m.phil_docox[1] (1)

NANO-BIOCHAR INDUCED GROWTH AND PHYSIO-
BIOCHEMICAL ALTERATIONS IN WHEAT (TRITICUM

AESTIVUM) AT VEGETATIVE STAGE
SUBMITTED BY:
HAFIZA RIDA QADEER
REG. # PBOT02193027
IN PARTIAL FULFILLMENTS FOR THE DEGREE OF MASTERS OF

PHILOSOPHY (M.Phil) IN BOTANY
INSTITUTE OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY

THE UNIVERSITY OF LAHORE
(2019-2021)
IN THE NAME OF ALLAH THE MOST BENEFICENT AND THE MERCIFUL
i
AUTHORS DECLARATION
I, Hafiza Rida Qadeer (REG No PBOT02193027) hereby state that my (M.Phil Botany)
thesis titled “NANO-BIOCHAR INDUCED GROWTH AND PHYSIO-
BIOCHEMICAL ALTERATIONS IN WHEAT (TRITICUM AESTIVUM) AT
VEGETATIVE STAGE” is my own work and has not been submitted previously by me
for taking any degree from this university “The University of Lahore” or anywhere else
in the country/world.
At any time if my statement is found to be incorrect even after my graduation, the
university has the right to withdraw my (M.Phil) degree.
__________________
______
HAFIZA RIDA
QADEER
PBOT02193027
ii
PLAGIARISM UNDERTAKING
I solemnly declare that research work presented in the thesis titled “NANO-BIOCHAR
INDUCED GROWTH AND PHYSIO-BIOCHEMICAL ALTERATIONS IN
WHEAT (TRITICUM AESTIVUM) AT VEGETATIVE STAGE” is solely my
research work with no significant contribution from any other person. Small
contribution/help wherever taken has been duly acknowledged and that complete thesis
has been written by me. I understand the zero-tolerance policy of the HEC and the
University “The University of Lahore” towards plagiarism. Therefore, I as an Author of
the above titled thesis declare that the thesis is complete with no material omitted.
Further, no portion of my thesis has been plagiarized and any material used as reference
is properly referred /cited.
I undertake that if I am found guilty of any kind of plagiarism in the above titled thesis
even after award of M.Phil ‘Botany’, the university reserves the right to withdraw/
revoke my M.Phil ‘Botany’ degree and that HEC and the University has the right to
publish my name on the HEC/University Website on which names of students are placed
who have submitted plagiarized thesis.
Official Name of the Student: HAFIZA RIDA QADEER
Registration No: PBOT02193027 Date:
Signature: ____________________________
This is to certify that research-work presented in the above mentioned thesis was
conducted under my supervision. I certify that the thesis is complete with no material
omitted. Further, no portion of the thesis has been plagiarized and any material used as
reference is properly referred /cited.
Official Name of the Supervisor: Prof. Dr Muhammad Yasin Ashraf TI
Employee No: 9024

Date: ________________
Signature: ________________________
iii
CERTIFICATE OF APPROVAL
This is to certify that research work presented in the thesis, entitled “NANO-BIOCHAR
INDUCED GROWTH AND PHYSIO-BIOCHEMICAL ALTERATIONS IN
WHEAT (TRITICUM AESTIVUM) AT VEGETATIVE STAGE” was conducted by
Hafiza Rida Qadeer under the supervision of Prof. Dr. Muhammad Yasin Ashraf TI.
No part of this thesis has been submitted anywhere else for any other degree. This thesis
is submitted to the COE-UOL in partial fulfillment of requirements for the degree of
Master of philosophy in the field of Botany, Institute of Molecular Biology and
Biotechnology, The University of Lahore.
Student Name: Hafiza Rida Qadeer Signature:

_________________
Supervisor Name: Prof Dr. Muhammad Yasin Ashraf TI Signature:
Examination Committee:
(a) External Examiner
Dr. Nudrat Aisha Akram Signature:
_________________
Associate Professor Date:
Department of Botany,
GC University,
Faisalabad, Pakistan
(b) Internal Examiner
Prof. Dr. M. Yasin Ashraf TI Signature: _________________
IMBB/CRiMM Date:
The University of Lahore
Lahore, Pakistan
(c) Prof. Dr. Muhammad Ashraf Signature:

_________________
Pro-Rector IMBB / CRIMM

Date:
The University of Lahore Lahore, Pakistan
iv
DEDICATION
I dedicated all my effort to ALLAH (most beneficent and the
merciful) the HOLY PROPHET (P.B.U.H) and my beloved
Mother and Father
v
ACKNOWLEDGEMENTS
All thanks to Allah Almighty for bestowing upon me the wisdom and potential for
successful completion of this manuscript. I offer my countless salutations to the Holy
Prophet (PBUH) who is the source of guidance for all humanity.
My special gratitude goes to my supervisor Prof. Dr. Muhammad Yasin Ashraf,
Professor, Institute of Molecular Biology and Biotechnology, the University of Lahore
for his valuable guidance, ever encouraging attitude, skilled advice and untiring help
during the course of my work.
I am greatful to my family members. First and foremost to my Father QADEER
AHMAD AWAN Mother SAJIDA PARVEEN then sister Ayesha Malik, brothers
Waleed Ahmad, Ahmad Awan, uncle Syed Abrar shah and grandfather Muhammad
Younas Awan for their moral support and prayers that enabled me to get through all the
ups and downs of my life and helped me to achieve this excellent goals. I am also
thankful to my dearest friends Umber Naeem and Samia Ahmad for their company and
timely support throughout my study duration.
Last but not the least; I feel scarcity of words to express my heartiest thanks to
MUHAMMAD TALHA YAQOOB who stood by me through thick and thin. His never-
ending love and understanding were the only things that kept me sane whenever I was
distressed.
vi
LIST OF ABBREVIATIONS
SYMBOLS NAMES
o
C centigrade
o
F fahrenheit
% percentage
TSS total soluble solids
CT color turning
g gram
mg mili gram
cm centimeter
mL mili liter
Fig figure
mg/g mili gram per gram
TFAA total free amino

acids
vii
TABLE OF CONTENTS
“Bismillah al rahman al rahim” II
AUTHOR’S DECLARATION III
PLAGIARISM UNDERTAKING IV
CERTIFICATE OF APPROVAL V
CERTIFICATION LETTER VI
DEDICATION VII
ACKNOWLEDGEMENT VIII
LIST OF ABBREVIATIONS IX
CHAPTERS DESCRIPTION PAGES #

ABSTRACT XIII
CHAPTER ONE INTRODUCTION
CHAPTER TWO REVIEW OF LITERATURE
CHAPTER THREE MATERIALS AND METHODS
CHAPTER FOUR RESULTS
CHAPTER FIVE DISCUSSION
CONCLUSION
REFERENCES
viii
LIST OF TABLES
Sr. # DESCRIPTION PAGE.
#
Table Physiochemical characteristics of the soil used for field experiment
3.1
Table Treatment detail
3.2
Table Effects of nano-biochar on total soluble sugars contents in wheat
4.1.1 leaves
Table Influence of nao-biochar on Nitrate Reductase Activity (NRA) in
4.1.2 wheat leaves
Table Effects of nano-biochar soil application on total free amino acids in
4.1.3 wheat leaves
Table Effects of nano-biochar on total soluble proteins in wheat leaves
4.1.4
Table Effect of nano-biochar on total phenol contents in wheat leaves
4.1.5
Table Influence of nano-biochar soil amendment on total flavonoid contents
4.1.6 in wheat leaves
Table Effect of nano-biochar soil amendment on chlorophyll “a” contents in
4.2.1 wheat leaves
Table Chlorophyll “b” contents of wheat leaves grown in soil amended with
4.2.2 nano-biochar
Table Effects of nano-biochar on total chlorophyll contents in wheat leaves
4.2.3
Table Influence of nano-biochar on carotenoid contents of wheat leaves
4.2.4
Table Effects of soil amended nano-biochar on shoot length of wheat leaves
4.3.1
Table Effects of nano-biochar on root length of wheat leaves
4.3.2
Table Effect of nano-biochar on the leaf area of wheat plants
4.3.3
Table Influence of nano-biochar application on number of leaves per plant
4.3.4 in wheat
Table Effect of NBC application on number of tiller per plant in wheat
4.3.5
Table Effect of nano-biochar on fresh weight per plant of wheat plants
4.3.6
Table Influence of nano-biochar on dry weight per plant of wheat grown
4.3.7 under different levels of NBC
ix
Table Sodium contents in wheat leaves grown under different levels of NBC
4.4.1
Table Analysis of variance for Potassium contents in wheat leaves grown
Table ANOVA showing the phosphorus contents in wheat leaves grown
Table Leaf nitrogen contents in wheat grown under different levels of NBC
4.4.4
Table Calcium contents in wheat leaves grown under different levels of
4.4.5 nano-biochar
Table Variations in magnesium contents of wheat leaves grown under
4.4.6 different levels of nano-biochar
x
LIST OF FIGURES
Sr. # DESCRIPTION PAGE.
#
Figure Field Experimental site of IMBB, The Univesity of Lahore 15
3.1
Figure During data collection 16-17
3.2
Figure During the analysis of Nitrate Reductase Activity (NRA) 21
3.3
Figure During the analysis of Chlorophyll contents 23
3.4
Figure Digestion of dry plants 24
3.5
Figure Working on spectrophotometer 27
3.6
Figure Effects of nano-biochar on total soluble sugars in wheat leaves 36
4.1.1
Figure Effects of nano-biochar on Nitrate Reductase Activity (NRA) in 37
4.1.2 wheat leaves
Figure Effects of nano-biochar on total free amino acids in wheat leaves 38
4.1.3
Figure Effects of nano-biochar on total soluble proteins in wheat leaves 39
4.1.4
Figure Effect of nano-biochar on total phenol in wheat leaves 40
4.1.5
Figure Effects of nano-biochar on total flavonoids in wheat leaves 41
4.1.6
Figue Effects of nano-biochar soil amendment on chlorophyll “a” in wheat 42
4.2.1 leaves
Figure Effects of nano-biochar on chlorophyll “b” in wheat leaves 43
4.2.2
Figure Variations in total chlorophyll contents in wheat leaves under 44
Figure Changes in carotenoid contents in wheat leaves grown under different 45
4.2.4 nano-biochar levels
Figure Nano-biochar induce changes in shoot length in wheat plants 46
4.3.1
Figure Root length variations in wheat plants grown on nano-biochar 47
4.3.2 amended soil
Figure Variations in leaf area of wheat plants grown in different levels of 48
4.3.3 nano-biochar
xi
Figure Variations in number of leaves per plant in wheat plant grown in soil 49
4.3.4 amended with different concentration of NBC
Figure Effects of nano-biochar amendment in soil on number of tillers per 50
4.3.5 plant in wheat grown under different levels of NBC
Figure Variations in fresh weight per plant of wheat plants grown under 51
Figure Variations in dry weight per plant of wheat plant grown under 52
4.3.7 different levels of NBC
Figure Sodium contents in wheat leaves grown under different levels of 53
4.4.1 Nano-biochar
Figure Variations in Potassium contents of wheat leaves grown under 54
4.4.2 different levels of Nano-biochar
Figure Variations in phosphorus contents in wheat leaves grown under 55
4.4.3 different levels of NBC
Figure Variations in nitrogen contents in wheat leaves grown under different 56
4.4.4 levels of NBC
Figure Calcium contents in leaves of wheat plant grown under different 57
4.4.5 levels of nano-biochar
Figure Variations in magnesium contents of wheat leaves grown under 58
xii
Abstract
Present investigation was conducted to study the effects of nano-biochar (NBC) on
growth and physio-biochemical activities of wheat. The aim of this study was to improve
the growth, biomass and physio-biochemical alteration in wheat plant with the
application of NBC and to achieve it a field experiment was conducted in the Botanical
area of the University of Lahore, Lahore, Pakistan. The experiment was comprised of 4
NBC treatments [0 (Control), 1, 3 and 5% NBC] with three replications in completely
randomized block design (CRBD). The soil amended with NBC significantly enhanced
the root and shoot lengths, fresh and dry biomass of root and shoot by improving
chlorophyll contents, carotenoids, total soluble sugar, phenol, flavonoids, Proteins, amino
acids and nitrate reductase. The nutrient uptake, like nitrogen, potassium, phosphorous,
calcium and magnesium was also increased by the soil amendment with NBC except
sodium which decreased due to NBC. The highest improvement in growth was noted in
plants grown under 3 % NBC which was closely followed by 5% NBC. So, 3% NBC is
the most effective rate of NBC application through soil.
xiii
Chapter 1
INTRODUCTION
Pakistan’s economy backbone is agriculture that influences the gross domestic

