Types of PCR

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Nutrient Availability and Plant Productivity

through PGPR: Mechanisms, Potential and
Constraints
Chapter · July 2022
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Agricultural Biotechnology
Volume - 5
Chief Editor
Dr. Sweta Mishra
Professor, Department of Genetics & Plant Breeding, PG College of
Agriculture, Dr Rajendra Prasad Central Agricultural University, Pusa,
Samastipur, Bihar, India
Co-Editor
Dr. Shailesh Kumar
Assistant Professor-cum Scientist, Department of Botany and Plant
Physiology, FBS & H, Dr. Rajendra Prasad Central Agricultural University,
Pusa, Samastipur, Bihar, India
AkiNik Publications
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Published By: AkiNik Publications
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Pages: 153
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Price: ` 741/-
Contents
Chapters Page No.

1. Gene Knockout: Is it Like Bandwagon in Plant Breeding 01-17
(Sudhakar Reddy B, Karthikeya Reddy S.G.P. and Sanjay Palakurthy)
2. Superiority of Black Rice over Normal Rice Variety 19-31

(Alija Priyadarshini, Amit Kumar Mohapatra and Abhilash Padhan)
3. Genome Editing: A Way Forward for Crop Improvement 33-48

(K. Bhargava, P. Sadhana, S. Ravali, P. Hima Bindu and R. Harisha)
4. Protein Bio-Synthesis/Translation in Prokaryotes & Eukaryotes 49-64

(Gita R. Chaudhari, Sheetal Patel, Shridhar Ragi and Krishna Prakash)
5. Role of Biological Ice Nucleation in Cloud Modification 65-74

(Guna M, Panneerselvam S, Subramanian K.S, Harinarayanan M.N, Govindaraj
T and Dharani C.)
6. Different Types of PCR and It’s Applications in Agricultural

Biotechnology 75-94
(Sonal Chavan)
7. Whole Genome Strategies for Marker Assisted Selection in Plant

Breeding 95-110
(Kankati Alekya, Vemula Anjula Krishna, Kommineni Jagadeesh, Pandiri
Pavani, Mondithoka Mounika and Vemula Srujana)
8. Earthworms Bio Markers in Soil Pollution Assessment 111-136

(Praveen Kumar, Rishbh Kumar Didawat, Arkaprava Roy, Priti Tigga, Koushik
Bag, Sandeep Kumar and Ritambhara)
9. Nutrient Availability and Plant Productivity through PGPR:

Mechanisms, Potential and Constraints 138-153
(Khushboo Rani, Priti Tigga, Arkaprava Roy, Abinash Das and Ankita Trivedi)
Chapter - 1
Gene Knockout: Is it Like Bandwagon in Plant
Breeding
Authors
Sudhakar Reddy B
Ph.D. Scholar, Department of Genetics and Plant breeding, G.B
Pant university of Agriculture and Technology, Pantnagar,
Uttarakhand, India
Karthikeya Reddy S.G.P.
Ph.D. Scholar, Department of Genetics and Plant breeding, G.B
Pant university of Agriculture and Technology, Pantnagar,
Uttarakhand, India
Sanjay Palakurthy
M.Sc. Student, Department of Genetics and Plant Breeding,
G.B Pant University of Agriculture and Technology, Pantnagar,
Uttarakhand, India
Page | 1
Page | 2
Chapter - 1
Gene Knockout: Is it Like Bandwagon in Plant Breeding
Sudhakar Reddy B, Karthikeya Reddy S.G.P. and Sanjay Palakurthy
Abstract
A gene knock-out (KO) is a genetic approach combined with a
biotechnological tool in which an organism is genetically modified to carry
inactive genes. These genes are known as knock-out organisms or simple
knock-outs, and they are used in the opposite of the gene knock-in approach
to give function to particular genes with unknown functions that have been
sequenced. The gene knock-out approach is a reverse genetics tool that uses
gene tagging, mutagenesis, and homologous recombination to discover the
target gene function, then studies the consequences of the changed
phenotype to create a recognizable gene function. Site-Specific Zinc fingers,
TALENS, and CRISPER are nucleases that are known to break DNA double
strands. After DNA damage, the cell's own repair process activates by
ligating two open ends through non-homologous end joining (NHEJ).
Because of repair process is incomplete, frame shift mutation occurs as a
consequence of insertion or deletion mutations. The gene produces non-
functional protein as a result of the mutation, resulting in a knockout for the
gene of interest. In the presence of a single gRNA, they are currently
employed to induce single point mutations. CRISPER/Cas9 is easier to
utilize than TALENs and ZNFs since just crRNA needed to be redesigned to
precisely target any gene. Furthermore, by creating libraries of big gRNA,
this approach allows for genome-wide gene function analysis. This method
was previously used to investigate, nullify, or modify gene function in
genetics. Increased agricultural productivity via novel breeding techniques is
critical for increasing global access to healthy food. Recent developments in
CRISPR/Cas genome editing have enabled effective targeted gene knockouts
in most crops, indicating that agricultural improvement can be accelerated.
Keywords: Gene, gene knockout, knock-out organism, recombination,
biotechnological tools, genome editing
Page | 3
Introduction
Gene targeting technologies are used to modify genomes of any living
organisms. When a mutation inactivates a gene function it is called as gene
knockout. By suppressing the operation of a single gene, gene knockout
approaches are used to identify a specific gene function. Gene knockout may
be used in traditional genetics as well as newer approaches like functional
genomics. Transposon mutagenesis was used to carry out gene knockout
during the early stages. The method's biggest flaw is the time-consuming
screening required to find the knocked-out gene.
Genetic engineering has been used to knock out other organisms. In
vitro techniques are used to modify genes on plasmids or Bacterial Artificial
Chromosomes (BACs), and then cell culture techniques are used to transfer
these modified constructs to the organism of interest. A combination of
genetic engineering and in vivo homologous recombination is used in quite a
few other ways. However, this combination is ineffective. The most recent
method for creating gene knockouts is recombineering. As a precursor stage,
it directly states bacterial chromosome or to edit any plasmid or BAC in
vivo. This method isn't dependent on restricted sites. A drug cassette can be
placed anywhere within a gene, or it can be used to replace the open reading
frame of the gene.
Technologies for gene-knockout
Homologous recombination is the most effective way of producing a
gene knockout, and gene knockout methods allow a single gene to be
removed without affecting all other genes in an organism. The organism is
known as a knockout organism when the gene of interest becomes inactive
with the aid of a gene knockout. When more than one gene in an organism is
knocked out, it is referred to as a double knockout (DKO), triple knockout
(TKO), or quadrule knockout (QKO), depending on the number of genes
involved.
Conditional gene knockout
Conditional knockouts are performed to delete a gene in a specific tissue
at a specific time. This method is required for the functional study of
individual genes in a living organism. In comparison to gene knockouts,
conditional knockouts are created in adult animals rather than in embryonic
stage where a mutation can show lethal effect. In mammalian cell
conditional knock outs are created through homologous recombination and
following strategies are used.
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Recombination with Cre-loxP-Cre, or cyclization recombination
enzyme, is capable of specifically recognizing and cutting DNA sequences,
which is then followed by recombination with the second enzyme, loxP.
Within DNA, Cre recombinase is able to bind to two loxP sites, resulting in a
recombination between them. Depending on the arrangement of the loxP
site, the recombination produces deletions or inversions of the target genes.
Due to the inducible nature of cre-loxP, the knockout can be induced by
chemicals such as tetracyclin and tamoxifane. Tetracyclin function in
activating the cre recombinase transcription whereas, tamoxifane is
responsible for nuclear transport.
Applications
Conditional genes have become widely used methods to study human
disease in different mammal models, such as cancer. Genes for breast cancer
like BRCA1 have been studied through gene knockout mice with BRCA1
deletion in tissues of the mammary gland to confirm the tumor suppression
role. Conditional mouse models are favoured for the study of human disease
as they show a resemblance in phenotype while specific genes are deleted.
Gene knock-in
Knock-in technology is different from knockout technology in that
knockout technology aims to either delete part of the DNA sequence or
insert irrelevant DNA sequence information to disrupt the expression of a
specific genetic locus. The gene knock-in technique, on the other hand,
modifies a genetic locus of interest by replacing DNA sequence information
one-for-one or by adding sequence information not present on the genetic
locus. A gene knock-in is therefore a gain-of-function mutation, while a gene
knock-out is a loss-of-function mutation. However, a gene knock-in may
also involve the replacement of a functioning gene locus with a mutant
phenotype that results in some loss of function.
Different methods for gene knockout
Homologous recombination
The traditional approach for gene knockout is homologous
recombination, which is commonly utilized in genome engineering. This
technique involves exchanging nucleotides across DNA sequences that are
either similar or identical. A DNA construct containing the desired mutation
and a drug resistance cassette to be swapped in lieu of the knockout gene are
used in the homologous recombination approach. In addition, the construct
has a roughly 2-kilobase homologous area with the target gene. The most
Page | 5
frequent ways of transferring the construct into the appropriate organism are
microinjection or electroporation.
The insertion of the vector construct into the target region is dependent
on the organism's DNA repair system. Once integrated, the vector construct
will cause the wild type gene to be altered, resulting in the synthesis of a
non-functional protein. The efficiency of homologous recombination, on the
other hand, only accounts for 10-2 to 10-3 DNA integration.
Site-specific nucleases
There are three ways for introducing double stranded breaks in DNA:
zinc fingers, TALENS, and CRISPER. Following DNA damage, the cells'
own repair machinery kicks in to ligate two open ends via non-homologous
end joining (NHEJ). Due to the repair mechanism's incompleteness, insertion
or deletion mutations occur, resulting in frame shift mutation. The gene
creates a non-functional protein as a result of the mutation, resulting in a
knockout for the gene of interest.
Zinc-fingers
Zinc finger nucleases (ZFNs) are restriction enzymes widely used in
genome engineering for initiating double stranded breaks. They are known to
act as site specific endonucleases, which have the ability to bind to DNA at a
specific site. Zinc finger nucleases (ZFNs) contain two parts: a zinc finger
DNA binding domain fused with a DNA cleavage part. The DNA bonding
domain consists of 3-6 zinc finger repeats, which can further recognize and
bind 9-18 base pairs in a specific DNA sequence.
The cleavage domain, on the other hand, is made up of FokI, a type II
restriction endonuclease. The zinc finger endonuclease protein works as a
very efficient genomic scissors when both domains are joined together. Zinc
finger nucleases (ZFNs) induce site-specific DNA double strand breaks
followed by homologous recombination when introduced into the cell.
Because ZFN plasmids can produce ZFN and successfully target a double
stranded break, they provide a great way to design therapeutic constructs in
advance at a specific site in the genome. ZFNs provide several benefits in
targeting genome editing, such as-
• ZFNs have the ability to quickly integrate or disrupt any part of the
genome.
• ZFN mutations are irreversible and can be passed down through
generations.
• Genome editing may be accomplished with a single transfection.
Page | 6
• They work with a broad variety of mammalian cell lines.
• Positive clone selection does not require antibiotic choices. Knock-
out/ knock-in effects have been seen to exist for nearly two months.
Applications
• In certain cell lines, zinc finger nucleases (ZFNs) are utilized to
create full knockout.
• They may be employed in cell-based screening techniques by
establishing knock-in cell lines in which endogenous target genes
can be tagged with promoter, reporter, or fusion tag proteins; they
can also be used to generate cell lines that produce more of a certain
protein or antibody.
Although Zinc finger nucleases (ZFNs) show several promising effects,
there is also a chance of having potential side effects. When the ZFN
construct do not target or not specific off target cleavage takes place. In such
cases, the DNA repair process is unable to regulate the overproduction of
double-stranded breaks, resulting in chromosomal rearrangement or cell
death. Off-target cleavage also causes random integration in the host
genome, which raises the risk of cells developing an immune reaction in
response to therapeutic agents.
TALENS
Transcription Activator-Like Effector Nucleases (TALENs) are
genetically modified enzymes that edit genes. These enzymes are primarily
isolated from Xanthomonas species bacteria, and they play a role in binding
and activating the host promoter in bacteria. TALENS, like ZFNs, is made
up of two groups that combine transcription activator-like (TAL) proteins
with Fok1 nuclease. The TAL protein is made up of 33-34 amino acid
repetitive motifs that can detect nucleotides of interest quite well. Because to
their variable nature, they're also known as "Repeat Variable Diresidue"
(RVD).
It is possible to build a particular domain to bind DNA by modifying the
combination of repeating motif with a certain RVD, as in TALEN, where
direct relationships between DNA and amino acid recognition exist. It can
successfully cut the genome by fusing TAL with FokI. FokI creates a double
stranded break in the presence of two TALEN domains. After synthesizing
the TALEN construct, a plasmid is used to transfect it into the host cell. The
construct is expressed within the host cell and gains access to the genome by
entering the nucleus. Furthermore, TALENs may be transferred as mRNA in
Page | 7
cells, bypassing the need for genomic integration. (ZFNs) TALENS shows
advantages, such as-
• The design of TALEN is much easier in comparison to ZFNs. Many
TAL repeats can be created with the RVD code, which will bind
strongly to the genomic DNA with high affinity.
• The number of TALE repeats can be extended based on the desired
length.
Applications
• TALENs are widely used for plant genome modification.
• They are useful for the production of biofuels.
• They have been used to generate knockouts in organisms such as
zebrafish, rats, mice or c. elegance.
• It has also been used to treat genetic diseases such as xeroderma
pigmentation of sickle cells.
However, like ZFNs, TALENs also display off-target effects. The off-
target effect will eventually generate chromosomal rearrangement due to the
presence of unwanted breaks in DNA double strands.
CRISPER/cas9
CRISPER/cas9 is a rapid genome editing method that is used to delete
or modify specific sequences of DNA. CRSIPER stands for Clustered
Regularly Interspaced Short Palindromic Repeats, which can be found
naturally in certain types of bacteria. When invaded by phage viruses,
bacteria use the CRIPER/Cas9 method to cut and disintegrate the viral DNA.
In bacteria, there are three types of CRISPER method, among them, type II
is the most widely studied. With this method, once cut into small parts, the
invading DNA gets incorporated into the CRIPER locus. Once transcribed,
small repeats of RNA or crRNA/ Crisper RNAs are generated.
CrRNA is next joined by another noncoding RNA known as trans-
activating CRISPR RNA or tracrRNA, which activates the endonuclease
Cas9 to target the invading viral DNA. CrRNA, together with tracrRNA,
forms a single guide RNA or sgRNA. CRISPER/Cas9 generates DNA
double-stranded breaks which can be repaired by two mechanisms:
i) Nonhomologous end joining or NHEJ, where homologous double
strands are absent.
Page | 8
ii) Homology-directed repair, which occurs in the presence of a
synthetic DNA repair template.
Applications
• The CRISPER/Cas9 gene editing technology may be used to
perform homology-directed repair (HDR).
• They may be used to silence genes, and they can also be employed
to temporarily activate endogenous genes.
• It may be used to create DNA-free gene editing approaches.
• They're designed to silence genes for a short period of time.
• Transgenic animals and embryonic stem cells are being prepared.
Following its discovery, the CRISPER/Cas9 system has been widely
employed for gene targeting in a variety of organisms, including plants,
humans, c. elegance, zebra fish, yeast, drosophila, and mice. When just a
single gRNA is available, they are now employed to induce single point
mutations. CRISPER/Cas9 is easier to utilize than TALENs and ZNFs since
just crRNA has to be redesigned to target any gene. This approach also
allows for genome-wide gene function research by generating massive
gRNA libraries.
Knockout-Mediated crop trait improvement
Negative gene deletion is a potential genetic enhancement technique. As
a result, the simplest and most popular use of CRISPR/Cas9 is to knock out
genes that impart undesired features. Yield, quality, and biotic and abiotic
stress tolerance are among the traits that have been enhanced using
CRISPR/Cas9. This strategy has also improved hybrid-breeding procedures
and many other crucial elements of agricultural yield.
Increasing yields
Yield is the major objective of gene editing for crop enhancement due to
the requirement for increased food security. Yield is a complicated attribute
that is influenced by a variety of conditions. Negative regulators known to
affect yield-determining factors like grain number (OsGn1a), grain size
(OsGS3), grain weight (TaGW2, OsGW5, OsGLW2, or TaGASR7), panicle
size (OsDEP1, TaDEP1), and tiller number (OsAAP3) were knocked out,
resulting in the expected phenotypes in plants with loss-of-function
mutations in these genes, demonstrating that CRISPR/Cas9 is an effective
tool for improving yield-related traits (Li et al. 2016, Liu et al., 2017, Lu et
al., 2018, Zhang et al., 2018, Zhang et al., 2016).
Page | 9
In rice, knocking down three grain weight-related genes (GW2, GW5,
and TGW6) at the same time resulted in trait pyramiding, which raised grain
weight significantly (Wang et al., 2017). Because most yield-related
variables are quantitative and regulated by quantitative trait loci, removing
individual components may not be enough to boost yield in the field. Huang
et al. (2018) have established a strategy that combines pedigree analysis,
whole-genome sequencing, and CRISPR/Cas9 technology to identify genes
that contribute to complicated quantitative variables like yield on a wide
scale.
The researchers analyzed 30 cultivars from the parents and offspring of
the Green Revolution miracle rice variety IR8 and selected 57 genes that
were maintained in all high-yielding lines for gene editing using Cas9 or
dCas9 deletion or knockdown. According to a phenotypic study, many of
these genes are crucial for rice yield. This research gave light on the process
of yield growth and might aid in the development of improved rice via
molecular breeding.
Improving quality
Quality characteristics differ depending on the requirements of the
breeding program. To date, genome editing has improved the quality of
starch content, fragrance, nutritional value, and storage quality of crops. Rice
with low amylose content was developed using CRISPR/Cas9 to knock out
Waxy, resulting in better rice eating and cooking quality (Ma et al., 2015;
Zhang et al., 2018). For commercial application, DuPont Pioneer developed
a CRISPR/Cas9 knockout waxy corn line with exceptional yields (Waltz
2016). By altering the starch branching enzyme gene SBEIIb, CRISPR/Cas9
was also utilized to make high-amylose and resistant starch rice; consuming
high-amylose meals could help patients with diet-related non-infectious
chronic illnesses (Sun et al., 2017).
Rice cultivars with desirable cooking aromas have higher economic
value. The production of 2-acetyl-1-pyrroline, the primary fragrance
compound in fragrant rice, is affected by a mutation in the betaine aldehyde
dehydrogenase 2 (BADH2) gene. They developed a fragrant rice line with a
similar 2-acetyl-1-pyrroline content (0.35–0.75 mg/kg) to the wild mutant
fragrant rice variety using TALEN-targeted disruption of OsBADH2 (Shan
et al., 2015) recently transferred the fragrance trait to more than 30 elite rice
cultivars in significant growing regions of China. Celiac disease is caused by
gluten proteins found in cereal grains and affects more than 7% of people in
Western countries. The primary gluten-encoding gene family in wheat, the α-
gliadin gene family, has almost 100 genes or pseudogenes.
Page | 10
CRISPR/Cas9 editing provides a novel technique to modify features
governed by large gene families with redundant roles. Researchers have
generated low-gluten wheat by knocking off most conserved domains of -
gliadin family members at the same time (Sanchez-Leon et al., 2018). Seeds
with high oleic acid oil in Camelina sativa (Jiang et al., 2017, Morineau et
al., 2017) and Brassica napus (Okuzaki et al., 2018), tomatoes with a long
shelf life (Ito et al., 2015, Lu et al., 2018), high-value tomatoes with
enhanced lycopene (Lu et al., 2018) or γ-aminobutyric acid content (Nonaka
et al., 2017), and potato (hairy roots) with reduced levels of toxic steroidal
glycoalkaloids (Nakayasu et al., 2018).
Biotic and abiotic stress resistance
Stress is the key element impacting crop yield and quality. The
CRISPR/Cas9 knockout has resulted in several plants with enhanced biotic-
stress tolerance, including resistance to fungal, bacterial, viral, and insect
diseases. Powdery mildew, for example, is a destructive fungal disease that
affects crops.
Wang et al. (2017) used TALEN and CRISPR/Cas9 to knock out all six
TaMLO alleles in wheat, resulting in plants that were more resistant to
powdery mildew. Similarly, Nekrasov et al. (2017) demonstrated that
knocking down MLO in tomato using CRISPR/Cas9 provides resistance to
powdery mildew. Rice blast is a deadly fungal disease, and blast-resistant
rice was created by knocking down the OsERF922 gene, which encodes an
ethylene response factor transcription factor (Wang et al., 2016).
Xanthomonas oryzae pv. oryzae causes bacterial blight in rice. The deletion
of the OsSWEET13 promoter resulted in the creation of disease-resistant
plants (Zhou et al., 2015).
In the case of viral diseases, CRISPR/Cas9 has generated tungro
disease–resistant eif4g rice (Macovei et al., 2018), broad potyvirus–resistant
eif4e cucumber (Chandrasekaran et al., 2016) and cotton leaf curl disease–
resistant clcud cotton (Iqbal et al., 2016). Lu et al. (2018) recently found that
altering OsCYP71A1 prevented serotonin production and dramatically
elevated salicylic acid levels, imparting resistance to plant hoppers and stem
borers, two of rice's most devastating pests.
Among abiotic stresses, contamination of arable lands has created a
need to avoid the build-up of hazardous heavy metals in crops. Rice strains
with low amounts of cadmium, radioactive cesium and arsenic have been
produced by knocking out OsARM1, OsNramp5 and OsHAK1 (Nieves-
Cordones et al., 2017, Tang et al., 2017, Wang et al., 2017). According to a
Page | 11
study published in 2018, pyl1/4/6 triple knockout rice produced by
CRISPR/Cas9 editing exhibited enhanced grain yield, better high-
temperature tolerance and less preharvest sprouting compared to wild type
rice (Miao et al., 2018).
Speeding hybrid breeding
Hybrid breeding is an effective way to boost agricultural output. A
male-sterile maternal line is required to produce a high-quality hybrid strain.
The thermosensitive male-sterile tms5 lines in rice (Zhou et al., 2016) and
maize (Li et al., 2017), photosensitive genic male-sterile csa rice (Li et al.,
2016), and ms45 wheat (Li et al., 2016) have all made significant progress
utilizing CRISPR/Cas-mediated gene knockout (Singh et al., 2018).
The greatest barrier to using heterosis in breeding is hybrid sterility.
SaF/SaM at the sterility locus Sa and OgTPR1 at the S1 locus (Xie et al.,
2017) were disrupted to overcome reproductive barriers in japonica-indica
hybrids. In japonica-indica hybrids, Shen et al. (2017) discovered that
knocking out one or two copies of the Sc gene in the indica allele Sc-I also
restored male fertility. Yu et al. (2018) found that knocking out the toxin
gene ORF2, which is involved in rice's newly identified selfish-gene suicide
mechanism, enhanced the fertility of japonica-indica hybrids. By knocking
off three essential meiotic genes, REC8, PAIR1 and OSD1, genome editing
was recently utilized to replace mitosis for meiosis in rice.
Two independent groups developed asexual propagation lines either by
simultaneous activation of BBM1 in the egg cell (Khanday et al., 2018) or
by knocking out MTL (Wang et al., 2019), enabling a fix of heterozygosity
of hybrids through seed propagation. Genome editing is also an effective
approach for enhancing many other traits, such as improving haploid
breeding (Dong et al., 2018, Yao et al., 2018), shortening growth times (Li
et al., 2017), increasing silique shatter resistance (Braatz et al., 2017), and
overcoming self-incompatibility in diploid potato (Ye et al., 2018), to meet
breeders’ requirements.
Conclusion
The development of several gene knockout methods has resulted in
significant progress in the research of gene functions. The present gene
knockout procedures are wisely executed in terms of their advantages.
CRISPR genome editing is now the quickest and most direct method of
accomplishing precise gene knockout. Gene knockout remains an important
method for studying biology. Scientists may learn more about a gene's
impacts and activities by altering it. This is important for studying signaling
Page | 12
pathways in many kinds of cells and is frequently required for large-scale
biology procedures like CRISPR screening.
As a sophisticated molecular biology technology, genome editing may
make precisely targeted alterations in any crop. The exceptional potential of
genome editing to produce targeted, sequence-defined, genome-wide genetic
diversity in plants has accelerated both basic plant research and crop
development. Because of the simplicity, versatility, and robustness of
CRISPR/Cas systems, genome editing is a powerful tool for precision crop
improvement via gene deletion, knock-in, replacement, point mutations,
fine-tuning of gene regulation, and other alterations at any gene locus in
crops.
It may also be used to generate antivirals and create substantial mutant
libraries. For efficiently transferring technologies from the bench to the field,
rapid discovery of the genetic bases of important traits, improved gene
targeting (gene insertion and replacement), effective delivery of
CRISPR/Cas reagents to plant cells and subsequent plant regeneration with
or without the need for tissue culture, and the availability of base editors
with improved targeting range and frequency are all required. Synthetic
biology and systems biology, as well as advances in functional genomics, in
conjunction with genome-editing technologies, next-generation sequencing,
and a number of other related methodologies, will allow the manufacture of
improved crops with much superior qualities.
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Chapter - 2
Superiority of Black Rice over Normal Rice
Variety
Authors
Alija Priyadarshini
Department of Botany, Utkal University, Bhubaneswar,
Odisha, India
Amit Kumar Mohapatra
School of Life Sciences, Sambalpur University, Sambalpur,
Odisha, India
Abhilash Padhan
Department of Fruit Science, Dr. YS Parmar University of
Horticulture and Forestry, Nauni, Himachal Pradesh, India
Page | 19
Page | 20
Chapter - 2
Superiority of Black Rice over Normal Rice Variety
Alija Priyadarshini, Amit Kumar Mohapatra and Abhilash Padhan
Abstract
Rice varieties with coloured pericarp (other than white and red) are
usually known as “black rice”. (Rice var with black or purple coloured outer
pericarp comes under “black rice” group). This rice is not only loaded with
powerful disease fighting antioxidants but also it contains anti-inflammatory,
anti-carcinogenic properties and has an ability to stop the development of
diabetes mellitus, cancer, heart disease, and even weight gain. In this field-
laboratory investigation during 2019-2021, two variety of rice samples were
selected based on geographical situation local data. In the laboratory, the
collected samples were processed and analysed. Data shows of black rice has
high protein which can be a great replacement for various expensive protein
sources. Various directions like metaboanalysis and genomic linkages can be
uncover with extensive research in future.
Keywords: Black rice, anthocyanin, protein, super food
Introduction
Rice is the foremost cereal food crop in many developing countries.
About half of the world population consumes rice as their major source of
carbohydrate. Almost 95% of the rice production is recorded in Asian
countries. White rice is the most commonly consumed rice, but there are
several rice cultivars which contain colour pigments, such as black and red
rice. Rice varieties with coloured pericarp (other than white and red) are
usually known as “black rice”. Black rice is a type of the rice species Oryza
sativa L.
Black rice is a type of the rice species Oryza sativa L. which is black in
colour, glutinous, packed with high level of nutrients and mainly cultivated in
Asia. It is actually heirloom rice means it is open pollinated (just check
heirloom var were too self-pollinated as cultivated var), was grown at earlier
times in the history and is not grown on a large scale in modern agriculture. It
also comes in a number of short grain, long grain and glutinous varieties
Page | 21
similar to brown rice with a slightly nutty flavour and its texture is smooth,
firm not at all sticky like most white rice. Black rice includes many more
varieties of dark colour rice like Forbidden rice, Purple rice, Japonica black
rice, Chinese black rice, Indonesian black rice and Thai black rice. The reason
they are grouped under the term “Black Rice” is the unusual dark/black colour
of the grain. Black rice now widely considered as a ‘super food’ by researchers
and scientists. The term ‘super food’ is used to describe food items with
extremely high nutritional value. Black rice is a super nutritious type of rice
that is high in fibers, antioxidants, vitamins B, vitamin E, iron, thiamine,
magnesium and niacin and phosphorous.
Historical background of black rice
Black rice has an incredibly rich cultural history. In ancient china and
Indonesia black rice was considered so superior, tasty and rare that it was
exclusively reserved for the emperor and used as a tribute food. Common
people were not allowed to have this. So, it was called “Emperor’s rice”. The
Emperor says, “Hands off the black rice! It’s mine”. Royal families and kings
of ancient China used to eat this special rice to ensure their longevity and good
health. It was also known as “Forbidden rice” as the name might imply,
consuming it without approval from the proper authorities can have life
threatening consequences for those involved. But times have changed and now
it is available to the different parts of world. Black rice is commonly cultivated
and eaten in Manipur, called “Chakho ambi” in, Manipuri language which is
commonly known as “Manipuri Chakho” which is eaten during community
feasts. “Chakho” means delicious and “amubi” means black, thus translating
the name to “delicious black rice”.
Origin and spread of black rice
Although the events surrounding the origin and spread of black rice traits
remain unknown, varieties with black grains due to anthocyanin accumulation
are distributed in various locations throughout Asia. A paper published in “The
Plant Cell” reveals the answer to the long-standing question of how black rice
became black and, moreover, traces the history of the trait from its molecular
origin to its spread into modern-day varieties of rice. Researchers from two
institutions in Japan collaborated to meticulously examine the genetic basis
for the black color in rice grains. They discovered that the trait arose due to a
rearrangement in a gene called Kala4, which activates the production of
anthocyanin. Both the Rc and Kala4 genes activate upstream flavonol
biosynthesis genes, such as chalcone synthase and dihydroflavonol-4-
reductase and downstream genes, such as leucoanthocyanidin reductase and
Page | 22
leucoanthocyanidin dioxygenase, to produce the respective specific pigments.
They concluded that this rearrangement must have originally occurred in the
tropical japonica subspecies of rice and that the black rice trait was then
transferred into other varieties by crossbreeding.
Black rice was actually only first introduced to United States in the 1990s
although it has been enjoyed in other part of the world for many more years.
Choudhury and Tran (2001) reported that black rice might be originated from
Sri Lanka, Philippine, Bangladesh, Thailand, Myanmar and Indonesia. China
is the richest country in the black rice resources (62%) followed by Srilanka
(8.6%), Indonesia (7.2%), India (5.1%) Bangladesh (4.1%) and few in
Malaysia.
Socio-economical value
Despite lower grain yield, there is close relationship between ethnic
cultural practices of Meiteis and black rice cultivation. Black rice dish (Chak)
is offered to the deities and ancestors in Usob (death ceremonies) and Kang-
pali (religious festival); while it is served as dessert (Kher) in Chakumba (first
rice-eating ceremony) by the Meitei people. The black rice also used
occasionally to prepare as Ethe tan (flatbread), Kabok (puffed rice), Kabok-
aafaba (puffed rice laddu), flakes (Chengpak), Utongchak (rice cooked in
bamboo stick) and Yu (alcoholic beverages). Traditional beer from the black
rice called ‘Chakhao-atingba’ is one of the most relished beverages among the
Meitei’s. However, black rice is not used as staple food as it takes more time
for cooking and feels rubbery while chewing due to high fiber content.
Method
Collection of seeds
The seeds were collected from Badturang (21º27ʹ27.4ʺN 84º9ʹ14.2ʺE)
Sambalpur, Odisha, India. Unharmed Seeds of uniform size, were selected and
processed for further research.
Page | 23
https://www.google.com/maps/place/Badturang
Experimental design
Whole experimental condition was directed towards comparative analysis
between control (white rice) and experimental (black rice).
Table 1: Experimental design
Kusumkali Manipuri chakhao

