i

DECLARATION

I, Ms. Mahima Misti Sarkar, hereby declare that the work presented in my

thesis entitled “Evaluation of silica nanoparticles and their functionalization

in the alleviation of salinity stress in two legumes – Lens culinaris and Glycine

max” has been carried out by me under the supervision of Dr. Swarnendu Roy,

Assistant Professor, Department of Botany, University of North Bengal for the

award of the degree of Doctor of Philosophy (Ph.D.) in Science (Botany). I
also declare that this thesis or any part of this has not been submitted for any

other degree/diploma either to this or other Universities.

(Mahima Misti Sarkar)

Date:

Place: Department of Botany,

University of North Bengal,

Siliguri, West Bengal - 734013



ii

Dr. Swarnendu Roy

Assistant Professor
Department of Botany, NBU

TO WHOM IT MAY CONCERN

It is certified that the work presented in the thesis entitled “Evaluation of silica

nanoparticles and their functionalization in the alleviation of salinity stress

in two legumes – Lens culinaris and Glycine max” by Ms. Mahima Misti
Sarkar, has been carried out under my supervision. I am forwarding her thesis

for the Ph.D. degree in Science (Botany) at the University of North Bengal. She

has fulfilled all the requirements according to the rules of the University of

North Bengal regarding the works existing in her thesis. This thesis or any part

of this has not been submitted for any degree or diploma either to this or other

Universities.

Name & Signature of Supervisor

Department of Botany
University of North Bengal
FEBRUARY 2024



iii

ANTI-PLAGIARISM REPORT



iv



ix

ACKNOWLEDEGEMENTS

It is a genuine pleasure to express my profound thanks and gratitude to my sir, mentor, supervisor,

and guide, Dr. Swarnendu Roy, Assistant Professor, Plant Biochemistry Laboratory, Department of

Botany, University of North Bengal. His timely advice, meticulous scrutiny, scholarly advice,

scientific approach, and overwhelming attitude to help everybody have always encouraged me to

accomplish my research work.

I express my sincere gratitude to Prof. Aniruddha Saha, HOD, Department of Botany, University

of North Bengal, for his kind advice in the journey of my research work.

I would like to extend my gratitude to all the faculty members, Prof. Subhas Chandra Roy, Prof.

Arnab Sen, Prof. Manoranjan Choudhury, Dr. Piyush Mathur, and Late Dr. Palash Mandal, of

the Department of Botany, University of North Bengal, for their constant encouragement and

suggestions.

I would also like to thank all the non-teaching staff of the Department of Botany, for their

administrative assistance throughout my research tenure.

I would like to convey my keen gratefulness to the DST-FIST-sponsored Central Instrumentation

Facility (CIF) - Department of Botany, Department of Chemistry, Department of Pharmaceutical

Technology, and University Science and Instrumentation Centre (USIC) of the University of North

Bengal. Additionally, Sophisticated Analytical Instrument Facility (SAIF), Indian Institute of

Technology, Bombay; Sophisticated Analytical Instrumentation Facility (SAIF), and Central

Instrumentation Facility (CIF) of Panjab University; Sophisticated Analytical Instrumentation Centre

(SAIC), Tezpur University; University Scientific and Instruments Centre (USIC), Karnataka

University, Dharwad; Central Instrumentation Facility (CIF), Lovely Professional University;

Nucleome Informatics, NKC Centre for Genomic Research, Hyderabad, for allowing me the access

to their instrumentation facility for all the nanoparticle sample characterization and RNA-Seq

analysis.

I would also like to acknowledge the University Grants Commission (UGC, New Delhi, Govt.

of India) for providing fellowship in the form of JRF and SRF (Award No. 16–6(DEC.

2018)/2019(NET/CSIR).

I also want to convey my gratitude to Dr. Soumya Mukherjee, Dr. Tarun Kumar Dua, and Dr.
Paramita Paul for their valuable inputs in my publications.



x

A special thanks to all my fellow scholars of the Plant Biochemistry Laboratory, including Mr.
Ashis Sarkar, Mr. Biswanath Karmakar, Mr. Dibakar Ghosh, Mr. Raja Ghosh, Mr. Bittu Paul,
Mr. Buddhadev Sarkar, Ms. Puja Saha, Md. Salman Haydar, and Mrs. Anindita Ghosh Basu
for their encouragement, help, and support during the tenure of my research work.