product (GDP), foreign exchange earnings and employment rate in the country.
Wheat (Triticum aestivum L.) is the main cereal crop belonging to the family Poaceae
(Zhang et al., 2010). It is an important staple food used in Asia and the rest of the
world. Wheat is the leading human food, which provides protein and more than 60%
calories in the daily diet (Rahman et al., 2014). Wheat is worldwide cultivated crop
with 650 million tons per year and is the second crop produced after rice (FAO,
2012). Over the past two decades the ongoing demand for food of ever growing
population of world is increased and to meet this demand, food crop production has to
be accelerated. Although worldwide the food grain production has increased by 70%
(FAO, 2016) but still it is not sufficient to fulfill food requirements of the world
population, so there is an urgent need to increase the crop productivity, especially
wheat grain production.
In Pakistan wheat is cultivated on an area of over 9.0 million hectares, i.e., 40 percent
of the entire arable land. In May 2019/20 wheat production was estimated at two
percent to be about 25.76 million tons than last year 25.25 million tons. The
government of Punjab, Pakistan fixed the wheat grain price Rs.1800/40 kg during
2021 (Foreign Agriculture Service, 2021). Due adverse climatic conditions during
2020-21, wheat grain yield severely affected and total grain production was not
enough to meet the domestic requirements of the country and Government asked the
agriculture sector to increase wheat grain production by adopting some cultural
practices like crop nutrient management or introducing new high yielding wheat
cultivars. Development of wheat varieties having high yield potential is a long term
process, however, grain yield of the existing wheat varieties can significantly be
improved by proper crop nutrient management.
Soil is the mainly essential source of nutrients. Because of the quick reduction of
agricultural areas and soil value by means of continuously increasing population and
an unnecessary addition of chemical fertilizers, an improved concentration is a need
to maintain sustainable approaches in agricultural crop production. Crop growth and
1
productivity are powerfully influenced by different biotic and abiotic stresses such as
drought, high salinity, excessive temperature, etc and the soil quality (Thalman and
Santelia, 2017). Soil is also contaminated by heavy metals through different human
activities (Moon et al., 2013) which affect plant growth and development and
eventually brings low yielding cropping systems (Al-Farraj et al., 2013).
Biochar is the best way to overcome all the problems being faced by nowadays.
Biochar reduces the carbon deficiency in the soils. It improves the fertility of soil and
also reduces the soil acidity. Biochar is an excellent practice for overcoming any
biotic stress in the soil. Biochar affects the interaction between water of soil plants
and helps to improve its photosynthetic activity, nitrogen availability and water
efficiency in plants (Rawat et al., 2019). After biochar implantation the carbon
content increases rapidly in the soil and also improves the texture of soil, composition
and porosity. Biochar also provides potassium, phosphorus, sulfur and other trace
elements in the soil (Keith et al., 2011). Soil and plant responses to biochar use also
varied with agricultural system, crop type and climatic conditions (Gundale and
DeLuca, 2007; Unger et al., 2011).
Biochar is a strong carbon blended with pyrolysis of different biomass. The thermal
degradation of biomass occurs at high temperatures where there is or lack of oxygen
producing coal as a biochar product. The carbon present in the biochar is mainly
carbon perfumed, and is formed by the extraordinary quantity of perfuse rings with
specific area ranged between 1.5–500 m2 g-1 (Suliman et al., 2016; Li et al., 2018).
Carbon-rich materials have a very oily structure and a high level of aroma (Chen et
al., 2013; Lehmann and Joseph, 2015). The carbon technology used in this process
and raw materials are closely related to biochar structures (Zhao et al., 2018).
Biochar contains many active groups like carboxyl, carbonyl and hydroxyl and it also
has excellent absorption capacity, hydrophilicity, and ion exchange power (Anton-
Herrero et al, 2018). Biochar has high carbon content, boiling point, hardness,
chemical decomposition and organic matter (Hammes et al., 2008; Leng et al., 2019).
Because of these factors, it can remain in the soil for thousands of years under natural
conditions (Sun et al., 2018). Biochar promotes plant growth and development,
increases the nutrient uptake and reduces the effects of harmful substances such as
heavy metals (Beluri et al., 2018). As compared to fertilizer, the reaction of the
2
biochar is more efficient to improve the growth and productivity of the crop. Biochar
is one of the most eco-friendly fertilizers, which are used to improve the soil fertility
(Dejene et al., 2019).
In addition to meet the growing demand for bioenergy, there will be an additional
mandate to improve agricultural productivity (Prasad et al., 2014). A major increase
in agricultural give way could be possible through using current policy of
nanotechnology to boost the rate of photosynthesis to develop competent nutrient
system in plants (Tarafdar et al., 2013).
Mainly the nanotechnology is used to enhance the crop production with appropriate
progress by improving the forming system (Chen et al., 2017). From Biochar, nano-
biochar is recently prepared by Chinese scientist and being used to enhance the crop
yield because it plays an important role in increasing water flow and reducing the
toxicity and reactivity of heavy metals in the soil. Shortage of carbon can be beat by
the use of nano-biochar and also improve the soil composition and porosity. (Kavitha
et al., 2018). Naturally, nano-biochar is usually synthesized in the form of
carbonaceous nano-particles (Ma et al., 2010).
Nano-biochar is the smallest particle of biochar and it is obtained by grinding and

crushing of biochar. The size of the nano-biochar is less than 100nm. Its use depends
on many factors but mainly on its size. Nano-biochar will inevitably interact with
natural minerals in the soil and environment (Wang et al., 2015). And nano-biochar
also has the potential to increase water mobility and reduce nutrient deficiencies
(Chen et al., 2017). Bulk-biochar has failed to transport a group of nutrients and other
substances whereas nano-biochar as a carrier can make possible the movement of
nutrients, natural solids and other substances (Lian and Xing, 2017). Nano-biochar is
used to improve the product quality (Brunner et al., 2006), increase soil fertility and
bio-availability to plant nutrients (Chhipa, 2017; Singh and Kumar, 2017).
In Pakistan, crop production is affected by the deficiency of nutrients and other

various factors. All the problems of crop assembly as well as the nutrient shortage can
be resolved by using nano fertilizer and it can be proved a very effective tool in
agriculture. Nano-fertilizer is also called nanomaterial, which can be a nutrient of its
own (micro- or macro-nutrient) or a transporter of plant material (DeRosa et al.,
2010). The crop yield depends on the application of fertilizers. Excess use of
3
fertilizers reduces fertility and growth of the crops (Kumar et al., 2019; Diatta et al.,
2020).
For the sustainable agriculture, environmental protection from pollution nano-

particles provide security for better planning and maintenance of plant production
inputs (Li et al., 2016; He et al., 2019). To keep in mind, the importance of wheat
grain to the people of Pakistan can increase crop growth and productivity; present
investigation was conducted to test the following hypothesis:
Hypothesis
“Application of nano-biochar may enhance the wheat crop growth by improving

nutrient uptake and altering physio-biochemical metabolic activities”.
Objectives:
Current study was conducted to achieve objectives given below:
 To investigate effects of nano-biochar on the physio-biochemical activities of

wheat.
 To estimate influence of nano-biochar which induce wheat plant growth at
vegetative stage.
4
Chapter 2
Review of Literature
Wheat is one of the most widely grown crops in the world, producing 650 million tons
per year. Wheat is the second produced cereal after rice (FAO, 2012). Wheat is the
grain crop of the Poaceae family (Zhang et al., 2010), which played a major role in
the Pakistani economy (Iqbal et al., 2005). It is one of the staple cereals grown
worldwide to meet nutritional needs. According to the 2019 Pakistan Economic
Survey, the wheat growth rate was 1.99%. Its total share in food grain production was
35.5% (Sturz et al., 2000).
Jasrotia et al. (2018) studied that wheat a staple food crop grown in 221.6 million
hectares in a variety of climates is expected to reach an annual production of 750.4
million metric tons in 2016-17 probably. To meet future food demands imposed by a
growing population that is expected to reach nine billion by 2050, global wheat
production must continue to grow at 2% per year. Among the advance technologies,
nanotechnology is gathered critical information due to its broad spectrum in
agriculture and allied fields. It has wide applications in the fields of crop production,
food security and sustainability.
Abid et al. (2018) analyzed the wheat crop trends and diversity in Pakistan. The study
found that the area under wheat cultivation in Punjab, Sindh and Balochistan
increased over time and the area under wheat cultivation in Khyber Pakhtunkhwa
province decreased slightly between 1981-85 and 2011-15. The results confirmed that
Balochistan recorded high level of variations in acreage in wheat crop and Punjab
region recorded very low level of variations in wheat crop production. They
concluded that many variations in wheat production were due to the variety of wheat
area and its yield.
Biochar is a solid carbon-containing product formed when biomass decomposes