Biochemical parameters
(Sample 1) (Sample 2)
Starch Control Test
Sugar Control Test
Protein Control Test
Chlorophyll Control Test
Flavonoids Control Test
Anthocyanin Control Test
Page | 24
Estimation of starch
Estimation of total soluble Starch by Phenol-sulphuric method (Dubois et
al. 1956). Seed coats were removed & and 1 gram of seeds were measured.
These seeds were homogenized in dist. water with the help of mortar and
pestle. The homogenized product was centrifuged for 10 minutes at 4000rpm,
supernatant was collected measured and stored. To the residue 3% HCL-
Methanol was added and allowed to water bath for 3 hours. After that samples
were again centrifuged for 10 minutes at 4000rpm. The supernatant was
collected & the volume was measured. A sires of test tubes were taken,
prepared and absorbance were measured at 490n.m.
Estimation of sugar
Estimation of total soluble Starch by Phenol-sulphuric method (Dubois et
al. 1956). Seed coats were removed & and 1 gram of seeds were measured.
These seeds were homogenized in dist. water with the help of mortar and
pestle. The homogenized product was centrifuged for 10 minutes at 4000 rpm,
supernatant was collected measured and stored. Test tubes were prepared as
per tabulation. Glucose standard were taken as standard. 18% of Phenol was
added to the supernatant followed by concentrated H2SO4. Then the solution
was allowed to stand for 20 minutes, pink color will be developed. Absorbance
was measured at 490nm.
Estimation of protein
Estimation of Protein done by the method as per Lowry et al. (1951).
Samples of about 1gram fresh weight was homogenized with 10% TCA with
mortar and pestle. The homogenate was centrifuged at 4000 rpm for 10 min.
The supernatant was discarded and pellet was taken and then dissolved in 1N
NaOH solution. A clear solution was then made with the help of homogenizer.
Total volume of the extract was measured. The extract was diluted 10 times
with 0.3N NaOH solution and the protein content was measured by taking OD
at 750 nm method.
Estimation of flavonoid
Estimation of Flavonoid be done by using Ethanol: HCL. 0.1 gram of
sample was grained with 3 ml HCL-Ethanol solution Kusirisin et al. 2009,
Yang et al. 2014). The sample was centrifuged at 5000rpm for 10 minutes.
The supernatant was boiled for 10 minutes in a water bath then volume was
measured. The sample was diluted 10 times, absorbance was measured at 270,
300, 330nm and concentration was measured.
Page | 25
Estimation of anthocyanin
Estimation of Anthocyanin be done by using 1% HCL-Methanol solution
(Kim et al. 2008). 0.1 gram of sample was taken and homogenized along with
1% HCL- Methanol solution. The supernatant was incubated at 4ºc for 4 hours
overnight. After incubation the sample was centrifuged at 5000rpm for 10
minutes, supernatant was collected and the volume was measured. The
supernatant was diluted by taking 0.1 ml of the extract to 10 ml HCL-Methanol
solution. The absorbance was measured at 530, 657nm and concentration of
anthocyanin was measured.
Results and Discussion
Parameters Kusumkali (Sample 1) Manipuri chakhao (Sample 2)
Starch (mg/g) 290.1 ± 9.899 408.7 ± 6.363
Sugar (mg/g) 161.7 ± 6.222 208.3 ± 8.202
Protein (mg/g) 17.14 ± 1.131 51.55 ± 2.192
Anthocyanin (µg/g) 16.5 ± 0.707 50 ± 1.414
20.10 ± 2.679 97 ± 1.414
Flavonoid(µg/g) 270nm
18.59 ± 2.347 72.5 ± 0.707
300nm 330nm
17.65 ± 3.323 59 ± 1.414
Fig 1: A graphical comparison of biochemical between sample 1 and 2
Page | 26
Data presented above shows a great advantage of black rice above the
white rice variety. High protein content of black rice makes it a great
replacement for various expensive protein sources. High content of
anthocyanics can make it a natural killer of cancer.
Conclusion
This above experiment shows that black rice has a great potential to
replace various supplements and be a super food. Various aspects are still there
which can be taken into consideration like
1) Increasing awareness for cultivation of black rice cultivar and its
usefulness.
2) As black rice is a wild rice variety (not hybrid) and naturally have
capacity to fight against several diseases, research should be done on
local black rice cultivar to develop a new link of knowledge.
3) As kala4 gene is known responsible for the development of
anthocyanin then maximum research must be done in order to
incorporate this gene into other rice cultivars, Whole genome
sequencing of rice varieties to be analyzed in order to know presence
of various SNPs (Single Nucleotide Polymorphism) in kala4 gene to
determine some gene silencer as well as enhancers exist in nature.
Acknowledgement
Alija Priyadarshini, Amit Kumar Mohapatra, done all the experiments
and analysis. Abhilash Padhan helped in manuscript preparation and samples
collection.
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Chapter - 3
Genome Editing: A Way Forward for Crop
Improvement
Authors
K. Bhargava
Ph.D. Scholar, Department of Genetics and Plant Breeding,
Professor Jayashankar Telangana State Agricultural
University, Hyderabad, Telangana, India
P. Sadhana
S. Ravali
Ph.D. Scholar, Department of Plant Pathology, College of
Agriculture, Professor Jayashankar Telangana State
Agricultural University, Hyderabad, Telangana, India
P. Hima Bindu
R. Harisha
Ph.D. Scholar, Division of Genetics and Plant Breeding, Indian
Agriculture Research Institute (ICAR-IARI) New Delhi, India
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Chapter - 3
Genome Editing: A Way Forward for Crop Improvement
K. Bhargava, P. Sadhana, S. Ravali, P. Hima Bindu and R. Harisha
Abstract
Crop improvement is sheer essential to meet the ever increasing global
food demand and enhance food nutrition. Conventional crop-breeding
methods have certain constrains such as taking lot of time and resources, and
causing biosafety concerns. These limitations could be overcome by the
recently emerged-genome editing technologies that can precisely modify
DNA sequences at the genomic level using sequence-specific nucleases
(SSNs). The artificially engineered SSNs such as zinc finger nucleases
(ZFNs), transcriptional activator-like effector nucleases (TALENs) and
clustered regularly interspaced short palindromic repeats (CRISPR)-
associated endonuclease Cas9 (CRISPR/Cas9) have proven to be highly
efficient in targeted mutagenesis in a wide range of plant species to
characterize gene functions and improve agricultural traits. Among these the
CRISPR/Cas9 is the most recently developed targeted genome modification
system and seems to be more efficient, inexpensive, easy, user-friendly and
rapidly adopted genome-editing tool. Currently genome editing technology
was indispensable in many crops for improvement several important traits.
Keywords: Genome editing technologies, sequence-specific nucleases, zinc
finger nucleases, transcriptional activator-like effector nucleases and
clustered regularly interspaced short palindromic repeats
Introduction
In today's world, nearly a billion people suffer from chronic
malnutrition, while at the same time our agricultural systems are degraded,
exacerbated by the loss of biodiversity and the growing uncertainties of
climate change [1]. With the world population projected to exceed 9 billion
by 2050, modern agriculture will face enormous challenges that will require
higher yields, better quality and fewer inputs [2]. Although conventional
farming is currently the most common approach to crop improvement, it is
labour-intensive and typically takes much longer time to develop new
varieties. Various technologies in crop breeding such as the use of semi-
dwarf cultivars, which allow the use of large quantities of agricultural inputs
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and hybrid breeding techniques, have contributed greatly to increasing
yields. In addition to these techniques, genetically modified crop technology
has been used to improve productivity since 1980s and genetically modified
crops are now grown on over 10% of the world's agricultural land. Its use is
hampered by largely unfounded environmental health and safety concerns.
New plant breeding techniques have recently been developed, including the
genome editing technique that uses artificial nucleases to modify specific
sequences in the genome.
Genome editing
Genome editing is defined as a collection of advanced molecular
biology techniques that allow precise, efficient and targeted modification of
genomic loci [3]. Genome editing using mega nucleases, zinc finger nuclease
(ZFN) and transcription activator-like effector nucleases (TALEN) has been
around for two decades, but it has recently come under the spotlight through
the development of clustered regularly interspaced short palindromic repeats
(CRISPR)/Cas systems which offers simplicity and ease of targeted gene
editing. All of these technologies use typical sequence-specific nucleases
(SSNs) that can be induced to recognize specific DNA sequences and to
generate double-stranded breaks (DSBs). The plant’s endogenous repair
systems fix the DSBs either by non-homologous junction (NHEJ), which can
lead to nucleotide insertion or deletion, leading to gene knockout, or by
homologous recombination (HR), which can lead to gene substitution and
insertion [4]. Many knockout gene mutants and gene insertion mutants have
been produced in various plants through the use of genome editing
technologies and many of these mutants have been shown to be useful for
plant improvement.
Genome editing tools
a) Mega nucleases
The first enzymes used for genome editing were mega nuclease enzymes
[5]
. The mega nucleases are categorised into four groups: LAGLIDADG, His-
Cys box, GIY-YIG, and the HNH family. The LAGLIDADG family
includes the popular mega nucleases I-CreI and I-SceI where they recognise
and cleave large DNA sequences (12-40 bps). It was originally developed for
animal and human systems, but it has also been used to edit the genomes of
plants such as Arabidopsis thaliana and Zea mays [6, 7]. A modified mega
nuclease, derived from the I-CreI homing nuclease was used to target MS26
gene of maize and create plants that were male sterile when homozygous for
the gene knockout [8]. However, DNA-binding and nuclease domains of
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homing endonucleases overlap, making it difficult to engineer them to target
different DNA sites and their use was also restricted due to patent issues.
b) Zinc finger nucleases
ZFNs were the first-generation genome editing tools and its utilization
in the genome editing in plants were started in 2005 [9]. These are fusions of
zinc-finger-based DNA-recognition modules and the FokI restriction
enzyme's DNA-cleavage domain. Each zinc finger recognizes and binds to a
nucleotide triplet and fingers are frequently grouped together to bind to
specific DNA sequences [10]. ZFNs has been used in Arabidopsis, Nicotiana,
maize, petunia, soybean, rapeseed, rice, apple and fig for manipulation of
many traits till to date. The endogenous maize gene ZmIPK1 was disrupted
by insertion of PAT gene cassettes is one example of ZFNs in crop breeding,
resulting in herbicide tolerance and a change in the inositol phosphate profile
of developing maize seeds [11]. Nevertheless, designing ZFNs is a time-
consuming and technically challenging process with a low efficacy rate.
c) Transcription activator-like effector nucleases
Transcription activator-like effector nucleases (TALENs) are the
second-generation genome editing tools and were first used for plant genome
editing in 2011 [12, 13]. TALENs like ZFNs are specific DNA-binding proteins
with a 33 or 34-amino-acid repeat array. TALENs are artificial restriction
enzymes created by fusing a nuclease's DNA cutting domain with TALE
domains that can be tailored to recognise a specific DNA sequence. These
fusion proteins act as "DNA scissors" for gene editing applications, allowing
for targeted genome modifications like sequence insertion, deletion, repair,
and replacement in living cells. TAL effectors, DNA-binding proteins
excreted by plant pathogenic bacteria Xanthomonas, provide the DNA
binding domains, which can be designed to bind any desired DNA sequence.
TAL effectors are made up of multiple domains, each of which contains a
highly considered sequence of 34-amino-acids and recognises a single DNA
nucleotide within the target site.
The nuclease can cause double strand breaks at the target site, which can
be repaired by error-prone non-homologous end-joining (NHEJ), causing
gene disruptions via small insertions or deletions. With the exception of the
so-called repeat variable di-residues (RVDs) at amino acid positions 12 and
13, each repeat is conserved. The RVDs will determine the DNA sequence,
the TALE will need bind to, because the TALE repeats and the
corresponding DNA sequence have a simple one-to-one correspondence,
assembling repeat arrays to recognise novel DNA sequences is simple. The
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catalytic domain of a DNA nuclease, FokI, can be fused to these TALENs to
create a transcription activator-like effector nuclease (TALEN). The TALEN
constructs that result combine specificity and activity, resulting in sequence-
specific nucleases that bind and cleave DNA sequences only at pre-selected
locations. TALEN can be performed within a 6 base pairs range of any
single nucleotide in the entire genome.
d) CRISPR/Cas9
CRISPR/Cas9 is considered a third-generation genome editing tool.
First used in 2013 to edit plant genes and it is currently the most commonly
and widely gene editing tool [14, 15]. CRISPR has been shown to be a
prokaryotic immune system that protects cells by selectively targeting and
destroying foreign DNA such as viruses and plasmids. The CRISPR/Cas9
system consists primarily of three components: the endonuclease Cas9,
crRNA (CRISPR RNA) and tracrRNA (transactivating crRNA). The role of
the Cas9 endonuclease is to break the invading phage DNA into small pieces
and integrate it into the CRISPR array as a spacer. Subsequently, crRNA and
a complementary tracrRNA are transcribed from the CRISPR array to form a
double-stranded RNA structure that recruits Cas proteins for cleavage. The
adjacent proto spacer motif (PAM) sequence (5'-NGG-3) located
downstream of the target DNA is a prerequisite for binding and cleavage of
the target DNA [16]. There are two nucleic acid binding grooves namely REC
lobe and NUC lobe in Cas9 endonucleases. The REC lobe is a Cas9-specific
functional domain, while the NUC lobe is composed of three components:
the RuvC, HNH and PAM interaction domains. The homology between
RuvC and HNH helps predict the nuclease domain structure. The Cas9
enzyme is activated upon contact with the sgRNA in the REC lobe. The
Cas9-sgRNA complex then scans the double-stranded DNA for PAM sites.
When the correct PAM is met, the HNH nuclease domain cleaves the RNA-
DNA hybrid and RuvC cleaves the other strand. In addition, the DSB is
modified by error-prone NHEJ or highly accurate HR routes. NHEJ usually
introduces indel mutations, but HR more accurately modifies DSB by gene
insertion or substitution [17].
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The basic mechanism of genome editing systems: ZFNs, TALENs and CRISPR/
Cas9
Source: Rukmini Mishra and Kaijun Zhao, 2018 [18]
Recent advances in genome editing
a) CRISPR-Cpf1: a new system for genome editing
CRISPR-Cpf1 is class II type V endonuclease, that recognizes a T-rich
PAM (5′-TTTN-3′) present in the 5′ end of the target site and requires a
single crRNA of ~44-nucleotide (nt) length for cleavage. It generates sticky
or cohesive ends unlike blunt ends in case of SpCas9, which enhances the
efficiency of gene insertion at a precise genome location. The advanced
features like lower rates of off-target activity make it a more desirable
editing system in plants as compared to SpCas9 [19]. CRISPR-Cpf1 system
was used for multiplexed gene editing in rice by editing four OsBEL genes
[20]
.
CRISPR-Cpf1 system has wide applications in plant genome editing like
functional screening based on gene knockouts, transcriptional repression
using catalytically inactivated Cpf1 (dCpf1) or transcriptional activation
using dCpf1 fused with a transcription activator domain, epi-genome editing
with dCpf1 fused to epi-genetic modifiers, and the tracking of cell lineages
with DNA-barcoding techniques. These advanced applications will improve
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crop yield and quality, assisting in the achievement of food security and
sustainability. To study the loss of function of genes, researchers used
CRISPR-Cpf1 technology to knock out EPFL9 (epidermal patterning factor
like-9) in rice [21]. Stomagen, also known as OsEPFL9, is a rice
developmental gene that regulates leaf stomatal density. The mutated plants
stomatal density was reduced by eightfold without any off-target activity.
The research not only aids in the understanding of gene function loss, but
also in the understanding of plant development in its early stages.
b) DNA-free genome editing in plants
The prevention of transgene assimilation and the minimization of Cas9
off-target activity are major objectives to consider during the escalation of
the CRISPR/Cas9 method. Accordingly, it is a goal to design a
CRISPR/Cas9 Ribo-nucleoproteins (RNP)-mediated genome editing
technology to generate adequate and absolute genome editing. In vitro
testing of CRISPR/Cas9 RNP revealed strong editing activity and a
significant decrease in off-target activity. CRISPR/Cas9RNPs have been
used to successfully edit a wide range of plants, including A. thaliana,
tobacco, lettuce and rice [22]. Korean scientists recently successfully
delivered CRISPR/Cas9 RNPs to the grape and apple protoplast systems,
resulting in transgene-free plants [23].
c) Base editing
Crop plants with single-base changes have elite trait variations, which
aids in crop improvement. Genome editing in plants using CRISPR–Cas9
system via homology directed repair (HDR) is quite challenging due to low
frequency and efficiency of delivery of template DNA; thus, efficient
techniques for producing precise point mutations in crops are urgently
needed. Base-editing technology mediated by CRISPR/Cas9 is a new
genome-editing technique that can accurately convert one DNA base into
another without the use of a DNA repair template. Cas9 nickase (nCas9) or
dead Cas9 (dCas9) fused to an enzyme with base conversion activity are
used in base-editing technologies. For example, cytidine deaminases convert
cytosine (C) to uracil (U), and the latter is treated as thymine (T) in
subsequent DNA repair or replication processes, so creating a C•G to T•A
substitution. Likewise, adenine deaminases convert adenine (A) to inosine
(I), which is treated as guanine (G) by polymerases, creating A•T to G•C
substitutions. Caixia Gao and group from China have successfully
demonstrated base editing in cereal crops like rice, wheat and maize plants
using a plant base editor named nCas9-PBE, composed of rat cytidine
deaminase APOBEC1 and a Cas9 variant [Cas9-D10A nickase (nCas9)] [24].
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Application of genome editing systems in crop improvement
Genome editing technologies have been successfully used in a wide
range of crops to target genes and improve varieties for higher yield, better
quality, increased nutritional value, and disease resistance. By focusing on
traits primarily controlled by negatively regulatory genes, genome editing
tools have shown great promise in improving crop resistance to a variety of
biotic and abiotic stresses [25].
a) Improved yield
Grain yield is primarily determined by grain number, size, and weight,
which are all quantitative traits, and many genes affecting crop yield have
been characterized. Knockout of genes such as GS3, DEP1, GS5, GW2,
Gn1a, and TGW6 in rice, is a simple and direct way to improve crops.
CRISPR/Cas9 was used to individually mutate GS3, DEP1, and Gn1a, and
some of the predicted phenotypes were observed [26, 27]. In rice, knocking out
GW2, GW5, and TGW6 all at the same time resulted in a 29.8% increase in
thousand-grain weight in the triple mutant [28]. In bread wheat, thousand-
kernel weight also exhibited an increase after the three homo-alleles of
GASR7, a negative regulator of kernel width and weight, were knocked out
using CRISPR/Cas9 [29].
b) Disease resistance
