EFFECTS OF Hg AND Zn ON GERMINATION AND

EARLY SEEDLING GROWTH OF

CHICKPEA (Cicer arietinum L )

B.Sc. Dissertation

By

PREXA JAYESHKUMAR MEHTA

151128102004

Under the supervision of

Mr. Gaurav Shrimali

May 2018

PARUL INSTITUTE OF APPLIED SCIENCES AND RESEARCH

FACULTY OF APPLIED SCIENCES

PARUL UNIVERSITY

BOPAL – GHUMA ROAD, GHUMA

AHMEDABAD – 380058, GUJARAT, INDIA

EFFECTS OF Hg AND Zn ON GERMINATION AND
EARLY SEEDLING GROWTH OF
CHICKPEA (Cicer arietinum L )
B.Sc.

Dissertation submitted in partial fulfilment of the requirements for the degree of

BACHELOR OF SCIENCE
IN

BIOTECHNOLOGY

By

PREXA JAYESHKUMAR MEHTA

15112810204

Under the supervision of

Mr. Gaurav Shrimali

May 2018

PARUL INSTITUTE OF APPLIED SCIENCE AND RESEARCH

FACULTY OF APPLIED SCIENCES

PARUL UNIVERSITY

BOPAL – GHUMA ROAD, GHUMA

AHMEDABAD – 380058, GUJARAT, INDIA

 CERTIFICATE

This is to certify that the dissertation entitled EFFECTS OF Hg AND Zn ON

GERMINATION AND EARLY SEEDLING GROWTH OF CHICKPEA (Cicer
arietinum L ) is a report of original work done by Prexa Jayeshkumar Mehta under
my supervision. The work has not been submitted for award of any other degree.
The extent of plagiarism does not exceed the permissible limit laid down by the
University.

Mr.Gaurav Shrimali

Faculty Supervisor

Date: 28th May 2018

Mr.Gaurav Shrimali Dr.Kuldeep R. Sharma Dr.Kuldeep R. Sharma

Name of HOD PG Coordinator Principal, PIASR

ACKNOWLEDGEMENTS

Myself, Prexa Mehta, I am thankful to god almighty for giving me whatever I have in
today’s day,and my parents who have faith in me all the time.

I express my deep sense of gratitude to Mr.Gaurav Shrimali, Assistant Professor,

Department of Biotechnology, Parul Institute of Applied Science and Research, Parul
University, Ahmedabad for his guidance, suggestions, help and friendly
encouragement are greatly acknowledged. His trust and support inspired me in
making correct decisions and I’m glad that I had such a great opportunity to work
under his guidance and he also provided exposure for knowledge.

I’m also thankful to Dr.Kuldeep Sharma, I/C principal of Parul Institute of Applied
Science And Research, for his constant support and advice

At last I would also like to thank Ms. Aakanksha Mishra who helped me in my
dissertation work and all other people also who helped me directly or indirectly.

Prexa Mehta
Table of Content
CHAPTER 12
1.1 Plant Cicer arietinum L. Classification4
1.1.1 common names of chickpea5
1.1.2 morphology...............................................................................................................6
1.1.3 synonyms of Cicer arietinum L.7
1.2 Distribution and status of crop in india and world wide8
1.3 chemical constituents of chickpea ...............................................................................13

1.4 Traditional medicinal use of chickpea...........................................................................14

1.4.1 nutrients and health benefits of chickpea.............................................................15

1.5 biotic and abiotic stress................................................................................................20

1.5.1 biotic stress.............................................................................................................21

1.5.2 abiotic stress...........................................................................................................21

1.6 effect of heavy metals on chickpea............................................................................21

CHAPTER 2 REVIEW OF LITERATURE........................................................................................27

CHAPTER 3 MATERIALS AND METHODS..................................................................................29

3.1 Laboratory experiment................................................................................................29

3.2 measuring physiological parameters...........................................................................30

3.3 biochemical study........................................................................................................31

3.3.1 starch estimation...............................................................................................31

3.3.2 total phenol estimation.....................................................................................32

3.3.3 estimation of sugar by DNS method..................................................................33

CHAPTER 4 RESULT AND CONCLUSION...................................................................................35

REFERENCES.............................................................................................................................43
 LIST OF TABLE
Table 1 clssification of Cicer arietinum L.................................................4
Table 2 common names of chickpea..........................................................4
Table 3 high yielding varieties of chickpea recommended for general
cultivation in different states....................................................................8
Table 4 effect of various metals on chickpea...........................................20
Tables 5 observation of seed germination................................................29
LIST OF FIGURE
Fig 1Morphology of chickpea....................................................................5
Fig 2Structure of gram seed.......................................................................6
Fig 3State wise production of chickpea in India......................................8
Fig 4Global production of chickpea.........................................................10
Fig 5Chart of seed germination................................................................30
Fig 6Chart of root length..........................................................................31
Fig 7Chart of dry weight..........................................................................31
Fig 8Chart of fresh weight.......................................................................32
Fig 9Graph of starch concentration..........................................................33
Fig 10Graph of total phenol.................................................................... 33
Fig 11Graph of sample sugar...................................................................34
Fig 12 Graph of standard sugar................................................................35
 CHAPTER 1

GENERAL INTRODUCTION

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 CHAPTER 1 General Introduction

Chickpea (Cicer arietinum L.), ranks among the world’s three most important
food legumes. It is commonly known as ‘Gram’. The crop play important role in
human diet and agricultural system. Chickpea is high protein, low fat and sodium,
cholesterol free, an excellent source of both soluble and insoluble fibers, complex
carbohydrates, vitamins, folate, and minerals, especially calcium, phosphorus, iron,
zinc, and magnesium.

Chickpea is an important ‘rabi’ pulse of India. India is an largest pulse

producing country and the total production of pulse in India was 13.1 million tons
against the requirement of 20 million tons indicating the shortfall which is to be
minimized either by increasing the area under pulses or by increasing the productivity
per unit area. It is grown in more than 35 countries but the main producing region are
Indo-Pakistan.