thermally under anoxic conditions. In recent years, with increasing demands for food
security, environmental protection and reduction of greenhouse gas emissions,
biochar gradually has become associated with soil management, sustainable
agricultural development and carbon sequestration (Lehmann et al., 2006). Biochar is
generally alkaline, related to inorganic minerals such as carbonates and phosphates
5
and formed during pyrolysis and carbonization (Yuan et al., 2011). At different
pyrolysis and carbonization temperatures, the pH usually rised with temperature due
to decomposition of acidic functional groups such as carboxyl and phenolic hydroxyl
and evaporation of organic acids (Novak et al., 2009; Yuan et al., 2011; Chintala et
al., 2014).
Biochar is stable black carbon that is obtained by the pyrolysis of carbonated material
in an anaerobic state (Qin et al., 2020). Biochar is widely used in the removal of
organic pollutants in the soil. The versatile properties of biochar make it suitable for
the removal of organic and inorganic pollutants in soil and water contaminated areas
with microporosity, surface functional group and high surface area (Ahmad et al.,
2014). Biochar exhibits different properties due to different raw materials such as
stalks, grain bran, hard wood, compost and sludge that are susceptible to pyrolysis
(Atkinson et al., 2010). The pyrolysis process in biochar production helps to remove
odor and moisture from the feedstock (McHenry, 2011) and determines the properties
of the biochar.
According to Ding et al., (2016) biochar improves soil physical conditions, texture,
structure, bulk density, porosity and particle size distribution. Crop residues
management has become a challenge for farmers to strike a balance between an
economic and sustainable environment. Therefore pyrolysis of crop residues may
become an alternative technology. The heterogeneous properties of biochar produced
by the pyrolysis process and used as soil modification. Biochar application solves
ongoing problems in the soil and enhances crop production. It greatly involved in
carbon sequestration by maintaining soil quality (Yazhini et al., 2020)
Abiotic stress drought that was severely reduced the wheat yields worldwide. Haider
et al. (2020) observed that biochar is a living soil supplement, improves soil carbon,
organic content, soil water retention capacity and also improved soil fertility. They
performed experiment using biochar during the stages of wheat growth, flowering and
grain filling stage to mitigate the adverse effects of drought. The results showed that
drought stress had a negative impact on wheat growth and all the stages of
development and the grain filling stage was identified as the most important stage,
resulting in a significant increase in yield. Biochar application has been used as an
6
effective strategy to achieve better wheat yields by minimizing the negative effects of
drought stress.
Biochar is produced from plant material and stored in the soil as a means of removing
carbon dioxide from the atmosphere (Jatav et al., 2016). Biochar improves soil health
and crop productivity (Bista et al., 2019). An experiment was performed to evaluate
the effects of biochar on wheat. Biochar applied with different percentages (11.2,
22.4, and 44.8 Mg ha-1) on wheat and to compare them with controls. Biochar
increased soil (SOM), soil pH, phosphorus, potassium, sulfur content, germination
and wheat source biomass. The results showed that biochar treatment plots had
greater soil moisture during the experiment which attributed to the high water
retention capacity of biochar 8 to 40% higher than the control plots (Glaser et al.
2002; Brockhoff et al., 2010; Makoto and Yasuyuki, 2010).
According to Shetty and Prakash, (2020) biochar is used to reduce the soil acidity and
for the improvement of plant growth. They conducted study to understand the effects
of biochar on soil and growth parameters of plants. The results showed that the
application of biochar reduced the solubility and decrease the acidity of the soil and in
turn increased the plant growth by improving soil fertility. They concluded that high
dose of biochar performed better and improve the productivity and growth of the crop.
Hamzah and Shuhaimi, (2018) performed experiment to evaluate the effectiveness on

rice growth by the different applications of biochar (300, 600 and 900 g) and compare
them with control plant. The results showed that growth based on plant height was
maximum by the application of biochar. In another trial biochar applied in soil with
different rates. The results showed that biochar applications improve the soil fertility
by reducing the acidity of soil. They concluded that soil enriched with biochar
increase growth rate by improving the soil fertility.
Jabborova et al., (2021) reported that biochar improve the plant growth and
productivity. They performed pot experiment to determine the effects of biochar on
the growth of spinach. They took four treatments of biochar and arranged them in
completely randomized block design. The result showed that biochar had a positive
effect on the growth of the plants and also significantly increased the all
morphological and physiological parameters of the plants as compared to control.
7
They concluded that biochar improve the soil microbial activity and growth rate of the
plant.
According to Jien and Wang (2013), biochar improved the physical and biological
properties of soil. In a study they used three different percentages (0%, 2.5% and 5%)
of biochar in soil. The experimental results showed that biochar applications
improved the soil structure, particle size, texture and density of soil and also increase
the pH and carbon biomass in the soil as compared to control plants. They concluded
that application of biochar was suitable for soil because biochar reduced the soil loss
and increase the physical properties of soil.
Githinji, (2014) stated that biochar is used as soil amendment for improving the soil
quality and long term carbon storage in the soil. He performed experiment to analyze
the effects of biochar on the soil physical and chemical properties. Biochar applied
with different rates on soil. The results showed that soil amended with biochar gives
the better growth and improve the soil fertility as compared to non-amended soil.
According to Alam, (2018) the study aimed to investigate the effects of biochar and
nitrogen fertilizer on soil physio-biochemical properties and wheat crop growth. He
conducted experiment on wheat and soil properties in randomized complete block
design with the applications of biochar and nitrogen fertilizers. Biochar was applied
before sowing, and later applications of nitrogen were carried out in splits in crops.
The soil structure, texture, density and particle size showed positive response due to
biochar application. The results of the experiment showed that biochar improve the
soil properties while the combine effects of biochar and nitrogen fertilizers might be
helpful in maintaining the soil fertility and to obtained the healthy soil for the better
crop growth and productivity.
Ali et al., (2015) investigated that biochar is important source to improve soil fertility
and increase the nutrients availability in the soil. They performed experiment to
evaluate the effects of biochar and synthetic fertilizers on wheat crop. The results
showed that biochar significantly increased soil carbon, phosphorus and potassium
contents in the soil. Similarly, nitrogen fertilizers improve wheat yield and
components of the yields. They concluded that biochar application alone and
combined with fertilizers increase the wheat crop growth, components of the yield
and improve the soil quality in wheat cropping system.
8
Xiao-Phi Tan et al., (2016) studied that synthesis of biochar-based nano-compounds
provides new structures and combines the benefits of biochar with nano-materials.
Emerging structures often showed significant improvements in functional groups,
pore structures, more active sites, reduced probability and ease of segmentation. The
results showed that these combinations have excellent abilities to absorb a wide
variety of impurities from powerful solutions. The integration of biochar-based nano-
alloys has been an important practice to maximize the natural use of biochar and
nanotechnology.
Biochar production and its use in agriculture played an important role in mitigate the
climate change and helped in the organization of waste from gardening and forest
management (Alburquerque et al., 2012). Biochar is a carbonaceous material obtained
by the thermal decomposition of residual biomass at relatively low temperatures and
under oxygen limiting conditions (pyrolysis). Biochar is currently the subject of active
research around the world as it is a viable alternative to sustainable agriculture
because of the long-lived source for soil and the helpful to crop (Alburquerque et al.,
2013).
Nano-biochar attracts growing interest due to its unique environmental behavior.

However, understanding of its structure, physico-chemical properties and stability of
NBC is limited. Liu et al., (2018) investigated that at 300–600℃ nano-biochar
formed from the bulk biochar i.e shell of peanut, cotton grass, sugar residues and hair
residues. The carbon solidification process and the nano peanut shell (NBC) process
were also observed. Nano-biochar is produced by digestion during biomass charging.
The amorphous fraction in bulk biochar is easily reduced to nano-biochar instead of
the graphic component (Lu et al., 2020).
Yang et al., (2020) studied that to improve soil quality, grain yield, nano-biochar was
applied on plant crop in different percentages. The effects of nano-biochar contents
(0, 0.1%, 0.5%, 0.7%, 1% in mass) were analyzed on soil moisture, nutrients, and
yields. The results showed that with the increased nano-biochar rates, soil bulk
density, soil particle size and moisture content also increased as compared to control.
They concluded that nano-biochar slowed down the transport rate of nitrogen from the
surface to the depth of the soil. The plants with higher nano-biochar rates significantly
increased the yield of the crop.
9
According to Chen et al., (2021) biochar used as soil amendment to improve soil
physical properties and also increased the soil fertility. They performed experiment to
influence the effects of nano-biochar on soil. The results showed that nano-biochar
changed the soil porosity, soil structure and density and also increased the water
holding capacity of the soil. They suggested that nano-biochar used for the
improvement of soil and increase the growth of the crop.
Biochar innovation and nano biotechnology may results in generating of carbon based
nano materials counting nano-biochar and biochar nano composites to revolutionize
the inquire about in concerned areas. Nano-biochar is nano sized biochar material
with better physical, chemical and surface properties. It has many benefits such as
enhanced plant growth, soil properties and plant disease management. It could also
change climate by carbon depletion. Nano-biochar has outstanding potential of
absorbing pollutants, nutrients and impurity as compared to biochar (Chausali et al.,
2021)
Liu et al., (2020) revealed that soil contaminated with cadmium poses a serious threat
to human health and food security worldwide. Biochar has been used to improve soil
fertility and reduced the toxicity of the metals in the soil. The Investigation was
carried out to determine effect of severe cadmium and nano-biochar with different
rates in plant growth. The results showed that plant hight, weight and biomass were
significantly reduced in cadmium contaminated soil as compared to nano-biochar
treated plants. The application of nano-biochar produces a cooperative effect on plants
by increasing soil alkalinization and thereby reducing Cd phytotoxicity in the soil.
Nanotechnology is one of the revolutionary fields in science and technology and is

expected to contribute to the advancement of sustainability, including energy
production, conservation, storage and exchange (Roco et al., 2011). Nanoparticles are
defined as particles not exceeding 100 nm at least, being applied in various industries
like cosmetics, pharmaceutical, food and packaging, bioremediation, paints and
coatings (Zhang et al., 2011). With the increasing use and variety of nano products,
the dangers of nanoparticles are attracting considerable attention. However,
information on interactions between nanoparticles and biological systems is still
lacking (Barrena et al., 2009). Currently, the most commonly released NPs into the
10
environment are carbonaceous nanoparticles, metal oxides, and nanopolymers (Ma et
al., 2010).
Olmo et al., (2014) investigated effect of slowly adding pyrolysis in a field

experiment during the growing season of wheat. Biochar supplementation includes
pH, organic C and N, ammonium, nitrate or microbial biomass. Biochar-treatment
plants showed higher relative growth and equilibrium, surface biomass and net levels
of yield compared to control. Grain or nutrient content levels were not significantly
affected by biochar conversion. The result of this experiment showed that the use of
biochar as a soil supplement in agriculture improved soil structure and increase
fertility, which was favorable for crop growth in low humidity conditions in the
mediterranean.
Bhattacharya et al., (2015) studied that biochar and crop residues in recent times have
attracted a lot of attention as a viable strategy for maintaining soil health. An
experiment was performed to identify effect of biochar in residue and compare them
control plants. The result showed that biochar application increased the wheat
production by 6.2% - 24.2% over control and found high index of C and N
multiplication in biochar. They concluded that biochar significantly increased
Nitrogen and Phosphorus availability beyond control and crop residues.
According to Tomczyk et al., (2020) the positive effect of biochar on soil quality and
subsequently, crop growth was volatile and climate appears to be the main reason for
the differences. Sun et al., (2019) conducted experiment on wheat to assess the
biochar effects of nitrogen utilization efficiency, grain yield and amino acids at
different rates (i.e., 0, 5, 10, 20, 30 t/ha). The nitrogen utilization efficiency and wheat
grain yield of wheat were increased by applied high amount of biochar. The high
biochar applications (more than 30 tons per hectare) had a negative effect on both
nitrogen utilization efficiency and grain yield. Whereas amino acids significantly
affected by biochar. The result showed the positive effect when the biochar was
applied at sensible rates, but the effect was negative when the biochar is overused.
Jatav et al., (2017) investigated that modern agriculture was leading to a decline in the
level of soil organic matter through nutrient mining and repeated cutting of crops. To
solve this problem, biochar was added to the soil for the improvement of soil health,
increased soil fertility and sequestering carbon. However, variable application rates,
11
uncertain feedstock effects, and early soil conditions offer wide prices for slightly
better yields from biochar additions, which was often economically feasible.
Saxena et al., (2014) worked on carbon nanoparticles of biochar. Water soluble