Genome editing has been applied to increase disease resistance by
editing disease-related genes. The rice OsSWEET14 gene is activated by a
type-III effector protein secreted by the bacterial rice pathogen Xanthomonas
Oryzae pv. Oryzae, which causes bacterial blight [30]. The use of TALEN to
disrupt the bacterial-derived protein binding sequence in the OsSWEET14
rice promoter increased resistance to bacterial blight. CsLOB1 is also a host
disease susceptibility gene that promotes pathogen growth and the formation
of erumpent pustules in citrus [31]. Two groups recently used CRISPR/Cas9
to modify the CsLOB1 promoter to create canker-resistant citrus cultivars
[32]
. Rice blast resistance was improved when the ERF transcription factor
OsERF922, a negative regulator of rice blast resistance, was knocked out.
Plant viruses, in addition to bacterial and fungal pathogens, have been
targeted using genome editing techniques. Virus-resistant tobacco and
tomato have been created by using CRISPR/Cas9 to target viral genomic
sequences directly [33].
c) Herbicide tolerance
Genome editing has been used to edit plant genes such as ALS and
EPSPS, providing a new approach for creating herbicide-tolerant crops. The
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acetolactate synthase enzyme, which is involved in the biosynthesis of
branched-chain amino acids like valine, leucine and isoleucine, is encoded
by the ALS gene. ALS inhibitors are used as herbicides, starving affected
plants of these amino acids over time, eventually inhibiting DNA synthesis.
Resistance to these herbicides can be conferred by specific point mutations
within the conserved region of the ALS gene. The first example, tobacco,
was reported in 2009 using ZFNs and donor templates to introduce precise
mutations in the ALS gene to produce herbicide-tolerant plants using
genome editing-based gene replacement. The EPSPS gene codes for a 5-
enolpyruvylshikimate-3-phosphate synthase, which is required for the
biosynthesis of aromatic amino acids, which are necessary for plant survival.
Glyphosate, a widely used herbicide that binds to EPSPS functional sites to
prevent its activity, is a target for EPSPS in plants. Flax (Linum
usitatissimum) genome editing was used to replace two nucleotides in the
EPSPS glyphosate binding site using CRISPR/Cas9 and single-stranded
oligo DNA repair templates. Glyphosate tolerance was higher in the EPSPS
edited flax than in the controls.
d) Healthy food
Genome editing can be used to modify plant components, resulting in
healthier foods.
i) Improved oil composition
Oils with high polyunsaturated fatty acid content, particularly linolenic
acid, have poor oxidative and frying stability, limiting their use. To change
the fatty acid composition and improve oil quality, Fatty Acid Desaturase
(FAD) genes have been targeted. The FAD2 gene family catalyses the
conversion of monounsaturated oleic acid to linoleic acid, whereas the FAD3
gene family encodes enzymes that catalyse the production of linolenic acid
from linoleic acid. TALENs used to knock out two soybean FAD2 genes,
FAD2-1A and FAD2-1B, resulted in vastly improved oil quality: oleic acid
increased from 20% to 80%, while linoleic acid decreased from 50% to 4%
[34]
. To improve oil composition even more, TALEN introduced mutations in
FAD3A into previously produced fad2-1a/fad2-1b soybean plants, resulting
in even higher levels of oleic acid and lower levels of linolenic acid.
ii) Healthy potatoes
Cold storage of potatoes reduces sprouting and ensures a steady supply,
but it also accumulates reducing sugars. During high-temperature processing,
reducing sugars react with free amino acids to produce brown, bitter-tasting
products and raise acrylamide levels, which is a suspected human carcinogen
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that has raised global safety concerns [35]. VINV is a vacuolar invertase that
catalyses the conversion of sucrose to glucose and fructose in cold-stored
potato tubers. TALENs were used to create a VINV mutation in a
commercial Ranger Russet potato variety, resulting in potatoes with
undetectable reducing sugar levels.
iii) Manipulating production of bioactive compounds
The CRISPR-Cas9 technology has also been used to manipulate
bioactive compound biosynthesis in plant cells. For example, by knocking
out the 4′OMT2 gene, it was able to effectively control the production of
bioactive alkaloids in the opium poppy, where it was used to regulate benzyl
isoquinoline alkaloids (BIAs) metabolism and biosynthesis [36]. GABA
production was effectively increased in Corynebacterium glutamicum using
CRISPRi-based gene regulation, while L-lysine and L-glutamate production
were also improved using a similar approach. Importantly, CRISPR/Cas
technology has been used to engineer industrial production hosts such as
yeast (S. cerevisiae) strains for brewing.
iv) Other examples
The rapid development of temperature-sensitive lines for use in hybrid
rice production was aided by CRISPR/Cas9 targeted mutation of the TMS5
gene in rice cultivars. The Waxy (Wx) gene in maize codes for a granule-
bound starch synthase (GBSS) that produces amylose in the kernel. Wx/wx
lines contain nearly 100% amylopectin and are referred to as waxy maize.
Wild type maize kernels contain 75 percent amylopectin and 25 percent
amylose, whereas wx/wx lines contain nearly 100 percent amylopectin and
are referred to as waxy maize. CRISPR-mediated Waxy gene knockout has
produced the economically valuable waxy maize. High-amylose rice, with
potential health advantages, was generated through CRISPR/Cas9-mediated
knockout of the starch branching enzymes genes, SBEI and SBEIIb [37].
Future prospects
The field of basic and applied biology has been transformed by recent
advances in genome editing techniques. However, certain issues and
challenges must be addressed for improved efficiency and output, such as
SSN/DNA delivery methods, off-target effects, and the balance between
HR/NHEJ pathways. Although off-target activity in plants is low in
comparison to animal systems, there is still a need to address off-target
mutations in major crops and other plant species in a systematic manner.
Furthermore, some aspects of the editing system remain unknown, such as
Cas9's catalytic activity, the identification of target sites, and the significance
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of PAM sites. A thorough understanding of these aspects will aid in the
resolution of issues and improve the editing system's efficiency.
Genome editing systems have primarily been used in plants to destroy
genes by inducing DSBs after NHEJ repair, which introduces Indels at the
target site. Because more desired traits rely on gain of function mutation,
precision editing by sequence replacement and fragment knock-in via
homologous recombination (HR) has more important implications for crop
improvement. Precision editing, on the other hand, is still difficult in plants
due to the low efficiency of HR. In the generation of point mutations in
crops, base editing has emerged as a sophisticated and precise tool.
However, improvisation and its application in major cereal crops and plants
still has a lot of room for improvement.
Conclusion
Over the past several decades, traditional breeding that depends on
access to plant populations with sufficient variability has made great
contributions to agriculture. However, this variability is mainly derived from
spontaneous mutations or from mutations that are induced by chemical
mutagens or physical irradiation. Such mutations are usually rare and occur
at random. Moreover, many types of variation might not arise in elite
varieties and consequently time-consuming, laborious breeding programs are
needed to introduce desirable alleles into elite crops. By contrast, genome
editing as an advanced molecular biology technique can produce precisely
targeted modifications in any crop. CRISPR/Cas9 has emerged as the most
promising approach due to its simplicity, ease of use, versatility and
tolerable off-target effects. The genome editing system has the potential to
create crop varieties with improved disease resistance, increased yield and
quality, and novel agronomic traits that will benefit farmers and consumers
alike. Targeted mutagenesis has been successfully implemented in a variety
of model and non-model crops. CRISPR–Cpf1 has recently been used as a
new and alternative method for plant genome editing, overcoming some of
the limitations of CRISPR/Cas9, such as the requirement for a PAM site, and
thus expanding the scope of genome editing in crops. Nevertheless, an
increasing number of case studies suggest that CRISPR/Cas9 is an effective
and widely used technology that can accelerate basic and applied research
towards crop improvement.
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gene knockout. Plant Biotechnol. J. 2015;14:169-176.
36. Alagoz Y, Gurkok T, Zhang B, Unver T. Manipulating the biosynthesis
of bioactive compound alkaloids for next-generation metabolic
engineering in opium poppy using CRISPR-Cas 9 genome editing
technology. Scientific reports. 2016;6:309-10.
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37. Sun Y, Zhang X, Wu C, He Y, Ma Y, Hou H, et al. Engineering
herbicide-resistant rice plants through CRISPR/Cas9-mediated
homologous recombination of acetolactate synthase. Molecular Plant.
2016;9:628-631.
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Chapter - 4
Protein Bio-Synthesis/Translation in Prokaryotes
& Eukaryotes
Authors
Gita R. Chaudhari
Assistant Professor, College of Horticulture, Anand
Agricultural University, Anand, Gujarat, India
Sheetal Patel
Assistant Professor, Regional Rice Research Station, Navsari
Agricultural University, Vyara, Gujarat
Shridhar Ragi
Ph.D. Scholar, Division of Genetics, Indian Agricultural
Research Institute, IARI, New Delhi, India
Krishna Prakash
Scientist, ICAR-Indian Agricultural Research Institute (IARI),
Goria Karma, Hazaribagh, Jharkhand, India
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Chapter - 4
Protein Bio-Synthesis/Translation in Prokaryotes &
Eukaryotes
Gita R. Chaudhari, Sheetal Patel, Shridhar Ragi and Krishna Prakash
Abstract
This chapter summarizes our current understanding of translation in
prokaryotes and Eukaryotes, focusing on the mechanistic and structural
aspects of each phase of translation: initiation, elongation, termination, and
ribosome recycling. The assembly of the initiation complex provides
multiple checkpoints for messenger RNA (mRNA) and start-site selection.
Correct codon–anticodon interaction during the decoding phase of
elongation results in major conformational changes of the small ribosomal
subunit and shapes the reaction pathway of guanosine triphosphate (GTP)
hydrolysis. The ribosome orchestrates proton transfer during peptide bond
formation, but requires the help of elongation factor P (EF-P) when two or
more consecutive Pro residues are to be incorporated. The nascent protein
begins to fold cotranslationally, in the constrained space of the polypeptide
exit tunnel of the ribosome. When a stop codon is reached at the end of the
coding sequence, the ribosome, assisted by termination factors, hydrolyzes
the ester bond of the peptidyl-tRNA, thereby releasing the nascent protein.
Following termination, the ribosome is dissociated into subunits and
recycled into another round of initiation. At each step of translation, the
ribosome undergoes dynamic fluctuations between different conformation
states. The aim of this chapter is to show the link between ribosome
structure, dynamics and function.
Keywords: mRNA, tRNA, ribosomes, Amino acids, Polypeptide chain,
Elongation factors
Introduction
 Genes produce their phenotypic effects by specifying the amino
acid sequence of specific proteins.
 Protein synthesis is the process by which the genetic information
stored in the sequence of nucleotide in an mRNA is translated,
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according to the specification of the genetic code, into the sequence
of amino acid.
 As a first step in gene expression, the gene is transcribed to yield,
ultimately an mRNA.
 Then the base sequence of this mRNA is converted into the amino
acid sequence of the protein specified by this gene this process is
known as translation.
 Translation is a highly dynamic process that entails four major
phases: initiation, elongation, termination, and ribosome recycling.
 During each phase, ribosomes form transient complexes with
auxiliary translation factors that facilitate protein synthesis.
 In addition to the compositional dynamics of translating ribosome
complexes, conformational fluctuations of the ribosome and the
translation factors play an important role in promoting the
directionality of the process.
 A major challenge is to understand how the loosely coupled
motions of the translational components lead to rapid and accurate
protein production.
 Here, we summarize recent results of biochemical, biophysical, and
structural work that dissected the order of events at each step of
translation in prokaryotes and eukaryotes, identified dynamic
components, and captured structures of individual intermediates.
The process of translation requires the following major components
 mRNA
 tRNA
 rRNA
 Many translation factors
Messenger RNA (mRNA)
 Messenger RNA (mRNA) is a single-stranded base-for-base
complementary copy of the antisense stand of a prokaryotic
transcription unit.
 But in case of the eukaryotes first the pre-mRNA is produced &
then processed by deleting introns, adding poly-(A) tails and by
capping to yield mature mRNAs.
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 The mRNA provides the information for amino acid sequence of
the polypeptide specified by the concerned gene, or genes
represented in the transcription unit.
 In prokaryotes translation proceeds along with transcription.
 But in eukaryotes transcription and RNA processing are completed
in nucleus, and mRNA is transported to cytoplasm for translation.
 Eukaryotic cell generally contain 5% mRNA in total RNA content
 Generally, a single prokaryotic mRNA molecule codes for more
than one polypeptide. such an mRNA is known as polycistronic
mRNA.
 On the other hand, all eukaryotic mRNAs are monocistronic i.e.,
code for protein specified by a single cistron.
 All mRNA molecules have a translation initiation site close to their
5’ end and a chain termination site towards the 3’ end.
 The chain initiation site consists of the codon AUG and an
unknown secondary structure of mRNA. Hence, they are read in 5’-
3’ direction.
 The chain termination site has one or two of the following three
nonsense codons, UAA, UAG and UGA.
 In prokaryotes, ribosomes attach to the 5’ end of mRNA as soon as
transcription begins. A bunch of ribosomes moves along with a
single mRNA molecule adding second to the polypeptide chain,
almost at the same speed at which RNA polymerase transcribes the
mRNA. The group of ribosomes together with the single mRNA
molecule they are translating is called polysome.
Fig 1: Structure of Prokaryotic and Eukaryotic mRNA
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The process of translation
The zero step or prerequisite is amino acid activation
1) Translation Initiation.
2) Translation Elongation.
3) Translation Termination.
Amino acid activation
 Each tRNA molecule has a cloverleaf secondary structure
consisting of three stem loops, one of which bears the anticodon at
its end.
 The amino acid is covalently bound to the 3’ OH group at the 3’
end by aminoacyl synthetase to form aminoacyl-tRNA.
 The synthesis of aminoacyl-tRNA is also referred to as amino acid
activation.
 Amino acids that are not linked to tRNAs cannot be added to the
growing polypeptide.
 Amino acid activation, occurs in two steps and requires ATP to
form an intermediate, aminoacyl-adenylate.
1) The first step is the reaction of an amino acid and ATP to form an
aminoacyl-adenylate (also known as aminoacyl-AMP)
2) In the second step, without leaving the enzyme, the aminoacyl
group of aminoacyl-AMP is transferred to the 3’ end of the tRNA
molecule to form aminoacyl-tRNA
Step 1)
Step 2)
Overall reaction
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Translation initiation in prokaryotes
 In prokaryotes, the initiation site has usually the codon AUG. But
codons GUG and UUG can also act as initiation codons.
 The 5' leader of bacterial mRNAs has a conserved hexamer
sequence, called ShineDalgarno sequence, which is located at 7
bases upstream of the AUG (initiation) codon; the sequence of this
consensus is 5’ AGG AGG 3’ which is complementary to part of
the 16S rRNA in the small ribosomal subunit.
 Therefore, this is the binding site for the 30S ribosomal subunit
which then migrates in a 3’ direction along the mRNA until it
encounters the AUG initiation codon.
 The bacterial rRNA has near it 3'-end a highly conserved hexamer
sequence 3’ UCC UCC 5', this sequence is complementary to and
base-pairs with the Shine-Dalgarno sequence of mRNA, this base
pairing allows the smaller subunit of ribosomes to bind mRNA
during translation.
Fig 2: Secondary Structure of Aminoacyl tRNA
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Each ribosome has three binding sites for tRNAs i.e. A site, P site & E
site
i) A site: The aminoacyl-tRNA binding site (or A site) is where,
during elongation, the incoming aminoacyl-tRNA binds.
ii) P site: The peptidyl-tRNA binding site (or P site) is where the
tRNA linked to the growing polypeptide chain is bound.
Fig 3: Translation initiation in Prokaryote
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iii) E site: The exit site (or E site) is a binding site for tRNA following
its role in translation and prior to its release from the ribosome.
 All three sites (A, P and E) are formed by the rRNA molecules in
the ribosome.
 The first codon translated in all mRNAs is AUG & it is called as
start codon or initiation codon.
 Here, it should be noted that AUG as start codon, codes for N-
formylmethionine, but as in-between codon, codes for methionine
in prokaryotes.
 It is essential that the correct AUG is used as the initiation codon
since this sets the correct reading frame for translation.
 For that A short sequence rich in purines (5’-AGGAGGU-3’),
called the Shine-Dalgarno sequence, lies 5’ to the AUG initiation
codon and is complementary to part of the 16S rRNA in the small
ribosomal subunit is essential.
 Initiation of protein synthesis requires proteins called initiation
factors (IFs).
 In prokaryotes, three initiation factors (IF-1, IF-2 and IF-3) are
essential.
 Translation in prokaryotes begins by the formation of a 30S
initiation complex between the 30S ribosomal subunit, mRNA,
initiation factors and fMet-tRNA.
 The 30S subunit binds to the Shine-Dalgarno sequence which lies
5’ to the AUG Start codon and is complementary to the 16S rRNA
of the small ribosomal subunit.
 The ribosome then moves in a 3’ direction along the mRNA until it
encounters the AUG codon.
 The 50S ribosomal subunit now binds to the 30S initiation complex
to form the 70S initiation complex. In this complex, the anticodon
of the fMet-tRNA is base paired to the AUG initiation codon (start
codon) in the P site.
Elongation
 The elongation cycle consists of three steps: aminoacyl-tRNA
binding, peptide bond formation and translocation.
 The aminoacyl-tRNA corresponding to the second codon binds to
the A site on the ribosome as an aminoacyl-tRNA/EF-Tu/GTP
complex. (EF-elongation factor).
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 After binding, the GTP is hydrolyzed and EF-Tu/GDP is released.
 The EF-Tu is regenerated via the EF-Tu–EF-Ts exchange cycle.
B) Peptide bond formation is catalyzed by peptidyl transferase between
the C-terminus of the amino acyl moiety in the P site and the amino
group of the aminoacyl-tRNA in the A site.
C) In the final (translocation) step, EF-G/GTP binds to the ribosome,
the deacylated tRNA moves from the P site to the E site, the
dipeptidyl-tRNA in the A site moves to the P site, and the ribosome
moves along the mRNA to place the next codon in the A site. EF-G
also called as translocase.
 The GTP is hydrolyzed to GDP and inorganic phosphate.
 When the next aminoacyl-tRNA binds to the A site in the next
round of elongation, the deacylated tRNA is released from the E
site.
Fig 4: Translation Elongation in Prokaryotes

