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 Advanced
Botany
Volume - 5

Chief Editor
Dr. Hanmant Raghunath Aglave
Principal & Head Department of Botany, Lokmanya Mahavidyalya Sonkhed,
Nanded, Maharashtra, India

Co-Editor
Dr. Sanjib Baruah
Assistant Professor, Department of Botany, Bodoland University, Assam,
India

®
AkiNik Publications
New Delhi
Published By: AkiNik Publications

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Chief Editor: Dr. Hanmant Raghunath Aglave

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Publication Year: 2024
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ISBN: 978-81-968391-7-8
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 Preface

Plants are a kingdom of life forms that includes familiar organisms such as
trees, herbs, bushes, grasses, vines, ferns and mosses. Plants are crucial to the
existence of all other living creatures on Earth, both through the systemic life-
support services they sustain and the food, medicine and other material
resources they provide. The total number of described plant species hovers
around 250,000.
Botany is a branch of biology that deals with the study of plants, including
their structure, properties, and biochemical processes. It included are plant
classification and the study of plant diseases and of interactions with the
environment. The principles and findings of botany have provided the base for
such applied sciences as agriculture, horticulture, and forestry. The early
recorded history of botany includes many ancient writings and plant
classifications. Modern botany is a broad, multidisciplinary subject with
contributions and insights from most other areas of science and technology.
This edited book, “Advanced Botany”, consists of seven articles written
by experts in the field of botany. This book will be helpful for students and
researchers who have a keen interest in knowing the current development of
the field of botany. The editors remain grateful to AkiNik publications and the
authors for being given the opportunity to review the present book.

Editors
 Contents

S. No Chapters Page No.

1. Antioxidants in Plants 01-34
(Navyashree R.)

2. Synthetic Promoters and Plant Development 35-52

(Kaveri Chawan)

3. Genetic Enhancement in Pre-Mendelian Era and 21st 53-63

Century
(A. Krishnamoorthi and V. Lakshmi Prasanna Kumari)

4. Fertility Variation in Forest Tree Seed Orchards: 65-76

Implications for Genetic Diversity and Conservation
(Rakesh M. Jaliya)

5. Molecular Characterization of Phytoplasma: An Overview 77-99

(Shoeb Ahmad, Shveta Malhotra, Syed Saad, Akil Ahmad Khan and
Azahar Sajjad)

6. Ethnobotanical Importance and Pharmacological Action of 101-111

Moringa oleifera
(Vidyan Kumari)

7. Diversity of Loranthaceae Juss. Members Occurring in 113-126

Chirang Reserve Forest, BTR, Assam, India
(Sanswrang Basumatary and Sanjib Baruah)
 Chapter - 1
Antioxidants in Plants

Author
Navyashree R.
Ph.D. Scholar, Department of Crop Physiology, University of
Agricultural Sciences, Dharwad, Karnataka, India

Page | 1
Page | 2
 Chapter - 1
Antioxidants in Plants
Navyashree R.

Abstract
Plants are necessary to sustain life on this planet for their oxygen-giving
qualities and as a key link in the food chain. Plants are rich sources of
phytoconstituents such as carotenoids, flavonoids, vitamins, alkaloids,
polysaccharides, antioxidants and active peptides. The antioxidants have
various benefits for human health, such as anti-inflammatory, anti-ageing,
anti-cancer, and neuroprotective effects. Considering their important health
effects, the efficient extraction methods of natural antioxidants, the
appropriate assessment of antioxidant activity, and their main resources from
food and medicinal plants, they are drawing great attention in food science
and nutrition. The present study deals with in-depth study of the antioxidant
activity of the plants, their mechanisms and importance.
Keywords: Antioxidants, chloroplast, oxidative stress, mitochondria, plants
Introduction
Antioxidants have essential roles in some plants' signalling and defence
mechanisms, where they serve as precursors to more complex compounds, as
a regulator of plant growth, and as a defence system against pathogenic
organisms and predators. The extraordinary variety of chemical structure and
substitution found in plant antioxidants makes them an inexhaustible source
of interesting compounds capable of combating reactive oxygen/nitrogen
species (ROS/RNS) and stimulating the activation of signal cascades within
cells. Antioxidants detoxify these hazardous chemicals through intricate
methods that entail either direct or indirect interaction with radicals.
Antioxidants prevent or quench free radical processes primarily by their
reducing or hydrogen atom-donating capability, solubility, and chelating
characteristics. Furthermore, their ability to control critical metabolic
enzymes and activate/block gene transcription is crucial.