I would like to express my heartfelt thanks to all the research scholars and project fellows of the

Department of Botany, University of North Bengal, for their cooperation and help throughout these

years.

I would like to show my love and care to all the dissertation students, including Mehebub Alom,

Jadav Debgupta, Ahona Guha, Kritika Pokhrel, Pritha Rudra, Sayani Sarkar, Sankhasubra
Ghosh, Tapas Das, Barnita Dey, Ankit Roy, Anuradha Adhikary, Barnali Mandal, Mitali Rava,

Shrijita Paul, Debojeet Choudhury, Akanksha Bhagat, Prasanti Karkidoli, Soumili
Chakraborty for their cooperation through my tenure.

I would like to thank all the administrative sections and Officers of the University of North
Bengal for their kind help and cooperation throughout my research work.

I want to convey my deepest gratitude to all the teachers who have embraced me with not only

the bookish knowledge but also the lessons for making me a better human being.

Finally, I express my profound gratitude to my parents, Mrs. Minati Sarkar and Mr. Mrinal
Kanti Sarkar, my elder brother Bulet Kanti Sarkar, and my full family for providing me with

unfailing support and continuous encouragement throughout my years of study.

Thank you

Mahima Misti Sarkar



xi

PREFACE

In the rapidly expanding world population, the necessity of “Sustainable Food Security” has been a

serious matter of concern. The accelerated environmental pollution and global warming have brought

forth various destructive forces engulfing global food production. Among those disparaging forces,

salinity has emerged as one of the crucial abiotic stresses that impede substantial damage to the

production of food crops. Salinity affects the overall plant health, resulting in poor growth and

development of plants along with loss in plant productivity and nutritional components. Mother Earth

was initially nurtured with excessive amounts of fertilizers and minerals to confront these challenges.

Instead of curing the complications, the over-application of these chemical fertilizers has worsened

the situation, resulting in the loss of soil fertility. Therefore, a consequential demand for alternative

sustainable strategy is a matter of high priority.

Nanotechnology has emerged as a viable solution in the past decade to address this crisis.

Nanomaterials having a high surface area to volume ratio, are easily uptaken by plants playing a more

interactive role with the cellular active components, and thus, their efficiency also gets amplified.

Silica is a semi-essential element; its deficiency can retard the essential plant responses. In this

connection, silica nanoparticles (SiNPs) possess great crop improvement potential because they can

translocate more silica to plants. Applying SiNPs has been manifested to improve plants’ tolerance

to salinity. Moreover, the surface functionalization of these nanomaterials increases their efficiency

and reduces the toxic effects at higher concentrations. Hence, this thesis explores the synthesis of

SiNPs and sugar-functionalized SiNPs and their implementation against the NaCl-induced salinity

stress in lentil and soybean plants.



xviii

LIST OF TABLES

Table no. Description Page no.

Table 2.1. Mixture design showing the variations in different factors (variables) for
the optimization of SiNPs. 23

Table 2.2. Actual or real values of size and surface charge of SiNPs obtained
through DLS and Zeta potential. 27

Table 2.3. Analysis of variance for DLS data. 27
Table 2.4. Analysis of variance for Zeta potential. 28
Table 2.5. Statistical analysis of the best-selected model for each parameter or

response. 31

Table 2.6. The ratio of TEOS:Ammonia:Ethanol to obtain the SiNPs of 50 nm size
based on the values of DLS. 33

Table 7.1. Primer sequence of the selected genes for RT-PCR. 188
Table 7.2. Quality check of the isolated RNA samples. 188
Table 7.3. List of top 50 DGEs for heatmap analysis showing the fold change

values of NaCl vs control, NaCl + TSiNPs vs control, and NaCl +
TSiNPs vs NaCl.

194

Table 7.4. List of genes related to the important cellular functions retrieved using
MapMan that are majorly related to stress responses in plants. 202

Table 7.5. List of selected genes for RT-PCR analysis from the RNA-Seq data 206
Table 8.1. Percentage of different stages of dividing cells of A. cepa root tip after

treatment with different concentrations of SiNPs (~50 nm) and control
solutions.