The Indian grams have been classified into two broader groups:

1. Desi or Brown Gram (Cicer arientinum L.): In this group the color of the seed
ranges from yellow to dark brown. Seed size is usually small. It is the most
widely grown group. Plants are small with good branching ability.
Chromosomes number is 2n = 14, 16.
2. Kabuli or White Gram (Cicer kabulium): In this group the color of the seed is
usually white. Grains are bold and attractive. Yield potential of this group is
poor as compared to desi or brown gram. Plants are generally taller than the
desi gram and stand more or less erect. The chromosome number is 2n = 16

Chick pea is a winter season crop but severe cold and frost are injurious to it.
Frost at the time of flowering results in the failure of the flowers to develop seeds or
in the killing of the seeds inside the pod. It is generally grown under rainfed
conditions but gives good returns in irrigated conditions as well. Excessive rains soon
after sowing or at flowering and fruiting or hailstorms at ripening cause heavy loss. It
is best suited to areas having moderate rainfall of 60-90 centimeters per annum.

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 The agronomic problems associated with chickpea crops differ little between
geographical areas; drought and nutrient deficiencies appear to be major limiting
factors. The area and production of chickpea show pronounced seasonal fluculation,
world wide, which are partly associated with the amount and distribution of rainfall.
Often, the yield attained by the farmers are considerably less than its potentially yield.
The low variable yields in many countries are due in part to the relegation of chickpea
to marginal lands and to adverse effect of environmental stresses on growth and
development. Limited availability of water and deficiencies of mineral nutrients both
contribute to poor and unstable yields and it is important to find ways to improve
yield stability.

Plant is affected by two types of stress. 1. Biotic stress. 2. Abiotic stress .

Biotic stress: plant is infected by viruses, fungi, bacteria or living microorganisms
which cause disease in plant. Abiotic stress: non-living things like drought,
temperature, pH, heavy metals, light which directly affect in plant growth and
seedling. Heavy metals are a serious ecological, nutritional and environmental
problem. Heavy metals can occur naturally in rocks; they are released during the
alteration of rocks, which become the geographical level but rarely at toxic level. The
natural concentration of heavy metals in soil varies with the nature of the rock, its
location and age. Mining , manufacture and the use of synthetic products (e.g.,
pesticides, paints, industrial waste, and applications of industrial or domestic sludge to
the land) were the major source of this kind of contamination. These elements have a
strong negative effect on ecosystem. They can be non-essential trace elements, which
are highly toxic and very harmful to all living organisms even in trace concentration
(Cd, Pb, Hg). While some of these elements such as Cu, Zn, and Fe are essential for
plant growth at low concentration.

Zinc is an important component of many vital enzymes having catalytic, co-

catalytic, and structural role as structural stabilizer for proteins, membrane, and DNA
binding protein. Zn is found to be involved in many cellular function such as protein
metabolism, photosynthetic carbon metabolism, and indole acetic acid metabolism,
yet its higher concentration cause toxicity.One of the first symptoms of Zn deficiency
is an inhibition of cell growth and proliferation. Zinc affect growth of shoots and
roots.

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 Zinc deficiency is considered the most widespread of the micronutrient
deficiencies with zinc-deficient soil being common in both tropical and temperate
climates. Zinc has a specific role in the integrity and functioning of the cell membrane
and zinc nutrition may affect the water relations of plant and plant that suffered from
zinc deficiency appeared tobe more sensitive to water defects. Zinc nutrition may be
important for maintaining productivity under drought stress.

1.1 Plant Cicer arietinum L. classification

Kingdom Plantae
Division Magnoliophyta
Class Magnoliopida
Order Fabales
Family Fabaceae
Subfamily Faboideae
Genus Cicer
Species Cicer arietinum
Table 1: Classification of Cicer arietinum L

1.1.1 Common names of chickpea (Cicer arietinum L.)

Arabic Hummu , hommos, lablabi

Chinese Ying zui dou
English Bengal gram, chickpea,
garbanzo
French Pois chiche
German Kichererbse
India Kala chana, Bengal gram
Italian Cece
Portuguese Graode-bico
Spanish Garbanzo
Swedish Kikart
turkish Nohut
Table 2: common names of chickpea

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1.1.2Morphology

The morphology and composition of seeds of desi and kabuli chickpea (Cicer
arietinum L.) genotypes were studied using light microscopy with differential staining
for protein, starch, β-glucans, and non fluorescing compounds. Kabuli seeds had a
thinner seedcoat due to thinner palisade and parenchyma layers which contained
fewer pectic polysaccharides and less protein. The outer palisade layer varied in
thickness from one to two cells, leading to a textured and sometimes wrinkled
appearance of the seed surface. In contrast, the desi palisade layers were rigid and
extensively thickened. Hourglass cells were homogeneous for both seed types, but not
in an interspecific desi line (containing Cicer echinospermum parentage), which had
heterogeneous cells. The inner surface of the seed coat contained both pectic and
proteinaceous materials. The cotyledon comprised a single outer epidermal layer of
protein-filled cells devoid of starch, with thickened outer cell walls; cell size and
shape differed on abaxial and abaxial faces. Sub epidermal cells on the abaxial face
were similar to epidermal cells. These findings help explain differences in the
processing behaviour between the major chickpea seed types.

Fig:1 Morphology of cicer arietinum (chickpea seed)

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 Fig:2 structure of gram seed

1.1.3 Synonyms of Cicer arietinum L.

• Cicer album hort.

• Cicer arientinium L. [Spelling variant]
• Cicer arientinum L. [Spelling variant]
• Cicer edessanum Bornm.
• Cicer grossum Salisb.
• Cicer nigrum hort.
• Cicer physodes Rchb.
• Cicer rotundum Alef.
• Cicer sativum Schkuhr
• Cicer sintenisii Bornm.
• Ononis crotalarioides M.E.Jones

1.2 Distribution and status of crop in India & world wide

Role of pulses in Indian agriculture needs hardly any emphasis; India is a

premier pulse growing country. The pulses are the integral part of the cropping
systems of the farmers all over the country because these crops fit in well in the crop
rotation and crop mixtures followed by them. Pulses are important constituents of the

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Indian diet and supply major part of the protein requirements. Pulse crops, besides
being rich in protein and some of the essential amino acids, enrich the soil through
symbolic nitrogen fixation from atmosphere.

In India, the total food production in 1999-2000 was about 209 million tones, out
of this only 13.4 million tones was contributed by pulses. The production of cereals
increased by 460 per cent since 1950-51 the production of pulses has increased only
178 per cent. There is a shortage of pulses in the country. The price has increased
considerably and the consumer is hard hit to buy his requirements. Thus, the
availability of pulse per capita per day has proportionately declined from 71 g (1955)
to 36.9 g (1998) against the minimum requirement of 70 g per capita per day. There is
not much possibility of the import of pulses in the country. The production of pulses
has to be increased internally to meet the demand.