Page | 3
 When present at lower quantities than the substrate, antioxidants
significantly delay or prevent oxidation of oxidizable substrates. 1.
Antioxidants can be produced in the body (e.g., reduced glutathione (GSH),
superoxide dismutase (SOD), and so on) or consumed as dietary
antioxidants. Exogenous (i.e., dietary) antioxidants have long been found in
plants. It is estimated that two-thirds of the world's plant species have
medicinal value, and nearly all of these have high antioxidant potential. 3.
The discovery and subsequent isolation of ascorbic acid from plants sparked
interest in exogenous plant antioxidants. 4. Since then, increasing oxidative
stress has been discovered as a major causal component in the development
and progression of various life-threatening disorders, including neurological
and cardiovascular disease. Furthermore, supplementing with exogenous
antioxidants or strengthening the body's natural antioxidant defences has
been demonstrated to be a viable approach of combating the negative
consequences of oxidative stress.
Why do all plants have antioxidant potential?
Within plant cells, the two primary powerhouses and locations of
reactive oxygen species (ROS) creation are chloroplasts and mitochondria.
These compounds are also engaged in maintaining a delicate balance
between energy-related functions and ROS production management.
Peroxisomes, solitary membrane-bound subcellular organelles, are a third
key location of ROS production within plant cells, producing hydrogen
peroxide (H2O2), superoxide (O2-), and nitric oxide (NO). Peroxisomes
contain fundamental enzymatic elements such as catalase (CAT) and flavin
oxidases that produce hydrogen peroxide (H2O2). ROS are generated within
the plant cell at photosystem I and II (PS I and PS II) of the chloroplasts, the
peroxisome membrane and matrix, and complex I, ubiquinone, and complex
III of the mitochondrial electron transport chain (ETC). Under normal
physiological conditions, electrons escape from the chloroplast PS I and PS
II membranes, the mitochondrial ETC membrane, and the peroxisome.
These electrons are then combined with molecular oxygen to form the
superoxide radical (O2). The superoxide radical is next transformed to the
hydroperoxyl radical (HO2) and then to H2O2. In addition to ROS, reactive
nitrogen species (RNS) such as nitric oxide radical (NO•) and peroxinitrite
(ONOO-) are generated in numerous cell compartments such as chloroplasts,
mitochondria, and peroxisomes. The third sort of free radical, reactive
sulphur species (RSS), is said to be produced by the interaction of thiols with
ROS. Figure summarises the overall process of free radical generation.

Page | 4
These free radicals are constantly created in live cells' subcellular organelles.
Because free radicals operate as signalling molecules, their production is
usually genetically programmed. Overproduction of free radicals, on the
other hand, can occasionally damage biomolecules such as DNA, proteins,
and lipids.

Oxidative stress
Oxidative stress is a complicated chemical and physiological
phenomenon that occurs in conjunction with practically all biotic and abiotic
stimuli in higher plants and results from the overproduction and
accumulation of reactive oxygen species (ROS). Oxidative stress occurs
when crop plants are subjected to harsh abiotic conditions, which cause an
increase in the generation and accumulation of reactive oxygen species
(ROS). Drought, high temperatures, heavy metals, salinity, and UV radiation
are examples of harsh abiotic conditions or stresses that induce yield and
quality losses in crops.

Page | 5
What are free radicals?
 Any molecule containing one or more unpaired electrons.
 These unpaired electrons readily form free radical molecules which
are chemically reactive and highly unstable.
An atom or molecule that has one or more unpaired electrons in the
valency shell or outer orbit and is capable of independent existence is
referred to as a free radical. A free radical is unstable, short-lived, and
extremely reactive due to its odd number of electrons.

Page | 6
Properties of free radicals
1. Highly reactive.
2. Very short half-life.
3. Generate new radicals by chain reaction.
4. Cause damage to biomolecules, cells and tissues.
Types of free radicals
1. Superoxide (O2-).
2. Hydrogen peroxide (H2O2).
3. Hydroxyl radical (OH-).
4. Singlet oxygen (1O2).
5. Hydroperoxi radical (HOO-).
6. Lipid peroxide radical (ROO-).
7. Nitric oxide (NO-).
8. Peroxinitrite (ONOO-).