227

Table 8.2. Percentage of different stages of dividing cells of A. cepa root tip after
treatment with different sizes of SiNPs. 230

Table 8.3. Percentage of different stages of dividing cells of A. cepa root tip after
treatment with different concentrations of bare and functionalized
SiNPs.

238

Table 8.4. Number of cells with chromosomal aberrations observed in A. cepa root
tip mitotic stages after treatment with different concentrations of SiNPs
(~50 nm) and control solutions (from 10 random microscopic fields).

241

Table 8.5. Number of cells with chromosomal aberrations observed in A. cepa root
tip mitotic stages after treatment with different sizes of SiNPs (from 10
random microscopic fields)

242

Table 8.6. Number of cells with chromosomal aberrations observed in A. cepa root
tip mitotic stages after treatment with bare and functionalized SiNPs
(from 10 random microscopic fields)

243

Table 9.1. Physical properties (pH, water absorption, and soil water retention
capacity) of formulated and non-formulated nanoparticles. 265

Table 9.2. Germination attributes of lentil seeds after pre-soaking in different non-
formulated nanoparticles and nano-formulations. 270

Table 9.3. Production cost of all formulated and non-formulated nanoparticles. 273



xix

LIST OF FIGURES

Figure no. Description Page no.

Figure 2.1. DLS graphs of the lowest values obtained from the ten runs of the
mixture design. 28

Figure 2.2. Zeta potential graphs of the lowest values obtained from the ten runs of
the mixture design. 29

Figure 2.3. Actual values vs the predicted value plots of – (a) DLS and (b) Zeta
potential; and the contour plots of (a) DLS and (b) Zeta potential. 30

Figure 2.4. SEM images of the SiNPs obtained using the parameters of mixture
design. 32

Figure 2.5. Corresponding size of the nanoparticles obtained using DLS (minimum
particle size was taken) and SEM (N =30) derived SiNPs and the
relationship between DLS and SEM.

33

Figure 2.6. Corresponding surface charge of the nanoparticles obtained using zeta
potential (maximum negative value was taken) and SEM (N = 30)
derived SiNPs and the relationship between zeta potential and SEM.

34

Figure 2.7. Characterization of SiNPs through different analytical procedures.
36

Figure 3.1. Effect of SiNPs on the germination parameters of lentil seedlings
exposed to NaCl stress. 57

Figure 3.2. Effect of SiNPs on the phenotype of lentil plants exposed to NaCl stress
during seedling and vegetative stage. 59

Figure 3.3. Effect of SiNPs application on the growth and physiological parameters
of lentil plants subjected to different concentrations of NaCl during the
seedling and vegetative stage.

62

Figure 3.4. Effect of SiNPs application on the silica content and ion accumulation
of lentil plants subjected to different concentrations of NaCl during the
seedling and vegetative stage.

63

Figure 3.5. Effect of SiNPs application on the osmolyte contents of lentil plants
subjected to different concentrations of NaCl during the seedling and
vegetative stage.

65

Figure 3.6. Effect of SiNPs application on the enzymatic antioxidant activity of
lentil plants subjected to different concentrations of NaCl during the
seedling and vegetative stage.

66

Figure 3.7. Visualization of the effect of SiNPs application on the antioxidant
isozymes, and proteins of lentil seedlings subjected to different
concentrations of NaCl through gel electrophoresis.

67

Figure 3.8. Effect of SiNPs application on the non-enzymatic antioxidants and
membrane integrity of lentil plants subjected to different concentrations
of NaCl during the seedling and vegetative stage.

69

Figure 3.9. Effect of SiNPs application on the ROS contents of lentil plants
subjected to different concentrations of NaCl during the seedling and
vegetative stage.

70

Figure 3.10. Effect of SiNPs application on the in situ localization of ROS in lentil
plants subjected to different concentrations of NaCl during the seedling
stage.

71

Figure 3.11. Effect of SiNPs application on the in situ localization of ROS in lentil
plants subjected to different concentrations of NaCl during the
vegetative stage.