Gram commonly known as 'chick pea' or Bengal gram is the most important pulse
crop in India. Chick pea occupies about 38 per cent of area under pulses and
contributes about 50 per cent of the total pulse production of India. It is used for
human consumption as well as for feeding to animals. It is eaten both whole fried or
boiled and salted or more generally in the form of split pulse which is cooked and
eaten. Both husks and bits of the 'dal' are valuable cattle feed. Fresh green leaves are
used as vegetable (sag). Straw of chick pea is an excellent fodder for cattle. The
grains are also used as vegetable (chhole). Chick pea flour (besan) is used in the
preparation of various types of sweets. Chick pea is considered to have medicinal
effects and it is used for blood purification. Chick pea contains 21.1 per cent protein,
61.5 per cent carbohydrates, 4.5 per cent fat. It is rich in calcium, iron and niacin.

➢ ORIGIN AND HISTORY

Chick pea has been known in this country for a long time. It is said to be one of
the oldest pulses known and cultivated from ancient times both in Asia and in Europe.
Its probable place of origin lies in south western Asia, which is in the countries lying
to the north-west of India such as Afganistan and Persia. According to Aykroid and
Daughty (1964) the centre of origin of chick pea is eastern Mediterranean. According
to De Candolle, the fact that gram gas a Sanskrit name would indicate that the crop
has been under cultivation in India longer than in any other country.

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 ➢ AREA AND DISTRIBUTION

Chick pea is one of the important pulse crop of the world cultivated over an area
of 12.0 million hectares with a production of about 9.2 million tones of grain (1999).
The important gram growing countries are India, Pakistan, Ethiopia, Burma and
Turkey. India ranks first in the world in respect of production as well as acreage
followed by Pakistan.

It is the most important pulse crop of India occupying an area of 6.3 million
hectares with production of 5.1 million tones. The average yield of chick pea is only
806 kg per hectare. The major chick pea production areas are situated in Madhya
Pradesh, Rajasthan, Uttar Pradesh, Haryana, Maharashtra and Punjab.

Fig 3: state wise production of chickpea in inida.

Unlike cereals high yielding photo-insensitive cultivars are not available in pulses
and this appears to be the most important reasons for low productivity of pulse crops
in the country. However, several improved varieties of chick pea have been evolved
in different chick pea growing states.

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State Recommended Varieties

Andhra Pradesh ICCV-2, ICCV-37, ICCV-4, ICCV-10

Assam KWR-108, BG-256, L-550, KPG-59

Gujarat Pusa-319, Vijay, ICCV-4, Pusa-240, GG-1, Pusa1053

Haryana Haryana Chana-1, GNG-469, Pusa-362, Gora Hisari,

Himachal Pradesh BBG-1, Haryana Chana-1, L-550

Jammu & Kashmir GNG-469, L-550, PBG-1, Haryana Chana-1

Karnataka BDN 9-3, ICCV-10, ICCV-2 Annegiri-1

Punjab PBG-1, GNG-469, Haryana Chana-1, Gaurav, L-550,

C235, G-543, Pusa-1053, GPF-2, PDG-3

Rajasthan GNG-416, GNG-469, GNG-663, PBG-1, L-550, Pusa-

256,

RSG-44, Pusa-1053, PDG 84-1

Tamil Nadu ICCV-10, BDN 9-3, CO-3, CO-4

Uttar Pradesh KWR-108, Avrodhi, BG-256, K-850, Pant G-186, Pusa-

372, Radhey, JG-315, Uday (KPG-75), Pusa-1003,

Pusa-1053

Madhya Pradesh JG-74, JG-315, Vijay, Pusa-256, Phule G-5, Pusa-1053

Maharashtra Vijay, Phule G-5, Vishal, ICCV-10, Pusa-1053

Orissa Radhey, ICCV-10,L-550, Pusa-372, Pusa-1003

West Bengal Pusa-372, KWR-108, KPG-59, BG-256, Pusa-1003

Bihar KWR-108, Avrodhi, BG-256, Pant G-114, Pusa-209,

L-550, Pusa-1003

North Eastern KWR-108, Avrodhi, KPG-59, BG-256

States

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Table 3 : High Yielding Varieties of Chick Pea Recommended for General
Cultivation in Different states

The chickpea is widely grown in Indian sub-continent which accounts for

almost 90% of the world's crop. The crop is also grown in Bangladesh, Mynamar,
Iran, Nepal, Pakistan, Syria and Turkey in Asia.

Africa, mainly grown in Algeria, Ethiopia, Malawi, Sudan, Tanzania and

Tunisia, accounts for 6% of the world's production.

The North and Central America produce 5% of the total chickpea of the world.
The major chickpea growing countries are Canada, and Mexico. In Europe,Oceania in
Australia. chickpea is grown mainly in Spain and Portugal. Chickpea is grown in
Oceania in Australia.

Fig 4: global production of chickpea

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1.3 Chemical constitutes of chickpea.

Chickpea seed has 38-59% carbohydrate, 3% fiber, 4.8-5.5% oil, 3% ash,

0.2% calcium, and 0.3% phosphorus.

Digestibility of protein varies from 76-78% and its carbohydrate from 57-
60%. (Hulse, 1991, Huisman and van der poel, 1994).

Raw whole seeds contain per 100 g: 357 calories, 4.5-15.69% moisture, 14.9-
24.6 g protein, 0.8-6.4 % fat, 2.1-11.7 g fiber, 2-4.8 g ash, 140-440 mg Ca, 190-382
mg P, 5.0-23, 9 mg Fe, 0-225 m g b-carotene equivalent, 0.21-1.1 mg thiamin, 0.12-
0.33 mg riboflavin, and 1.3-2.9 mg niacin (Duke, 1981; Huisman and van der Poel,
1994).

"Boiled and roasted seeds contain similar amounts. Sprouting is said to

increase the proportionate amounts of ascorbic acid, niacin, available iron, choline,
tocopherol, pantothenic acid, biotin, pyridoxine, inositol, and vitamin K. Malic and
oxalic acid exudation from the leaves of the plant may soil and damage trousers and
shoes. Wild species often have similar glandular secretions" (Duke, 1981).