carbon nanoparticles were applied on wheat plants to improve growth rate. On wheat
crop the water soluble carbon nanoparticles were used in the range of 10 to 150 mg-1
L. Seeds were treated with carbon nanoparticles that showed high growth rate as
compared to control plants. The results showed that water soluble nano particles
positively promoted the growth of the plants. Nanoparticles would better choice than
the fertilizer and manure, because they would be helpful for better plant growth.
Salim, (2016) conducted research to determined the concentration of macro and micro
nutrients in floral parts of the plant by applied biochar. After the application of
biochar he took samples. On the first day of sampling , plant height, number of tillers
/ plants, root length, chlorophyll, number of spikes per plant, maximum spike length
and and micro nutrients were determined on the roots and leaves. On the second day
of the sample (harvest time), spike weight, number of grains per spike, were also
recorded. The result showed that plants with biochar application had greater
morphological and stimulating effects than control.
According to Praveen et al., (2017) application of nanoparticle (NPs) is very effective

in reducing metal toxins. They performed experiment to identify the effects of iron
oxide nanoparticles (Fe3O4 NP) on reducing arsenic toxicity in Indian mustard
(Brassica juncia v. Pusa jagannath). Toxicity was observed in seed germination and
root-shoot length also effects under arsenic (As) pressure, but with the use of Fe3O4
NPs plant growth was improved. The result showed that nanoparticles as nano-
absorbents used in reducing the toxins in a plant and increasing / decreasing various
parameters.
Seleiman et al., (2020) studied the effects of zinc oxide nanoparticles and biochar on
the soil. Biochar significantly reduced the availability of heavy metals in the soil as
compared to ZnO NPs and control treatment. As compared with the untreated site,
single application of biochar improved the soil fertility, availability of nutrients in the
soil. The positive effect of biochar is that it improves the production and yield of the
crop. Biochar treatment increase the plant growth and biomass yield in low nutrient
12
soils. However, the combination of compost and biochar could improve the soil
properties and plant productivity over time (Trupiano et al., 2017).
According to Akhtar et al., (2015) biochar application decreased negative effect of

salinity. An experiment was carried out to identify the effect of biochar in wheat
plant. Biochar supplementation significantly reduced the salinity in plants and
increase the mineral nutrients (especially K+, Ca+, Mg ++) in soil solution. The results
showed that wheat crop growth was particularly positively affected by biochar
processing and significantly reduced the salinity in the soil.
Kubar et al., (2019) reported that through the application of potassium fertilizers plant
growth, yield and nutrient uptake can be obtained. An experiment was performed to
study the effects of macronutrients gathering of wheat about potassium. The
potassium was applied at different percentages on wheat crop. The results showed that
application of potassium increased the potassium contents in grains as compared to
control. The plants without the application of potassium improve the phosphorus
contents in wheat crop. They concluded that application of potassium nutrition
increased the yield components, growth and accumulation of nitrogen, phosphorus
and potassium contents in wheat crop.
Liu et al., (2019) conducted experiment to determine the effects of zinc application on
wheat root and shoot. The results of this study showed that zinc fertilization increased
all the parameters of root as compared to shoot. Shoot biomass, shoot length increased
as zinc application increased on the top layer of soil. They concluded that in order to
get the better yield and grain zinc concentration soil should be fertilized to 30 cm
depth with about 11.4 kg ha−1 zinc.
In emergent nations, the deficiency of zinc caused by the insufficient food intake is a
worldwide alimentary problem. Chattha et al., (2017) in another study performed to
evaluate the effects of zinc application on different parameters of grain. They took
three different wheat cultivars and they were treated with five different applications of
zinc. The results revealed that grain yield and zinc concentration in grain were
positively affected and increased from 2013-2014 and 2014-2015 respectively.
13
Chapter 3
Methodology
3.1. Materials and Methods
To estimate the effects of nano-biochar in enhancing wheat crop growth by improving

nutrients uptake and physio-biochemical processes. A field experiment was conducted
in the experimental area of IMBB, The University of Lahore, Lahore Pakistan with
wheat variety Zincol. The experiment was comprised of 4 nano-biochar treatment i.e
control, 1, 3 and 5% nano-biochar (Table 3.2.) as soil amendment. Each treatment had
3 replications and experiment was conducted in randomized complete block design
(RCBD).
3.1.1. Experimental Site and Conditions
The field experiment was laid out in randomized complete block design
(RCBD) with three repeats in clay loam soil at Agricultural Farm of The University of
Lahore, Lahore Pakistan. The soil texture was determined with the hygrometer
method (Dewis and Freitas, 1970). The physiochemical characteristics (Electrical
conductivity, pH and ion contents) of the soil extract (field experiments) were
determined according to the methods described by Jackson (1962) and are presented
in Table 3.1.
Table 3.1: Physiochemical characteristics of the soil used for field experiments
Soil Characteristics Values
Physical
Soil texture Clay loam
Chemical
Saturation percentage (%) 40.4
ECe (dS m-1) 1.72-1.92
14
Soil pHs 7.5-7.8
Organic matter (%) 0.1-0.3
Ca+Mg (meq L-1) 2.57-4.78
CO3 (meq L-1) Nil
HCO3 (meq L-1) 2.5-4.8
NO3-N (mg kg-1) 3.5- 4.5
Available K (mg kg-1) 75- 80
Available P (mg kg-1) 1.5- 2.8
Fig.3.1. Field Experimental site of IMBB, The University of Lahore
3.1.2. Nano-biochar Treatments
The nano-biochar (NBC) was obtained from Prof. Dr. Lixin Zhang, College of
Life Sciences, Northwest A & F University, Yaangling, Shaanxi, China and applied as
soil amendment 0, 1, 3 and 5%. Treatment detail is summarized below:
15
Table.3.2. Treatment Detail
Sr.No. Treatment Treatment description
1. Control or Without amendment of Nano-Biochar

0 % Nano-biochar
2. 1 % NBC With 1 % NBC as soil amendment
3. 3% NBC With 3 % NBC as soil amendment
4. 5 % NBC With 5 % NBC as soil amendment
3.1.3. Wheat Seeds and Layout
Seed of wheat variety Zincol was obtained from National Agricultural Research
Council (NARC), Islamabad. When soil was at field condition the seeds were sown in
plots (10’x5’ each) for each treatment. Three blocks were maintained during course of
study each meant for each replication in which four NBC treatments were randomly
selected. When plants were of 50 days old their root length and shoot length were
measured using measuring scale. And by using digital electrical scientific balance
fresh weight was recorded. Then plants were oven dry at 720 C for 48 hours. After
that dry weight was recorded in grams. The fresh leaves were preserved for the
following chemical analysis.
16
Fig.3.2. During data collection
3.2. BIOCHEMICAL PARAMETERS
For biochemical analysis, fresh leaves of the plants were collected and chopped and
1g of each sample was added in 10 ml of 0.2 M phosphate buffer solution and mixed
them well. For further analysis the extract was placed in refrigerator.
3.2.1. Total Free Amino Acids (TFAA)
Using the procedure described by Hamilton and Van Slyke (1943) total free amino
acids was analyzed from the above extract brief details are as under:
Ninhydrin solution
Ninhydrin solution was prepared by dissolving 1g of ninhydrin in 100 ml distilled

water (DW).
Pyridine solution
This solution was prepared by dissolving 10 ml of pyridine in 100 ml of distilled

water.
Procedure
In 25 ml test tube 1ml of leaf extract one ml of ninhydrin and 1ml of pyridine solution
was added. Then heat the test tubes in boiling water bath for 20 minutes and allowed
to cool it. The color of the solution was turned into blue and if the color was dense,
then 20 ml of distilled water was added to dilute the color. By using the HALO-SB 10
17
UV-VIS single beam spectrometer the optical density of total free amino acids at 570
nm was measured. Total free amino acids were observed by using the following
formula.
TFAA= Sample reading x sample volume x dilution factor

Leaf fresh weight (µg/g fresh weight) x 1000
3.2.2. Total Soluble Sugar Estimation
By using the method of Riazi et al,(1985) total soluble sugar was estimated.
Anthron Reagent
Anthron reagent was prepared by dissolving150 mg of anthron in 72% of

H2SO4solution. Whenever needed the anthron solution was freshly prepared.
Procedure
One ml of leaf extract was poured in 25 ml test tube and 3 ml of anthron reagent was
also added. After that, in boiling water heat the tubes were incubated for 10 minutes
and then allowed to cool. As a result, green color appeared. Optical density of total
soluble sugars was measured at 625 nm in HALO-SB 10 UV-VIS single beam
spectrometer.
3.2.3. Estimation of Total soluble Proteins
Total soluble protein was fixed by using a process presented by Lowry et al., (1951).
Reagents
1. Phosphate buffer was prepared by using sodium dihydrogen phosphate and

disodium hydrogen phosphate as follows: NaH2PO4.2H2O and
Na2HPO4.2H2O was prepared in stock.
2. Three solutions were prepared for copper reagent. Solution A was prepared
and making the volume of 100ml by adding 2 g of Sodium Carbonate, 0.2 g
of sodium hydroxide and one g of sodium potassium tartarate in pure water.
For solution B, 0.5g of CuSO4.5H2O was added in 100 ml of distilled water.
18
50 ml of solution A and one ml of solutions B were mixed together for
preparing solution C.
3. Folin-Ciocalteu phenol reagent was prepared and to make volume of twenty
ml by adding two ml of Folin-Ciocalteu phenol with eighteen ml of distilled
water.
Procedure
Fresh leaves 0.2 g were taken in 5ml of 0.2 M phosphate buffer then in different
test tubes 1 ml of leaf extract was added .1ml of phosphate buffer was included for
blank. In tubes 1ml of solution C was also added. Then mix them thoroughly and
incubate at room temperature for 40 minutes. After that 1ml of Folin reagent was
added then mixed well and allowed to stand at room temperature for 20 minutes.
Color of the solution tuned blue. After twenty minutes, add 10 ml of distilled
water. On spectrophotometer the optical density was recorded at 620 nm.
Protein (mg/g FW) = Sample Reading x concentration of standard x dilution factor

Weight of fresh leaf x 1000
3.2.4. Estimation of total phenols
According to Julkunen-Titto (1987) total phenols were determined.
Reagents
1. Methanol 80% or 80% acetone
2. HCl 0.1 N in 80% Methanol
3. Sodium Carbonate (20%)
4. Folin-Denis reagent / Folin-Ciocalteu Phenol reagent
Extraction
1 g of oven dried extract was taken and milled the leaves in 10 ml 80% acetone
solution. The sample was added in 50 ml test tube with 10 ml of 80% acetone,
dissolved it thoroughly for 2 hours. During the extraction solvent was changed 3 times
(3 x 10). The extracted mixture was centrifuged for ten minutes with 10,000 rpm at
300C. The supernatant was cooled then made the volume up to 100 ml and it is used
for total phenol estimation. The extract was called crude extract.
Total phenol estimation
19
In ten ml test tube 1ml of crude extract was taken with 2 ml water. One ml of
Folin-Denis reagent was added and the test tube was continuously shaking.
Immediately, 5 ml of twenty percent sodium carbonate solution was added and made
the volume of the mixture up to 10 ml by shaking thoroughly again. After twenty
minutes read the absorptivity of the mixture at 700 nm and 735 nm without
background measurements. Against the air, spectrophotometer was set to zero before
measuring the samples, for precipitate formation centrifuge is necessary. For standard
curve tannic acid was used.
3.2.5. Nitrate reductase activity (NRA)