72



xx

Figure 3.12. Effect of SiNPs application on the phenotype of lentil plants subjected
to different concentrations of NaCl during the reproductive stage. 73

Figure 3.13. Effect of SiNPs application on the morphological and physiological
parameters of lentil plants subjected to different concentrations of NaCl
during the reproductive stage.

74

Figure 3.14. Effect of SiNPs application on the yield of lentil plants subjected to
different concentrations of NaCl during the reproductive stage. 76

Figure 3.15. Effect of SiNPs application on the yield parameters of lentil plants
subjected to different concentrations of NaCl during the reproductive
stage.

77

Figure 3.16. Effect of SiNPs application on the nutrient content of seeds obtained
from lentil plants subjected to different concentrations of NaCl during
the reproductive stage.

78

Figure 4.1. Effect of SiNPs on the germination parameters of soybean seedlings
exposed to NaCl stress. 97

Figure 4.2. Effect of SiNPs on the phenotype of soybean plants exposed to NaCl
stress during seedling and vegetative stage. 99

Figure 4.3. Effect of SiNPs application on the growth and physiological parameters
of soybean plants subjected to different concentrations of NaCl during
the seedling and vegetative stage.

100

Figure 4.4. Effect of SiNPs application on the silica content and ion accumulation
of soybean plants subjected to different concentrations of NaCl during
the seedling and vegetative stage.

102

Figure 4.5. Effect of SiNPs application on the osmolyte contents of soybean plants
subjected to different concentrations of NaCl during the seedling and
vegetative stage.

104

Figure 4.6. Effect of SiNPs application on the enzymatic antioxidant activity of
soybean plants subjected to different concentrations of NaCl during the
seedling and vegetative stage.

106

Figure 4.7. Visualization of the effect of SiNPs application on the antioxidant
isozymes, and proteins of soybean seedlings subjected to different
concentrations of NaCl through gel electrophoresis.

107

Figure 4.8. Effect of SiNPs application on the non-enzymatic antioxidants and
membrane integrity of soybean plants subjected to different
concentrations of NaCl during the seedling and vegetative stage.

109

Figure 4.9. Effect of SiNPs application on the ROS contents of soybean plants
subjected to different concentrations of NaCl during the seedling and
vegetative stage.

110

Figure 4.10. Effect of SiNPs application on the in situ localization of ROS in
soybean plants subjected to different concentrations of NaCl during the
seedling stage.

111

Figure 4.11. Effect of SiNPs application on the in situ localization of ROS in
soybean plants subjected to different concentrations of NaCl during the
vegetative stage.

112

Figure 4.12. Effect of SiNPs application on the phenotype of soybean plants
subjected to different concentrations of NaCl during the reproductive
stage.

113

Figure 4.13. Effect of SiNPs application on the morphological and physiological
parameters of soybean plants subjected to different concentrations of
NaCl during the reproductive stage.

114

Figure 4.14. Effect of SiNPs application on the yield of soybean plants subjected to
different concentrations of NaCl during the reproductive stage. 115



xxi

Figure 4.15. Effect of SiNPs application on the yield parameters of soybean plants
subjected to different concentrations of NaCl during the reproductive
stage.

117

Figure 4.16. Effect of SiNPs application on the nutrient content of seeds obtained
from soybean plants subjected to different concentrations of NaCl
during the reproductive stage.

118

Figure 5.1. Randomized complete block design employed to prepare three
biological replicates of the experimental design. 131

Figure 5.2. Schematic representation of the experimental setup for evaluating
uptake and translocation of silica and glucose obtained from SiNPs and
GSiNPs.

133

Figure 5.3. Characterization of GSiNPs along with SiNPs and APTES-SiNPs.
137

Figure 5.4. Characterization of GSiNPs along with SiNPs and APTES-SiNPs.
138

Figure 5.5. Lentil seedlings exposed to different treatments of nanoparticles and
NaCl. 139

Figure 5.6. Soybean seedlings exposed to different treatments of nanoparticles and
NaCl. 140

Figure 5.7. Changes in the plant height and RWC in saline-stressed lentil and
soybean seedlings after the application of SiNPs and GSiNPs. 141

Figure 5.8. Changes in the photosynthetic pigment contents in saline-stressed lentil
and soybean seedlings after the application of SiNPs and GSiNPs. 143

