants can be subsequently harvested, processed and disposed.

Plants have evolved a great diversity of genetic adaptations to handle the accumulated pollutants
that occur in the environment. Growing and, in some cases, harvesting plants on a contaminated
site as a remediation method is a passive technique that can be used to clean up sites with
shallow, low to moderate levels of contamination. Phytoremediation can be used to clean up
metals, pesticides, solvents, explosives, crude oil, polyaromatic hydrocarbons, and landfill
leachates. It can also be used for river basin management through the hydraulic control of
12 |3 7
contaminants. Phytoremediation has been studied extensively in research and small-scale
demonstrations, but full-scale applications are currently limited to a small number of projects.
Further research and development will lead to wider acceptance and use of phytoremediation.

OBJECTIVE OF THE STUDY

This study mainly focuses on the following facts:

1. To standardize the method of hydroponics for experimental plant Pisum sativum.

2. To evaluate the preliminary effect of zinc metal on plant system.
3. To compare the plant under stress condition with control plant set.

13 |3 7
 Chapter 2

LITERATURE REVIEW

Slinkard et al, 1994; He stated that as a winter annual , pea tolerates frost to -20 C in the seedling
stage, although top growth may be affected at -60C . Winter hardly peas can withstand -100 C,
and the snow cove protection, tolerance can be increased to -400C. The optimum temperature
levels for the vegetative and reproductive periods of peas were reported to be 21 0C and 160C and
160C and 100C (day and night), respectively.

Huisman and Poli, 1994; reported that the natural protein of the pea and faba beans is almost
entirely digested in the small intestine and the impaired performance in the literature was
attributed to the increased secretion of the endogenous protein.

Jambunathan et al., 1994; stated that cultivars such as “ Alaska” ,” Super Alaska”, “Supergreen”
and “Alaska Wilt Resistance” have long been the standard type of canning pea.

Duke, 1981; Peas are cultivated for the fresh green seeds, tender green pods, dried seeds and
foliage. He also reported that the oil from the ripened seeds has antisex harmonic effects;
produces sterility and antagonizes the effect of male hormone. He also reported that the seeds
contain trypsin and chymotrypsin which could be used for contraceptive, ecbolic, fungistatic and
spermicide. According to his report the seeds are globose or angled, smooth or wrinkled,
exalbuminous, whitish, grey, green or brownish; 100 seeds can weigh from 10-36 grams;
germination cryptocotylar. He also mentioned in his report that temperatures above 270 C
shortens the growing period and adversely affect the pollination. A hot spell is more damaging to
peas than a light frost. Peas can be grown successfully during midsummer and early fall I those
areas having relatively low temperatures and a good rainfall, or where irrigation is practiced for
very early crops, a sandy loam is preferred; for large yields where earliness is not a factor, a
well-drained clay loam or silt loam is preferred. In his studies he also mentioned that the peas are
propagated from seeds only. He also explain that pea vines are hauled to a vining station, where
pods are separated from vines, after which seeds are separated from pods. In his report he also
said that throughout temperate regions both green and dried peas are an important garden and
field crop. In the United States, ca. 550,000 MT are produced commercially for food annually,
and ca. 200,000 MT of field peas for feed.

14 |3 7
Davies , 1985; he stated that the green peas are eaten cooked as vegetables and are marketed
fresh , canned or frozen while ripe , dried peas are used as whole, split or made into flour. he also
stated that in some part of world dried peas are consumed split dahl ,roasted, parched or boiled
.Green peas are the number one processed vegetable specifically in UK and USA .Green foliage
or garden pea is also used as vegetable in some parts of Asia and Africa. He also mentioned in
his report that the leaf type could be conventional, semi-leafless and leafless. Peas reduced the
fertilizer requirements of maize b 20- 32 Kg/ha in India compared with wheat or fallow, or
respectively; in France it was estimated that about 50Kg/ha of Nitrogen are returned to the soil
by peas. Pea growing seasons vary from 80- 100 days in semi-arid regions and up to 150 days in
humid and temperate areas.

Kay, 1979; he reported that the leaves of Pisum sativum are used as a pot herb in Burma and
parts of Africa.

Smart, 1990; reported that there are no significant amounts of toxicity or anti-metabolites in
peas.

Zeiev and Zohary, 1973; and Pohill , Maesen et al, 1985; stated that pea comprises only two
species named as Pisum sativum and Pisum fulvum .

Sibeth and Smith, 1981; reported that pea is self pollinated annual herb, bushy or climbing,
glaucous; stems weak, round and slender, 30-150 cm long; leaves alternate, pinnate with 1-3
pairs of leaflets and a terminal branched tendril leaflets ovate or elliptic, 1.5-6 cm long.