The limiting amino acid concentrations are 0.52 for methionine, 1.45 for
lysine and cystine, 0.71 for threonine and 0.16 for tryptophan (Williams et al., 1994).
The amino acid composition of seeds with 19.5% protein, 5.5% oil is (per 16 g N): 7.2
g lysine, 1.4 g methionine, 8.8 g arginine, 4.0 g glycine, 2.3 g histidine, 4.4 g
isoleucine, 7.6 g leucine, 6.6 g phenylalanine, 3.3 g tyrosine, 3.5 g threonine, 4.6 g
valine, 4.1 g alanine, 11.7 g aspartic acid, 16.0 g glutamic acid, 0.0 g hydroxyproline,
4.3 g proline, and 5.2 g serine" (Duke, 1981; Huisman and van der poel, 1994; and
Williams et al., 1994).

"Percent fatty acid compositions are: 'Desi': oleic 52.1, linoleic 38.0, myristic
2.74, pactic 5.11, and steatic 2.05; 'Kabuli': oleic 50.3, linoleic 40.0, myristic 2.28,
palmitic 5.74, stearic 1.61, and arachidic 0.07%. The leaves contain 4-8% protein"
(Duke, 1981).

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 1.4 Traditional Medicinal Uses

Among the food legumes, chickpea is the most hypocholesteremic agent;

germinated chickpea was reported to be effective in controlling cholesterol level in
rats (Geervani, 19991).

"Glandular secretion of the leaves, stems, and pods consists of malic and
oxalic acids, giving a sour taste. In India these acids used to be harvested by spreading
thin muslin over the crop during the night. In the morning the soaked cloth is wrung
out, and the acids are collected in bottles.

Medicinal applications include use for aphrodisiac, bronchitis, hypertension,

hyperlipidemic, diabetes, itchy skin, inflammation, catarrh, cutamenia, cholera,
constipation, diarrhea, dyspepsia, flatulence, snakebite, sunstroke, and warts. Acids
are supposed to lower the blood cholesterol levels. Seeds are considered antibilious"
(Duke, 1981).

1.4.1 Nutrients and health benefits of chickpea

Originally cultivated in the Mediterranean and the Middle East, chickpeas, also
known as garbanzo beans, have spread their culinary influence across the world. They
also come with a range of potential health benefits.Though the most common type of
chickpea appears round and beige, other varieties can be black, green, and red.Like
other legumes, such as beans, peas, and lentils, chickpeas are high in fiber and
protein, and contain several key vitamins and minerals..

Fast facts on chickpeas:

Here are some key points about chickpeas. More detail and supporting information is
in the main article.

• Chickpeas are sometimes known as garbanzo beans.

• They are featured extensively in the Mediterranean diet and Middle-Eastern food.

• They are a good source of protein, carbohydrates, and fiber.

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• Soak them in water for 8 to 10 hours before cooking for the best results.

Benefits

Though the most common type of chickpea appears round and beige, other
varieties include colors such as black, green, and red.

Chickpeas have been associated with a number of possible health benefits.

1) Diabetes

Chickpeas are particularly high in fiber. Studies have shown that people with type 1
diabetes who consume high-fiber diets have lower blood glucose levels.

For people with type 2 diabetes, higher fiber intake may improve blood sugar, lipid,
and insulin levels.

The Dietary Guidelines for Americans recommends a minimum of 21 to 25 grams (g)

of fiber per day for women and 30 to 38 g per day for men.

2) Bone health

The iron, phosphate, calcium, magnesium, manganese, zinc, and vitamin K in

chickpeas all contribute to building and maintaining bone structure and strength.

Though phosphate and calcium are both important in bone structure, the careful
balance of the two minerals is necessary for proper bone mineralization - consumption
of too much phosphorus with too little calcium intake can result in bone loss.

Bone matrix formation requires the mineral manganese, and iron and zinc play crucial
roles in the production and maturation of collagen.

Adequate vitamin K consumption is important for good bone health because it

improves calcium absorption and may reduce urinary excretion of calcium, making

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sure that enough calcium is available for building and repairing bone. Low intake of
vitamin K is associated with a higher risk for bone fracture.

3) Blood pressure

Maintaining a low-sodium (low-salt) intake is essential for maintaining a low blood

pressure, however increasing potassium intake may be just as important because of its
vasodilation effects. According to the National Health and Nutrition Examination
Survey, fewer than 2 percent of United States adults meet the daily 4,700-milligram
recommendation.

4) Heart health

The high fiber, potassium, vitamin C, and vitamin B-6 content all support heart
health. Chickpeas contain significant amounts of fiber, which helps lower the total
amount of cholesterol in the blood, thereby decreasing the risk of heart disease.

In one study, those who consumed 4,069 milligrams of potassium per day had a 49
percent lower risk of death from ischemic heart disease compared with those who
consumed less potassium (about 1,000 mg per day).

5) Cancer

Although the mineral selenium is not present in most fruits and vegetables, it can be
found in chickpeas. It helps the enzymes of the liver to function properly and detoxify
some cancer-causing compounds in the body. Additionally, selenium
prevents inflammation and decreases tumor growth rates.

Chickpeas also contain folate, which plays a role in DNA synthesis and repair, and so
helps prevent the formation of cancer cells from mutations in the DNA. Saponins,
phytochemicals present in chickpeas, prevent cancer cells from multiplying and
spreading throughout the body.

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High-fiber intakes from chickpeas and other legumes, whole grains, fruits, and
vegetables are associated with a lowered risk of colorectal cancer. Vitamin C
functions as a powerful antioxidant and helps protect cells against free radical
damage.

6) Cholesterol

Research shows that including chickpeas in the diet lowers the amount of low-density
lipoprotein, or bad cholesterol, in the blood.

7) Inflammation

The choline in chickpeas helps with sleep, muscle movement, learning, and memory.
Choline also helps to maintain the structure of cellular membranes, aids in the
transmission of nerve impulses, assists in the absorption of fat and reduces chronic
inflammation.

8) Digestion and regularity

Because of their high fiber content, chickpeas help to prevent constipation and
promote regularity for a healthful digestive tract.

9) Weight management and satiety

Dietary fibers function as "bulking agents" in the digestive system. These compounds
increase satiety (a feeling of fullness) and reduce appetite, making people feel fuller
for longer and thereby lowering overall calorie intake.