Termination
 Eventually, one of three termination codons i.e. UAG, UAA and
UGA (also called Stop codons) becomes positioned in the A site.
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 Unlike other codons, prokaryotic cells do not contain aminoacyl-
tRNAs complementary to Stop codons. Instead, one of two release
factors (RF-1 and RF-2) binds instead.
 RF-1 recognizes UAA and UAG whereas RF-2 recognizes UAA
and UGA. A third release factor, RF-3, is also needed to assist RF-1
or RF-2 interaction with the ribosome.
 The release factors trigger peptidyl transferase to transfer the
polypeptide to a water molecule instead of to aminoacyl-tRNA.
 The polypeptide, mRNA, and free tRNA leave the ribosome and the
ribosome dissociates into its subunits ready to begin a new round of
translation.
Fig 5: EF-Tu-Ts exchange cycle

Translation initiation in eukaryotes
 Eukaryotic ribosome has a sedimentation coefficient of 80S with
subunits of 40S and 60S. The composition of eukaryotic ribosomal
subunits is more complex than prokaryotic subunits but the function
of each subunit is essentially the same as in prokaryotes.
 Initiation of protein synthesis in eukaryotes requires at least nine
distinct eukaryotic initiation factors (eIFs).
 In eukaryotes, the initiating amino acid is methionine.
 The 40S ribosomal subunit attaches at the 5’ end of the mRNA and
moves downstream (i.e. in a 5’ to 3’ direction) until it finds the
AUG initiation codon. This process is called scanning.
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Fig 6: Translation Termination in Prokaryotes
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The full details of initiation in eukaryotes are still not fully known but
the process occurs broadly as follows
 The first step is the formation of a pre-initiation complex consisting
of the 40S small ribosomal subunit, Met-tRNA, eIF-2 and GTP.
 The pre-initiation complex now binds to the 5’ end of the
eukaryotic mRNA, a step that requires eIF-4F (also called cap
binding complex) and eIF-3. The eIF-4F complex consists of eIF-
4A, eIF-4E, and eIF-4G; eIF-4E binds to the 5’ cap on the mRNA
(an essential step) whilst eIF-4G interacts with the poly(A) binding
protein on the poly(A) tail. Thus this complex checks that both the
5’ and 3’ ends of the mRNA are intact.
 The eIF-4A is an ATP-dependent RNA helicase that unwinds any
secondary structures in the mRNA, preparing it for translation.
 The complex now moves along the mRNA in a 5’ to 3’ direction
until it locates the AUG initiation codon, this process is called as
Scanning Model for initiation.
 Any secondary structures found during scanning are removed.
 The initiation codon is usually recognizable because it is often (but
not always) contained in a short sequence called the Kozak
consensus (5’-ACCAUGG-3’).
 Once the complex is positioned over the initiation codon, the 60S
large ribosomal subunit binds to form an 80S initiation complex, a
step that requires the hydrolysis of GTP and leads to the release of
several initiation factors.
Elongation
 The elongation stage of translation in eukaryotes requires three
elongation factors, eEF-1A, eEF-IB and eEF-2, which have similar
functions to their prokaryotic counterparts EF-Tu, EF-Ts and EF-G.
 Although most codons encode the same amino acids in both
prokaryotes and eukaryotes, the mRNAs synthesized within the
organelles of some eukaryotes use a variant of the genetic code.
 During elongation in bacteria, the deacylated tRNA in the P site
moves to the E site prior to leaving the ribosome. In contrast,
although the situation is still not completely clear, in eukaryotes the
deacylated tRNA appears to be ejected directly from the ribosome.
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Termination
 In eukaryotes, eukaryotic release factor eRF-1 recognizes all three
termination codons (UAA, UAG and UGA) and with the help of
protein eRF-3, terminates translation.
Fig 7: Translation initiation and elongation in Eukaryotes
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Fig 8: Translation termination in Eukaryote
Table 1: Comparison of Protein Synthesis Factors in Prokaryotes and Eukaryotes
Prokaryotic Eukaryotic Function

Initiation factors
At least 9 initiation factors Individual factors have functions
Example: that differ between prokaryotes and
IF-1, IF-2, IF-3
eIF-1, eEF-2, eIF-2B, eEF- eukaryotes.
3, eIF-4
Elongation factors
Aminoacyl tRNA delivery to
EF-Tu eEF-1A
ribosome
EF-Ts eEF-1B Recycling of EF-Tu or eEF-1A
EF-G eEF-2 Translation
Termination factors
RF-1, RF-2, RF-3 eRF-1, eRF-3 Polypeptide chain release
Table 2: Comparison of Translation Process in Prokaryotes and Eukaryotes
Prokaryotes Eukaryotes
Polycistronic RNA. Monocistronic RNA.
Initiation codon AUG will code for N- Initiation codon AUG will code for
formyl methionine. methionine.
3 initiation factors are required. Atleast nine initiation factors are there.
Ribosomes are 70 S (50 S + 30 S). Ribosomes are 80 S (60 S + 40 S).
Transcription & Translation Occurs Transcription & Translation are separate
simultaneously in the cytoplasm. process, transcription occurs in nucleus
whereas translation occurs in the cytoplasm.
5’ end of mRNA is Directly available It is available after post translational
for transcription. modification.
mRNA can act as the template for the mRNA act as polypeptide for single
synthesis of many polypeptides. polypeptide.
mRNA have many start sites Shine- Always have one start site located towards
Dalgarno sequences. the 5’ region.
Shine-Dalgarno sequence present 8 Shine Dalgarno sequence is absent.
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nucleotide upstream of start codon & it
act as ribosome binding site.
Ribosome mass is appx. 2700 kd. Ribosome mass is appx. 4200 kd.
ER is absent and hence protein ER is present and hence protein synthesizing
synthesizing ribosomes are distributed ribosomes are usually attached to ER.
freely in cytoplasm.
References
1. Marina Vand Rodnina. Translation in Prokaryotes, Cold Spring Harb
Perspect Biology, 2018. Doi: 10.1101/cshperspect.a032664.
2. Goss DJ, Domashevskiy AV, Messenger RNA (mRNA): The Link
between DNA and Protein, In: Ralph A Bradshaw and Philip D Stahl
(Editors-in-Chief), Encyclopedia of Cell Biology, Waltham, MA:
Academic Press. 2016;1:341-345.
3. John W Pelley. Protein Synthesis and Degradation, Elsevier's Integrated
Review Biochemistry (Second Edition), 2012, 149-16.
4. Doherty M Guo. Transfer RNA, Encyclopedia of Cell Biology.
2016;1:309-340.
5. "Translation (Protein synthesis)" by Ross Hardison, LibreTexts, (The
Pennsylvania State University), 2021.
https://bio.libretexts.org/@go/page/384.
6. Carla Schmidt, Victoria Beilsten-Edmands, Carol V Robinson. Insights
into Eukaryotic Translation Initiation from Mass Spectrometry of
Macromolecular Protein Assemblies, Journal of molecular biology
2016;428(2):344-356.
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Chapter - 5
Role of Biological Ice Nucleation in Cloud
Modification
Authors
Guna M
Agro Climate Research Centre, Tamil Nadu Agricultural
University, Coimbatore, Tamil Nadu, India
Panneerselvam S
Water Technology Centre, Tamil Nadu Agricultural
Subramanian K.S
Nano Science and Technology, Tamil Nadu Agricultural
Harinarayanan M.N
Govindaraj T
Dharani C.
Page | 65
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Chapter - 5
Role of Biological Ice Nucleation in Cloud Modification
Guna M, Panneerselvam S, Subramanian K.S, Harinarayanan M.N, Govindaraj T and
Dharani C.
Abstract
Bioprecipitation or “Biological Precipitation” cycle is when the
bacterium starts forming colonies on the phyllosphere of plants and wind
speed sweep up the Ice nucleation bacteria into the atmosphere. INA bacteria
forms Ice crystals around them, clouds particles clump with INA bacteria
and making drop larger. Ice crystals formed as a snow or rain and reaches
onto the earth surface. When bioprecipitation occurs, the bacteria got
opportunity to reach the earth surface. Even one bacteria fall on surface of
the plant, it can multiply and form bacterial colonies. A bacterial colony
causes the cycle to repeat the process of bio-precipitation. Mostly rain and
snow forms Ice crystals in the clouds before producing precipitation. Soot
and dust particles act as Ice nuclei. The bacterial nucleation different from
soot and dust particles, because these biological nuclei can forms Ice crystals
at higher temperatures (-4 to -2 °C). The idea of rain-making bacteria has
been around since the 1980’s, but it hasn't been revised due to a lack of study
data. More research on “Bacteria as a Rainmaker” might lead to a better
understanding of its significant role in the water cycle.
Keywords: Bioprecipitation, Ice nucleation, condensation nuclei, bacteria
1. Introduction
Rainfall is a major source of water to the Earth’s planet. It provides
water for all creatures in the world including humans, plants and animals.
The rain is formed as a result of condensation of water vapour in the
atmosphere. In India, rains are received as precipitation as a result of water
currents passing the sub-continent in a unique weather pattern as South-West
and North-East Monsoons. Apart from monsoon rainfall, there are
atmospheric disturbances and convectional processes leading to the
formation of rain. In all the cases, there is a need for a nucleus in the form of
dirt or microorganism facilitates cloud condensing nuclei. The evaporated
liquid condenses in the cold air, forming moisture-filled rain clouds. In the
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climate change scenarios and extreme weather events such as Elnino and La
Nino that are known to impede the rainfall pattern and cause irregular and
erratic precipitation. Since, the rainfall pattern in highly erratic and
occurrence of frequent droughts is quite common, there is an urgent need to
develop a strategy to develop artificial rain to protect agriculture from
devastation. Artificial rain was first reported in the globe in the year 1940
(Schaefer, 1946) and it was demonstrated in India during 1983 (CASWMT,
1983). Silver iodide, sodium chloride and dry Ice were well known
substances used for artificial rain (Rao., 2015). The condensation of water
vapour led to the formation of Ice crystals at extremely low sub-zero
temperatures of below 40 oC under natural conditions. Such condensation
reaction will take place at -20 oC when artificial nucleating agents such as
Ag ions are used to develop the rain water droplets (anonymous. 1956).
Despite the fact that chemicals have been successfully demonstrated for the
production of artificial rains, the excessive quantities may cause associated
ill-effects such as toxicities and salinity (Shaista Malik et al., 2018).
Continuous use may lead to accumulation in the natural ecosystems affecting
the flora and fauna.
Among the microbial communities, bacteria are well known to be
involved in Ice nucleation. Some bacteria's outer cell membrane contains a
protein that can function at temperatures as warm as −1 oC as an effective Ice
nucleator. A few bacterial species generate INA (Ice Nucleation Active
proteins). The INA bacteria were widely found in plants (as saprophytic
epiphytes and/or as plant pathogens) that include Pseudomonas syringe, P.
viridiflava, P. fluorescens, Panton agglomerans (formerly known as Erwinia
herbicola), bacillus subtilis and Xanthomonas campestris (Wolber., 1993;
Gaignard and Luisetti., 1993).
This review mainly focuses on a bacterium. It includes from
identification to possible future of bacteria in causing rainfall.
1.1 Biological component of aerosols
In urban regions, the concentration of aerosols was around 3 x 109 to 5 x
1010 particles per cubic meter. An atmospheric aerosol contains more
minerals dusts. Soot particles are more in the polluted area. But, anyone
knows that pollens are the only biological components in atmospheric
aerosols. In fact, a bacterium covers around 25 per cent of atmospheric
aerosols (Matthias-Maser et al., 2000). Pollen is the only plant particle that
contributed in the atmospheric aerosols. Other abiotic biological aerosols are
surrounding our planet including bacteria; spores of fungi, mosses, ferns and
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protozoa; virus particle; algae and pollen grains; dust mites and part of
insects and biotic aerosols are dead debris that sweeps up into the
atmosphere. Below 15m MSL altitude, there are thousands of biological
particles per cubic meter of air. In suburban region the quantity of
microorganisms, in the air has been observed to be 1500 cultural bacteria per
cubic meter and 7000 cultural of fungi (Jones and Cookson, 1983).
The impact of biological particles in the atmosphere is only aspect of
aerobiology. For bacterium, Ice nucleation active proteins are originates
from the soil or plants. For example, above the frost and wheat fields have
been detected thousands of bacteria per cubic meter in the air (Lighthart,
1997). Agricultural practices such as bailing and combining are shoots the
value up to 105 to 109 bacteria per cubic meter (Lighthart, 1984). Plant
canopy emits flux of bacteria have been measured. For example alfalfa,
wheat and bean canopies are emits the bacteria upward rate 50 to 500
culturable cell/m2/sec (Lindemann et al., 1982). The statistical simulation
using global climate model results convey that average bacteria emission rate
over land was about 200 m2/s (Burrows et al., 2009). The average bacterial
emission rate was reliable with observed rate of over continental zones
(Huffman et al., 2013); (Šantl-Temkiv et al., 2013).
1.2 Microorganism on plants
The surface of plant parts has larger number of microorganisms (shoot,
leaves, flowers and fruits), mostly bacteria also have fungi, protozoa and
yeasts. In this study we focusing on bacteria, some bacteria involve in
decomposition activities but also plays role in primary producers in food
web. Pseudomonas is typically rod-shaped bacterium with the size of 1 to 3
µm length and 0.2 to 0.5 µm wide. They are capable to move one place to
another place with the present of liquid by helping of flagella (Morris et al.,
2004). A healthy mature leaves of plant may carry 108 bacteria per cm2,
small amount of fungi and other organisms. If we think about number of
plant on earth, then the plant kingdom has significant habitat for bacteria at
the global scale. Researchers found that the 1030 bacterial inhabitants on
earth (Whitman et al., 1998). In that about 1024-26 are found in only on leaf
surfaces (Morris, 2001).
1.3 Ice nucleation activity
Several pathogenic and non-pathogenic bacteria can produce Ice
formation (Lindow, 1986). Ice formation can cause damage to sensitive
plants (Lindow et al., 1982; Lindow, 1983b) and can inhibit the entry of
phytopathogenic microbes in to plant species. The Ice nucleation is a
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conditional virulence factor in temperature (Lindow, 1983b). When
maintaining the ability to generate Ice nuclei should have metabolic and
biological costs, it was not found that the production of Ice nuclei can grant
any positive benefit on the proteins.
P. syringae and P. fluorescens Ice mutants are equally balanced on plant
surfaces to the parental Ice+ strains (Vasebi et al., 2019). Likewise, P.
syringae Ice mutants grew and escaped as parental strain on all species of
plants examined in the green house under wet and dry conditions (Lindow,
1985a). Finally, in their ability to live frequent freezing and thawing cycles
in an aqueous environment and on leaves, Ice mutants are comparable to the
parental strains (Lindow, 1985b). The bacterial strain of Lysinibacillus was
recently characterized as first Ice nucleating gram positive bacterium (Failor
et al., 2017). Some of the fungal species have Ice nucleation active protein
viz., F. sporotrichioides (Huffman et al., 2013), Isaria farinosa, and
Acremonium implicatum, (Huffman et al., 2013), Mortierella alpine
(Fröhlich-Nowoisky et al., 2015). The pollen also added as biological Ice
nucleation active particle (Diehl et al., 2001). Therefore, microorganism’s
Ice nucleation activity is continuous process that coupled with global
circulation.
1.4 Application of biological Ice nucleators
Pure liquid water is metastable only at low temperatures and is
sometimes referred to as supercooled water. Nucleation is called the
initiation of conversion from supercooled water to Ice crystals. The term
temperature of nucleation actually describes the temperature at which
nucleation occurs (Knight, 1968). Homogenous crystallization is described
with an embryo crystal formed by the water molecules. As temperature
drops, water molecules cluster to form variable-size Ice-like water clusters.
A rapid growth of Ice crystals occurs whenever an aggregate reach a critical
size. The likelihood that a water aggregate will reach the crucial size is
dependent upon temperature. Due to temperature dependence, homogeneous
nucleation generally occurs at -40 °C. A factor of 50 tends to increase the
crystallization rate of each 1 DC reduction in temperature. A liquid
aggregate's critical size is 70 molecules at -40 °C and -20 °C 650 molecules
(Vali, 1995). Heterogeneous crystallization is defined by clustering water
molecules on a substance other than water's surface. It is the most
appropriate form of nucleation in living organisms in which water contacts a
variety of substances in direct contact. An Ice nucleator or Ice nucleation site
is called a place which enables Ice-like clustering of water molecules. A
nucleator site could comprise of one molecule or may occur from the
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aggregation of many molecules to form the nucleation site. Since other
compounds could promote the nucleation of Ice at lower temperatures, it is
convenient to limit the use of word Ice nucleators to such compounds which
promote nucleation at fairly high temperatures, such as above -l2 °C.
Inorganic Ice nucleators such as silver iodide have been known for a
long time (Vonnegut, 1947). Most of the previous work to define Ice
nucleators was done by meteorologists concentrating on molecules which
induce Ice nucleation in clouds. Many popular Ice nucleators, almost all of
them mineral particles, were identified by meteorologists. In 1970, Russell
C. Schnell found that microbial origin was the influential Ice nucleators
originating from decomposing leaves (Upper and Vali, 1995) and in 1972
Pseudomonas syringae was recognized as the high nucleation temperature
organism (Maki et al., 1974). The existence of Ice nucleators was reported in
various clades organisms including Fungi (Lundheim, 1997). Ice nucleators
were categorized by temperature of nucleation and chemical properties
(Yankofsky et al., 1981). Studies shows that Ice nucleators from P. syringe,
E. herbicola, P. fluorescens, P. viridiflava and transgenic Escherichia coli
have separated Ice nucleators into three main classes, based on the
nucleation temperatures. Namely the Ice nucleator classes A (nucleating > -
4.4°C), B (nucleating at -4.8°C to -5.7°C), and C (nucleating < -7.6°C)
(Turner et al., 1990).
2. Conclusion
The recent studies shows that rain making bacterium are most effective
at forming Ice nuclei then the dust and soot particles because of the their
surface area as well larger in size. Dust and soot particles are orienting less
water molecules then the bacterial Ice nucleation proteins. Bacterial proteins
are bigger in size by that it can orient more water molecules in clouds. Past
60 years silver iodide has been used as artificial cloud seeding particle which
chemically synthesized. A decreasing amount of bacteria on crop ecosystem
could affect the climate variability. Research shows that even in Antarctica
rain making bacteria was found, it’s possible to turn on Ice nucleation
proteins into the atmosphere and again returned to the earth surface.
Microbes being natural with no-side effects, scientists are looking for a
biological strategy to produce artificial rains using aerobic microbes. The
biologically enabled cloud condensing nuclei is one of the emerging areas of
interest for meteorologists to explore as the receipt of rainfall is high erratic
and unpredictable. Since more than 60% of our agriculture is rainfall
dependent, biologically enabled rain may be of practical significance.
Page | 71
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Robinson NH, et al. High concentrations of biological aerosol particles
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13. Lighthart Bruce. The ecology of bacteria in the alfresco atmosphere.
FEMS Microbiology Ecology. 1997;23(4):263-274.
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Christen D Upper. Plants as sources of airborne bacteria, including Ice
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of Ice-deletion mutants constructed in vitro for plant frost control.
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Society for Microbiology, Washington, DC, 1985a, 23-35.
17. Lindow SE. Integrated control and role of antibiosis in biological
control of fireblight and frost injury, 1985b.
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19. Lindow Steven E, Deane C Arny, Christen D Upper. Bacterial Ice
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Caldwell. Ice nucleation induced by Pseudomonas syringae. Appl.
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distribution of primary biological aerosol particles in cloud water on the
mountain Kleiner Feldberg/Taunus (FRG). Atmospheric Research.
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23. Morris CE. Encyclopedia for Life Sciences. Nature Publishing Group,
London, 2001. http://www.els.net [doi: 10.1038/npg.els.0000400].
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bacteria and their potential role in precipitation. Journal de Physique IV
(Proceedings), 2004.
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25. Rao, GSLHV Prasada. Agricultural meteorology: PHI Learning Pvt.
Ltd., 2015.
26. Šantl-Temkiv, Tina, Kai Finster, Thorsten Dittmar, Bjarne Munk
Hansen, Runar Thyrhaug, et al. Hailstones: a window into the microbial
and chemical inventory of a storm cloud. PloS one. 2013;8(1):e53-550.
27. Schaefer Vincent J. The production of Ice crystals in a cloud of
supercooled water droplets. Science. 1946;104(2707):457-459.
28. Shaista Malik, Haleema Bano, Rauoof Ahmad Rather, Shakeel Ahmad.
Cloud Seeding; Its Prospects and Concerns in the Modern World -A
Review. Int. J Pure App. Biosci. 2018;6(5):791-796.
29. Upper CD, Vali G. The discovery of bacterial Ice nucleation and its role
in the injury of plants by frost. Biological Ice nucleation and its
applications, 1995, 29-39.
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LV (ed.), Biological Ice nucleation and its applications. American
Phytopathological Society, St. Paul, 1995, 1-28.
31. Vasebi Yalda, Marco E, Mechan Llontop, Regina Hanlon, David G
Schmale, Russell Schnell, et al. Comprehensive characterization of an
aspen (Populus tremuloides) leaf litter sample that maintained Ice
nucleation activity for 48 years. Biogeosciences. 2019;16(8):1675-1683.
32. Vonnegut, Bernard. The nucleation of Ice formation by silver iodide.
Journal of applied physics. 1947;18(7):593-595.
33. Wolber, Paul K. Bacterial Ice nucleation. In Advances in microbial
physiology, Elsevier, 1993, 203-237.
34. Yankofsky SA, Levin Z, Bertold T, Sandlerman N. Some basic
characteristics of bacterial freezing nuclei. Journal of applied
meteorology. 1981;20(9):1013-1019.
35. Turner MA, Arellano F, Kozloff LM. Three separate classes of bacterial
Ice nucleation structures. Journal of Bacteriology. 1990;172(5):2521-
2526.
Page | 74
Chapter - 6
Different Types of PCR and It’s Applications in
Author
Sonal Chavan
University, Rajendranagar, Hyderabad, Telangana, India
Page | 75
Page | 76
Chapter - 6
Different Types of PCR and It’s Applications in
Sonal Chavan
Abstract
Polymerase Chain Reaction (PCR), an in vitro technique that amplifies a
DNA sequence to billions fold amplitude is a widely useful technique in
biotechnology that works on similar process of DNA replication as in all
cellular organisms. The standard PCR is the qualitative PCR and in order to
overcome the limitations of this conventional PCR, different types of PCR
have been developed such as the Real-time PCR or quantitative PCR (qPCR),
Asymmetric PCR, Multiplex PCR, Nested PCR, Hot start PCR, Touchdown
PCR, Colony PCR, Assembly PCR, Degenerate PCR, Methylation-specific
PCR, Reverse Transcription PCR, Inverse PCR, Miniprimer PCR, Allele-
specific PCR, Thermal asymmetric interlaced PCR (TAIL-PCR), Linear-
After-The-Exponential (LATE) PCR, Nanoparticle assisted PCR (NanoPCR),
Suicide PCR and Single specific primer-PCR (SSP-PCR) that can be applied
for various studies in agricultural biotechnology.
Keywords: Polymerase chain reaction, PCR, types of PCR, DNA
amplification
Introduction
The invention of polymerase chain reaction (PCR) by Kary Banks Mullis
in the year 1983 for which he was awarded Nobel prize in 1993, has been a
milestone in the history of biological and medical sciences as it underlies
almost all of modern molecular cloning. PCR is an in vitro technique that
mimics the natural process of DNA replication occurring in all cellular
organisms enabling replication and amplification of a DNA sequence to
billions fold amplitude. In contrast to entire DNA sequence in case natural
DNA replication, here in case of PCR, a defined target sequence of a DNA
molecule is rapidly and selectively amplified in a quasi-exponential chain
reaction. It has been used in a wide variety of tasks such as cloning of cDNA
and genomic DNA, DNA sequencing, in vitro mutagenesis, mutation
Page | 77
detection and allelotyping, owing to its simple set up, speed, sensitive, robust
and flexible nature.
This process of selective duplication uses temperature cycling to initiate
and end bursts of enzyme-catalyzed DNA synthesis to generate two copies of
the original target double-helix molecule in each cycle of PCR is divided into
3 phases: Denaturation, Annealing and Extension.
Essential components of PCRs
The following components are required to set up PCRs:
1) Template DNA: It consists of target DNA sequence to be amplified,
which is a part of large and complex DNA of a genome. It can be
either a DNA or RNA.
2) Primers: A pair of primers namely forward and reverse primers
which are synthetic oligonucleotides usually 16-30 nucleotides in
length, limit the DNA sequence to be replicated and also effect the
specificity and efficiency of the amplification reaction. Thus, making
the designing of primers a crucial step.
3) Deoxy nucleotide triphosphates (d NTP’s): These are dATPs,
dTTPs, dCTPs and dGTPs present in equal amounts in a Standard
PCR, which are added by the polymerase enzyme to the primer in
extension step of the reaction.
4) DNA polymerase: To catalyze template-dependent synthesis of
DNA a thermostable DNA polymerase is required and for routine
PCRs, Taq polymerase obtained from Thermus aquaticus is widely
preferred.
5) Divalent and monovalent cations: Mg+2 is the divalent cation
required by all thermostable DNA polymerases in order to react with
dNTPs to form complexes that are the substrates for Taq polymerase
and to stabilize the primer-template complexes. Monovalent cations
are provided by KCl in standard PCR.
6) Buffer: Tris-Cl is the commonly used buffer to adjust pH between
8.3 and 8.8 at room temperature.
7) Water
8) Thermal cyclers (thermocyclers): The instruments used for
amplification that raises and lowers the temperature of the samples
in pre-programmed steps.
Page | 78
Three phases of the PCR
1) Denaturation: It is the first step in which the two complimentary
strands of template DNA are separated by heating at a temperature of
94 ˚C for 2 minutes in the first PCR cycle, followed by 1 minute in
subsequent cycles.
2) Annealing: In this step the mixture is cooled to a temperature of in
between 40-60 oC that allows the primers to anneal to the
complementary 3’ ends of the target DNA segment to be amplified.
The duration of this step 1 minute.
3) Extension: It is the final step where the primers bound to 3’ ends are
extended by addition of nucleotides (dNTPs) catalyzed by a
thermostable DNA polymerase for synthesis of a new strand. This
step of extension is carried out at a temperature of 72 oC for 2
minutes.
One cycle will yield two daughter DNA strands, which act as templates
for next cycle. This process is repeated for about 35-40 times in a thermal
cycler, a programmable device that controls temperature and time of each step
in the cycle. Thus, with doubling of target sequence in each cycle, PCR
generates millions of copies of it suitable for further use.
Types of PCR
1. Conventional (Qualitative) PCR
Conventional PCR is a normal PCR process used for detecting a specific
DNA segment with the help of a set of primers used as forward and reverse
primer that limit sequence to be replicated and makes billions of copies of that
particular DNA segment. It is also referred to as standard or qualitative PCR.
Page | 79
It requires a post-PCR step for detection or visualization of the DNA amplified
for assessing quality of the DNA amplified.
2. Real-time PCR or quantitative PCR (qPCR)
Real-time PCR also referred to as quantitative PCR (qPCR) is type on
polymerase chain reaction (PCR) that was introduced with the concept of
monitoring DNA amplification in real time. This method uses fluorescent
dyes, such as Sybr Green, or fluorescence-containing DNA probes, such as
TaqMan, to measure the amplification of DNA at each cycle of PCR in real-
time by the use of Special thermal cyclers that monitor the amount of product
during amplification by measuring the fluorescence after each cycle. Increase
in emitted fluorescence reflects the increase in DNA in each cycle.
qPCR has advantage of eliminating the need for post amplification
processing of the sample as it combines amplification and detection into a
single step, in addition with advantages such as real time detection of reaction
progress, sensitivity, speed of analysis and precise measurement of sample.
qPCR is widely used in gene expressions studies due to its ability to provide
semi-quantitative results without standards, but using reference material. It
expresses results as higher or lower multiples with reference to control.
Page | 80
3. Asymmetric PCR
Asymmetric PCR is designed to preferentially amplify only one strand of
the target DNA molecule. In this variant of PCR, primers are added in unequal
molar ratio to favor the synthesis of the desired DNA strand as replication
occurs arithmetically by using the excess primer. This kind of PCR has
applications in molecular techniques such as DNA sequencing and
hybridization probing where amplification of one of the complementary
strands is required.
There are many limitations for this technique such as low reaction
efficiency, frequent generation of non-specific product, complicate primer
designing as melting-temperature reduces below optimal reaction annealing-
temperature due to limited concentration of one primer.
Source: Tolnai et al. 2019.