Oxidative stress in plants and its consequences

An oxygen-containing unstable molecule that readily interacts with
other molecules in a cell. Free radicals (O2.-, OH.) and non-free radicals
(H2O2, 1O2, which are particularly reactive) in the biological system are
collectively referred to as reactive oxygen species in a wide sense. Cells,
proteins, and DNA can be harmed by oxidative stress, which can speed up
ageing.
However, excessive ROS can result in oxidative stress, a condition
where there is an imbalance between the production of ROS and the
antioxidants' ability to neutralise free radicals. This imbalance damages
cellular components like lipids, nucleic acids, metabolites, and proteins and
ultimately causes plant cell death.

Page | 7
Page | 8
How are reactive oxygen species produced
The majority of reactive oxygen species are produced during
mitochondrial electron transport as byproducts. Additionally, ROS are
produced as essential by products of oxidation reactions that are catalysed by
metals.
Mitochondria
The release of electrons from donor redox centres to molecular oxygen
is what causes ROS to form. The molecular oxygen that is reduced by four
electrons to water at complex IV is the ultimate acceptor of an electron's
charge in the mitochondrial electron transport chain, which is made up of
various complexes with numerous redox centres. Superoxide radical (O2) is
created by premature single electron reduction of molecular oxygen earlier in
the cycle, whereas H2O2 is created by divalent reduction. Superoxide is
changed to H2O2 by superoxide dismutase-2 in the matrix, which may then
escape and be measured in the surrounding medium. Complex I and complex
III produce the majority of mitochondrial ROS. Because the Q cycle permits
superoxide escape into the intermembrane space, whereas Complex I ROS
remain in the lumen where they can be detoxified by antioxidant enzymes,
Complex III is the most likely source of cytosolic ROS.

Page | 9
 In addition to possessing an O2 and carbohydrate-rich environment,
plant mitochondria also participate in photorespiration, setting them apart
from their animal counterparts. The primary offender is the mitochondrial
ETC (mtETC), which contains enough energetic electrons to degrade O2 and
produce ROS. Complex I and Complex III are the two main elements of the
mtETC that produce ROS. In its flavoprotein region, Complex I, also known
as NADH Dehydrogenase, directly converts O2 to O•2. Due to a paucity of
NAD+-linked substrates, the ROS generation at Complex I is further
increased when there is a reverse electron flow from Complex III to
Complex I. ATP hydrolysis regulates the flow of electrons in reverse. In
Complex III, ubiquinone contributes an electron to Cytochrome c1 in its
fully reduced form, leaving behind an unstable ubisemiquinone semi-radical
that encourages electron leakage to O2 and the production of O•2.
The different enzymes found in the mitochondrial matrix are additional
sources of ROS generation in the mitochondria. This includes both enzymes
that directly make ROS, such as aconitase, and those that produce ROS
indirectly, such as 1-Galactono--lactone dehydrogenase (GAL), which does
this by providing electrons to the ETC. O•2 is the main ROS in the
mitochondria, however the Mn-SOD and the APX turn it into H2O2.
According to estimates, the mitochondria redirect 1–5% of their total O2
consumption to produce H2O2. When there is abiotic stress, mitochondrial
production of reactive oxygen species (ROS) is significantly increased. Such
demanding circumstances have an impact on the close linkage between ETC
and ATP synthesis, which results in an excess reduction of electron carriers
like the ubiquinone (UQ) pool and the production of ROS. Due to the
increased respiratory rate brought on by drought, there is an increase in
mitochondrial ATP synthesis to make up for the decreased rate of
chloroplast ATP synthesis, which increases the formation of mitochondrial
ROS. Two enzymes, Mitochondrial Alternative Oxidase (AOX) and
Mitochondrial SOD (Mn-SOD), are essential for reducing this oxidative
stress in the mitochondria. The AOX reduces ROS production while
maintaining the UQ pool's decreased condition.