Consuming fruits and vegetables of all kinds has long been associated with a reduced
risk of many lifestyle-related health conditions. Many studies have suggested that
increasing consumption of plant foods like chickpeas decreases the risk of obesity,
overall mortality, diabetes, heart disease, promotes a healthful complexion, healthful
hair, increased energy, and overall lower weight.

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 10) Irritable bowel syndrome

Although chickpeas do not ease the symptoms of irritable bowel syndrome, they can
be helpful to people affected by the condition.

Patsy Catsos, a registered dietitian and author of "IBS - Free at Last!" suggests that
increasing fiber consumption in individuals who have irritable bowel syndrome (IBS)
can be a challenge. However, chickpeas offer a source of fiber that is well-tolerated
by some IBS patients.Unfortunately, people with IBS who are following a low-
FODMAP diet do have to restrict chickpeas.

Nutrition

Raw chickpeas should not be consumed due to the harmful substances found
in uncooked legumes.

One cup of cooked chickpeas contains:

• 269 calories

• 45 g of carbohydrate

• 15 g of protein

• 13 g of dietary fiber

• 4 g of fat

• 0 g of cholesterol
Note: it is not recommended to eat raw chickpeas, or any other pulses due to the
content of toxins and anti-nutrients. These components are reduced with sprouting and
cooking.

Additionally, chickpeas contain vitamin K, folate, phosphorus, zinc, copper,

manganese, choline, and selenium.Besides being an excellent vegan and gluten-free
source of protein and fiber, chickpeas also contain exceptional levels of iron, vitamin
B-6, and magnesium.

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1.5 Biotic and Abiotic stress

Diseases are the most important stresses and have received about 60% of the
total research effort. Fortunately, disease-resistance breeding has been mostly
successful . On the other hand, drought is the second most important stress but only
modest success has been achieved through breeding . Breeding coldtolerant chickpeas
also has been successful (Singh, 1990). There has been little or no success with all
other stresses .

1.5.1 Biotic stress

Disease Among 50 diseases affecting chickpea, Ascochyta blight and

Fusarium wilt are the most destructive and widespread . Other diseases are either of
regional importance or of no significance .

1.5.2 Abiotic stress

Abiotic stress is defined as the negative impact of non-living factors on the

living organisms in a specific environment.

➢ Drought Stress

Drought stress is one of the main causes of crop loss within the agricultural
world. This is due to water's necessity in so many fundamental processes in plant
growth. It has become especially important in recent years to find a way to combat
drought stress. A decrease in precipitation and subsequent increase in drought are
extremely likely in the future due to an increase in global warming].

1.6 Effect of heavy metals on chickpea.

Heavy metals are a serious ecological, nutritional and environmental problem.

Heavy metals can occur naturally in rocks; they are released during the alteration of
rocks, which become the geographical level but rarely at toxic level. The natural
concentration of heavy metals in soil varies with the nature of the rock, its location
and age. Mining, manufacture and the use of synthetic products (e.g., pesticides,
paints, industrial waste, and applications of industrial or domestic sludge to the land)
were the major source of this kind of contamination. These elements have a strong

17 | P a g e
negative effect on ecosystem. They can be non-essential trace elements, which are
highly toxic and very harmful to all living organisms even in trace concentration (Cd,
Pb, Hg). While some of these elements such as Cu, Zn, and Fe are essential for plant
growth at low concentration.

Heavy metals (HMs) exists in the environment in both forms as essential and
non-essential. These HM ions enter in soil biota from various sources like natural and
anthropogenic. Essential HMs such as cobalt (Co), copper (Cu), iron (Fe), manganese
(Mn), molybdenum (Mo), nickel (Ni), and zinc (Zn) plays a beneficial role in plant
growth and development. At optimum level these beneficial elements improves the
plant's nutritional level and also several mechanisms essential for the normal growth
and better yield of plants. The range of their optimality for land plants is varied. Plant
uptake heavy metals as a soluble component or solubilized them by root exudates.
While their presence in excess become toxic for plants that switches the plant's ability
to uptake and accumulate other non-essential elements. The increased amount of HMs
within the plant tissue displays direct and indirect toxic impacts. Such direct effects
are the generation of oxidative stress which further aggravates inhibition of
cytoplasmic enzymes and damage to cell structures. Although, indirect possession is
the substitution of essential nutrients at plant's cation exchange sites. These ions
readily influence role of various enzymes and proteins, arrest metabolism, and reveal
phytotoxicity. On account of recent advancements on beneficial HMs ions Co, Cu, Fe,
Mn, Mo, Ni, and Zn in soil-plant system, the present paper: overview the sources of
HMs in soils and their uptake and transportation mechanism, here we have discussed
the role of metal transporters in transporting the essential metal ions from soil to
plants. The role played by Co, Cu, Fe, Mn, Mo, Ni, and Zn at both low and high level
on the plant growth and development and the mechanism to alleviate metal toxicity at
high level have been also discussed. At the end, on concluding the article we have
also discussed the future perspective in respect to beneficial HM ions interaction with
plant at both levels.

Heavy metal like cobalt (Co), copper (Cu), iron (Fe), manganese (Mn),
molybdenum (Mo), nickel (Ni), and zinc (Zn) enters in soil from various sources such
as mining, foundries, smelters, combustion, and agriculture .Plant genomes encode a
number of transporters that are specific in their substrate specificities, expression, and

18 | P a g e
in cellular localization to manage the translocation of these metals into the whole
plant . These metals are acting a beneficial role for plant growth, development, and
productivity at an optimum concentration in the form of the essential micronutrient.
To grow and complete the life cycle plants use these essential micronutrients. The
plant takes these essential heavy metals like iron, zinc, copper, and manganese from
the soil due to concentration gradients and selective uptake of these metals. These
ions enthusiastically affected the function of many enzymes and cellular metabolism.
These metals also play a prominent role in the synthesis of protein, nucleic acids,
photosynthetic pigment, and it also take part in the structural and functional integrity
of cell membranes.

Copper is an essential heavy metal which actively takes part in the

photosynthesis. Manganese is an important constituent of various metabolic enzyme
like mallic dehydrogenase and oxalosuccinic decarboxylase , Cobalt found in the form
of Vitmain B12, while Fe act as a cofactor in cytochrome. Although the concentration
of these heavy metal ions is severely altered by the arbitrary human activities and
through the various natural process. The enhanced concentration of these beneficial
ions poses a toxic effect into the plant cells. These effects may be substituted of
essential functional groups, cellular damage, generation of reactive oxygen species
(ROS), disturbance in the various metabolic reaction by altering the enzymatic
activity. Regarding the above facts, it is noticeable that only a limited amount of these
beneficial heavy metals is essential for the plant growth and metabolic function.