To estimate nitrate reductase activity (NRA), the method of Sym (1984) was
followed. For the analysis, randomly collected fresh leaves were used.
Reagents
 0.02 M Phosphate buffer of pH 7
 Substrate 0.02 M KNO3solution in pH 7 phosphate buffer.
 0.02 g N (I -naphthyl) ethylene diaminedihydrochloride
 1 g sulphanilamide solution in 2 N HCl
Preparation of reagants
Phosphate Buffer (pH 7.0):

By dissolving sodium dihydrogen phosphate (NaH2P04) Phosphate buffer 0.02 M (pH
7.0) was prepared and disodium hydrogen phosphate (Na2HP04) containing 0.1 %
triton X-100.
Substrate KNO3 Solution (0.02M)

In phosphate buffers of pH 7.0, 2.2 g of KNO3 was dissolved for substrate solution.
Sulphanilamide (1%)
One g of sulphanilamid dissolved in 100 ml of 2NHCl.
N (I-Naphthyl) ethylene diaminedihydro chloride (0.02%)

It was prepared by dissolving 0.02 g in 100 ml of distilled water.
20
Procedure
Leaf samples (0.5 g) widthwise chopped in 4.5 ml of 0.2 M phosphate buffer (pH 7.0)
containing 0.5 ml of 0.02 MKNO3, in twenty five ml test tube and incubated in dark
for one hour at 32 °C. After the incubation period, 1ml of reaction mixture was taken
in another test tube containing 0.5 ml of 1% sulphanilamide. After shaking 0.5 ml of
N (1-naphthyl)-ethylene diaminedihydro chloride immediately added. A pink colored
complex was formed with nitrogen dioxide. After twenty minutes dilution was made
by adding measured amount of distilled water to dilute color and centrifuge for five
minutes at 2000 rpm to remove the present turbidity. By spectrophotometer
absorbance was read at 542 nm. The nitrate reductase activity in the form of µ mol of
NO2 g-1 fresh weight h-1 was calculated from the standard graph developed with
different concentrations of sodium nitrate. Use blank consisting of one ml of
incubated buffer with all chemicals.
Fig.3.3. During the analysis of Nitrate Reductase Activity

(NRA)
3.2.6. Estimation of Flavonoid contents

By using the method given by Pekal and Pyrzynska (2014) flavonoide contents were
dertermined.
21
Reagents
 80% methanol
 10% Aluminium chloride
 4% NaOH
 5% sodium nitrus oxide
Procedure
Plant material was mixed in 10 ml of 80% methanol and then filter plant material was
added in test tube having 50 ml capacity.In test tube 5ml of methanolic extract 150µl
of 0.5 molar sodium nitrus oxide and 150 µl of 0.3 molar aluminium chloride was
added mixed it and incubate for five minutes and then added 1 ml of sodium
hydroxide. The obsorption was measured at 510 nm with UV via spectrophotometer.
3.2. PIGMENT ANALYSIS

For the determination for chlorophyll and carotenoid contents the method given
by Arnon (1949) and Davies (1976) was used. Using fresh leaves the chlorophyll
contents were studied. Required Reagents
 Acetone 80%
 Pure water
Procedure
In 5ml of 80% acetone 0.2g fresh leaves were chopped. The extraction was done
overnight at 10℃. The optical density was measured at 480 nm, 645nm and
663nm wavelengths on spectrophotometer (HALO SB-10, UV-VIS single beam
spectrophotometer, Dynamica, UK).
22
Fig.3.4. During the analysis of chlorophyll contents
Calculations
For the calculation of chlorophyll (chlorophyll a, b, total chlorophyll) and carotenoid

contents (Davies, 1976) the following formula were used.
Chl. a = [12.7 (OD663) - 2.69 (OD645)] xV
1000 x W
Chl b = [22.9 (OD645) – 4.68 (OD663)] xV
1000 x W
Total Chl = [20.2 (OD645) +8.02 (OD663)] xV
1000 x W
Carotenoids (g mL-1) = Acar / Em x 100
23
Where
A car= OD480 + 0.114 (OD663) - 0.638 (OD 645) E100 % cm =2500
V = Volume of sample extract
W = Weight of sample
3.3. Nutrient Analysis
Digestion
By using the method given by Wolf (1982) digestion of dried plant material was
carried out. In digestion tubes dry ground plant material (0.2 g) and 3 ml of
concentrated sulphuric acid was poured to each tube. All the tubes were incubated for
whole night at room temperature. Then 0.5 ml of H2O2 (35%) was added along the
sides of the tubes and transfer them in a digestion block and heated at 350oC until
fumes were produced. Again tubes were continuous heated for 30 minutes then
digestion tubes were split and allowed to cool down for 5 minutes. Then 0.5 ml of
H2O2 was added slowly and heated the tubes by placing them back into the digestion
block. The above step was repeated until the digested material became colorless. The
volume of the extract was made up to 40 ml in volumetric flasks. This extract was
filtered and used for determining sodium, potassium, calcium, magnesium,
phosphorus and nitrogen.
24
3.4.1. Estimation of Nitrogen
Total nitrogen (N) was determined by micro–Kjeldhal’s method proposed by Bremner

(1965). For N analysis, following reagents was prepared.
Reagents Required
 Boric acid solution (2%)

 Sulphuric acid standard (0.01 N)
 Mixed indicator of bromocresol green and methylene red
Procedure
The above mention aliquot (used for the determination of Na+, K+, Mg2+, Ca2+ and P)
5 ml was performed in Kjeldhal’s flask which was placed on Kjeldhal’s ammonia
distillation unit. Then 5 ml of 40% sodium hydroxide were poured to each tube. In a
conical flask nitrogen in the form of ammonia was taken out and boric acid solution
(5ml) with few drops of mixed indicator was also added. When the distillate was
about 40 ml in the flask, then distillation was stopped. For a few minutes the distillate
was cooled and titrate it with 0.01 N standards sulphuric acid till the appearance of
pink color (end point). A blank was also used in the same way. Total Nitrogen was
estimated using following formula:
(V2-V1) x N x 0.014 x 100

Nitrogen (N) % = ________________________
W
Where
V2 = Volume of standard H2SO4 required to titrate the sample
solution.
V1 = Volume of standard H2SO4 required to titrate the blank
solution.
N = Normality of H2SO4
W = Weight of the sample
25
3.4.2. Estimation of sodium (Na), Magnesium (Mg) and potassium
(K)
In digested leaf samples of Sodium (Na), Magnesium (Mg) and potassium (K) were
analyzed by flame photometer (Jenway, PFP)
3.4.3. Estimation of phosphorous
Phosphorus (P) estimation was done by a spectrophotometer (Jackson, 1962).
Barton’s Reagent
1. For the preparation of solution A 25 g of ammonium molybdat was dissolved

in 400 ml of pure water.
2. For the preparation of solution B 1.25 g of ammonium metavandate was
dissolved in boiling water (300 ml), cooled and mixed with 250 ml of conc.
HNO3 .Then the solution was cooled again at the room temperature.
To get ready working Barton’s reagent, solution A and B were mixed and made the
volume up to l liter. It was placed at room temperature.
Procedure
2 ml of above mentioned aliquot (used in Na, K, Mg2+, Ca2+ and P determination) was
liquefy in 2 ml of Barton’s reagent and made the total volume up to 50 ml. This
mixture was kept for thirty minutes at room temperature for the development of color.
The O.D of samples was measured at 470 nm wavelength on a spectrophotometer
(Hitachi-20). The concentration of phosphorus was determined by using standard
curve.
3.4.4. Estimation of Calcium

Estimation of calcium was done according to the method in Hand Book-60 by US
Salinity Laboratory Staff, 1962. Details are as under:
Solution B: Sodium hydroxide (approx.4N)
For the preparation of solution B, 160 g of sodium hydroxide (4 N) was dissolved in

one liter of water.
26
Solution E: (Ammonium pupurate indicator)
Ammonium pupurate 0.5 g was mixed with 100 g powederd potassium sulphate.
Solution F: (0.01N ethylene diamine tetra acetate ‘vesenate’ solution)
Dissolved disodium dihydrogen ethylene diammine tetra acetate (2.0 g) and

magnesium chloride hexa hydrate (0.05) in distilled water and made its volume up to
1 liter.
Procedure
For the estimation of Calcium, in a conical flask five ml of aliquot was taken and
diluted it to the volume of approximate twenty five ml. Then 0.25 ml (5 drops) of
reagent B and 2 drops of reagent E were added. Reagent F was used to titrate it using
10 ml micro-burette. The color of the solution was change from orange red to the
purple. Having close to the end point, after every 5 to 10 seconds one drop of solution
F was added. Immediately, the color was not changed, blank contained solution B and
one or 2 drops of solution E, titrated against 0.01 N EDTA, helped in distinguishing
the finishing line. Calcium contents were determined using following formula:
Ca (meq L-1) = ml of EDTA x Normality (0.01) x 1000