Gritton, 1980; leaf size in most cases increases up to the first node bearing the first flower.
Stipules are large, leaf like and up to 10 cm long. The inflorescence of pea is a raceme arising
from the axil of the leaf. Corolla white or pink or purple; pods swollen or compressed, short-
stalked, straight or curved, 4-15 cm long, 1.5-2.5 cm wide, 2-10 seeded, 2-valved, dehid=scent
on both sutures.

Duke, 1981; Davies, 1985; peas require a cool, relatively humid climate and are grown at higher
altitudes in tropics with temperatures from 70C to 300C and production is concentrated between
the Tropics of Cancer and 500 N.

Muehlbauer et al, 1983; He stated that in the Palouse region of eastern Washington and northern
Idaho peas are sown between March 25that lower elevation to May 10th at higher elevation when
15 |3 7
the soil temperature is above 40 C for planting winter peas, September 15th to the 30th has been
recommended. The plant densities vary tremendously on soil type, cultivar, seed, size and biotic
factors, particularly diseases. He also illustrated that peas are relatively unresponsive to
fertilizers, particularly nitrogen; additions are necessary when nodulation is poor or fails
completely. When the seeds are treated with the Rhizobium, care must be taken in the choice of
fungicide seed treatments to prevent potential toxicity and if the pea crop is over mature,
harvesting early in the morning or during the evening when relative humidity is low will
minimize shattering and seed breakage.

Hulse, 1994; in his report he described that pea is among the four important cultivated legumes
next to soyabean, groundnut and beans.

Oram and Agcaoili, 1994; They reported that the pea is the predominant export crop in world
trade and represent about 40% of the total trade in pulses.

Muehlbauer et al., 1983; Davies et al., 1985; van Emden et al., 1988; The comparative studies of
these three scientists states that peas are adversely affected by Pea Early Browning Virus
(PEBV), Pea Enation Mosaic virus (PEMV), Pea Mosaic Virus (PMV), Pea top yellows (PTY),
Pea seed-borne Mosaic Virus (PSbMV) and Pea Streak Virus (PSV) that constitute diseases
caused by viruses, while the most important bacterial disease is caused by Pseudomonas
pisi (bacterial blight).

Emden et al., 1988; Muehlbauer et al., 1983; They stated that the insect pests such as pea aphids ,
pea triphs, pea seed beetle, adzuki bean seed beetle, seed weevil, bean weevil, pea midge,
African bollworm, pod borer, cut worms, pea moth, leaf minor, American bollworm, lima bean
pod borer, bean fly, bean seed fly, spider mites, root lesion nematodes, stem nematodes, pea cyst
nematodes, root knot nematodes also damage the standing crop to the great extent.

16 |3 7
 Chapter 3

MATERIALS AND METHODOLOGY

Materials
(i). PLANT SAMPLE
1. Dry seeds of Pisum sativum.
(ii) INSTRUMENTS
1. Weighing Machine.
2. pH strips

(iii) EQUIPMENTS

1. Beaker
2. Distilled water
3. Petri plates
4. Wooden box
5. Measuring cylinder
6. Black plastic cups ( of volume up to 200 ml)
7. Pipettes
8. Test tubes
9. Muslin cloth

(iv) REAGENTS

1. KNO3 ( 10.11 gm/100 ml)

2. Ca (NO3). 4H2O (23.62 gm/100 ml)
3. MgSO4. 7 H2O (24.65 gm/100 ml)
4. NH4 H2PO4( 11.51 gm/100 ml)
5. KCl (0.186 gm/100 ml)
6. H 3BO3 (0.77gm/100ml)
7. MnSO4.H 2O (0.169 gm/100ml )
8. ZnSO4.7H 2O (0.0288 gm/100ml)
9. CuSO4.5H 2O (0.0062 gm/100ml)

17 |3 7
 10. H 2MoO4 ( 0.00040 gm/100ml)
11. NaFe EDTA (3 gm/100ml)
12. Sodium Hypochloride (4 ml)
13. Triton X 100
14. Ethanol (70%)
15. Zinc Solution

METHODOLOGY:-

(A). Soaking of pea (Pisum sativum) seeds after sterilization.

STERILIZATION

Dry pea (Pisum sativum) seeds were taken into a sterilized beaker in a sufficient quantity. The
mouth of a beaker is then wrapped tightly with a muslin cloth. The beaker containing the dry
pea seeds wrapped with a muslin cloth is then placed under a running tap water for its thorough
rinsing (for 15-20 minutes).

These seeds after rinsing thoroughly with tap water were treated with 4 ml of Sodium
hypochloride solution in 25 ml of distilled water for 2-3 minutes. After the treatment with
Sodium hypochloride solution the beaker is again placed under the running tap water for 15-20
minutes.

The last and the final step of sterilization is followed by adding 2-3 drops of Triton X 100 to the
beaker containing pea (Pisum sativum) seeds and rinsing it again with tap water for another 15 -
20 minutes.