19 | P a g e
 Metal Effect

• Zinc Important component of many

vital enzyme having catalytic,
co-catalytic.

Structural component of
proteins, membranes, dna
binding protein.

Affects growth of shoots and

roots. Help in seed
germination.

• Cadmium Toxic in seed germination.

• Arsenic Inhibit the growth of shoots

and roots

• Boron Important for cell division,

elongation, translocation, and
cell wall development

• Manganese Important in nitrogen

metabolism and
photosynthesis. Improve
seedling establishment and
growth.

• Mercury Not phytotoxic, but some time

inhibit the growth of seedling.

• Cobalt Vitamin B12 formation

• Copper an essential heavy metal

which actively takes part in
the photosynthesis

Table 4: effect of various metals on chickpea

20 | P a g e
 CHAPTER 2

REVIEW OF LITERATURE

21 | P a g e
 CHAPTER 2

REVIEW OF LITERATURE

Taylor C. Wallace et al, (2016) reported the nutritional value and Mohsen seyedi et
al, (2011) suggested the effect of seed priming with zinc sulphate on germination
characteristics and seedling growth of cicer arietinum L.

Morad shaban et al, (2012) reported response of chickpea cultivars to integrated

application of zinc nutrient with water stress.Prof Dr Ali Esmail Al-Snafi (2016)
coined the medical importance of cicer arietinum.

Dayanand et al, (2013) reported seed yield and economics of chickpea influence by
foliar application of agrochemicals. Nabakumar mondal et al, (2013) reported effect
of varying cadmium stress on chickpea seedling.

Kuldeep balai et al, (2017) suggested effect of phosphorus and zinc on growth, yield
an economics of chickpea.Girish Chandra pathk et al, (2012) reported improving
reproductive efficiency of chickpea by foliar application of zinc.

Igor S. KRYVORUCHKO (2017) suggested zn use efficiency for optimization of

symbiotic nitrogen fixation in chickpea.Legesse Hidoto et al, (2017) reported towards
zinc biofortification in chickpea

Habib ur rahman khan (1998) reported responses of chickpea to zinc supply and water
deficits.Taoufik El Rasafi et al,(2016-17) reported the effect of Cd, Zn, and Fe on
seed germination and early seedling growth of cicer arietinum L

Hatice bozoglu et al, (2007) suggested the response of chickpea to zinc and
molybdenum stress

22 | P a g e
 CHAPTER 3

MATERIALS AND METHODS

23 | P a g e
 CHAPTER 3
Material and Methods
.

3.1 Laboratory experiment:

A laboratory experiment to find out effectsof heavy metal on germination of

chick pea was conducted at Department of biotechnology, PIASR Ahmedabad.

Uniform seeds were kept for germination in appropriate heavy metal

concentration and control seeds were treated with distilled water. The experiment
detail is as follows.

➢ Effect of zn and Hg on germination and seedling growth of chickpea was

investigated in laboratory conditions in petri plates.
➢ To avoid contaminations of bacteria and fungi, the seed were surface sterilised
for 10 min with 1% sodium hypochloride solution, rinsed in distilled Water
(1time) and soaked in 70% ethanol for 1 min followed by 5 times washing
with distilled water.to create metallic stress prepare 0.1%, 0.01% and 1%
concentrated solution of znso4 and Hgcl2.
➢ 10 healthy seeds of species were grown in each petri plates containing one
whatman filter paper in the bottom soaked with 5 ml distlilled water.
➢ Add 2 ml concentrated metallic solution only in 1st day than gave distilled
water as a control for soaking the filter paper for the germination of seed
about 10 days and measure the germination. (0.1% hgcl2: 0.1 gm hgcl2 in 100
ml.).
➢ After 10 days physiological and biochemical parameters were measured.

3.2 Measuring Physiological parameters.

Following physiological parameters were recorded.

24 | P a g e
 1.germination percentage

2. shoot length

3..root length

4.dry weight

5.fresh weight

➢ Germination % of seeds :
Germination percentage is an estimate of the viability of a population of seeds.

Gp = seeds germinated / total seeds × 100

➢ Shoot length and root length:

How to measure?

Take a piece of thread and germinated seed .put the thread at the site of root initiate
and measure at the end of root and than measure with scale

➢ How to calculate average length?

Average length = length of all seed / number of see

➢ Dry weight:
Measuring of weight without the excess fluid .
➢ Fresh weight
Measuring of weight with the excess fluid like apply the water treatment on the
normal seed.

3.3 Biochemical study

Biochemical study means study of chemical constituents of livin organisms

such like sugar, starch, amino acid, phenol etc.

25 | P a g e
 3.3.1 Starch estimation:

➢ Method :

100 mg plant material was weighed Homogenated with 10 ml 80% ethanol I

Centrifuged at 5,000-10,OOOg for 10 minutes Supernatant I . Residue + 10 ml 80%
ethanol f Supernatant II Supernant I+II Discarded 'i' Centrifuged at 5,000-10,OOOg
for 10 minutes --------i Residue was dissolved in 20 ml 0.7% KOH and boiled it for
gelatinization for 40 minutes After cooling Centrifuged at 5,000-10,OOOg for 10
minutes I 1 ml aliquot + 0.5 ml 20% acetic acid + 1 ml citrate buffer (0.05 M, pH 5.0)
+ 1 ml I2KI I Incubated for 10 minute I Optical density was taken at 600nm 29

➢ Preparation of reagents for Starch estimation

(1) 80% Ethanol: 80 ml Ethanol was diluted up to 100 ml with DW.

(2) 0 7% Potassium hydroxide (KOH) 700 mg KOH was dissolved into 100 ml with
DW.

(3) 20% Acetic acid: 20 ml Glacial acetic acid was diluted up to 100 ml with DW.