Figure 5.9. Changes in silica and ion accumulation in saline-stressed lentil and
soybean seedlings after the application of SiNPs and GSiNPs. 144

Figure 5.10. Changes in ROS accumulation in saline-stressed lentil and soybean
seedlings after the application of SiNPs and GSiNPs. 145

Figure 5.11. Changes in antioxidant enzyme activities in saline-stressed lentil
seedlings after the application of SiNPs and GSiNPs. 147

Figure 5.12. Evaluation of silica and glucose uptake and translocation from the
applied SiNPs and GSiNPs. 148

Figure 6.1. Randomized complete block design employed to prepare three
biological replicates of the experimental design. 159

Figure 6.2. Schematic representation of the experimental setup for evaluating the
uptake and translocation of silica and trehalose from SiNPs and TSiNPs. 160

Figure 6.3. Characterization of TSiNPs along with SiNPs and APTES-SiNPs.
162

Figure 6.4. Characterization of TSiNPs along with SiNPs and APTES-SiNPs.
163

Figure 6.5. Lentil seedlings exposed to different treatments of nanoparticles and
NaCl. 165

Figure 6.6. Soybean seedlings exposed to different treatments of nanoparticles and
NaCl. 166

Figure 6.7. Changes in the plant height and RWC in saline-stressed lentil and
soybean seedlings after the application of SiNPs and TSiNPs. 167

Figure 6.8. Changes in the photosynthetic pigment contents in saline-stressed lentil
and soybean seedlings after the application of SiNPs and TSiNPs. 169



xxii

Figure 6.9. Changes in silica and ion accumulation in saline-stressed lentil and
soybean seedlings after the application of SiNPs and TSiNPs. 170

Figure 6.10. Changes in ROS accumulation in saline-stressed lentil and soybean
seedlings after the application of SiNPs and TSiNPs. 172

Figure 6.11. Changes in antioxidant enzyme activities in saline-stressed lentil
seedlings after the application of SiNPs and TSiNPs. 173

Figure 6.12. Evaluation of silica and trehalose uptake and translocation from the
applied SiNPs and TSiNPs. 174

Figure 7.1. Raw transcript trimming and fold change analysis of the DGEs of NaCl
vs control, NaCl + TSiNPs vs control, and NaCl + TSiNPs vs NaCl. 190

Figure 7.2. GO enrichment analysis of the DGEs of NaCl vs control, NaCl +
TSiNPs vs control, and NaCl + TSiNPs vs NaCl, emphasizing the
expressions of biological process, cellular components, and molecular
functions.

191

Figure 7.3. KEGG enrichment pathway analysis of the DGEs of NaCl vs control,
NaCl + TSiNPs vs control, and NaCl + TSiNPs vs NaCl, emphasizing
the expressions of different pathways.

193

Figure 7.4. Diven plot analysis of DGEs of NaCl vs control, NaCl + TSiNPs vs
control, and NaCl + TSiNPs vs NaCl, emphasizing their interrelation in
context to significant gene expressions.

196

Figure 7.5. Heat map analysis of the top 50 DGEs of NaCl vs control, NaCl +
TSiNPs vs control, and NaCl + TSiNPs vs NaCl genes. 198

Figure 7.6. Transcription factors associated with the expression of DGEs of NaCl
vs control, NaCl + TSiNPs vs control, and NaCl + TSiNPs vs NaCl. 199

Figure 7.7. Overview of the different cellular functions using the DGEs of NaCl vs
control, NaCl + TSiNPs vs control, and NaCl + TSiNPs vs NaCl using
MapMan.

200

Figure 7.8. Overview of the regulatory pathways using the DGEs of NaCl vs
control, NaCl + TSiNPs vs control, and NaCl + TSiNPs vs NaCl using
MapMan.

201

Figure 7.9. Network analysis (Sample–Gene–Pathway) of the DGEs of NaCl vs
control, NaCl + TSiNPs vs control, and NaCl + TSiNPs vs NaCl. 204

Figure 7.10. PPI interaction of the expressed proteins.
205

Figure 7.11. RT-PCR analysis of the expression of the selected genes for the
validation of RNA-Seq data. 207

Figure 8.1. A. cepa bulbs, along with the emerging roots, after being soaked with
different concentrations of SiNPs (~50 nm) and control solutions for
evaluating the concentration-dependent toxicity of the nanoparticles.