4. Multiplex PCR
Multiplex PCR is a variant of PCR where multiple primer sets are used
for multiple targets at once in a single test run. This kind of PCR is mostly
used to detect multiple pathogens such as viral/bacterial and other infectious
agents. Here each primer set will target a particular pathogen and will produce
amplicons of varying sizes which are specific for different sequences of DNA.
It is also used to detect deletions or duplications in a large gene, mutations and
polymorphisms and to identify exonic/intronic sequences in specific genes.
It has the advantage of reducing need of reagents for several times and
less time to perform as it targets multiple genes in a single run. The base pair
Page | 81
length of the amplicons should be different to form distinct bands when
visualized by gel electrophoresis. But, there is a risk of primer-dimer
amplification due to presence of one or more primer pairs in this type of PCR.
Source: Chang-Hui Shen, 2019.

5. Nested PCR
Nested PCR is a modification of PCR used to increase the specificity of
DNA amplification by minimizing amplification of non-specific and spurious
PCR products, which may be due to binding of primer to unwanted sites
similar to the target DNA. The specificity is increased by using two sets of
primers in two successive runs of the PCR reaction for a single locus point
nested (located) within the first set.
In first PCR, one set of primers are used to amplify the target DNA
products. The second set of primers binds to a secondary target within the
sequence amplified by the first set of primers and thus reduces the
contaminations in products as it is unlikely that the unwanted sequence will
have binding site for both the sets of primers. This type of PCR has application
in detection of pathogens occurring in less amounts. It has drawbacks such as
primer dimerization.
Page | 82
Source: Jeanne Carr et al. 2010.
6. Hot start PCR
Hot start PCR is a modified form of conventional PCR designed to
overcome the problem of spurious or undesired product and primer dimer
formation due to non-specific DNA amplification at room temperature or on
ice in a nonhot-start PCR. Here in conventional PCR during the denaturation
stage when temperature is raised to 94 °C, the temperature of the sample
passes through 72 °C on the way and as this 72 °C is the optimal temperature
for primer elongation (Taq polymerase) it leads to generation of nonspecific
PCR product due to base pairing at imperfect sites.
In case of Hot start PCR, Taq polymerase is added after the rest of the
PCR components are heated to the DNA melting temperature (94 °C), so as to
keep the enzyme inactive until the temperature of the reaction is higher than
the primers melting temperature thereby avoiding non-specific amplification
at lower temperatures.
Page | 83
7. Touchdown PCR
Touchdown PCR is designed to decrease off-target priming and thus to
increase the specificity of PCRs by increasing the annealing temperature by
3-5 °C above the standard Tm of the primers used during early cycles which
favors perfect primer-template hybrids formation. In subsequent cycles, the
annealing temperature is gradually decreased allowing for more efficient
amplification by end of the PCR. touchdown PCR should always be performed
in conjunction with a hot start protocol to minimize mispriming during early
stages of the PCR. This PCR is useful when the target DNA is of a different
species from that used to design the primers.
Page | 84
8. Assembly PCR
Assembly PCR, also known as Polymerase Cycling Assembly (PCA) is a
flexible technique for producing novel gene sequences from multiple
overlapping oligonucleotides that may substitute for Ligation-based assembly
as the overlapping sequences can be joined without the need for restriction
sites. By performing PCR, long DNA structures are synthesised by assembling
two or more long oligonucleotides with short overlapping segments. It is a
two-step approach where primers having an overlap are involved in the initial
PCR and the second PCR uses products as the template that generates the final
full-length product. This type of PCR is beneficial for assembling constructs
with modular elements, such as antibodies.
9. Colony PCR
Colony PCR is a method to quickly screen colonies of yeast or bacteria
for determining the presence or absence of insert DNA in plasmid constructs
Page | 85
that have grown up on selective media following a transformation step. It is
used in addition to the traditional blue white screening method so as to avoid
sequencing of the false positive clones. It helps in determining the insert size
by designing primers that target vector DNA flanking the insert to determine
whether or not the insert is the correct molecular size. Both the specificity and
size of the insert DNA can be determined by use of Insert specific primers,
while simultaneous screening of multiple constructs is possible by the use of
vector specific primers. Apart from these uses, it helps to determine insert
orientation by using an insert specific primer paired with a vector specific
primer that produces an amplicon of a specific size only if the insert is in the
correct orientation and it also helps in generation of sufficient amount of
desired PCR product for sequencing.
Source: Megan Bergkessel and Christine Guthrie, 2013.

10. Degenerate PCR
Degenerate PCR is a PCR method which employs degenerate primers (a
mix of primers with similar sequences and not specific) to amplify unknown
DNA sequences, related to a known DNA sequence or to amplify a mixture of
related sequences in one PCR reaction. Degenerate primers are designed on
the basis of known and sequenced gene homologs. It allows identification of
orthologous genes from different organisms and new members of a gene
family.
Page | 86
Source: Bartl S, 1997.
11. Methylation-specific PCR
Methylation-specific PCR (MSP) is a method to identify patterns of DNA
methylation at cytosine guanine islands (C&G islands) in genomic DNA
which are concerned in regulation of gene expression. In this PCR, two
amplifications are carried out on the bisulfite-treated DNA which converts
unmethylated cytosine bases to uracil making it complementary to adenosine
in PCR primers. Two sets of primers are used among which one set of primer
detects methylated DNA by annealing with cytosine and the other set detects
unmethylated DNA by annealing to DNA with uracil.
Page | 87
Source: Bryzgunova and Laktionov, 2017.
12. Reverse transcription PCR
Reverse Transcription-PCR (RT- PCR) is a type of PCR which allows to
reverse transcribe and amplify RNA to cDNA by the help of reverse
transcriptase enzyme, followed by further amplification of cDNA using
standard PCR enabling quantitative detection of levels of RNA expression.
Source: Shen, 2019.
Page | 88
13. Inverse PCR
Inverse PCR also known as inverted or inside-out PCR is a type of PCR
that allows amplification of DNA when sequence information is known for
only one side of the target region, in contrast to standard PCR where two
inward-pointing primers having sequence information for both the 3’ ends of
the target DNA are used.
In this type of PCR, it involves digestion with a restriction enzyme of a
preparation of DNA containing the known sequence and its flanking region
that does not cut within the stretch of known DNA. The restriction fragments
with compatible sticky ends produced by digestion are ligated and converted
into circles which are then used as a template in PCR. PCR amplifies the
unknown DNA by designed primers those recognize the end regions of the
known sequence and bind to a different strand of the circular DNA pointing
“outward” into the unknown DNA. The amplified product has short stretches
of known DNA at the ends with restriction enzyme site in the middle.
Source: Clark and Pazdernik, 2015.
Page | 89
14. Miniprimer PCR
Miniprimer PCR is a modification of standard PCR, here to improve the
efficiency of the PCR an engineered Taq Polymerase and 10-nucleotide
"miniprimers" are used improving the scope of detectable sequences beyond
those detected by standard methods using Taq polymerase and longer primers
(20-30 nucleotides). This PCR is used to identify the conserved DNA
sequences such as 16S rRNA in microbial biology ecology to reveal the
diversity of microbial populations in environmental samples.
15. Allele-specific PCR
Allele-specific PCR (AS-PCR) is a technique based on allele-specific
primers that allows to analyze single nucleotide polymorphism (SNP)
effectively in a single reaction with standard PCR conditions using a common
reverse primer and two forward allele-specific primers with different tails that
amplify two allele-specific PCR products of different lengths, which are
further separated by ethidium bromide-stained agarose gel electrophoresis or
polyacrylamide gel. It effectively analyses SNPs including the
insertion/deletion, transition and transversion.
Source: Pignatelli et al. 2019.
Page | 90
16. Thermal asymmetric interlaced PCR (TAIL-PCR)
Thermal asymmetric interlaced PCR (TAIL-PCR) is a method to amplify
unknown sequences adjacent to known insertion sites. It uses nested known
sequence-specific primers (Tm = >65 °C) in consecutive reactions along with
a short arbitrary degenerate (AD) primer of 15-16 nucleotides having Tm of
about 45 °C and 64-256 folds of degeneracy, so that the relative amplification
efficiencies of target and nontarget products can be thermally controlled.
Specific products bordered by an insertion-specific primer on one side and an
AD primer on the other are yielded by using alternate cycles of high and low
annealing temperature.
With its simplicity and high efficiency, this type of PCR and its
modifications are used in isolation of upstream (promoters) and downstream
sequences of the known coding sequences, and large-scale determination of
transposon insertion sites and T-DNA in Arabidopsis and rice.
Source: Radhamony et al. 2005.

17. Linear-After-The-Exponential (LATE) PCR
Linear-After-The-Exponential (LATE) PCR generates single-stranded
products with predictable kinetics for many cycles beyond the exponential
phase as efficient as symmetric PCR assays regardless of the primer ratio. It
overcomes the problems of asymmetric PCR and is useful for real-time
quantitative analysis of target numbers in small samples.
18. Nanoparticle assisted PCR (NanoPCR)
To overcome the problems of poor sensitivity, low specificity, false
positive results, etc in case of conventional PCR, Nanoparticle assisted PCR
(NanoPCR) was developed to improve the productivity and quality of PCR by
the use of metal nanoparticles, carbon nanomaterials, quantum dots and nano
Page | 91
metal oxide. the major factor in improving the PCR specificity, selectivity and
efficiency are excellent surface effect and heat transfer property of the
nanoparticles.
19. Suicide PCR
Suicide PCR is used in studies where avoiding false positives and
ensuring the specificity of the amplified fragment is the highest priority. The
primers used in this PCR that target a genomic region are designed such as it
had never been amplified before using this particular primer or any other set
of primers and not been used in any positive control PCR reaction. It is widely
used in paleogenetic studies.
20. Single specific primer-PCR (SSP-PCR)
Single specific primer-PCR (SSP-PCR) is used for selective amplification
of genes for which only a partial sequence information is available, allowing
unidirectional genome walking from known into unknown regions of the
chromosome.
Application of PCR technology in Agricultural biotechnology:
• For gene discovery, vector construction, gene cloning, transformant
identification, screening and characterization, and seed quality
control.
• PCR testing to verify the presence or absence of GM content in food
and feed for the purpose of mandatory labelling by most countries.
• PCR testing for import to GM non-approved countries from GM
approved countries.
• To test for quantifying the improved quality of the commodity.
References
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to Isolate Genes with Minimal Sequence Similarity. PCR Cloning
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methylation analysis. Molecular Biology. 2017;51(2):167-183.
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12. Jeanne Carr, Donna G Williams, Randall T Hayden. Molecular Detection
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Page | 94
Chapter - 7
Whole Genome Strategies for Marker Assisted
Selection in Plant Breeding
Authors
Kankati Alekya
M.Sc. Genetics and Plant Breeding, Agricultural College,
Jagtial, PJTSAU, Hyderabad, Telangana, India
Vemula Anjula Krishna
M.Sc. Genetics and Plant Breeding, RLBCAU, Jhansi,
Uttar Pradesh, India
Kommineni Jagadeesh
Ph.D. Scholar, Genetics and Plant Breeding, PJTSAU,
Hyderabad, Telangana, India
Pandiri Pavani
M.Sc. Genetics and Plant Breeding, PJTSAU, Hyderabad,
Telangana, India
Mondithoka Mounika
M.Sc. Genetics and Plant Breeding, College of Agricultural,
Rajendranagar, Hyderabad, Telangana, India
Vemula Srujana
Ph.D. Scholar, Genetics and Plant Breeding, PJTSAU,
Hyderabad, Telangana, India
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Chapter - 7
Whole Genome Strategies for Marker Assisted Selection in
Plant Breeding
Kankati Alekya, Vemula Anjula Krishna, Kommineni Jagadeesh, Pandiri Pavani,
Mondithoka Mounika and Vemula Srujana
Abstract
The whole-genome strategies can be defined as a full package of
functional tools and methodologies required for molecular plant breeding at
the level of the whole genome. The development of strategies includes
complete genomic sequences for all germplasm accessions, molecular
markers covering important genomic regions, genes and functional alleles,
high-precision phenotyping system for various target traits (measured under
multiple environments) and integration of information on relevant
environmental factors influencing genes, genotypes and the whole-plant
performance. Some basic strategies include marker trait association analysis,
Seed DNA based genotyping, Haplotypes and tag single nucleotide
polymorphisms using breeding platforms like high throughput genotyping,
phenotyping and E-typing and selection strategies like MAS and GS.
Keywords: Whole-genome strategies, marker trait association analysis, Seed
DNA based genotyping, Haplotypes and tag single nucleotide polymorphisms.
Introduction
Molecular breeding for complex traits in crop plants requires
understanding and manipulation of many factors influencing plant growth,
development and responses to an array of biotic and abiotic stresses.
The ultimate goal of whole-genome strategies is to help bring out the best
combinations of genotypes/genes, alleles or haplotypes, linkage
disequilibrium (LD) blocks, optimised gene networks, and specific genomic
regions into breeding products with desirable phenotypes at the level of the
whole genome (Xu et al. 2012).
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Marker trait association analysis
For marker trait association analysis, various methods have been
developed. Thousands of studies on mapping phenotypic QTL (phQTL) in
crop plants have been reported to date. The current trend in QTL analysis is to
quantify gene transcript levels, or expression QTLs, in order to better
understand the genetic regulation of gene expression (eQTL). A system-wide
analysis can reveal the impact of DNA sequence variation across multiple
levels; that is, eQTL at the gene expression level, pQTL for protein abundance
or activity traits, mQTL for metabolite abundances and/or phQTL for
morphological traits.
Biparental population-based strategies
Marker-trait association analysis has been undertaken largely using
biparental populations such as F2/ F2:3, BC1, doubled haploid (DH), and
recombinant inbred line (RIL), where only a few target traits can be mapped
with each population, fine mapping and gene discovery requires numerous
rounds of MAS and population development, which takes time. As a result,
association mapping was developed to map quantitative trait loci (QTLs).
Association mapping (AM)
The premise in association studies is that a marker locus is close enough
to a trait locus for some marker allele to go together with the trait allele
through several generations of recombination.
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Major goal
To find inter-individual genetic variants, mostly single nucleotide
polymorphisms (SNPs), that have the strongest association with the phenotype
of interest, either because they are causal or, more likely, because they are
statistically correlated or in linkage disequilibrium (LD) with an unobserved
causal variant (s).
Multiparent population-based strategies
Nested association mapping
Nested Association Mapping was created by combining the benefits of
both Linkage mapping and Association mapping. It has high power (Linkage
mapping) and high resolution (Association mapping) (Association mapping).
JLLDM stands for Joint linkage and linkage disequilibrium mapping.
Creation of the maize NAM population
Twenty-five different corn lines were chosen as the NAM population's
parental lines in order to capture maize's extraordinary variety while
preserving historic linkage disequilibrium. To establish the F1 population,
each paternal line was crossed to the B73 maize inbred. After that, the F1
plants were self-fertilized for six generations, yielding 200 homozygous
recombinant inbred lines (RILs) per family, for a total of 5000 RILs in the
NAM population. The lines are publicly available through the USDA-ARS
Maize Stock Center.
Each RIL was then genotyped with the same 1106 molecular markers (for
this to be possible, the researchers selected markers for which B73 had a rare
allele), in order to identify recombination blocks. After genotyping with the
1106 markers, each of the parental lines was either sequenced or high-density
genotyped, and the results of that sequencing/genotyping overlaid on the
recombination blocks identified for each RIL. The result was 5000 RILs that
were either fully sequenced or high density genotyped that, due to genotyping
with the common 1106 markers, could all be compared to each other and
analyzed together (Yu et al. 2008 and McMullen, M.D. et al. 2009).
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Implications
For the study of agronomic features in maize and other species, nested
association mapping has enormous potential. NAM has the ability to detect
QTLs for agriculturally relevant traits and link them to homologs and
candidate genes in non-maize species, as demonstrated in the initial flowering
time study. Furthermore, the NAM lines serve as a valuable public resource
for the maize community, allowing for the exchange of maize germplasm as
well as the findings of maize studies through common databases, aiding future
maize agricultural trait research. Given that maize is one of the most important
agricultural crops worldwide, such research has powerful implications for the
genetic improvement of crops, and subsequently, worldwide food security.
Wheat, barley, sorghum, and Arabidopsis thaliana are all undergoing similar
developments.
Multi-parent Advanced Generation Inter-Cross (MAGIC)
The multiparent advanced generation inter-cross (MAGIC) is another
important multi-parental population-based approach that is expected to
improve the precision with which QTL can be mapped, improving the outlook
for QTL cloning by incorporating multiple parents ensures population
segregation for multiple QTL for multiple traits, overcoming the drawbacks
of AM and BM populations.
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Multiple founder parents were selected for at least one significant trait in
an enhanced background and intercrossed, yielding a funnel of lines with
genomes containing contributions from each of the founders. To enhance the
number of recombinations in the population and boost QTL mapping capacity,
mixed lines from different funnels are randomly and sequentially intercrossed
as in the advanced intercross. MAGIC provides several advantages over
existing techniques, including the ability to more precisely identify genes
responsible for improved quality features and the ability to build new genetic
combinations by reshuffling the starting types.
Use of MAGIC lines in breeding programs
MAGIC populations can be utilised directly as source materials for
extracting and developing breeding lines and varieties with a wide genetic
foundation and multiple agronomically useful features. It has the ability to
solve a variety of production issues (particularly stress tolerance).
Genome coverage
Table 1: Comparison of platforms and tools between regional and whole genome
strategies in marker-assisted plant breeding
Regional genome analysis Whole genome analysis