(4) I2KI Solution 200 mg Iodine crystal and 2g KI were dissolved up to 100 ml with
DW (5) Citrate buffer: (0.05 M, pH-5.0) Citrate ‘X’. 0.1 M Citric acid (2.19 g Citric
acid was dissolved in 100 ml with DW.) Citrate ‘Y’: 0.1 M Sodium citrate (2.94 g
Sodium citrate was dissolved in 100 ml with DW.) Citrate buffer: 20.5 ml Citrate ‘X’
and 29.5 ml Citrate ‘Y’ were diluted up to 100 ml with DW.

➢ . OD at 600 nm

3.3.2 Total phenol estimation

• Method

100 mg plant material was weighed Homogenated with 10 ml 80% ethanol

Centrifuged at 5,000-10,OOOg for 10 minutes supernatant I , Residue + 10 ml 80%
ethanol I Centrifuged at 5,000-10,OOOg for 10 minutes. Supernatant II Residue.
Supernatant I+II 1 Discarded 1 ml Alcoholic aliquot + 1 ml20% Na2CO3 + 0.5 ml
Folin-ciocalteau’s reagent I Boiled for 10 minutes at 100°c in water bath 4- Final
Volume was made up 20 ml with DW and Optical Density was noted at 660nm

26 | P a g e
Calculation for phenols was done using the regression formula of Tannic Acid. X =
400 Y - 0.001 Result was expressed as mg Phenols / g Plant Material.

Preparation of reagents for Phenol estimation

(1) 80% Ethanol: 80 ml Ethanol was diluted up to 100 ml with DW.

(2) 20% Sodium carbonate (NaaCo3): 20 g Na2Co3 was dissolved m 100 ml DW.

(3) Folin-ciocalteau’s reagent (IN): Commercially available (2N) reagent was diluted
with an equal volume of DW.

3.3.3 Estimation of sugar by DNS method

1) weigh 100 mg of the sample and extract the sugar with 80% alcohol twise
(5 ml each time).
2) Collect the supernant and evaporate on water bath.
3) Add 10 ml of water and dissolve sugar.
4) Pipette our alcohol free extract 0.5 ml nd add distilled water up to 3 ml of
total volume
5) Add 3 ml DNS reagent and mix
6) Heat for 5 min in waterbath
7) After the color has developed add 1 ml of 40% of Rochelle salt solution
and mix
8) Measure od at 510 nm.
➢ Preparation of reagent
DNS reagent:1 g of dinitrosalicylic acid, 200mg of crystalline phenol, and
50mg of sodium sulphite in 100 ml of 1% NAOH mix it. store the reagent at
40c
Standard glucose solution: 100 mg in 100 ml distilled water
40% Rochelle salt solution( sodium potassium tartrate solution)

27 | P a g e
 CHAPTER 4

RESULT AND CONCLUSION

28 | P a g e
 CHAPTER 4

RESULT AND CONCLUSION

➢ The effect of heavy metals like zinc and mercury are observed in the
germination, length, dry weight, the fresh weight, and in biochemical analysis (starch,
total phenol and sugar test) of chickpea are as follows. The result of physiological
study like seed germination, root length, dry weight, and fresh weight noted as
follows. The results obtained shows that the percentage of germination of seed tested
was related to metal kind, the concentration used and the species tested (fig 4)
➢

Treatments Day 1 Day2 Day3 Day4 Day5

Dist.water 0% 30% 40% 40% 40%

0.01%znso4 0% 20% 30% 70% 80%

0.1%znso4 0% 30% 50% 90% 90%

1%znso4 0% 30% 30% 80% 80%

0.01%Hgcl2 0% 0% 0% 50% 50%

0.1%Hgcl2 0% 0% 0% 10% 10%

1%Hgcl2 0% 0% 0% 10% 20%

Table 5: observation of seed germination

29 | P a g e
 germination percentage%

day 1
day2
day3
day4
day5

treatments

Fig 5: chart of seed germination

➢ The chart shows that the maximum germination occur in 0.1% Znso4 plate in
day 4 and day 5 (90%), where the minimum germination occur in 0.1%Hgcl 2
(10%) till the day 5. Chart show that the plates of Znso4 are shows the
maximum germination compare to controland the seeds which are treatment
by the Hgcl2 concentration has less germination.Observation of root length
shows in fig 5. Chart shows that seeds which are treatment by 0.1%znso4 has
maximum root length (4.93cm) because of its maximum germination
percentage compare to control. Another side the seed which are treatment by
1%Hgcl2 has minimum root length (0.66cm) because of its less seed
germination compare to control (3.25cm).

30 | P a g e
 Chart Title

control
0.01% znso4
root length

0.1%znso4
1%znso4
0.01%Hgcl2
1% Hgcl2

treatments

Fig 6: chart of root length

Chart Title

control
znso4 0.01%
dry weight

znso40.1%
znso4 1%
hgcl2 0.01%

treatments

Fig 6: chart of dry weight

➢ Observation of dry weight recorded in Fig 6. Chart shows that the 0.1% znso4
seeds have maximum dry weight (0.096g) compare to control seeds (0.082g).
Minimum dry weight is present in 1% znso4 seed (0.069g)

31 | P a g e
 Chart Title

control
0.01% znso4
0.1% znso4
1% znso4
0.01% Hgcl2

Fig 8: chart of fresh weight

➢ The graph shows that seeds of 0.1% znso4 have highest fresh weight (0.759g)
and the 0.01% znso4 seeds have low fresh weight (0.550g) compare to control
(0.624g).
➢ The results of biochemical study mentioned as follows. Result of Estimation
of starch recorded in chart .Graph (fig 8) shows that starch present in 0.1%
znso4 treated seed has maximum (0.338) compare to control (0.0451). Where
the 1% Hgcl2 treated seeds has lowest starch concentration (0.0015) compare
to control

32 | P a g e
 Chart Title

control

starch concentraion
0.01% znso4
0.1% znso4
1% znso4
1 % Hgcl2
0.01% Hgcl2

treatment

Fig 9: graph of starch concentration

➢ Result of phenol estimation mentioned in chart.The graph (fig 9) shows that

1% znso4 treated seeds contain highest phenol concentration (0.3000)
compare to control, and the 0.01% Hgcl2 treated seeds contain lowest phenol
concentration (0.1226) compare to control.

Chart Title

control
phenol concertration

hgcl2 0.01%
hgcl2 1%
znso4 0.01%
znso4 0.1%
znso4 1%

Treatments

Fig 10: graph of Total phenol.

➢ Estimation of sugar resulted (sample sugar) and (standard sugar) in graoh.