221

Figure 8.2. A. cepa bulbs along with the emerging roots after being soaked with
different concentrations of SiNPs (50 g/L, 100 g/L, 250 g/L, 500 g/L) of
different sized SiNPs (~30 nm, ~50 nm, ~100 nm) for evaluating the
size-dependent toxicity of the nanoparticles.

222

Figure 8.3. A. cepa bulbs along with the emerging roots after being soaked with
different concentrations of SiNPs and sugar-functionalized SiNPs, i.e.,
GSiNPs and TSiNPs (100 g/L, 150 g/L, 200 g/L, 250 g/L, 500 g/L) for
evaluating the effect of surface functionalization on the toxicity of the
nanoparticles.

223

Figure 8.4. Stages of A. cepa root tip mitotic cells (Magnification ×2025) after
treatment with different concentrations of SiNPs (~50 nm) and control
solutions.

228



xxiii

Figure 8.5. Genotoxicity indices for the assessment of concentration-dependent
toxicity of SiNPs (~50 nm). 229

Figure 8.6. Cell membrane integrity was visualized through Evan’s blue staining of
A. cepa root tips after treatment with different concentrations of SiNPs
(~50 nm) and control solutions.

231

Figure 8.7. Stages of A. cepa root mitotic cells (Magnification ×2025) after
treatment with different sizes of SiNPs. 232

Figure 8.8. Genotoxicity and cytotoxicity studies for the assessment of size-
dependent toxicity of SiNPs. 234

Figure 8.9. Membrane integrity of A. cepa root tip from different treatment sets
analyzed using Evan’s blue staining. 235

Figure 8.10. Stages of A. cepa root mitotic cells (Magnification ×2025) after
treatment with different concentrations of SiNPs, GSiNPs, and TSiNPs. 236

Figure 8.11. Genotoxicity and cytotoxicity studies for the assessment of toxicity of
SiNPs, GSiNPs, and TSiNPs. 239

Figure 8.12. Membrane integrity of A. cepa root tip from different treatment sets
analyzed using Evan’s blue staining. 239

Figure 8.13. Different types of chromosomal aberrations observed in the A. cepa root
tip mitotic stages owing to the genotoxic effects of benzene treatment
and also on the treatments with toxic concentrations of SiNPs.

240

Figure 8.14. Effect on the different bacterial communities obtained from the control
soils treated with dH2O, SiNPs, GSiNPs and TSiNPs. 244

Figure 8.15. Effect on the different bacterial communities obtained from the 300 mM
NaCl treated soils with dH2O, SiNPs, GSiNPs, and TSiNPs. 245

Figure 8.16. Number of different bacterial colonies from the soil treated with 0 mM,
and 300 mM NaCl in combination with dH2O, SiNPs, GSiNPs, and
TSiNPs treatment.

246

Figure 9.1. SEM images of the different non-formulated, and formulated silica
nanoparticles. 261

Figure 9.2. FTIR spectra of the different non-formulated, and formulated silica
nanoparticles. 262

Figure 9.3. Morphological attributes of the nano-formulations.
263

Figure 9.4. Time dependent release of sugar/silica from the non-formulated
nanoparticles and nano-formulations. 266

Figure 9.5. Effect of pH on the release of silica/sugar from the non-formulated
nanoparticles and nano-formulations. 268

Figure 9.6. Effect of temperature on the release of silica/sugar from the non-
formulated nanoparticles and nano-formulations. 269

Figure 9.7. Effect of non-formulated nanoparticles and nano-formulations on the
phenotype of lentil seedlings exposed to NaCl stress. 271

Figure 9.8. Growth parameters of lentil seedlings after the application of different
non-formulated nanoparticles and nano-formulations under unstressed,
and saline stressed conditions.

272



xxiv

LIST OF APPENDICES

Appendix
no. Description Page no.

Appendix-A List of Thesis-Related Publications A 1-A 52

Appendix-B List of Presentations in National and International
Seminars/Conferences A 53-A 57

Appendix-C List of Abbreviations A 58

Appendix-D List of Chemicals A 59