DNA sampling Leaf Seed and leaf
Biparental populations and Combining use of all kinds of
Population
independent association populations including NAM,
management
panels MAGIC and natural populations
Genotyping by markers or Genotyping by sequencing and
Genotyping
chips high-density marker chips
Phenotyping individual target High-throughput precision
Phenotyping
traits phenotyping for all traits
Marker-trait Association with markers or GWAS using high density markers
association selected candidate genes or GBS
Based on significantly Based on all markers with estimated
Selection
associated markers effects
Evaluated based on Evaluated by both phenotypic and
Environmental
phenotypic data without use environmental data collected
effect
of environmental data through years and locations
Information Two-dimensional: G-P, Three-dimensional: G-P-E, through
management through Excel and databases Web-based tools or networking
Decision support Individual decisions Collective decision supported by
tools supported by separate tools integrated tools with global thinking
Population size
Many fundamental elements of marker-assisted breeding are influenced
by population size. The population size necessary to transfer important gene-
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controlled traits will be determined by the distance between markers and target
gene, recombination frequency, and degree of dominance (trait). For
complicated traits (minor), some additional parameters such as gene influence,
number, interactions, and their relative placements on chromosomes, cost-
effective analysis of enormous populations sizes is now possible, thanks to
drastically lower high-throughput genotyping costs. Two approaches have
also made it easier to analyse large populations in molecular breeding. (1) seed
DNA-based genotyping can successfully replace leaf DNA-based genotyping,
lowering breeding costs and enhancing scale and efficiency; (2) selective
genotyping and phenotyping, in combination with pooled DNA analysis, can
capture the most relevant contributing components.
Seed vs leaf DNA-based genotyping
Seed DNA-based genotyping aids in effectively replacing the leaf DNA-
based genotyping, because sampling and DNA extraction can be easily
automated with seed samples, as practiced by several multinational
corporations, managing large-sized populations has become much more
practical (Gao et al., 2008)
The costs of planting and sampling can be greatly lowered because
sampling and DNA extraction can be easily automated using seed samples and
selections can be made before planting. When utilising this method for
monocots, however, caution should be exercised because the genotype
established using seed endosperm may differ from that of the plant produced
from the embryo.
Number of markers
The number of markers needed to cover the entire genome is determined
by the genome size. To quantify genetic variation inside a gene and its
surrounding regions, the marker density must be high enough to encompass
all allelic variation, which would necessitate tens to hundreds of markers per
gene. In the case of association mapping, a whole-genome scan will require at
least thousands of markers for species with moderate LD decay (such as rice),
but millions of markers for species with rapid LD decay (such as maize).
Haplotypes and tag single nucleotide polymorphisms (SNPs)
The fact that SNPs are not independent of one another is ignored in single
SNP association analysis. However, critical values of sequence variation must
be determined in the context of LD between SNPs. Because DNA sequence
diversity in a population is the result of previous transmission, the historical
past can be extremely useful in achieving the primary goal of identifying
related genes. There are three reasons why haplotype-based analysis is
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preferable. First, the potential genes' protein products are polypeptide chains
whose folding and other properties may be influenced by certain amino acid
combinations. Second, population genetic principles reveal that haplotypes are
fundamentally organised in populations. Third, independent of population
genetic causes, haplotypes minimise the dimensionality of the problem of
evaluating association, potentially increasing the test's power.
Maize is the first plant species to be given a haplotype map (HapMap).
Several million sequence polymorphisms were revealed among 27 maize
inbred lines, and the genome was found to be characterised by extremely
varied haplotypes. Depending on how a haplotype is produced, it can be used
to replace individual marker-based mapping to improve mapping power and
find specific alleles within a gene or allele combinations at other loci that
contribute to the same target characteristic. Tag SNPs, each representing a
haplotype fragment, can be created. The entire genome is covered by all tag
SNPs combined. The usage of haplotypes containing all SNPs within 10-kb
intervals enhanced QTL mapping performance in maize (Clark, 2004)
Molecular networks
Plant epigenetics has recently attracted a lot of attention, not only as a
topic of basic research but also as a potential new source of advantageous
features for plant breeding. Epigenetic processes (DNA methylation, histone
variations and modifications, nucleosome placement, and small RNA) have
recently been discovered to be significant regulators of plant development and
reproduction. Epigenetic markers control a variety of elements of plant
development, including flowering time, gametogenesis, stress response, light
signalling and morphological change.
Because epigenetic regulation mechanisms are responsible for the
formation of heritable epigenetic gene variants (epialleles) as well as
transposon mobility, both aspects could be used to broaden plant phenotypic
and genetic variation, improving plant adaptation to environmental challenges
and thus increasing productivity. Epigenomic research is expanding our
knowledge of plant genomes while also offering crucial insights about the
mechanics and functions of pathways that may be examined further using
genetic and biochemical methods.
Sequencing quality and quantity
When creating a whole genome sequence, both quality and quantity
should be considered. Rice researchers seek to develop a high-quality
reference genome for indica rice, similar to what they have for japonica rice.
The first (B73) and second (Mo17) reference maize genomes are temperate
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types, and researchers are now hoping to sequence at least one tropical maize
inbred in high quality.
When a high-quality reference genome is available, resequencing a panel
of various germplasm can be utilised to provide a complete picture of genetic
diversity as well as the process of domestication and improvement, as
demonstrated in rice, soybean and maize.
Genotyping by sequencing
Genotyping by sequencing (GBS) is a method for finding single
nucleotide polymorphisms (SNPs) in order to perform genotyping studies,
such as genome-wide association studies, employing restriction enzymes to
reduce genome complexity and genotype multiple DNA samples in the field
of genetic sequencing. Following digestion, PCR is used to increase the
number of fragments in the pool, and GBS libraries are analysed using next-
generation sequencing techniques.
This GBS approach was proven using RIL populations of maize and
barley, with approximately 200,000 and 25,000 sequence tags mapped,
respectively.
Genome wide association studies
GWAS is used to find the genes that are responsible for the phenotypic
differences that we are interested in by first identifying people who differ in
the trait of interest (genotype) and then looking for SNPs where two
populations have different allele frequencies.
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Whole genome resequencing
This is commonly done to determine the differences between a specific
individual's genome and a so-called reference genome, which is a genomic
sequence derived from thousands of individuals and intended to contain as
many variations as feasible.
A catalogue of mutations specific to each sequenced individual, usually
single nucleotide polymorphisms (SNPs) and insertions-deletions (indels), is
obtained by comparing the sequenced genomes to the reference, which can
provide extremely valuable insight into the genetic background of the
individuals. This is frequently linked to specific phenotypes based on which
individuals are chosen. Substantial rearrangements (e.g., translocations,
inversions, large copy number changes) can also be targeted by WGR with
carefully prepared studies.
WGR uses massively parallel sequencing technology to extract enough
DNA fragments to cover the entire length of the genome of interest. Different
sequencing methods, such as Illumina, PacBio, Oxford Nanopore and
Bionano, might be utilised depending on the size of the genome and the sort
of mutations being found.
Next generation sequencing
Next-generation sequencing (NGS), often known as massively parallel or
deep sequencing, is a catch-all phrase for a variety of current sequencing
methods. These technologies have revolutionised genomics and molecular
biology by allowing for considerably faster and cheaper sequencing of DNA
and RNA than previously possible using Sanger sequencing. Illumina (Solexa)
sequencing is one of these technologies:
Illumina sequencing works by recognising DNA bases and adding them
to a nucleic acid chain at the same time. Each base emits a distinct
fluorescence signal.
454 sequencing by Roche: This technology is based on pyrosequencing,
which uses fluorescence to detect pyrophosphate release after polymerase
incorporates nucleotides into a new strand of DNA.
Ion Torrent: Proton / PGM sequencing: Ion Torrent sequencing does not
measure light and instead measures the direct release of H+ (protons) from the
incorporation of individual bases by DNA polymerase.
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Plant high-throughput phenotyping
The Arabidopsis phenomics project resulted in a first GWA investigation
on 191 inbred lines for 107 primarily quantitative phenotypes, including
disease resistance, flowering characteristics and life history features. Because
genetic variation is subject to natural selection, automated platforms like these
could be particularly valuable for phenotyping plant populations in the wild.
It is far more difficult to develop methods for huge plants with longer life
cycles, such as maize and sorghum. The fundamental prerequisites for a
successful phenomics effort are simple to define but complex to achieve.
Because of the high expenditures and restricted phenotyping capabilities, none
of the pioneering phenome projects came close to attaining the complete
phenomic vision.
E-typing or environmental assay
As a third dimension of the profile for current plant breeding,
environment-typing (e-typing) or environmental assay is used. E-typing
combines genotypic and phenotypic information by providing a
comprehensive set of environmental data required to define a plant.
Because of increased data collecting via advanced systems such as
geographic information systems, large-scale e-typing is now conceivable
(GIS). Finally, a genotype can be constructed to generate an optimised
phenotype with the best fit to certain environmental conditions using the 3-D
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profile (genotype, phenotype, and environment). Following is the 3-D profile
of molecular plant breeding with the concept evolving from point to line, plane
and space. Selection in early plant breeding was performed based on single
desirable phenotypes one at a time (‘‘point’’). Classical breeding has been
based on selection of multiple phenotypes (‘‘line’’). Marker assisted breeding
now uses selection criteria determined by both phenotypes and genotypes
(‘‘plane’’), while our future breeding research will be built upon the
knowledge generated by genotyping, phenotyping and e-typing
(environmental assaying) (‘‘space’’)
Selection strategies
Marker assisted selection
Molecular breeding has gone through two significant stages in terms of
selection strategies: MAS and genomic selection (GS). Only substantial
association markers and target qualities are utilised for selection in the first
stage, which is based on significant associations markers and target traits.
Marker assisted backcross breeding (MABB), a conservative strategy that
improves the recurrent only to the amount defined by the introgressed
genes/QTLs, is a representative method for the initial step. It does not generate
new gene combinations, and the MARS approach is based on markers that
show a substantial connection with the trait(s), making it inefficient.
The genomic selection (GS) scheme compensates for the shortcomings of
the MAS and MARS techniques. The GS approach makes use of data from
genome-wide markers to see if their relationships with the qualities in question
are substantial. The second stage is based on all markers that cover the entire
genome, which is represented by GS, and includes all markers for model
construction and progeny prediction. As a result, GS is a significant step
toward whole-genome approaches.
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Marker assisted backcross breeding
In plant breeding, backcrossing is done to introduce desirable features
from a donor plant into an elite genotype (recurrent parent). The original cross
is backcrossed with the recurrent parent until most of the donor genes are
removed in successive crossings. Even after many backcrossing generations,
the donor segments connected to the target allele can stay rather big. In order
to minimize this linkage, drag, marker assays can be of advantage. Markers
can be used in the context of MABC to either control the target gene
(foreground selection), to accelerate the reconstruction of the recurrent parent
genotype (background selection) or to select backcross progeny with the target
gene and recombination events between the target locus and linked flanking
markers (recombinant selection). According to Tanksley et al. 1898, standard
backcross breeding requires more than six generations to recreate the recurrent
parent genotype, but MABC requires only three generations.
MABC is currently and will most likely remain the primary method of
backcrossing transgenes into elite inbred lines, which contributes to its
popularity. If a single allele is to be transplanted into a new genetic
background, such as to improve an existing variety for a specific trait, MABC
is especially effective. However, if a plant's performance is governed by a
complicated genotype, the perfect genotype is unlikely to be achieved only
through MABC. Other approaches, such as marker assisted recurrent selection
(MARS), must be considered to address the problem of only being able to
improve existing elite genotypes. MAB will almost certainly become more
popular in the future, mostly for the same reasons.
Marker-assisted recurrent selection (MARS)
The enhancement of complex features through phenotypic recurrent
selection is generally attainable, but the extended selection cycles limit this
breeding method's applicability. Recurrent selection can be significantly
accelerated with the use of markers. Pre-flowering genotypic information is
employed in continuous nursery operations for marker-assisted selection and
controlled pollination. As a result, many selection cycles within a year are
conceivable, accumulating advantageous QTL alleles in the breeding
population (Eathington, S.R. et al. 2007).
Furthermore, an ideal genotype can now be defined as a pattern of QTLs,
with all QTLs containing advantageous alleles from different parents. After
numerous generations of crossings, it may be possible to come close to the
optimum genotype if individuals are crossed based on their molecular marker
genotypes. It is expected that a MARS breeding strategy will yield more
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genetic gain than an MABC breeding scheme. Multi-trait selection indices
have been used to build concepts for achieving the perfect genotype.
(Peleman, J.D and Vander Voort, J.R. 2003)
Genomic selection
Steps in genomic selection
GS is a specialised version of MAS in which genotype data on marker
alleles encompassing the entire genome is utilised to inform selection utilising
highly saturated genetic maps or a larger number of markers to calculate
GEBV's of various lines, which is used to inform individual selection.
Training population: One of the most difficult decisions in the GS
approach is the training population composition. We choose highly different
lines based on cluster analysis. Individuals or lines in the training population
should have gone through multiple rounds of recombination, and the training
population should be typical of the breeding populations. For training the GS
model, genotyping is done for a large number of markers evenly dispersed
across the genome, and phenotyping is done at many locations.
Breeding population: In the case of breeding populations, phenotypic
evaluation is not performed. GEBVs for individuals/lines are calculated using
marker data. Finally, superior lines are chosen based on GEBV values.
In the case of plants, several types of real training populations can be
created e.g., biparental crosses, doubled haploid testcrosses and intermated
inbred lines. Ideally, a new training population should be generated for each
breeding population that increases high accuracy in GEBV prediction.
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References
1. Clark AG. The Role of Haplotypes in Candidate Gene Studies. Genetic
epidemiology. 2004;27:33.
2. Eathington SR, Crosbie TM, Edwards MD, Reiter RS, Bull JK. Molecular
markers in a commercial breeding program. Crop Science.
2007;47(3):S154.
3. Gao S, Martinez C, Skinner DJ, Krivanek AF, Crouch JH, Xu Y.
Development of a seed DNA-based genotyping system for marker-
assisted selection in maize. Molecular Breeding. 2008;22:477-494.
4. McMullen MD, Kresovich S, Villeda HS, Bradbury P, Huihui Li, Qi Sun,
et al. Genetic properties of the maize nested association mapping
population. Science. 2009;325(737):737-740.
5. Peleman JD, Vander Voort JR. Breeding by design. Trends Plant Science.
2003;8(7):330-334.
6. Tanksley SD, Young ND, Paterson AH, Bonierbale MW. RFLP mapping
in plant breeding: new tools for an old science. Biotechnology.
1989;7(3):257-264.
7. Xu Yunbi, Lu Yanli, Xie Chuanxiao, Gao Sh, Wan J, Prasanna B. Whole-
genome strategies for marker-assisted plant breeding. Molecular
Breeding. 2012;29:833-854.
8. Yu J, Holland JB, McMullen MD, Buckler ES. Genetic design and
statistical power of nested association mapping in maize. Genetics.
2008;178:539-551.
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Chapter - 8
Earthworms Bio Markers in Soil Pollution
Assessment
Authors
Praveen Kumar
Ph.D. Research Scholar, ICAR-Indian Agricultural Research
Institute, New Delhi, Delhi, India
Rishbh Kumar Didawat
Arkaprava Roy
Priti Tigga
Koushik Bag
Sandeep Kumar
Ritambhara
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Chapter - 8
Earthworms Bio Markers in Soil Pollution Assessment
Praveen Kumar, Rishbh Kumar Didawat, Arkaprava Roy, Priti Tigga, Koushik Bag,
Sandeep Kumar and Ritambhara
Abstract
Over the last two decades, soil pollution has exploded and it has now
become a global problem. Soil pollution has immediate and/or long-term
negative consequences for living creatures and ecosystems. Assessing soil
pollution is a crucial step in preserving living beings and the ecosystem.
Traditional physicochemical approaches to evaluate the danger of soil
contamination are based on pollutant concentrations in soil, but they are
insufficient and do not reflect the harmful impacts of pollutants on biota.
Earthworms are the most common fauna in terrestrial ecosystems, and they
have a positive impact on a soil's physical, chemical, and biological qualities.
Earthworms have long been utilized as a bio-indicator for soil health due to
their sensitivity to harmful substances and other unique biological benefits.
The histopathological biomarkers like bacterial and fungal populations in the
gut and intestine of earthworm are effective tool in assessment of
monocrotophos pollution in the soil. The metallothioneins (MT) could be
developed as a more effective biomarker for determining soil cadmium
contamination levels. Pd-contaminated ferrosol affects the different metabolic
pathways like alanine-aspartate-glutamate, purine, glutathione and valine-
leucine-isoleucine biosynthesis and degradation and nicotinate and
nicotinamide metabolism. Cu accumulation in earthworms was considerable
after 6 and 9 days, and at the highest dose of 120 mg Cu kg-1 soil, compared
to untreated controls, indicating that worms are bio-accumulators of heavy
metals in both soluble and bioavailable forms. Earthworms in the microfiber
MF 1.0% treatment showed a 1.5-fold lower cast 32 production, a 24.3-fold
increase in expression of mt-2 and a 9.9-fold decline in 33 hsp70 expression.
Earthworm biomarkers an important tool in soil pollution assessment. It can
be used for early-warning or diagnosis of soil pollution.
Keyword: Soil pollution, pollution assessment, earthworms, pollutants
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1. Introduction
A biomarker is defined as a "variation in biochemical, cellular,
physiological or behavioural parameters that can be tested in tissue or body
fluid samples, or at the level of whole organisms, to offer proof". Biomarkers
of exposure and/or effects from one or more contaminants are defined as
"biochemical, cellular, physiological or behavioural variations that can be
measured in tissue or body fluid samples or at the level of whole organisms,
to provide evidence of exposure and/or effects from one or more
contaminants" [1]. Contaminants' impacts at lower levels of biological
organisation (e.g. biochemical, cellular, physiological) occur more quickly
than at higher levels (e.g. ecological effects), and so may provide a more
sensitive early warning of toxicological effects within populations. Any
changes in the molecular, cellular, biochemical and physiological processes
that occur within an organism as a result of pollution exposure could
potentially be employed as biomarkers. Biomarkers are divided into two
categories: exposure biomarkers and impact biomarkers. The former indicate
that an organism has been exposed to a pollutant and provide an early warning
of micro-pollutant exposure. They can evaluate exposure to numerous
substances in both qualitative and quantitative terms. However, the degree of
deleterious effects on the organism or in the population may not be predicted
by changes in these biomarkers. Effect biomarkers are linked to the toxicant's
mechanism of action and are well-characterized enough to link the degree of
biomarker alteration to the severity of adverse effects [2]. Exposure biomarkers
and impact biomarkers can both help with environmental assessments.
Biomarkers of exposure may have the potential to provide a more
physiologically meaningful indicator of exposure than some chemical tests or
to evaluate the impacts of short-lived substances [3]. Biomarkers of effect, on
the other hand, can help with qualitative aspects of hazard identification by
establishing that a danger exists and clarifying the mechanisms that cause it.
Depending on the degree of specificity of the biomarker with the pressure and
the degree to which its expression to higher order effects is understood, these
biomarkers can provide insights into both the primary aspects of the hazard
and its ecological repercussions.
2. Role of earthworm biomarker in soil pollution assessment
The ability of the soil to retain the biota is one of the factors for soil health
quality. Because the earthworm is such an important part of the soil
ecosystem, if it cannot easily live or survive in it, the soil may be at risk or
unhealthy. As a result, the earthworm biomarker can provide some
information about the environmental quality of the soil and can be useful in
detecting specific soil pollution.
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Using sophisticated physicochemical analysis at the preliminary stage
without understanding the specific pollutant in soil is time consuming and
costly in large-scale soil pollution surgery. Under the following criteria (Fig.
1), a bioassay approach may be the best option right now:
 If preliminary toxicity testing reveal that the soil causes earthworm
mortality or reduces reproduction, the soil is in danger. The species
and concentration of the contaminant must subsequently be
determined using more precise chemical analysis (s).
 If standard earthworm toxicity tests reveal that soil may support
earthworms, an earthworm biomarker may be required to determine
whether the soil can have a variety of effects at different levels
(specifically, the molecular, cell, biochemical and physiological
level, etc.). Currently, a three-tier technique may be effective for
accurately eliciting the issue. Because single biomarker responses to
environmental stress are not always reliable, integrative biological
response index is calculated after a low-cost initial screening, such as
the neutral red assay in earthworm coelomocytes, to obtain an
objective (scored) assessment of the induced stress syndrome. If the
integrative biological response index is positive, the soil is likely to
be contaminated. When combined with other data, such as the
presence or absence of a pollution source, it can be determined
whether a chemical study is required to ascertain the risk factors.
 Chemical analysis is unnecessary if the earthworm biomarker
response is normal and the soil is clean and healthy. To summarize,
such a technology approach might be both cost-effective and
efficient, as well as integrative and compelling. Earthworm
biomarkers can also be valuable indices in determining the objective
of soil pollution remediation and ecological restoration evaluation,
because the ultimate goal of contaminated soil remediation is
harmlessness.
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Fig 1: Use of earthworm biomarker in soil pollution assessment survey
The earthworm biomarker could also be useful in determining soil
contamination levels. The usual method for monitoring soil contamination is
to look at overall pollutant concentrations in the soil. Soil pollution levels are
defined by comparing with particular threshold values and computing the
pollution index (PI) or integrated pollution indices (IPIs). IPIs can classify
pollution levels as low (IPI 1), moderate (1 IPI 2) or high (IPI 2), for example
[4]
. Traditional evaluation, on the other hand, cannot offer sufficient
information on the harmful effect of pollutants on biota since it ignores the
interaction of multiple pollutants as well as the bioavailability of pollutants [5],
and hence may not always be valid. The bioassay, which includes the
biomarker method, is extremely compensatory and necessary in this situation.
Furthermore, because in ecotoxicology, there is a tiered cascade in the
biological response to increased chemical exposure, earthworm biomarkers
may be useful in measuring soil contamination and grading soil pollution
levels. The bioassay approach of assessing soil pollution is depicted in (Fig.
2). According to the diagram, physicochemical analysis can be used in
conjunction with soil pollution assessment to gather more specific information
on sites/fields that have already shown risk or warning signs (due to moderate
or high pollution levels) and to aid in soil pollution repair. Furthermore, light
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pollution levels could be graded further by integrating with AOP. The word
stress magnitude, which encompasses the confounding factors affecting
biomarker response, may be required in this situation.
Three levels of stress magnitude can be classified based on biomarker
responses:
i) Low (e.g., biomarker responses abnormal only at the molecular
level).
ii) Moderate (e.g., biomarker responses abnormal at the molecular,
biochemical, and physiological level).
iii) High (e.g. biomarker responses abnormal at all sub-organism and
behavioural level).
Fig 2: The role of earthworm biomarkers in assessment of soil pollutant
3. Biomarkers in earthworms
The earthworm biomarker could be used to assess soil pollution. Because
changes in any of the molecular, cellular, biochemical and physiological
processes that occur within earthworms or as a whole after pollutant exposure
could be employed as biomarkers.
Overall, the use of earthworm biomarkers is becoming more relevant for
assessing the effects of pollutants on soil organisms. Acetylcholinesterase,
metallothionein, biotransformation enzymes and antioxidant defenses are
among the most commonly utilized biomarkers because of their critical roles
in neuro cholinergic transmission and cell homeostasis, which protects cells
from chemical toxicity. The study of novel biomarkers in earthworms, on the
other hand, is gaining popularity due to its potential for soil pollution
monitoring and assessment.
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4. Classification of earthworm biomarkers
The earthworm biomarker could be used to assess soil pollution. Because
changes in any of the molecular, cellular, biochemical and physiological
processes that occur within earthworms or as a whole after pollutant exposure
could be employed as biomarkers.
Overall, the use of earthworm biomarkers is becoming more relevant for
assessing the effects of pollutants on soil organisms. Acetylcholinesterase,
metallothionein, biotransformation enzymes, and antioxidant defences are
among the most commonly utilized biomarkers because of their critical roles
in neuro cholinergic transmission and cell homeostasis, which protects cells
from chemical toxicity. The study of novel biomarkers in earthworms, on the
other hand, is gaining popularity due to its potential for soil pollution
monitoring and assessment.
Table 1: Classification of earthworm’s biomarkers
Classification Earthworm biomarkers
Antioxidant defence system, cytochrome P450, GST
Physiological and (glutathione-S-transferase), AChE (acetylcholinesterase),
biochemical carboxylesterase, metallothioneins (mts), Hsp (heat shock
protein), glycogen stores.
Avoidance behavior, burrowing behavior, secrete of mucus, cast
Behavioral
production.
Histopathological Integrality of morphology, wound healing.
Molecular genetic Comet assay.
Immunological Total immune activity, granulocyte.
5. Soil pollution assessment using earthworm
Fig 3: Flow chart showing effect of soil pollutants on biochemical indices of the
earthworms
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6. Soil pollution assessment by earthworm biomarkers
6.1 Pesticides contamination.
6.2 Heavy metals and other inorganic metalloid.
6.3 Organic contamination.
6.4 Plastic and microplastic.
6.1 Pesticides contamination
6.1.1 Biochemical responses of Eudrilus eugeniae to chlorpyrifos,
cypermethrin and their combination
Chlorpyrifos, cypermethrin and its combination (chlorpyrifos +
cypermethrin) significantly inhibited the specific activity of AChE in different
body segments (pre-clitellar, clitellar and post-clitellar) of earthworms. In all
of the segments tested, the 10 percent LC50 of a pesticide combination showed
the greatest suppression of AChE activity, followed by chlorpyrifos and
cypermethrin. The activity of AChE (nM ATI hydrolyzed/min/mg protein) in
distinct body segments of earthworms (Pre-Clitellar, Clitellar and Post-
Clitellar) in response to pesticides chlorpyrifos, cypermethrin and their
combined exposure is shown in Fig 4. AChE activity decreased in a dose-
dependent and region-specific way. The fact that brain/ganglionic structures
(dorsal brain) are located in the prostomium of earthworm may be the
explanation for the greatest drop in AChE activity in pre-clitellar followed by
other locations. These pesticides' varying levels of inhibition could be related
to their various mechanisms of action. By covalently phosphorylating the
serine residue within the active site group, chlorpyrifos, an organophosphate,
limits enzyme activity. The irreversible inhibition of AChE causes an excess
of acetylcholine to accumulate, resulting in hyperactivity and, as a result,
damage of the neuronal and motor systems. However, pyrethroid
(cypermethrin) inhibits AChE activity by blocking sodium channels and
affecting the action of GABA-receptors in nerve filaments. Another cause for
differences in AChE inhibition levels could be related to pesticide buildup or
interaction with their metabolite in various organs. The combination pesticide
treatment has a greater impact on AChE activity than a single pesticide
treatment [6].
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Fig 4: Effect of chlorpyrifos, cypermethrin and their combined effect on AChE
activity. Here, PC: Pre-clitellar, C: Clitellar and PtC: Post-clitellar regions of
earthworm
6.1.1.2 Morphological changes in earthworm E. eugeniae exposed to

pesticide
With increasing doses of chlorpyrifos, morphological changes such as
coiling, clitellar enlargement, mucus secretion, and bleeding, followed by
body segmentation, were observed in chlorpyrifos-treated earthworms.
However, cypermethrin caused more noticeable morphological changes than
chlorpyrifos, while the effect of co-exposed pesticides on morphological
changes was determined to be intermediate (Fig. 5). During the trial, there
were no morphological changes in the control group of earthworms [6].
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Fig 5: Morphological changes in earthworm E. eugeniae exposed to different
concentrations of chlorpyrifos, cypermethrin and their combination. WBC: Whole
Body Coiling, BC: Body Constriction, FB: Fragmentation of Body, TB: Thinning of
Body, CS: Clitellar Shrinkage, BC: Body coiling
6.1.2 Assessment of herbicide and fungicide toxicity by Eisenia fetida

biomarkers
Table 2: The herbicide tribenuron-methyl and fungicide tebuconazole toxicity by
Eisenia fetida
Combined toxicity (1:1