Graph shows that (fig 10) 0.1% znso4 treated chickpeas contain highest sugar

33 | P a g e
 concentration (0.7039) compare to control (0.652) and 0.01% Hgcl2 treated
seeds contain lowest sugar concentration (0.0266) compare to control.
➢ Fig 11 shows the result of standard sugar. Fig 11 shows that 1ml aliquote
contain test-tube has maximum sugar concentration (0.9887) compare to
control (0.3958) and the 2ml aliquote contain test-tube has minimum sugar
concentration compare to control.

Chart Title

control
sugar concertration.

znso4 0.01%
znso4 0.1%
znso4 1%
hgcl2 1%
hgcl2 0.01%

treatments

Fig 11: sample sugar concentration

34 | P a g e
 Chart Title

0.0ml
sugar concertration

0.5ml
1ml
1.5ml
2ml
2.5ml

aliquotes

Fig 12: standard sugar concentration

Zinc (Zn) is one of the vital micronutrients for plants as it has many critical
functions. It acts as a cofactor for more than 300 enzymes and is also required for the
production of tryptophan which is a precursor of auxin. Zinc also plays a key role in
maintaining the integrity of biological membranes, protein synthesis, photosynthesis,
pollen formation and disease resistance . Moreover, more than 3000 proteins contain
Zn prosthetic groups in their structure . Zinc deficiency affects the productivity of
about 30% of cultivated soils around the globe .

Approximately 70% of cultivated soils of Pakistan are Zn-deficient . This

problem is more severe in rice-wheat cropping systems as half of the area grown
under this cropping system is severely affected by Zn-deficiency . Among the cereals,
wheat is more prone to Zn-deficiency than other micronutrient-deficiencies. ZINC
SEED PRIMING IN WHEAT Micronutrients, including Zn, can be supplied through
soil fertilisation, foliar spray and seed treatment. However, soil fertilisation and foliar
spray have limited effectiveness and some environmental concerns. In this regard,
micronutrient delivery as seed treatment (priming or coating) is a cost-effective and
pragmatic option. For priming, seeds are soaked in aerated of water or nutrient
solution for specific periods and then dried back to their initial weight . Nutri-priming
is a type of seed priming in which seeds are soaked in aerated solutions of nutrients .
However, as Zn is needed in very small quantities, priming in solution with higher
concentration may cause seed damage and poor germination. Therefore, Zn source
35 | P a g e
 and the concentration of nutrient solution should be optimised before field testing.
Since, no information is available regarding seed priming using different sources of
Zn, this study was conducted to optimise Zn application through seed priming using
different sources for improving the germination and seedling growth of wheat.

Among heavy metals, an zinc are actively involved in cellular metabolism

since both of them, especially zinc are present in the structure of many proteins. Zinc
as an element of the non-oxidative micronutrient has a structural and catalytic role in
many proteins and enzymes related to energy metabolism. Zinc is effective in
nitrogen metabolism of plants, increasing the uptake of elements at low temperatures
during the growing season and increasing the production of Indole Acetic Acid (IAA)
which causes cell elongation. High levels of zinc concentration can reduce plant
growth, stop the metabolic activity of soil, damage the microorganisms and
earthworms and slow the decomposition of organic matters which have roles in the
supply of root food.

Mercury is classified as a micronutrient. Mercury is very essential for plant nutrution

at low level because, it is vitally involved in a number of metalo enzymes to provide
stability to cytoplasm and ribosomes.

➢ Conclusion
Result obtained from this lab experiment illustrated seed germination with Zn , Hg,
and water in different concentration had different effect on seedling. By the
observation its noted that lower concentration of zinc is essential for germination of
chickpea while mercury is decrease seed germination. Zinc treated seeds contain high
amount of starch, phenol ,and sugar proven by biochemical analysis.
➢ References

Alireza Houshmandfar1* and Farhang Moraghebi2 2011. To study the effect of mixed
cadmium, copper, nickel and zinc on seed germination and seedling growth of
safflower. In department of Agronomy and Plant Breeding, Islamic University,
Saveh Branch, Saveh, Iran. Dep of Biology, Islamic Azad University, Shahre
Rey Branch, Tehran, Iran. pp. 1463,18.

Dayanand, S.M. Mehta and R.K. Verma 2013. To study SEED YIELD AND
ECONOMICS OF CHICKPEA AS INFLUENCED BY FOLIAR

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 APPLICATION OF AGROCHEMICALS. Krishi Vigyan Kendra, Jhunjhunu,
Rajasthan.pp:359-362

HABIB UR RAHMAN KHAN 1998. RESPONSES OF CICKPEA (CICER

ARIETINUM L.) TO ZINC SUPPLY AND WATER DEFICITS. University of
Agriculture, Faisalabad, Pakistan.

Hamideh Shirvani Sarakhsi, Ebrahim Khalilvand Behrouzyar 2014. Effect of seed

priming with Zn, Mn, and B in different concentrations on yield and yield
components of wheat. Department of Agronomy and Plant Breeding.
International Journal of Bioscience, vol.5, No.9, p.332-339.

J.B. Valenciano, J.A.Boto and V.Marcelo 2010. Response of chickpea (Cicer

arietinum L.) yield to zinc, boron and molybdenum application under pot
condition. Department of Agrarian Engineering and Sciences. Spanish Journal
of Agricultural Research, 8(3),797-807.

Kuldeep Balai , Y. Sharma , M. Jajoria , P.Deewan and R. Verma 2017. Effect of

Phosphorus, and Zinc on Growth , Yield and Economics of Chickpea (Cicer
arietinum L.). int.J.Curr.Microbiol.App.Sci 6(3) , 1174-1181.

Prof Dr Ali Esmail Al-Snafi 2016. The Medical Importance of Cicer arietinum L.
Department of pharmacology. IOSR Journal og Pharmacy vol.6, pp.29-40.

TAOUFIK EL RASAFI, MOHAMED NOURI, SAID BOUDA, ABDELMAJID

HADDIOUI 2016-17. THE EFFECT OF Cd, Zn, AND Fe ON SEED
GERMINATION AND EARLY SEEDLING GROWTH OF WHEAT AND
BEAN.vol.35, pp.213-223.

Taylor C. Wallace, Robert Murray and Kathleen M.Zelman 2016. The Nutritional
Value and Health Benefits of Chickpeas and Humus. Department of Nutrition
and Food studies, Human Sciences.

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