ml of aliquot (sample)
Fig.3.5.Working on Spectrophometer
27
Chapter 4
Results
4.1. BIOCHEMICAL PARAMETERS
4.1.1. Total Soluble Sugars
Total soluble sugars estimated in leaves of wheat plants grown on soils amended with
nano-biochar (NBC) showed that NBC significantly affected total sugars content
(Table 4.1.1). The highest sugars content (5.56 mg g-1 F.W) were found in those
plants which were treated with 5% nano-biochar, followed by 3% nano-biochar (4.72
mg g-1 F.W), 1% nano-biochar (4.48 mg g-1 F.W) and control plants (2.68 mg g-1 F.W)
(Fig. 4.1.1). The minimum values for total sugars content were observed in those
plants which were grown under normal conditions. The values for sugars content
differed significantly in all the treatments.
4.1.2. Nitrate Reductase Activity
The wheat leaves analysis for the estimation of nitrate reductase activity (NRA)
indicated that NRA changed due to soil amendment of nano-biochar and variations for
NRA among all the nano-biochar treatment were significant (Table 4.1.2).The highest
value for NRA(26.65 µmol g-1 F.W) was recorded in those plants which were grown
on the soil amended with 5% nano-biochar, followed by 3% nano-biochar (26.11
µmol g-1 F.W), 1% nano-biochar (20.94 µmol g-1 F.W) and control plants (11.91
µmol g-1 F.W) (Fig. 4.1.2). The minimum value for NRA was observed in those wheat
plants which were grown under 0% NBC. The soil application of NBC was effective
in enhancing the NRA of wheat.
28
4.1.3. Total Free Amino Acids
Data regarding total free amino acids (TFAA) contents in wheat leaves clearly
showed that TFAA were significantly influenced by nano-biochar applications (Table
4.1.3). The highest values for TFAA (2.71 mg g-1FW) were recorded in those plants
which were grown on the soil amended with 3 % nano-biochar, followed by 5%
nano-biochar (2.66 mg g-1 F.W), 1% nano-biochar(1.94 mg g-1 F.W) followed while,
the minimum values for TFAA (1.21 mg g-1 F.W) under control plants (Fig. 4.1.3).
The values for total free amino acid differ significantly in all treatments.
4.1.4. Total Soluble Proteins
Chemical analysis regarding total soluble proteins (TSP) contents indicated that soil
amendment with nano-biochar significantly affected this parameter (Table 4.1.4). The
highest values for total soluble proteins (4.77 mg g-1FW) were exhibited in the plants
grow on that soil which was amended with 3 % nano-biochar, followed by 5% nano-
biochar (4.56 mg g-1F.W), 1% nano-biochar (4.31 mg g-1F.W) followed and control
plants (1.61 mg g-1F.W).The lowest values for TSP were observed in plants which
were grown under normal conditions (Fig. 4.1.4).
4.1.5. Total Phenols
Data regarding chemical analysis for phenols from wheat leaves indicated that
application of nano-biochar in soil did not influence this parameter (Table 4.1.5). The
greatest value for phenols contents(5.38 µg g-1 F.W) were found in those plants which
were applied with 3 % nano-biochar, followed by 1% (5.38µg g-1 F.W) nano-biochar,
control plants (5.24µg g-1 F .W) and 5% (5.03 µg g-1F.W) (Fig. 4.1.5). The minimum
values for phenol were noticed in that plants which were grown in soil amended by
29
5% nano-biochar. The values for total phenol differ non-significantly in all
treatments.
4.1.6. Total Flavonoids
Analysis of variance for flavonoids contents in wheat leaves revealed that this
parameter was significantly affected by the soil amendment of nano-biochar (Table
4.1.6). The maximum flavonoids contents (5.45µg-1 F.W) was found in that plant
which was grown with 5 % nano-biochar, followed by 1% (4.69 µg g-1 F.W) nano-
biochar, 3% (4.51 µg g-1 F.W) and control plants (2.66 µg g-1 F.W).The lowest values
for flavonoids contents were noticed in that plants grown in normal conditions (Fig.
4.1.6). The values for total flavonoids contents differed significantly among different
nano-biochar treatments.
4.2. PIGMENT ANALYSIS
4.2.1. Chlorophyll a
Data shown in Table 4.7 clearly indicated that soil amendment with nano-biochar
significantly affected the chlorophyll “a” contents in wheat leaves. The maximum
chlorophyll “a” content (1.66 mg g-1 F.W) was recorded in that plant which was
grown on soil amended with 3 % nano-biochar, followed by 5% nano-biochar (1.63
mg g-1 F.W), 1% nano-biochar (1.41 mg g-1 F.W) and control plants (1.40 mg g-1
F.W).The minimum value of chlorophyll “a” contents were noticed in that plants
which were grown under normal conditions.
30
4.2.2. Chlorophyll b
Data about chlorophyll “b” contents in wheat leaves clearly indicated that soil
amendment with nano-biochar significantly influenced this parameter (Table
4.2.2).The highest chlorophyll “b” content (0.79 mg g-1 F.W) was found in that plant
which was grown in soils amended with 5 % nano-biochar, followed by 3% (0.61 mg
g-1 F.W), 1% nano-biochar (0.49 mg g-1 F.W) and control plants (0.27 mg g-1 F.W).
The lowest values for chlorophyll “b” contents were noted in plants subjected to
under normal condition (Fig. 4.2.2).
4.2.3. Total chlorophyll contents
Total chlorophyll contents estimated in the leaves of wheat plants that grown on soil
amended with nano-biochar showed that nano-biochar significantly affected the total
chlorophyll (Table 4.2.3).The highest value for total amount of chlorophyll content
(2.47 mg g-1 F.W) was observed in those plants which grown on soil amended with 5
% nano-biochar, followed by 3% nano-biochar (2.27 mg g-1 F.W), 1% nano-biochar
(1.89 mg g-1 F.W) and control plants (1.66 mg g-1 F.W) (Fig.4.2.3 ).The minimum
value for total chlorophyll content were noticed in those plants which were grown
under normal conditions.
4.2.4. Carotenoids
Data regarding total carotenoid contents in the wheat leaves clearly indicated that
wheat plants grown on soil containing nano-biochar were significant influenced
(Table 4.2.4). Variations in carotenoid contents in plants grown under different levels
of nano-biochar varied significantly in all the nano-biochar treatments (Table 4.2.4).
The maximum carotenoid contents (41.44 µg g-1 F.W) were found in those plants
31
which were grown on soil where 3 % nano-biochar was applied, followed by 5%
nano-biochar (34.34 µg g-1 F.W), 1% nano-biochar followed (33.94µg g-1 F.W ) and
control plants (30.1µg g-1 F.W) (Fig. 4.10). The smallest values for carotenoid were
noticed in those plants which were grown under normal conditions.
4.3. PHYSICAL PARAMETERS
4.3.1. Shoot length
Data regarding shoot length recorded for wheat plants showed that this parameter was
significantly influenced by soil amendment with nano-biochar (Table 4.3.1). The
maximum shoot length (37 cm) was found in that plant which was grown on soil
amended with 3 % nano-biochar, followed by 1% nano-biochar (32.33cm), 5% nano-
biochar (31.16 cm) and control plants (28.33 cm) (Fig.4.3.1).The minimum length of
shoot was noted in those plants which were grown under normal conditions.
4.3.2. Root length
The root length of wheat plants was significantly influenced by soil amendment of
nano-biochar (Table.4.3.2).The variations for shoot length among nano-biochar
treatments was significant. The maximum root length (7.56 cm) was found in those
plants which were grown on soil amended with 3% nano-biochar, followed by 5%
nano-biochar (7.10 cm), 1% nano-biochar (5.66 cm) and control plants (5.50 cm)
(Fig.4.3.2 ).The minimum root length was found in that plants which were grown
under normal conditions.
32
4.3.3. Leaf Area
Leaf area in the plants of wheat was influenced significantly by soil amendment of
nano-biochar (Table.4.3.3). The maximum leaf area (15.46 cm) was observed in that
plants which were grown in 5% nano-biochar amended soil, followed by 3% (15.18
cm), 1% nano-biochar (14.03 cm) and 0 % NBC amended soils (11.65 cm)
(Fig.4.3.3). The minimum leaf area was noted in plants which were grown in normal
condition.
4.3.4. Number of leaves per plant
Data regarding number of leaves per plants indicated that this parameter was also
significantly influence by soil amendment of nano-biochar (Table.4.3.4). The plants
grown on soil amended with 3% NBC were successful in maintain greater number of
leaves per plant (6.5) closely followed by 1% NBC (6.4), 5% NBC (6.2) and 0% NBC
(4.41) (Fig.4.3.4). The differences for number of leaves per plant under NBC 1, 3 and
5 % were significant, but plants grown under 0% NBC or normal conditions showed
significantly lower number of leaves per plant than all other NBC treatments.
4.3.5. Number of tillers per plant
Data regarding number of tillers per plant in wheat plants showed significant variation
due to nano-biochar application in soil (Table 4.3.5). Wheat plants grown on soil
having 3% NBC contents maintained maximum number of tillers (1.9) followed by
1% nano-biochar (1.533),5% nano-biochar (1.4) and control plants (1.166). The
differences among all the treatments of NBC were significant. The minimum value
for number of tillers was found in those plants which were grown under normal
conditions (Fig. 4.3.5).
33
4.3.6. Fresh weight per plant
Analysis of variance (Table 4.16) clearly indicated that plant fresh weight was
significantly influenced by soil amendment with nano-biochar (Table.4.3.6).The
maximum value for fresh weight per plant (5.38 g) was maintained by those plants
which was grown on soil amended with 3 % nano-biochar, followed by 5% nano-
biochar (5.36 g), 1% nano-biochar (4.74 g) and control plants (4.30 g) (Fig.4.3.6).The
variations for fresh weight between NBC 3% and NBC 5% were significant; same
was the case with NBC 1% and NBC 0%.
4.3.7. Dry weight per plant
Statistical analysis regarding dry weight per plant of wheat plants shown in Table
4.3.7, revealed that plant dry weight per plant of wheat plants significantly influenced
by soil amendment with nano-biochar. Plant grown in soil amended with 5% nano-
biochar maintained the highest value for dry weight per plant (0.69 g), while plants
grown on soil amended with 1% nano-biochar have statistically lower fresh weight
per plant (0.57 g). The lowest fresh weight per plant was recorded for those plants
which grown on soils amended with 0% nano-biochar (0.43 g) and was statistically
recorded to those plants grown on soil amended with 3%NBC (0.47 g) (Fig.4.3.7).
4.4. NUTRIENT ANALYSIS
4.4.1. Sodium
Analysis of variance regarding sodium (Na) contents in wheat leaves was

significantly affected by nano-biochar (Table 4.4.1). Sodium contents values for NBC
treatments revealed that the highest Na contents were recorded for plants grown under
0% NBC (2.90 mg g-1) ) followed by 5% nano-biochar (2.33 mg g-1), 1% nano-
34
biochar (2.30 mg g-1 ) and 3% nano-biochar (2.10 mg g-1) (Fig. 4.4.1). The variations
among all the NBC treatment were significant.
4.4.2. Potassium
Data regarding potassium (K) contents indicated that it was significantly influenced
by soil amendment with nano-biochar (Table 4.4.2). Plants grow under 5% NBC were
successful in maintaining the highest value for K content (9.03 mg g-1) followed by
3% NBC (8.13 mg g-1), 1% NBC (7.92 mg/g) and 0% NBC (7.2 mg/g). The lowest
value for K contents was noted in that plant which was grown under normal
conditions. The variations for K contents among all the treatment were significant
(Fig. 4.4.2).
4.4.3. Phosphorus
Data regarding phosphorus contents in wheat leaves showed that it was influenced
significantly by soil amendment with NBC (Table 4.4.3). The plants grown on soil
amended with 5% NBC were successful in maintaining the greatest value of
phosphorus contents (2.21 mg g-1 ), followed by 3% nano-biochar (1.92 mg g-1), 1%
nano-biochar (1.666 mg g-1) and control plants or 0% NBC (1.343 mg g-1). The lowest
value for phosphorus contents was noted in those plants which were grown under 0%
NBC (Fig. 4.4.3). The variations for phosphorus contents among all the treatments of
NBC were significant.
4.4.4. Nitrogen
Data for nitrogen contents in wheat leaves grown on soil amended with nano-biochar
showed that it was significantly affected (Table 4.4.4). Plants grown under 5% NBC
maintained the highest value for nitrogen contents (37.33 mg g-1), followed by 3%
nano-biochar (29.67 mg g-1 ),control plants (24.27 mg g -1) and 1% nano-biochar
(21.85 mg g-1) (Fig. 4.4.4). The lowest value for nitrogen contents was observed in
those plants which were grown at 1% nano-biochar. Variations for nitrogen contents
among all the NBC treatments were significant.
35
1
) (Fig. 4.4.5). The lowest value for Ca contents was noted in those plants which were grown
under normal conditions. The variations among all the NBC treatment were significant.
Table. 4.1.1. Effects of nano-biochar on total soluble sugars content in wheat
leaves
Source DF SS MS F P
Treatment 3 13.1689 4.38963 263 0.0000***
Error 8 0.1334 0.01667
Total 11 13.3023
Note: *** =P<0.001; **P<0.01; *P<0.05; S=significant
Fig. 4.1.1. Effects of nano-biochar on total soluble sugars in wheat

leaves
6
5
Total Soluble Sugars (mg g-1FW)
0
0 1 3 5
Treatment (Nano biochar %)
36
Table. 4.1.2. Influence of nano-biochar on Nitrate Reductase Activity (NRA) in
wheat leaves
Source DF SS MS F P
Treatment 3 419.959 139.986 181 0.0000***
Error 8 6.190 0.774
Total 11 426.150
Fig. 4.1.2. Effects of nano-biochar on Nitrate Reductase Activity (NRA)

in wheat leaves
30
Nitrate Reductase (µ mole g-1 FW)
25
20
15
10
0
0 1 3 5
37
Table. 4.1.3. Effects of nano-biochar soil application on total free amino acids in
wheat leaves
DF SS MS F P
Source
3 4.51103 1.50368 87.5 0.0000***
Treatment
Error 8 0.13752 0.01719
Total 11 4.64855
Fig.4.1.3. Effects of nano biochar on total free amino acids in wheat leaves
3
Total Free Amino Acids (mg g-1FW)
2.5
1.5
0.5
0
0 1 3 5
38
Table.4.1.4. Effects of nano-biochar on total soluble proteins in wheat leaves
Source DF SS MS F P
Treatment 3 19.7706 6.59019 187 0.0000***
Error 8 0.2820 0.03525
Total 11 20.0526
Fig.4.1.4. Effects of nano biochar on total soluble proteins in wheat leaves