Individual toxicity LC50 (95%
toxicity) LC50 (95%
Toxicity confidence interval) (mg kg−1)
Time confidence interval) (mg kg−1) Combined
method action
Tribenuron- Tribenuron-
Tebuconazole Tebuconazole
methyl methyl
Contact 24 h 377.9 9.4 202.8 8.5
Antagonism
filter paper (195-526) (8-10) (168-251) (7-10)
test 135.6 5.7 114.2 4.7
(μg cm−2) 48 h (103-150) (3-7) (94-141) (3-5)
Antagonism
1007.3 746.3 926.7 478.7

Artificial 7 d (898-1235) (690-806) (802-1091) (414-563)
Antagonism
soil test
(mg kg−1) 14 d 511.0 287.9 479.8 247.9
Antagonism
(401-623) (119-415) (242-560) (125-367)
Eisenia fetida, a soil earthworm, was treated with tribenuron-methyl

(TBM) and tebuconazole (TEB). In the contact filter paper and artificial soil
tests, the TBM showed modest toxicity to E. fetida, with median lethal
concentrations (LC50) of 135.6 g cm-2 at 48 h and 511 mg kg-1 on day 14,
respectively. In the artificial soil test, TEB likewise demonstrated low toxicity
to E. fetida, with an LC50 of 287 mg kg-1 on day 14. In the contact filter paper
test, TEB was highly poisonous to earthworms, with an LC50 of 5.7 g cm-2
after 48 hours. The earthworm was affected negatively by the combination of
two insecticides. In the early days of earthworm exposure, single or
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combination pesticide applications produced oxidative stress and reduced
cellulase activity under 0.1 LC50 of TBM and TEB. Both insecticides,
however, had no effect on earthworm DNA. The findings imply that pesticides
can harm soil earthworms and provide important information on how soil
biological engineers react to harmful agrochemicals [7].
Fig 5: Effects of tribenuron-methyl (A), tebuconazole (B) and mixture of both

pesticides (C) on cellulose activity in E. fetida. Each column represents the mean of
three replicates, and the error bars represent the standard deviations (SD)
The differences in cellulase activity in the earthworms exposed to
individual and combined applications of TBM and TEB (Fig. 5). The cellulase
activity was inhibited at only high concentration of TBM on day 3 and 7
compared to the control treatments (Fig. 5A). The cellulase activity did not
differ in control and TEB exposure experiments during the whole period (Fig.
5B). Under high doses of pesticides mixture, the cellulose activity was
significantly lower than that of control in the first 14 days and then it recovered
to the control level (Fig. 5C) [7].
6.1.3.1 Lampito mauritii sensitivity to monocrotophos pollution on
bacterial and fungal populations in the gut
Using the lower sub-lethal concentration of monocrotophos (T1), there
was no significant change in the bacterial population (3%) on day 1. The
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population significantly decreased by 23% and 34% as compared to the
controls on days 5 and 15, respectively. Similarly, using the higher sub-lethal
concentration (T2) the bacterial population did not change significantly on day
1. The population significantly decreased by 39% and 49% on days 5 and 15,
respectively. On day 30, the bacterial population increased from 313 ± 8.67
CFU (as determined on day 15) to 351 ± 8.08 CFU and from 239 ± 5.49 CFU
(as determined on day 15) to 316 ± 5.20 CFU in T1 and T2, respectively. Upon
exposure to the lower sub-lethal concentration (T1) of monocrotophos, the
fungal population significantly decreased by 23%, 27% and 31% on days 1, 5,
and 15, respectively, as compared to that in the control troughs. Using the
higher sub-lethal concentration (T2), fungal populations significantly
decreased by 26%, 31%, and 36% on days 1,5 and 15, respectively. On day
30, the fungal population increased from 125 ± 3.18 CFU (as determined on
day 15) to 161 ± 2.33 CFU and from 117 ± 2.31 CFU (as determined on day
15) to 153 ± 2.89 CFU in the T1 and T2 troughs, respectively. On day 30, the
number of bacteria and fungi were less in T1 and T2 than in the control troughs
(Table 1). Fig. 6 shows the maximum reduction of bacterial and fungal
populations on day 15 of the experiment these eight bacterial species
(Klebsiella pneumonia, Proteus vulgaris, Proteus mirabilis, Pseudomonas
aeruginosa, Enterobacter aerogenes, Enterobacter cloacae, Morganella
morganii and Bacillus subtilis) were observed in the gut of the control L.
mauritii. Among these species, K. pneumoniae and P. vulgaris were absent in
the earthworms from the T1 and T2 troughs. However, Aspergillus fumigatus,
A. niger, A. flavus, Mucor plumbeus and Rhizopus sp. were observed in the
gut of the control L. mauritii. Among these five fungal species, M. plumbeus
and Rhizopus sp. we’re not observed from the gut of L. mauritii exposed to
low and high concentrations of monocrotophos [8].
Fig 6: The bacterial and fungal cultures of the gut contents of earthworm exposed to
monocrotophos on day 15
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6.1.3.2 Histology of Lampito mauritii intestine as biomarkers in response
to pesticide
On the 1st day of the experimental period, loss of compact structure of
epithelial lining, damaged villi, fusion of cells and pyknotic nuclei in some
regions were observed in the intestine (Fig. 7b). On 5th day, vacuoles formed
in the cytoplasm and villa damaged due to degenerated nuclei, space formation
and congestion of blood sinuses. The monocrotophos was highly affected the
intestine in the 5th day of exposure (Fig. 7c). At the end of 30th day, the
epithelial cells are renewed and more or less compactly arranged in the
epithelial layer (Fig. 7). The results showed that the prominent damages were
observed on 1st and 5th day thereafter slowly recovered 30th day [8].
Fig 7: Histology of Lampito mauritii intestine. (a) Cross section of control Lampito
mauritii intestine. (b) Cross section of intestine of L. mauritii exposed to sublethal
lower concentration of monocrotophos for 1st day.; (c) Cross section of intestine of L.
mauritii exposed to sublethal lower concentration of monocrotophos for 5th day. (d)
Cross section of intestine of L. mauritii exposed to sublethal lower concentration of
monocrotophos for 30th day. V-villi, EL-epithelial layer, TY-typhlosole, CH-
chloragogen cells, VO-vacuolations, S-blood sinuses
6.2 Heavy metals and other inorganic metalloid

6.2.1 Accumulation of copper in earthworm
During the genotoxicity experiment, copper residues in soil and
earthworms results of accessible Cu in soil after 3, 6 and 9 days of treatment.
A significant amount of accessible copper was identified in soil during the
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experiment at all sample intervals when compared to control soil without
copper addition. The mean Cu concentration at each sampling period was
always lower than the Cu applied: only 59.14 percent and 57.04 percent of the
applied dosages, respectively, were found in soil on day 3 following treatment
for 60 and 90 mg Cu kg-1 soil, whereas the lowest dose of 30 mg Cu kg-1 soil
was 84.13 percent. In our experimental context, the concentration used
resulted in no worm death. After 6 and 9 days, the copper buildup in entire
worms was measured at 120 mg/kg. Copper levels in the control, unexposed
animals remained unchanged for the whole 9-day period. After 6 days of
treatment, a substantial quantity of Cu was accumulated in whole earthworms,
which increased further after 9 days of exposure (Fig. 8) as compared to the
unexposed control [9].
Fig 8: Copper accumulation in entire worms after 6 and 9 days of exposure to 120
mg Cu/kg soil. The data is provided in nanograms of copper per milligramme of
worm wet weight
6.2.1.2 Eisenia Andrei’s genotoxicity biomarkers sensitivity to copper

exposure
The Comet test was used on coelomocytes taken from organisms exposed
to copper (30 mg/kg, 60 mg/kg, and 120 mg/kg) for 3, 6, and 9 days to evaluate
copper-induced double-strand DNA break formation (Fig. 9). The genotoxic
effect in the overall population was calculated using the median values of
DNA damage indexes. Furthermore, the fraction of cells with a higher degree
of DNA damage was calculated using the 75th percentile of the same
indicators. At 30 mg/kg, no significant increase in DNA-damage indexes was
observed, whereas at 60 mg/kg, significant damage was observed after 6 days
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(median TI + 47 percent, p 0.05; 75th percentile TI + 52 percent, p 0.01), with
a significant increase in Tail moment observed exclusively in the fraction of
more sensitive cells (75th percentile TM+ 13 percent, p 0.01). When animals
were given 120 mg/kg copper sulphate, median TI increased significantly after
3 days (+41 percent) increased even more after 6 days (+56 percent) (Fig. 9)
[9]
.
Fig 9: Following in vivo exposure to copper at escalating concentrations, DNA

damage was measured as median TI (a); 75th percentile TI (b); median TM (c); 75th
percentile TM (d)
6.2.1.3 Eisenia Andrei’s molecular biomarkers sensitivity to copper
exposure
We studied the impact of copper exposure at 120 mg kg-1 Cu on gene
expression in E. andrei immune cells, which monitor the expression of genes
encoding for metallothioneins (MET), which play a key role in trace metal
homeostasis and detoxification. Proteins involved in antioxidant response
(SOD, CAT), as well as a number of genes that operate as immune response
modulators. Were also looked into. The up-regulation of MET expression seen
in our work (Fig. 10) is consistent with other studies showing similar
dynamics, indicating that this protein is involved in Cu homeostasis and
protection against oxidative damage. The antioxidant enzymes' overall
adaptive response is not triggered, which could be owing to increased
copper/mediated oxidative stress and damage, as seen by DNA oxidative
damage data. Lack of induction of the cellular antioxidant enzymatic
machinery, on the other hand, could be attributed to a purposeful optimization
of gene expression patterns by selective activation of metal-binding proteins
[9]
.
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Fig 10: Heat Shock Protein 70 (HSP70), superoxide dismutase, Catalase (CAT) and
Metallothionein (MET) mRNA levels in coelomocytes after 6 and 9 days of exposure
to CuSO4 300 mg/kg
6.2.2.1 Biomarkers responses (total proteins) (Allolobophora caliginosa)
to cadmium contaminated soil
By monitoring some physiological and antioxidant responses of worms
exposed to a battery of high cadmium concentrations, the toxicity of cadmium
on the earthworm Allolobophora caliginosa was evaluated. Biomarkers can
be utilized as early warning indicators of environmental contamination and
population-wide consequences. Metal buildup is controlled by two coexisting
intracellular routes in earthworms. Because proteins called "MTs" are engaged
in the transport and regulation of essential metal ions and serve as intracellular
distributors of copper and zinc, they also play a role in non-essential metal
regulation and detoxification. They are known to protect cells from Cd toxicity
and oxidative stress in particular. In this study, worms treated with the lowest
concentration of cadmium 25 mg/500g dry weight showed a significant
increase in total proteins rate over a 7-day period, while worms treated with
the highest concentration of cadmium 150 mg/500g dry weight showed a very
highly significant increase in total proteins rate. However, the author observed
a dose-dependent modulation of total protein rate over a 14-day period (Fig.
11). The remarkable endurance of earthworms to cadmium poisoning is likely
attributable to detoxification by metallothionein proteins in the posterior
alimentary canal [10].
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Fig 11: Effect of cadmium stress on the total proteins rate in Allolobophora
caliginosa
6.2.2.2 Effect of cadmium stress on GST/GSH level in Allolobophora

caliginosa
Fig 12: Effect of cadmium stress on the total proteins rate in Allolobophora
caliginosa
Glutathione is the most important non-enzymatic radical scavenger in
animal cells, and Glutathione-s-transferase (GST) is one of the phase II
enzymes. Enzymes aid in the elimination of reactive electrophiles by
catalysing the conjugation reaction of GSH and electrophilic xenobiotics. This
enzyme also helps to protect cells from oxidative damage. The author
observed a dose-dependent rise in GSH level along with a significant drop in
GST activity after 7 days of exposure, which could be attributable to
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upregulation of the GSH synthesis pathway and a protective mechanism
against the metal's harmful effects indicating that the earthworms' detoxifying
and antioxidant processes failed to deal with the toxic stress caused by Cd
exposure during a brief exposure period. The current study clearly shows a
robust dose-response relationship between cadmium and GSH expression
after 14 days of exposure (Fig. 12). GSH expression appears to be susceptible
to cadmium: it was significantly reduced even at low cadmium concentrations,
suggesting that its depletion may be attributable to its widespread
consumption via the oxidation of two molecules of GSH to one molecule of
GSSG. GSH can also scavenge reactive oxygen species (ROS) and it can
sequester Cd to prevent it from interacting negatively with biomolecules. This
revealed a large decrease in GSH concentration in paramecia exposed to
pesticide, as well as a considerable rise in GST activity, which is known to
perform detoxifying functions. GSH depletion usually triggers GST
activation, which is followed by GSH synthesis to restore the normal level. As
a result, it plays an important role in metal metabolization by organisms and
provides the cell with a state of balance and protection against oxidised
reactive species [10].
6.2.3.1 Lumbricus terrestris biomarker in assessment of chemical
pollutants
Fig 13: Lysosomal membrane stability and AChE activity in control and treated
animals during the exposure to copper sulfate and methiocarb
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6.2.3.2 Assessment of chemical pollutants by Lumbricus terrestris as
biomarkers
Fig 14: (A) Earthworm weight, (B) mortality, (C) reproduction rate measured in
control and treated animals during the exposure to copper sulfate and methiocarb.
Reproduction rate was measured as new born production per box after 4 weeks from
mature earthworm removal at time 30 and 14 days (for copper sulphate and
methiocarb exposure respectively
In a variety of organisms, including earthworms, lysosomal membrane
stability is commonly employed as an early predictor of contamination
impacts. Following 14 days of treatment to either copper sulphate or
methiocarb, lysosomal membrane stability in L. terrestris coelomocytes
varied by 30-40 percent. After 30 days of exposure to copper sulphate, the
percentage variance climbed to almost 70%. These findings show that
lysosomes are a subcellular target for heavy metals and pesticides in L.
terrestris coelomocytes. In both exposure trials, however, lysosomal
membrane stability showed a lesser variation in pollution treated organisms
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than granulocyte enlargement. AChE levels in methiocarb-exposed rats were
lowered by around 70% after 7 days and by about 80% after 14 days. The
AChE inhibition statistics were matched by approximately 30% mortality after
7 days of exposure and 40% mortality after 14 days of exposure (Fig. 14). In
general, high order level ecotoxicological metrics revealed the same pattern
(growth, mortality, reproduction). These death rates (below 50%) are lower
than those reported in vertebrates, where AChE inhibition greater than 50% of
normal is considered permanent and considered to be in the deadly range.
Other earthworm species have lately demonstrated a lesser susceptibility to
AChE inhibition than vertebrates, suggesting that the harmful impact of
pesticide on earthworms may include other molecular or cellular targets
beyond AChE. This is supported by the fact that AChE percentage variation
did not alter between 7 and 14 days of exposure, whereas mortality percentage
variation after 14 days of exposure doubled the action under either copper
sulphate or methiocarb exposure [11].
6.2.4.1 Earthworm biomarkers in arsenate pollution assessment by using
burrowing behavior responses
Fig 15: Burrowing activity of earthworm in control (CK) and exposed to As-V at 25,
50 and 100 mg/kg. Burrowing behavior parameters after 14 d As-V exposure with the
cumulative and the maximum depth of burrow systems recorded over 72 h.
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6.2.4.2 Earthworm biomarkers in arsenate pollution assessment by using
biochemical responses
Fig 16: Biochemical responses of earthworms in control (CK) and exposed to As-V
at 25, 50 and 100mg/kg. (a) TAs-E: total arsenic content in earthworm, SOD:
superoxide dismutase, (e) ATP: adenosine triphosphate content
There was an increase in arsenic concentration in earthworms (TAs- E)
over time, particularly for the 100 mg As-V/kg treatment (Fig. 1a). Partial
correlation analysis showed that total arsenic in earthworms (TAs-E) (Fig. 15-
16) was significantly increased with exposure time and concentrations.
Correspondingly, arsenic concentrations in soil (TAs-S) were slightly
decreased with exposure time, resulting in the average BAF in earthworms
from the 25, 50 and 100 mg As-V/kg concentration As-V treatments were
2.26, 2.46 and 3.58 respectively at day 28. ATP content showed a general
concentration and exposure time dependent decrease, except that in 25mg As-
V/kg treatment group at day 3. Multiple linear regression analysis revealed a
significant linear relationship, showing that ATP production was roughly
inhibited by As-V concentration but upregulated by ROS. ATP inhibition with
increasing As-V concentrations and exposure time indicates the toxicity of
mitochondrial damage. The findings of a significant linear relationship
between increased As-V concentrations and increased ATP concentrations
and increased ROS provide an explanation for the observation of up-
regulation of antioxidant enzymes with increased ATP content in earthworms
exposed to hexabromocyclododecane by 3.2. As-V up-regulates the
expression of antioxidant related proteins and mRNA [12].
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6.3 Organic contamination
6.3.1 Persistent organic pollutants assessment genotoxicity biomarkers
Because of their high toxicity, persistence and bio magnification across
food chains, persistent organic pollutants (POPs) pose a potential concern to
ecosystems. Ecosystem bio indicators have been developed to assess the
impact of environmental contaminants. In terrestrial environments,
earthworms are among the most frequent bio indicator organisms. The goal of
this study was to assess the genotoxicity of POP exposure in wild earthworms
caught at various levels of urbanization across the Coatzacoalcos River's lower
basin (industrial, urban and rural areas). POP concentrations in soil and
earthworm tissue were determined using Gas-Mass Chromatography, and
earthworm DNA damage was assessed using the comet assay. Industrial
locations had the highest amounts of POPs, DDT and HCH, followed by urban
and rural areas (504.68, 383.10, 298.16; 22.6, 4.6, 2.6 and 433.7, 364, 255.6
mg/kg, respectively). Unlike other contaminants, industrial soil samples had
the highest mean PCBs concentrations, followed by rural and urban soil
samples (41.10, 33.97 and 12.44 mg/kg, respectively) (Fig. 17). Individuals
from industrial sites had the highest concentrations of POPs in all earthworm
tissue studies, followed by those from urban and rural settings [13].
Fig 17: Comparison of Olive tail moment and tail length between earthworms to
different zones of Coatzacoalcos River. PCBs, HCHs, DDT, POPs. The greatest
concentrations of POPs, DDT and HCH were found in soil from industrial sites,
followed by urban and rural areas (504.68, 383.10, 298.16; 22.6, 4.6, 2.6 and 433.7,
364, 255.6 mg/kg, respectively)
6.4 Plastic and Microplastic