(NOTE: Refer figure below for reference ).

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Figure: Sterilization of dry pea (Pisum sativum ) seeds

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SEED SOAKING

After the surface sterilization by the above procedure the sterilized pea seeds were then soaked
into a clean and dry petri plates on the moist double layered filter paper.

After placing the sterilized seeds on the moist double layered filter paper at the optimum distance
from each other; they were sprinkled with adequate amount of distilled water and were covered
with the lid of a petri plates. These petri plates with the pea seeds were then placed in dark in a
thermostatically controlled culture room for their sprouting.

Figure: Soaking of the pea seeds after sterilization for the sprouting.

20 |3 7
(B). Germination of seeds in thermostatically controlled culture room.

The seeds after their transfer in the petir dishes on the moist double layered filter paper were kept
in dark under thermostatically controlled condition at 300C with humidity up to 50 %.The
sprouting is then observed on a regular time intervals .

DAY 1

DAY 7

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DAY 14

DAY 16

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(C) Partially developed plantlets punch with Zn [zinc] solution.

The partially developed plants of Pisum sativum is punch by using syringe with containing
different stock of hoagland’s media on one leaf of each plant. 5 pots each of which contains 3
plantlets were set up under the thermostatically controlled culture condition for their optimal
growth to check the zinc stress. The Hoagland‘s medium was punch with the syringe on every
3rd days.

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Table: Composition of Hoagland’s nutrient medium.

Stocks Reagents Amount (g/100ml)

Stock 1 16mM KNO3 10.11

Stock 2 4 mM Ca(NO3)2.H2O 23.62

Stock 3 2mM NH4H2PO4 11.51

Stock 4 1mM MgSO4.7H 2O 24.65

Stock 5 50µM KCl 0.186

25 µM H 3BO3 0.77

2µ M MnSO4.H 2O 0.169

2µM ZnSO4.7H 2O 0.0288

0.5 µM CuSO4.5H 2O 0.0062

0.5µM H 2MoO4 0.0040

Stock 6 30µM NaFe EDTA 3

(D). Treatment of Zinc at different concentrations to study the stress on pea (Pisum
sativum ) plant.

The stress on the Pisum sativum (pea) plant was studied at the different levels of concentration of
zinc metals. So, the four containers were adjusted at 100µM, 200µM, 500µM, 700µM while one
container is set as a control. Thus, in all five containers were set on experimentation to study the
zinc stress on the Pisum sativum (pea) plant.

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In this procedure Zinc sulphate (ZnSO4) solution was prepared by dissolving 1.61 gm of ZnSO4
into 100 ml of distilled water at the concentration of 700µM which is used as a main stock in
whole treatment process.

Therefore, for 100µM, 200µM, 500µM, 700µM the quantity of main stock used was 1.42 ml,
2.84 ml, 7.14 ml and 10 ml respectively.

The volume of the above mentioned concentrations were made up to 200 ml in each plastic
containers.

EFFECT OF
ZINC ON
THE LEAF
OF PLANT.

25 |3 7
 Chapter 4

RESULT AND DISCUSSION

The zinc meal stress was studied under different parameters that were fresh weight of the treated
plant, shoot length of the treated plant, root length of the treated plant and dry weight of the
treated plant. All these parameters were carefully observed during the experiment and were
precisely analyzed against the untreated plants which were set as a control for the experiment.
Later on in the experiment the tolerance index, relative shoot length, relative root length and
relative dry weight were also calculated separately.

FRESH WEIGHT

The fresh weight was calculated as an average weight of the treated plant at different
level of concentrations that is 100µM, 200µM, 500µM, 700µM and also for the control.
Tolerance index = Average weight of the treated plants 100
Average weight of the control

Table 1 : The average fresh weight of the control and treated plants with their respective
tolerance index at different level of concentrations of zinc metal.

FRESH WIEGHT (in g)

CONCENTRATION AVERAGE TOLERANCE INDEX
Control 2.31 100
100 µM 2.30 99.54
200 µM 2.17 93.93
500 µM 2.09 90.47
700 µM 1.61 69.69

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 AVERAGE
2.5

1.5
AVERAGE

0.5

0
control 100µm 200µm 500µm 700µm

Figure1. 1. Graph of average fresh weight v/s concentration of zinc metal.

TOLERANCE INDEX

CONTROL 100µm 200µm 500µm 700µm

Figure1.2. Tolerance index of the treated plant with reference to the control plant.

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SHOOT LENGTH

The shoot length was calculated as an average length of the treated plant at different level
of concentrations that is 100µM, 200µM, 500µM, 700µM and also for the control.

Relative shoot length = Average shoot length of the treated plants 100
Average shoot length of the control

Table 2 : Average shoot length of control and treated plant with their respective relative
shoot length against different level of concentrations of zinc metal.