6
5
Total Soluble Proteins (mg g-1 FW)
0
0 1 3 5
39
Table. 4.1.5. Effects of nano-biochar on total phenol contents in wheat leaves
Source DF SS MS F P
Treatment 3 0.24667 0.08222 0.69 0.5814NS
Error 8 0.94860 0.11857
Total 11 1.19527
Note: *** =P<0.001; **P<0.01; *P<0.05; N S= non-significant
Fig. 4.1.5. Effects of nano biochar on total phenol in wheat leaves

5.6
5.4
5.2
Total Phenols (µg g-1 FW)
4.8
4.6
4.4
4.2
0 1 3 5
40
Table.4.1.6. Influence of nano-biochar soil amendment on total flavonoid
contents in wheat leaves
Source DF SS MS F P
Treatment 3 12.6305 4.21016 34.9 0.0001**
Error 8 0.9662 0.12077
Total 11 13.5967
Fig.4.1.6. Effects of nano-biochar on total flavonoids in wheat leaves

6
5
Total Flavonoids (µg g-1 FW)
0
0 1 3 5
41
Table. 4.2.1. Effects of nano-biochar soil amendment on chlorophyll “a” contents
in wheat leaves
Source DF SS MS F P
Treatment 3 0.18112 0.06037 5.11 0.0290*
Error 8 0.09451 0.01181
Total 11 0.27563
Fig.4.2.1. Effects of nano-biochar soil amendment on chlorophyll

"a" in wheat leaves
1.8
1.6
Chlorophyll "a "(mg g-1 FW)
1.4
1.2
0.8
0.6
0.4
0.2
0
0 1
Treatment (Nano biochar %) 3 5
42
Table. 4.2.2. Chlorophyll “b” contents of wheat leaves grown in soils amended
with nano-biochar
Source DF SS MS F P
Treatment 3 0.44896 0.14965 25.4 0.0002**
Error 8 0.04709 0.00589
Total 11 0.49605
Fig.4.2.2 . Effects of nano-biochar on cholorophyll "b" in

wheat leaves
0.9
0.8
Chlorophyll "b" (mg g-1 FW)
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0 1 3 5
43
Table. 4.2.3. Effects of nano-biochar on total chlorophyll contents in wheat leaves
Source DF SS MS F P
Treatment 3 1.10239 0.36746 13.6 0.0017**
Error 8 0.21597 0.02700
Total 11 1.31836
Fig.4.2.3. Variations in total chlorophyll contents in wheat

leaves under different levels of nano-biochar
3
2.5
Total chlorophyll
1.5
0.5
0
0 1 3 5
Treatment ( Nano-biochar %)
44
Table. 4.2.4. Influence of nano-biochar on carotenoid contents of wheat leaves
Source DF SS MS F P
Treatment 3 201.870 67.2901 39.2 0.0000***
Error 8 13.735 1.7169
Total 11 215.606
Fig.4.2.4. Changes in carotenoid contents in wheat leaves grown

under different nano-biochar levels
45
40
35
Carotenoids (µg g-1 FW)
30
25
20
15
10
0
0 1 3 5
45
Table.4.3.1. Effect of soil amended nano-biochar on shoot length of wheat plants
Source DF SS MS F P
Treatment 3 118.190 39.3967 24.2 0.0002**
Error 8 13.007 1.6258
Total 11 131.197
Fig.4.3.1. Nano-biochar induce changes in shoot length in wheat

plants
40
35
30
Shoot length (cm)
25
20
15
10
0
0 1 3 5
46
Table. 4.3.2. Effects of nano-biochar on root length of wheat plants
Source DF SS MS F P
Treatment 3 10.0533 3.35111 50.3 0.0000***
Error 8 0.5333 0.06667
Total 11 10.5867
Fig.4.3.2. Root length variations in wheat plants grown on nano-

biochar amended soil
9
6
Root length (cm)
0
0 1 3 5
47
Table. 4.3.3. Effect of nano-biochar on the leaf area of wheat plants
Source DF SS MS F P
Treatment 3 18.4820 6.16068 56.9 0.0000***
Error 8 0.8669 0.10837
Total 11 19.3490
Fig.4.3.3. Variations in leaf area of wheat plants grown in different

levels of nano-biochar
18
16
14
12
Leaf Area (cm)
10
0
0 1 3 5
48
Table. 4.3.4. Influence of nano-biochar application on number of leaves per plant
in wheat
Source DF SS MS F P
Treatment 3 0.84667 0.28222 8.91 0.0063**
Error 8 0.25333 0.03167
Total 11 1.10000
Fig.4.3.4. Variations in number of leaves per plant in wheat plant

grown in soil amended with different concentration of NBC
8
6
Number of leaf per plant
0
0 1 3 5
49
Table. 4.3.5. Effects of NBC application on number of tiller per plant in wheat
Source DF SS MS F P
Treatment 3 8.72490 2.90830 20.9 0.0004**
Error 8 1.11407 0.13926
Total 11 9.83897
Fig.4.3.5. Effects of nano-biochar amendment in soil on number of

tillers per plant in wheat grown under different levels of NBC
2.5
2
Number of tillers per plant
1.5
0.5
0
0 1 3 5
50
Table. 4.3.6. Effects of nano-biochar on fresh weight per plant of wheat plants
Source DF SS MS F P
Treatment 3 2.41609 0.80536 16.4 0.0009**
Error 8 0.39210 0.04901
Total 11 2.80818
Fig.4.3.6. Variations in fresh weight per plant of wheat plants grown

under different levels of nano-biochar
6
5
Fresh weight per plants (g)
0
0 1 3 5
51
Table. 4.3.7 Influence of nano-biochar on dry weight per plant of wheat grown
under different levels of NBC
Source DF SS MS F P
Treatment 3 0.12002 0.04001 8.83 0.0064**
Error 8 0.03625 0.00453
Total 11 0.15627
Fig.4.3.7. Variations in dry weight per plant of wheat grown under

different levels of NBC
0.8
0.7
0.6
Dry weight per plant (g)
0.5
0.4
0.3
0.2
0.1
0
0 1 3 5
52
Table. 4.4.1. Sodium contents in wheat leaves grown under different levels of
NBC
Source DF SS MS F P
Treatment 3 1.07390 0.35797 73.7 0.0000***
Error 8 0.03887 0.00486
Total 11 1.11277
Fig.4.4.1. Sodium contents in wheat leaves grown under

different levels of Nano-biochar
3.5
2.5
Sodium (mg g-1)
1.5
0.5
0
0 1 3 5
Treatment ( Nano biochar %)
53
Table. 4.4.2. Analysis of variance for Potassium contents in wheat leaves grown
Source DF SS MS F P
Treatment 3 5.30620 1.76873 24.2 0.0002**
Error 8 0.58480 0.07310
Total 11 5.89100
Fig.4.4.2. Variations in potassium contents in wheat leaves

grown under different levels of Nano-biochar
10
7
Potassium (mg g-1)
0
0 1 3 5
54
Table. 4.4.3. ANOVA showing the phosphorus contents in wheat leaves grown
Source DF SS MS F P
Treatment 3 1.22377 0.40792 56.7 0.0000***
Error 8 0.05753 0.00719
Total 11 1.28130
Fig.4.4.3. Variations in phosphorus contents in wheat leaves

grown under different levels of NBC
2.5
2
Phosphorus (mg g-1)
1.5
0.5
0
0 1 3 5
55
Table. 4.4.4. Leaf nitrogen contents in wheat grown under different levels of
NBC
Source DF SS MS F P
Treatment 3 424.012 141.337 39.2 0.0000***
Error 8 28.855 3.607
Total 11 452.867
Fig.4.4.4. Variations in nitrogen contents in wheat leaves grown

45
40
35
30
Nitrogen (mg g-1)
25
20
15
10
0
0 1 3 5
56
Table. 4.4.5. Calcium contents in wheat leaves grown under different levels of
nano-biochar
Source DF SS MS F P
Treatment 3 1.56900 0.52300 30.6 0.0001**
Error 8 0.13667 0.01708
Total 11 1.70567
Fig.4.4.5. Calcium contents in leaves of wheat plants grown

under different level of nano-biochar
4
3.5
3
Calcium (mg g-1)
2.5
1.5
0.5
0
0 1 3 5
Treatment ( Nano-biochar %)
57
Table. 4.4.6. Variations in magnesium contents of wheat leaves grown under
different levelss of nano-biochar
Source DF SS MS F P
Treatment 3 0.28716 0.09572 45.2 0.0000***
Error 8 0.01693 0.00212
Total 11 0.30409
Fig.4.4.6. Variations in magnesium contents of wheat leaves

grown under different levels of nano-biochar
4.5
3.5
3
Magnisium (mg g-1)
2.5
1.5
0.5
0
0 1 3 5
Treatment (Nano-biochar%)
58
Chapter 5
Discussion
In present study, effects of nano-biochar were studied on growth and physio-