6.4.2.1 Molecular biomarkers in assessment of microplastic pollution in
soil
Microplastics are little plastic fragments with a particle size of less than
5 mm that are found in both marine and terrestrial habitats. Earthworms
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Eisenia fetida were given 100 and 1000 g of fluorescent polystyrene
microplastics (PS-MPs) per kg of artificial soil for 14 days in this
investigation. To determine the toxicological effects of PS-MPs on E. fetida,
researchers looked at the uptake or accumulation of PS-MPs in earthworm
intestines, histopathological alterations, oxidative stress, and DNA damage.
The average accumulated concentrations in the earthworm intestines were
higher for 1300 nm PS-MPs (0.084 28 0.005 and 0.094 0.003 g/mg for 100
and 1000 g/kg, respectively) than for 100 nm PS-MPs (0.015 0.001 and 0.033
0.002 g/mg for 100 and 1000 g/kg, respectively) than for 100 nm PS-MPs. In
addition, histological research revealed that PS-MP exposure caused damage
to intestine cells. PS-MPs also modified glutathione (GSH) levels and
superoxide dismutase (SOD) activity considerably. The GSH levels were
86.991 ± 7.723, 165.436 ± 4.256-167.767 ± 18.642, and 93.590 ± 4.279-
173.980 ± 15.523 μmol/L in the control, 100 nm, and 1300 nm PS-MPs
treatment groups. Furthermore, the SOD activity for the control, 100 nm, and
1300 nm PS-MPs treatment groups were 10.566 0.621, 9.039 0.787–9.408
0.493, and 7.959 0.422–9.195 0.327 U/mg protein, respectively, showing that
oxidative stress was produced following PS-MPs exposure. In addition, the
comet assay suggested that PS-MP exposure caused DNA damage in
earthworms. Overall, 1300 nm PS-MPs were more hazardous to earthworms
than 100 nm PS-MPs, revealing new information about the toxicological
effects of low microplastic concentrations on earthworms, as well as the
ecological dangers of microplastics to soil animals [14].
7. Conclusions
Biomarkers an important tool in soil pollution assessment. It can be used
for early-warning and diagnosis of soil pollution. In response to pesticides,
they help in assessment of soil pollution and their toxicity levels. Also, plastic
and micro plastic soil pollution can be evaluated with help of earthworm and
its biomarkers. Moreover, morphological biomarker of earthworm give
significant result after exposure inorganic contamination. Thus, they play
important role in assessment of heavy metal toxicity as early warning in soil
environment.
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Chapter - 9
Nutrient Availability and Plant Productivity
through PGPR: Mechanisms, Potential and
Constraints
Authors
Khushboo Rani
Scientist, ICAR-Indian Institute of Soil Science, Bhopal,
Madhya Pradesh, India
Priti Tigga
Institute, New Delhi, India
Arkaprava Roy
Institute, New Delhi, India
Abinash Das
Scientist, ICAR-Indian Institute of Soil Science, Bhopal,
Madhya Pradesh, India
Ankita Trivedi
Scientist, ICAR-Indian Agricultural Research Institute,
New Delhi, India
Page | 138
Page | 139
Chapter - 9
Nutrient Availability and Plant Productivity through
PGPR: Mechanisms, Potential and Constraints
Khushboo Rani, Priti Tigga, Arkaprava Roy, Abinash Das and Ankita Trivedi
Abstract
Intensive chemical based agricultural practices have inevitably fulfilled
food demands of growing human populations but at the cost of soil quality and
health. The current focus should be shifted towards more sustainable options.
In this regard, one alternative can be the use of plant growth promoting
rhizobacteria (PGPR). They are known to enhance plant growth directly as
well as indirectly. This chapter explains the various mechanisms of action of
PGPR which has been exploited in the field of agriculture and use of PGPR as
a potential biofertilizer, phyto-stimulant and as a bio-control agent. Use of
PGPR can be an ecofriendly approach towards sustainability. Many PGPR
genera have been effectively tested for their performance in biological control
as well as plant growth promotion. However, their use for increasing
agricultural yields has several pros and cons. Inconsistent field level results,
high cost of production, short shelf life and lack of awareness about PGPR bio
formulations are some of the many reasons which have restricted its wide scale
usage among farmers. Successful commercialization of these strains needs
improvements and advancements in microbiological and biotechnological
research, large-scale production, better and effective formulation methods,
product marketing and awareness among the end users.
Keywords: Plant growth promoting rhizobacteria; Biofertilizer; Phyto-
stimulant; Biocontrol agents
Introduction
Currently the world is treading a rocky path amidst challenges of land
degradation, poor quality and air, soil and water, changing climate, etc.
Adding to these challenges, reduction in agricultural productivity and soil
health due to various biotic and abiotic stresses is posing a major threat
towards sustainable agriculture. Promoting sustainable agriculture through
utilization of the biowaste-derived substances at the same time exploring the
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genetic potential of crop plants and microorganisms may be an effective
strategy to counter the rapid soil and environmental deterioration while
promoting better agricultural productivity soil health [1]. The 68th UN General
Assembly declared 2015 as the International Year of Soils (IYS). The UN
stated that “soils constitute the foundation for agricultural development,
essential ecosystem functions and food security and hence are a key to
sustaining life on Earth”. In the assembly, the UN declared that the
sustainability of soils is key to addressing the pressures of a growing
population and the sustainable management of soils can contribute to healthy
soils and thus to a food-secure world and to a stable and sustainably used
ecosystems. Aiming towards sustainability, one of the most promising
solutions can be the expanded use of plant growth promoting rhizobacteria
(PGPR) as assistance to plants in preventing or minimizing the environmental
stresses [2]. In support of role of PGPR in agriculture, Glick (2014) [3] quoted
“Scientists have dramatically increased our knowledge of the mechanisms
employed by PGPR in the past 15-20 years, additional understanding of the
fundamental mechanisms employed by these bacteria will likely hasten the
acceptance of these organisms as suitable and effective adjuncts to agricultural
practice”. To understand the role of PGPR, it is important to understand the
different mechanisms of action of PGPR, its potential in the field of agriculture
and the constraints associated with its large-scale usage among the farming
population. All these aspects have been discussed concisely and holistically in
this chapter.
Plant growth promoting rhizobacteria
The term rhizobacteria was used for the first time by Kloepper and
Schroth (1978) during the Fourth International Congress of bacterial plant
pathogen held at France. Based on their microbiological experiment on radish
crop, they used the term ‘rhizobacteria’ for those bacterial species which were
able to compete with other microbes and efficiently colonized the root surfaces
followed by growth stimulation and reduced incidence of diseases. Later in
1981, Kloepper and Schroth renamed it as ‘Plant growth promoting
rhizobacteria’ (PGPR). Plant growth promoting rhizobacteria is an
indispensable part of rhizosphere biotic community expressing positive root-
microbe interaction and can be defined as, a heterogeneous group of bacteria
that can be found in the rhizosphere and can improve the extent or quality of
plant growth directly and/or indirectly. Out of the 15% bacterial species
occupying the root volume, PGPR share is 2-5% as not all bacteria are having
plant growth promoting traits [4]. There are certain criteria which a bacterial
species must fulfill in order to be grouped under PGPR such as
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i) They should be able to compete with other rhizospheric
microorganisms.
ii) They should be compatible with other microbes.
iii) They should be easy to multiply.
iv) They should be safe to the environment and above all.
v) They should have the potential to enhance plant growth and
productivity [5].
Many bacterial genera are known to possess these characteristics. Both
gram positive and gram-negative genera are known to behave as PGPR.
Further PGPR can be divided into two different classes based on its site of
existence i.e., extracellular PGPR (e-PGPR) and intracellular PGPR (i-PGPR).
e-PGPR are found in the rhizosphere, on the rhizoplane or may be inside the
spaces between the cortical root cells whereas, i-PGPR are having intracellular
occurrence inside the specialized nodular structures of root cells. Some
bacteria genera belonging to e-PGPR are Agrobacterium, Arthrobacter,
Azotobacter, Azospirillum, Bacillus, Burkholderia, Erwinia, Flavobacterium,
Micrococcus, Pseudomonas, Serratia etc. whereas examples of i-PGPR are
nodule forming genera of Allorhizobium, Bradyrhizobium, Mesorhizobium,
Rhizobium, etc. Microscopic view of some potentially used rhizobacteria for
plant growth and stimulation has been presented in Figure 1.
Fig 1: Microscopic view of different genera of PGPR used in plant growth promotion
and stimulation
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Direct and indirect mechanism of action of PGPR
It is very important to understand the different mechanisms through which
PGPR affects plant growth. Broadly, these mechanisms can be divided into
two categories i.e., direct and indirect based on the kind of interaction of these
PGPR with their host plants (Figure 2). Direct mechanism include those that
influence the balance of plant growth regulators either because the micro-
organisms release growth promoting substances which are integrated into
plant or help in mobilizing nutritionally important elements which in turn
induce better metabolism of plant [6]. Indirect mechanisms on the other hand
are those in which participation of the plant defensive metabolic process is
required which may be in form of signal sent from bacteria which influences
the plants. Direct mechanisms includes nitrogen (N) fixation, solubilization of
phosphorus (P) and potassium (K), release of plant growth stimulating
hormones and production of siderophore. Indirect mechanisms also include
production of siderophore, antibiotics, exopolysaccharides, hydrolytic
enzymes and mechanism of induced systemic resistance.
Fig 2: Different mechanism of action of PGPR

Based on the different mechanism of action of PGPR, we can emphasize
on the role of PGPR as potential biofertilizers, phytostimulant and biopesticide
which have been discussed in the following sections and defined in Table 1.
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Table 1: Role of plant growth promoting rhizobacteria
Term Definition Mechanism References

A substance which contains live Biological nitrogen
microorganisms which when fixation
applied to the seed, plant or the Utilization of
soil, colonizes the rhizosphere or insoluble forms of [7]
Biofertilizer
the interior of the plant and phosphorus.
promotes the growth through
increased supply/or availability of
primary nutrients to the host plant.
Microorganism with the ability to Production of
produce or change the phytohormones
[8]
Phytostimulation concentration of growth regulators (auxins, cytokinins
such as IAA, GA, cytokinins and and GA).
ethylene.
Microorganisms that promote Production of
plant growth through the control of antibiotics and
phytopathogenic agents, mainly production of
for the production of antibiotics hydrolytic
and antifungal metabolites. enzymes [7, 8]
Biopesticides
Competitive
exclusion, acquired
and induced
systemic
resistance.
Role of PGPR as a potential biofertilizer

Biofertilizers, are any product or substances carrying one or more strains
of live microorganisms which when applied to plant surface, soil or seed
surface helps in mobilizing nutritionally important elements from unusable
form to usable form either by nitrogen fixation, solubilization of P and K and
other essential elements. Here, it is important to understand that biofertilizers
and PGPR are two different terms though it is used interchangeably in many
literatures. The subtle difference between the two lies in their domain. Plant
growth promoting rhizobacteria includes only the beneficial bacteria annd
hence only those bacteria which help in nutritionally enriching the soil by one
or more mechanisms, may be included under biofertilizers wheras, the broad
categorization of biofertilizers also includes Arbuscuar Mycorrhiza (AM) and
actinomycetes (e.g. Frankia) other than bacterial population.
Some PGPR strains which are able to fix atmospheric N either
symbiotically in assocaition of host plant or asymbiotically in free living
condition, are of great importance as nitrogenous biofertilizer. Rhizobium sp.
are able to form nodule in the roots of compatible leguminous plants (cross
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inoculation group) as a result of some chemical signals secreted by plants
(flavonoids). The atmospheric N is taken up and reuced to ammonia by these
bacteria. The bacterium grows at the expense of energy supplied by
carbohydrates from the host, and provides fixed nitrogen for amino acid
biosynthesis in return [9]. Other examples of N fixing stains are Azotobater,
Azosprillum, Herbaspirillum, Bukholderia, Bacillus, and Penicillus [10].
Besides, the studies on enhaced nitrogen availability by PGPR in leguminous
crop [11] N fixation have also been studied in many non leguminous crops [12].
Phosphorous (P) is another macronutrient which is poorly available to plants
due to formation of insoluble complexes of P with elements such as Fe, Ca,
Al. Only monobasic (H2PO4-) and dibasic (HPO42-) forms of P are absorbed
by plants. In this respect, PGPR possessing the ability of P solubilization and
mineralization can play vital role in biochemical cycles and determining the
overall P supply to plants. Solubilization of P by microorganisms was
reported by Pikovskaya in the year 1948. Many bacterial genera like
Azotobacter, Bacillus, Beijerinckia, Burkholderia, Enterobacter, Erwinia,
Flavobacterium, Microbacterium, Pseudomonas, Rhizobium Serratia and
Azospirillum are reported as the most significant PSB [12, 13]. Phosphorus
solubilizing bacteria are reported to release low molecular weight organic
acids e.g., oxalic acid, malic acid, citric acid, acetic acid, lactic acid etc. [14] in
the soil environment which in turn dissociates to form H+ and organic anions.
The proton released reduces the pH of the soil and helps in dissolution of
insoluble phosphates and the organic anions forms chelate with Fe, Ca, Al and
helps in release of bound P. Similarly, there are certain groups of
microorganism which have the potential to solubilize unavailable forms of K
to available forms. The main mechanism involved in this is the release of low
organic weight organic acids which help in reducing the pH of the soil solution
and also the anion part of these organic acids are capable of forming chelate
with Si, Al and Fe of the mineral thereby releasing K in soil solution and
enhancing its availability to plants [15]. A wide range of bacteria namely
Pseudomonas sp., Burkholderia sp., Acidithiobacillus ferrooxidans, Bacillus
mucilaginosus, Bacillus edaphicus, B. circulans and Paenibacillus sp., etc.
has been reported to release potassium in accessible form from potassium-
bearing minerals in soils [16]. They are also known to improve availability of
micronutrient such as iron. Many strains of PGPR are known to produce
siderophore which is a low molecular weight organic compounds (500-1000
Da) produced by microorganisms under iron-limiting conditions that enhance
iron uptake capacity [17]. The role of siderophores is primarily to scavenge Fe,
but they also form complexes with other essential elements (i.e., Mo, Mn, Co
and Ni) in the environment and make them available for microbial cells [18].
These PGPR acting as biofertilizers affect plant growth and growth related
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parameters which ultimately have its influences crop yield. Some of the PGPR
and their effect on crop growth have been enlisted in Table 2.
Table 2: Growth promoting effects of PGPR on different crops
Crop PGPR Response Reference

Pseudomonas
Phaseolus fluorescens P-93; Increased nodule number seed [18]
vulgaris L Azospirillum lipoferum S yield and protein content.
21; Rhizobium sp.
Saccharum Increased plant uptake of N, P [14]
Klebsiella variicola
officinarum and K
Klebsiella sp. Br1;
Klebsiella pneumoniae Fr1; Improved ear yield and [19]
Zea mays
Bacillus pumilus S1r1 nitrogen fixation
and Acinetobacter sp. S
Azospirillum sp.
Triticum Increased root growth and [20]
Azoarcus sp.
aestivum nitrogen accumulation
and Azorhizobium sp.
Oryza [21]
Bacillus and Citrobacter Enhanced growth
sativa
Brassica [22]
Paenibacillus polymyxa Enhanced N-fixation and groth
napus
Increase in plant growth,
nodulation and enzyme activity
over Rhizobium-inoculated
and uninoculated control pla
increase in plant growth,
Azotobacter chroococcum;
Cajanus Azospirillum brasilense;
over Rhizobium-inoculated [23]
cajan Pseudomonas
fluorescens, Pseudomonas
increase in plant growth,
putida and Bacillus cereus
over Rhizobium-inoculated
Enhanced plant growth,
nitrogenase activity nodulation
Azospirillum lipoferum,
Curcuma Increased yield and plant [24]
Trichoderma viride,
longa growth
Bacillus megaterium
Increased root and shoot dry
Cicer [25]
Mesorhizobium ciceri weight, chlorophyll content,
arietinum
nodulation
Role of PGPR as potential phytostimulator

Plant growth regulators (phytostimulators) are organic molecules that
affect growth and development of plants in low concentrations. Common
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groups of phytohormones along with its functions in plant are presented in
Table 3. Some bacterial species are also known to modify the concentration
of these phytohormones and affect plant growth. The PGPRs reported to act
as phytostimulators includes the genera Rhizobium, Bradyrhizobium,
Pseudomonas, Paenibacillus, Mesorhizobium, Bacillus, Rahnella, Pantoea,
Arthrobacter Pseudomonas, Herbaspirillum, Enterobacter, Brevundimonas
and Burkholderia [8].
Table 3: Common groups of phytohormones along with its functions in plant
Plant growth
Functions
hormone
Auxins or Indole- Promotes stem elongation, adventitious root initiation and fruit
3-acetic acid growth, inhibit lateral bud outgrowth.
Promotes seed germination and emergence, floral induction, flower
Gibberellin (GA)
and fruit development, and stem and leaf growth.
Stimulate cell division, inhibit leaf senescence and vascular
differentiation, induce the proliferation of root hairs, formation of
Cytokinin embryo vasculature, nutritional signaling, leaf expansion,
branching, chlorophyll production, promotion of seed germination,
and delay of senescence.
Promotes fruit ripening, seed germination, flowering, root and
Ethylene
shoot initiation, elongation and branching.
Role of PGPR as a potential biocontrol agent

Plant pathogens are microbes that affect growth of plant and reduce the
desired yield. In natural environment, plants always remain in contact with
pathogenic microbes (bacteria, fungi, viruses, etc.) but all of them do not cause
disease to the plants because plant growth conditions are not favorable for
many pathogens and plant's defense system also protects the plant from
pathogen's attack. The protection of crop against plant pathogens is generally
done by application of chemical pesticide and fungicide, but the application
of these toxic chemical agents for disease control causes serious
environmental impacts and results in development of pathogen resistance
against these chemicals. It has been reported that rhizospheric microbes
(PGPR) play key role in protection of host plants against pathogens [26] and are
addressed as biocontrol agents. Common methods for microbial-based
pathogen control are antagonism, indirect inhibition by competition,
activation of plant's defense system, etc. Rhizospheric microbes release some
antagonistic compounds that causes direct inhibition of plant pathogens [27].
For example, Bacillus, Aeromonas, Alcaligenes, Pseudomonas, and
Rhizobium have been reported to produce compounds like hydrogen cyanide
(HCN) that inhibits growth of pathogens causing bacterial canker and black
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rot in tomato and tobacco [28]. Pseudomonas strains GRP3A and PRS9
compete with other disease-causing microbes through production of iron
chelating siderophores in maize that help to acquire iron in ferric ion form,
and the iron became unavailable to other microorganisms [29].
Commercialization of PGPR and related constraints
Many researchers have studied the potential of bacterial communities to
highlight the effectiveness of PGPR over conventional fertilizers and the
extent to which the use of conventional fertilizers could possibly be replaced.
Sood et al. (2018) [30] showed positive effects of combined application of
indigenous plant-growth promoting rhizobacteria (PGPR) and chemical
fertilizers on productivity of wheat and soil properties. Three PGPR isolates
(B2, SIR1 and BIS2) with maximum PGP traits were screened at different
doses of nitrogen (N) and phosphorus (P) (80%, 60% and 40% of
recommended fertilizer dose, RFD) under net-house conditions followed by
field experimentation. Application of 80% RDF of NP with PGPR (B2)
significantly increased wheat yield, number of tillers per plant, 1000-grain
weight and biomass relative to the un-inoculated control with 100% RFD thus
suggesting reduction of chemical fertilizers by 20% without hampering the
soil fertility and productivity of wheat. Use of microorganisms in agriculture
have shown comparative advantages over agricultural chemicals as these are
safe to the environment, safe to use and effective even in less amount and they
may be applied along with chemical fertilizers [31]. However, most often the
results of laboratory experiments are not found to be consistent with field
performance, which is a major hindrance for commercialization of these
microbes. Apart from this, there are other factors which make
commercialization of PGPR a tough job. The poor shelf life of PGPR strains
is a major setback. For most of the commercially available PGPR strains, the
expiry date is of 6 months however, the population declines rapidly just after
about 3 months. The demand for PGPR formulation is often inconsistent and
seasonal and so proper storage condition is required. Moreover, use of PGPR
is a cheaper source of improving plant growth than chemical fertilizers.
However, due to less demand its overall production cost increases. Also,
PGPR strains are considered to have the feature of host specificity and so the
combination of bacterial strains used on one crop as PGPR may not perform
equally well when inoculated on another crop because of crop incompatibility.
Strict and complicated regulatory issues also make commercialization of
PGPR very challenging. In every country, there are different risk assessments
and efficacy testing policies to avoid unregulated release of potentially
damaging biological organisms. Above all, lack of awareness among our
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farmer friends and lack of information on field performances created
uncertainty in farmer’s community about its use and performance [32].
In order to ensure successful effect of PGPR inoculation detailed
investigation of various factors such as plant/crop type, soil type, nutrient, soil
microbiome, ambient environment, agronomic practices and type of
inoculants and carrier used for crop improvement needs to be considered. All
these factors together regulate the effectiveness of inoculants and therefore
needs to be considered. Besides, efficient delivery procedure and carriers must
be selected as per the need which may help in fast growth of inoculants. It has
been also reported that use of indigenous microbes can be a better option as
they possess good adaptation and competitive power [33]. It is also
recommended to use bio inoculants that perform wide functions like
solubilization of P, fixation of N, and promoting growth of roots; biocontrol
agents as a mixture for better results. While commercializing rhizobacteria for
plant growth in mixture, in combination with chemical fertilizer and chemical
pesticide, many ecological aspects need to be considered like sensitivity of bio
inoculant with pesticide, shelf life, cost of production, application procedure,
and regulatory aspects [34]. Delivery of microorganisms is performed by
sprays, root dips, and dry in furrow as the time of planting, but large-scale
application will require large amount of inoculums. Pretreatment of seeds with
inoculum is a good approach for transfer of inoculum to soil for better
colonization.
Conclusion
In today’s scenario, in order to fight the major challenges, PGPR may be
considered an ecofriendly and sustainable approach. Various PGPR strains
have been reported till date which can significantly increase acquisition of
essential plant nutrients along with crop productivity. The performance of
crops under stress condition can be improved through the use of PGPR
inoculation which in turn can also increases crop productivity through various
mechanism of interaction with host. However, for various shortcomings and
constraints in microbial inoculation, this technology is not popularized on a
large scale. The current need is high output yield and enhanced production of
the crop at the same time maintaining soil health and improving fertility of
soil in a sustainable manner. Hence, the future research has to be focused on
the new concept of rhizo-engineering which create a unique setting for the
interaction between plant and microbes. Future research in rhizosphere
biology will depend on the development of molecular and biotechnological
approaches to enhance our knowledge of rhizosphere biology and to aim
towards better management of soil microbial populations. Fresh alternatives
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should be explored for the use of bio inoculants for other high value crops
such as vegetables, fruits, and flowers especially indigenous microbial
populations should be explored as potential PGPR.
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Nutrient Availability and Plant Productivity

Chapter · July 2022

Khushboo Rani Arkaprava Roy

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Abinash Das Ankita Trivedi

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Chief Editor: Dr. Sweta Mishra

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Chapters Page No.

2. Superiority of Black Rice over Normal Rice Variety 19-31

3. Genome Editing: A Way Forward for Crop Improvement 33-48

4. Protein Bio-Synthesis/Translation in Prokaryotes & Eukaryotes 49-64

5. Role of Biological Ice Nucleation in Cloud Modification 65-74

6. Different Types of PCR and It’s Applications in Agricultural

7. Whole Genome Strategies for Marker Assisted Selection in Plant

8. Earthworms Bio Markers in Soil Pollution Assessment 111-136

9. Nutrient Availability and Plant Productivity through PGPR:

Kusumkali Manipuri chakhao

Fig 1: A graphical comparison of biochemical between sample 1 and 2

Fig 1: Structure of Prokaryotic and Eukaryotic mRNA

Fig 2: Secondary Structure of Aminoacyl tRNA

Fig 3: Translation initiation in Prokaryote

Fig 4: Translation Elongation in Prokaryotes

Fig 5: EF-Tu-Ts exchange cycle

Fig 7: Translation initiation and elongation in Eukaryotes

Prokaryotic Eukaryotic Function

Table 2: Comparison of Translation Process in Prokaryotes and Eukaryotes

Source: Tolnai et al. 2019.

Source: Chang-Hui Shen, 2019.

Source: Megan Bergkessel and Christine Guthrie, 2013.

Source: Shen, 2019.

Source: Clark and Pazdernik, 2015.

Source: Pignatelli et al. 2019.

Source: Radhamony et al. 2005.

Regional genome analysis Whole genome analysis

Fig 2: The role of earthworm biomarkers in assessment of soil pollutant

5. Soil pollution assessment using earthworm

6.1.1.2 Morphological changes in earthworm E. eugeniae exposed to

6.1.2 Assessment of herbicide and fungicide toxicity by Eisenia fetida

Combined toxicity (1:1

1007.3 746.3 926.7 478.7

Eisenia fetida, a soil earthworm, was treated with tribenuron-methyl

Fig 5: Effects of tribenuron-methyl (A), tebuconazole (B) and mixture of both

6.2 Heavy metals and other inorganic metalloid

6.2.1.2 Eisenia Andrei’s genotoxicity biomarkers sensitivity to copper

Fig 9: Following in vivo exposure to copper at escalating concentrations, DNA

6.2.2.2 Effect of cadmium stress on GST/GSH level in Allolobophora

6.4 Plastic and Microplastic

Fig 2: Different mechanism of action of PGPR

Term Definition Mechanism References

Role of PGPR as a potential biofertilizer

Crop PGPR Response Reference

Role of PGPR as potential phytostimulator

Role of PGPR as a potential biocontrol agent

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