SHOOT LENGTH (in cm)

CONCENTRATION AVERAGE RELATIVE SHOOT LENGTH
Control 19.66 100
100 µM 16.72 85.04
200 µM 16.6 84.43
500 µM 15.86 80.67
700 µM 12.13 61.69

SHOOT LENGTH [in cm]

25

20
average

15

10

0
control 100µm 200µm 500µm 700µm

Figure2. 1. Graph of average shoot length v/s zinc metal concentration.

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 RELATIVE SHOOT LENGTH

control 100µm 200µm 500µm 700µm

Figure2.3. Relative shoot length of the treated plant with reference to control.

ROOT LENGTH

The root length was calculated as an average length of the treated plant at different level
of concentrations that is 100µM, 200µM, 500µM, 700µM and also for the control.
Relative root length = Average root length of the treated plants 100
Average root length of the control

Table 3 : Average root length of the control and the treated plants with their relative root
length against different level of concentration of the zinc metal.

ROOT LENGTH(in cm)

CONCENTRATION AVERAGE RELATIVE ROOT LENGTH
Control 13.6 100
100 µM 12.2 89.70
200 µM 11.53 84.77
500 µM 10.23 75.22
700 µM 9.7 71.32

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 ROOT LENGTH [in cm]
16

14

12

10

8
average
6

0
CONTROL 100µm 200µm 500µm 700µm

Figure.3.1. Average root length V/S zinc metal concentration.

RELATIVE ROOT LENGTH

control 100µm 200µm 500µm 700µm

Figure.3.2. Relative root length of the treated with reference to the control.

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DRY WEIGHT

The fresh weight was calculated as an average weight of the treated plant at different level of
concentrations that is 100µM, 200µM, 500µM, 700µM and also for the control.

Relative dry weight = Average weight of the treated plants 100

Average weight of the control

Table 4 : Average dry weight of the control and the treated plants with their relative dry
weight at the different level of concentration.

DRY WEIGHT(in g)
CONCENTRATION AVERAGE RELATIVE DRY WEIGHT
Control 0.19 100
100 µM 0.177 93.15
200 µM 0.152 80
500 µM 0.13 68.42
700 µM 0.10 52.63

DRY WEIGHT [in g]

0.2
0.18
0.16
0.14
0.12 AVERAGE
0.1
0.08
0.06
0.04
0.02
0
control 100µm 200µm 500µm 700µm

Figure.4.1. Graph of average dry weight V/S zinc metal concentration.

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 RELATIVE DRY WEIGHT

control 100µm 200µm 500µm

Figure.5.2. Relative dry weight of the treated with reference to the control.

32 |3 7
 CHAPTER 5

CONCLUSION

On the basis of above drawn result this report can be concluded as the growth parameters such as
tolerance index (relative fresh weight), relative shoot length, relative root length and relative dry
weight constantly decreases with the increasing concentration of the zinc metal in the plant. This
happens because the increased concentration of the zinc metal posses a high rate of stress to the
plant.

SHOOT LENGTH AFTER STRESS

ROOT LENGTH AFTER STRESS

33 |3 7
 Chapter 6

Summary:-

The aim of this report was to check the zinc metal stress at different levels of concentration on
the pea (Pisum sativum) plant grown with zinc solution under the controlled temperature
conditions in the thermostatically controlled culture room.

The pea is most commonly the small spherical seed or the seed-pod of the pod fruit Pisum
sativum. Each pod contains several peas. Pea pods are botanically fruit, since they contain seeds
and developed from the ovary of a flower.

The pea (Pisum sativum) plants were grown in the step by step manner from the dry seed until
they were developed up to the optimum level.

These plants were than treated with the zinc metal at the four different levels of the concentration
that is 100µM, 200µM, 500µM, 700µM and one was set as control.

Zinc plays as an essential micronutrients that affects several metabolic processes of plants and
has a long biological half life. The phytotoxicity of zn is indicated by decreases in growth and
developments, metabolism and an induction of oxidative damage in various plant species such as
pisum sativum. High level of zn in soil inhibit many plant metabolic functions, result in retarded
growth and cause senescence .zinc toxicity in plants limited the growth of both root and shoot.

Zinc toxicity lso cause chlorosis in young leaf, which can extend to older leaves after prolonged
exposure to high soil zn level. Excessive zn also give rise to manganese and copper deficiencies
in plant shoots. Phytoremediation method where plants, for being cost effective and non
hazardous to ecosystem are used to cured the heavy metal contaminated environments.

In the end the result was drawn on different parameters that were fresh weight of the treated
plant, shoot length of he treated plant, root length of the treated plant and dry weight of the
treated plant.

It was concluded that there was a constant decline in the above mentioned parameters with the
increase in the zinc metal concentration.

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 CHAPTER 7

REFERENCE:-

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