biochemical attributes of wheat grown in field condition. Results indicated that soil
amendment with nano-biochar significantly increased the root length (Fig.4.3.2),
shoot length (Fig.4.3.1) their fresh (Fig.4.3.6) and dry weight (Fig.4.3.7). The growth
parameters were significantly improved under all the nano-biochar treatments.
However, the more prominent results were noted in 3% NBC amendment in soil,
except dry weight which was higher in plants grown under 5% NBC. The literature
also indicated that application of biochar or amendment of nutrients or effective in
promoting the growth of the plants under normal stress condition (Marimuthu &
Surendran, 2015; Alam et al., 2020; Khan et al., 2021). Applied nano-biochar is a rich
source of carbon and other macro- and micronutrients and in nano-particles form,
which were easily entered in plant body and promoted the different metabolic
activities. As nano-biochar is the major source of carbon, so, it took part in improving
the photosynthetic activities i.e Calvin cycle in plants. This improvement in
photosynthesis activity reflected an increase in growth and productivity of plants.
Many studies are available on growth and plant productivity that confirming the
above findings (Ramanayakaet al., 2020; Ramadan & Abd-Elsalam, 2020; Chausaliet
al., 2021).
In this investigation some physiological processes were also studied and results
indicated that physiological parameter like leaf area (Fig.4.3.3), necessary to optimize
the rate of photosynthesis because plants having the higher leaf area had higher rate of
photosynthesis consequently maintained higher growth and productivity (Bote &
Struik, 2011; Thakur et al., 2011; Honda etal., 2021). Present study confirmed that
leaf area significantly enhanced by the soil amendment of nano-biochar (Table.4.3.3)
it meant that plant treated with nano-biochar can capture higher light energy which is
used in the fixation of carbon to prepare the carbohydrates (Stitt, 2013) (chemical
energy) available for plants to provide energy required for different metabolic
activities (Linsmayer et al., 2020; Lan et al., 2021) and to store as a food for the use
of human being and animals in the fruits or grains (Molina et al., 2017; Horton et al.,
2021). Maximum leaf area of the wheat plant was observed under 5% level of nano-
59
biochar which showed that this level of nano-biochar effective in promoting growth,
so, for a good growth of plant application of nano-biochar is necessary (Chen et al.,
2020; Das et al., 2020; Ramadan et al., 2020). But in present the maximum growth as
a biomass was observed under 5% level of nano-biochar. While maximum shoot
length, root length, number of tillers were recorded in those plants, where 3% nano-
biochar was applied through soil. It means that 3% level was more appropriate for
growth (Chen et al., 2020).
Different biochemical changes due to application of nano-biochar were also

investigated and results indicated that total soluble sugar, nitrate reductase activity,
total free amino acids, soluble proteins, total phenol, flavonoids, chlorophyll a, b, total
chlorophyll and carotenoids significantly enhanced by the application of nano-
biochar. Sugar contents of leaves of wheat were maximum under 5% nano-biochar
(Yang et al., 2020; Ahangeret al., 2021; Khan et al., 2021). Same was the case with
nitrate reductase (Ahanger et al., 2021; Fenget al., 2021), flavonoids (Ajenget al.,
2020; Deyet al., 2021), chlorophyll b and total chlorophyll (Shen et al., 2020) which
clearly indicated that highest level of nano-biochar (5%) enhanced the above
mentioned biochemical activities which are necessary to promote the plant
productivity and growth (Nawaz et al., 2016; Kapoor et al., 2020). Many earlier
findings are inconformity that application of nutrients either folliarly or through soil
enhanced the growth and productivity of plants (Leghariet al., 2016; Meenaet al.,
2017; Souri & Hatamian, 2019) by altering the physio-biochemical processes in a way
to favor the improvements in growth and plant productivity (Hidang mayum et al.,
2019; Meena et al., 2020).The earlier investigations on biochar and nano-biochar also
supported the present findings (Ali et al., 2019; Sabagh et al., 2020; Shekhawat et al.,
2021). Their results indicated that application of biochar and nano-biochar effective in
increasing the sugar contents (Akinyemi, & Adesina, 2020; Ardebili, et al., 2020;
Kumar et al., 2020), nitrate reductase activity (Lu et al., 2020) and other biochemical
parameters under normal as well as in stressed environment (Mamoudet al., 2020;
Chausaliet al., 2021; Khare, 2021).
The results of present study also indicated total amino acids, proteins, phenols,
chlorophyll a and carotenoids were higher at 3% nano-biochar which showed that this
level was appropriate to to have optimum plant growth productivity (Gautam et al.,
2021; Radziemska et al, 2021). Although, all the nano-biochar level enhanced the
60
above mentioned biochemical parameters however, a few exceptions in nano-biochar
actions regarding biochemical attributes were noted by many workers (Qin et al.,
2020; Shi et al., 2020). However, all are agreed that nano-biochar application is
effective in regulating the biochemical activities in favor of growth enhancement.
It is well documented that proper nutrient is for optimal plant growth and productivity
(Malhotra et al., 2018; Nair, 2019; Bakhshandeh et al., 2020). In present study,
nitrogen, potassium, phosphorus and sodium contents in plants significantly enhanced
by the soil application of nano-biochar. All these nutrients were higher at the highest
level of nano-biochar. These are the basic nutrients which required in higher amount
to regulate metabolic activities in plants (Razaq et al., 2017; Gouda et al., 2018;
Mitran et al., 2018). Nitrogen is the basic constituents of the proteins and all the
enzymes are protein in nature (Kuypers et al., 2018). These are also one of the major
constituents of the plant body required in higher amount (Leghari et al., 2016). So the
maximum availability of nitrogen is necessary to maintain higher plant growth and
productivity which is very clear from the present study (Fig.4.4.4) that the application
of nano-biochar maintain the N:C ratio in higher site and helpful in enhancing the
growth and productivity (Xiao et al., 2020). Literature also indicated nano-biochar
application significantly improve the nitrogen uptake in plants which enhanced the
plant growth and productivity (Shi et al., 2020). Potassium is also required in higher
amount because it regulates the opening and closing of stomata and more than 60
metabolic enzymes so any inheritance in its uptake may cause severe growth and
yield loses in crop plants (Sharma et al., 2020). Reports in the literature also indicated
that application of potassium promotes the growth and productivity of the plant
(Imran et al., 2020; Hafez et al., 2021). In present study, nano-biochar at all its level
was effective in enhancing the potassium uptake (Fig.4.4.2), consequently, higher
plant growth and biomass was recorded under the nano-biochar treatments (Shen et
al., 2020; Shi et al., 2020). Phosphorus is the major constituents of DNA, RNA and
phospholipids required phosphorylation reactions (Nam et al., 2017; Pasek et al.,
2017). So its optimum supply of phosphorus is necessary to maintain the plant growth
and proper transfer of genetic material (Meena et al., 2018). The supply of nano-
biochar enhanced phosphorus uptake which altered the plant metabolism in favor of
better growth and productivity (Ajeng et al., 2020; Ahanger et al., 2021). There are
many reports which indicated that application of nano-biochar is effective in
61
enhancing the phosphorus uptake in plants (Das et al., 2020; Liu et al., 2020; Shen et
al., 2020). In present study, sodium contents significantly decrease with the
application of nano-biochar (Fig.4.4.1) which showed that application of nano-biochar
is beneficial for plant growth if plants were grown under saline condition (Yue et al.,
2019; Shen et al., 2020). Sodium causes toxic effects on plant growth and
productivity under saline condition reports indicated that higher concentration of
sodium severely reduced the growth productivity of potassium, calcium uptake
(Weisany et al., 2014). Fortunately, in present study nano-biochar reduced an uptake
of Na (Fig. 4.4.1).
Application of nano-biochar promotes the uptake of calcium and magnesium

(Chausali et al., 2021). Calcium regulates different metabolic activities e.g synthesis
of cell wall in which calcium is a major constituent of cell wall (Wu et al., 2018).
Similarly, magnesium is an integral part of chlorophyll so its proper supply is
necessary to enhance the photosynthetic activities. Different reports are available
which suggested nano-biochar applications are beneficial in enhancing calcium and
magnesium uptake (Waraich et al., 2011). However, there are some contrasting
reports which showed that that nano-biochar did not influence the uptake of
magnesium (Yue et al., 2019; Shen et al., 2020; Liu et al., 2022).
62
Conclusion
It can be concluded from the present findings that soil amendment with nano-biochar
is effective in improving the growth and biomass of the wheat by altering physio-
biochemical metabolic activities.
Recomendation
From this study, the 3% nano-biochar can be recommended to enhance the plant
growth. However, to have higher plant productivity 5% nano-biochar is more reliable.
63
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NANO-BIOCHAR INDUCED GROWTH AND PHYSIO-

BIOCHEMICAL ALTERATIONS IN WHEAT (TRITICUM

HAFIZA RIDA QADEER

IN PARTIAL FULFILLMENTS FOR THE DEGREE OF MASTERS OF

INSTITUTE OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY

thesis titled “NANO-BIOCHAR INDUCED GROWTH AND PHYSIO-

BIOCHEMICAL ALTERATIONS IN WHEAT (TRITICUM AESTIVUM) AT

At any time if my statement is found to be incorrect even after my graduation, the

university has the right to withdraw my (M.Phil) degree.

Official Name of the Student: HAFIZA RIDA QADEER

Registration No: PBOT02193027 Date:

Official Name of the Supervisor: Prof. Dr Muhammad Yasin Ashraf TI

Employee No: 9024

Student Name: Hafiza Rida Qadeer Signature:

Supervisor Name: Prof Dr. Muhammad Yasin Ashraf TI Signature:

(b) Internal Examiner

Prof. Dr. M. Yasin Ashraf TI Signature: _________________

The University of Lahore

(c) Prof. Dr. Muhammad Ashraf Signature:

Pro-Rector IMBB / CRIMM

I dedicated all my effort to ALLAH (most beneficent and the

merciful) the HOLY PROPHET (P.B.U.H) and my beloved

Mother and Father

successful completion of this manuscript. I offer my countless salutations to the Holy

Prophet (PBUH) who is the source of guidance for all humanity.

My special gratitude goes to my supervisor Prof. Dr. Muhammad Yasin Ashraf,

Professor, Institute of Molecular Biology and Biotechnology, the University of Lahore

during the course of my work.

I am greatful to my family members. First and foremost to my Father QADEER

timely support throughout my study duration.

TSS total soluble solids

mg/g mili gram per gram

TFAA total free amino

CHAPTERS DESCRIPTION PAGES #

Present investigation was conducted to study the effects of nano-biochar (NBC) on

NBC treatments [0 (Control), 1, 3 and 5% NBC] with three replications in completely

the most effective rate of NBC application through soil.

Pakistan’s economy backbone is agriculture that influences the gross domestic

Nano-biochar is the smallest particle of biochar and it is obtained by grinding and

In Pakistan, crop production is affected by the deficiency of nutrients and other

For the sustainable agriculture, environmental protection from pollution nano-

“Application of nano-biochar may enhance the wheat crop growth by improving

Current study was conducted to achieve objectives given below:

 To investigate effects of nano-biochar on the physio-biochemical activities of

Biochar is a solid carbon-containing product formed when biomass decomposes

Hamzah and Shuhaimi, (2018) performed experiment to evaluate the effectiveness on

Nano-biochar attracts growing interest due to its unique environmental behavior.

Nanotechnology is one of the revolutionary fields in science and technology and is

Olmo et al., (2014) investigated effect of slowly adding pyrolysis in a field

Saxena et al., (2014) worked on carbon nanoparticles of biochar. Water soluble

According to Praveen et al., (2017) application of nanoparticle (NPs) is very effective

According to Akhtar et al., (2015) biochar application decreased negative effect of

3.1. Materials and Methods

To estimate the effects of nano-biochar in enhancing wheat crop growth by improving

3.1.1. Experimental Site and Conditions

Soil Characteristics Values

Soil texture Clay loam

Saturation percentage (%) 40.4

ECe (dS m-1) 1.72-1.92

Organic matter (%) 0.1-0.3

Ca+Mg (meq L-1) 2.57-4.78

CO3 (meq L-1) Nil

HCO3 (meq L-1) 2.5-4.8

NO3-N (mg kg-1) 3.5- 4.5