Page | 10
 During the process of oxidative phosphorylation, the electron transport
chain, which is located on the inner mitochondrial membrane, is primarily
where mitochondrial ROS are produced. Superoxide is created when oxygen
is partially reduced as a result of electron leakage at complexes I and III of
the electron transport chains. Two dismutases, superoxide dismutase (SOD)
in the mitochondrial matrix and superoxide dismutase (SOD1) in the
intermembrane space, then promptly dismutate superoxide to hydrogen
peroxide. Superoxide and hydrogen peroxide produced during this process
are regarded as mitochondrial ROS collectively.

Page | 11
Chloroplast
ROS are continuously produced by photosynthesizing chloroplasts as a
result of many electron transfer processes when oxygen is present. The
production of ROS in chloroplasts is influenced by a number of
physiological processes, including as photosynthesis, gene expression, the
synthesis of chlorophyll (tetrapyrrole), and hormone regulation. For instance,
the amount of O2 O2•/H2O2 produced by photosystem I (PS I) varies in
response to changes in the rate of CO2 fixation and photosynthetic electron
transfer. Chloroplastic actions can be quickly regulated by extracellular
inputs, such as the plasma membrane receptors' detection of bacterial
components. The cues recognised by the apoplast during plant-pathogen
interactions set off MAPK cascades, which quickly down regulate
photosynthetic genes and cause H2O2 to build up in chloroplasts. Another
illustration is the momentary reduction in PS II's capacity to emit excessive
light energy as heat through non-photochemical quenching (NPQ) during the
beginning of pathogen detection. This drop in NPQ increases the likelihood
that ROS will be produced by chloroplasts, which may act as a priming
mechanism for chloroplast ROS signalling.

Page | 12
 Both biotic and abiotic stimuli cause the apoplast to produce ROS,
which is then transmitted to the interior of the cell, where the signal causes a
rise in chloroplastic ROS production. The signal can then be amplified by
the chloroplast and sent to the nucleus via a variety of cytosolic signalling
networks. The nucleus can also be directly contacted by apopplastic ROS
signalling via cytosolic pathways. Red arrows indicate long-distance
("systemic") signalling occurring across the entire plant, whereas yellow
arrows show intracellular transmission of apoplastic and chloroplastic ROS-
induced signals where they link up neighbouring cells (local signalling).
Reactive oxygen species (ROS) signalling networks connecting apoplast,
chloroplast and nucleus

Extracellular peroxidases (H2O2; hydrogen peroxide) and plasma

membrane-bound NADPH oxidases (Rboh) both generate apoptotic ROS.
After that, superoxide (O2O2•) is changed into H2O2. H2O2 (and possibly
O2O2•) may enter the cell through aquaporins in the plasma membrane
and/or may interact with apoplastic protein (AP) or transmembrane sensor
proteins (e.g., receptor-like kinases, RLKs) to change the expression of
certain genes through intracellular signalling pathways, such as those
involving MAPKs (mitogen-activated protein kinases). The chloroplast
detects extracellular ROS production by as-yet unidentified processes, and
the electron transfer chain (ETC) then produces more ROS as a result.

Page | 13
Singlet oxygen (1O2) is created in several ETC domains; O2O2•/H2O22O
should be modified to H2O2. Through known (such as EXECUTER1/2,
EX1/EX2, rupture of the chloroplast envelope) and unknown components of
the retrograde signalling, as well as hormone signalling, such as increased
production of the stress hormone salicylic acid (SA), elevated ROS inside
the chloroplast leads to transcriptional reprogramming. ROS may potentially
leak from the chloroplast to the cytoplasm through channel proteins (AQP).
The apoplast and chloroplast both use calcium (Ca2+) to control the
formation of ROS. However, the exact mechanisms are yet unknown. In the
latter instance, it functions via the sensory protein Calcium-Sensing Receptor
(CAS).
Mehler reaction in chloroplast
Alan H. Mehler, who reported findings indicating that isolated
chloroplasts convert oxygen to generate hydrogen peroxide (H2O2) in 1951,
is credited with the invention of the Mehler reaction. Mehler noted that the
H2O2 produced in this method is not an active intermediate in
photosynthesis; rather, as a reactive oxygen species, it can operate as an
oxidising agent and be harmful to nearby biological processes. In scientific
writing, the creation of H2O2 via photosynthesis is frequently referred to as
the Mehler reaction or the Water-Water Cycle. Sensu stricto, the Water
Water Cycle includes the Mehler Reaction, which reduces oxygen to make
H2O2, the Hill reaction, which splits water to create oxygen, and the
antioxidants' scavenging of this H2O2 to create water.

When there is a lack of CO2 fixation due to stress, the Calvin cycle
produces less carbon, which reduces the amount of oxidised NADP+ that can
be used as an electron acceptor during photosynthesis. Additionally, when
ferredoxin (Fd) is over reduced during photosynthesis electron transfer, an

Page | 14
electron may be transferred from photosystem I (PSI) to O2 to form O2,
which is a process known as the Mehler reaction.

Peroxisomes
Due to their intrinsic oxidative metabolism, peroxisomes single-
membrane-bound spherical microbodies are the primary sources of
intracellular H2O2 generation. During numerous metabolic processes, they
similarly to mitochondria and chloroplasts also create O•2. O•2 is produced in
two separate places. Both xanthine and hypoxanthine are converted into uric
acid by the Xanthine Oxidase, which is found in the peroxisomal matrix, and
O•2 is produced as a byproduct. The second is the peroxisomal membrane-
located NADPH-dependent small ETC, which uses O2 as the electron
acceptor and releases O•2 into the cytosol. It is made up of NADH and Cyt b.
Additionally, the three integral membrane proteins in charge of O•2 synthesis
are peroxisomal membrane polypeptides (PMPs) with molecular weights of
18, 29, and 132 kDa. The 18 and 32 kDa PMPs employ NADH as an
electron donor, while the 29 kDa PMP reduces cytochrome c using NADPH
as an electron donor. The ratio of CO2 to O2 decreases noticeably under
stressful circumstances, when water is scarce and stomata are closed, which
increases photorespiration and results in the synthesis of glycolate. This
glycolate is the main source of H2O2 during photorespiration because it is
oxidised by the glycolate oxidase in peroxisomes. Additionally, there are
additional metabolic pathways such the flavin oxidase pathway, the
disproportionation of O•2 radicals for peroxisomal ROS generation, and the -
oxidation of fatty acids.

Page | 15
 System for scavenging ROS in peroxisomes. ROS/RNS and
macromolecular ROS damage are indicated by red letters. Nonenzymatic
antioxidants are indicated with blue letters. For information, see the text.
GPX, glutathione peroxidase; GR, glutathione reductase; GSH, reduced
glutathione; GSSG, oxidized glutathione; H2O2, hydrogen peroxide; iNOS,
inducible nitric oxide synthase; L., lipid radical; LOO., lipid peroxyl radical;
LOOH, lipid peroxide; LOH, lipid alcohol; LONP2, Lon protease 2; NO,
nitric oxide radical.
Antioxidants
Antioxidants found in plants operate as a natural storehouse for
bioactive substances. They are crucial for the acclimatisation and
environmental adaption of plants.
 Antioxidants are substances that prevent oxidation or a substance
that shields cells from damage brought on by free radicals, unstable
molecules produced by the oxidation process during regular
metabolism.
They could be thought of as free radical scavengers. Plants have a robust
antioxidant defence system made up of enzymes and metabolites to stop
oxidative damage. The two main water-soluble antioxidant metabolites are
ascorbate (AsA, vitamin C), and glutathione (GSH), but secondary
metabolites such polyphenols, flavonoids, and terpenoids also contribute to
the detoxification of ROS under various environmental stressors.

Page | 16
Classification of antioxidants
There are numerous subcategories of antioxidants. They can be divided
into enzymatic and non-enzymatic antioxidants based on their action.
Antioxidants that use enzymes neutralise and eliminate free radicals to
operate. In a multi-step process, the antioxidant enzymes convert harmful
oxidative products to hydrogen peroxide (H2O2) and then to water when
cofactors like copper, zinc, manganese, and iron are present. Non-enzymatic
antioxidants function by halting the chain reactions of free radicals.
Examples of non-enzymatic antioxidants include glutathione, vitamin C,
vitamin E, plant polyphenols, and carotenoids.
The other method of classifying antioxidants is based on how soluble
they are in lipids or water. The antioxidants fall into two categories: lipid-
soluble antioxidants and water-soluble antioxidants. The cytosol and
cytoplasmic matrix of cells include water-soluble antioxidants, such as
vitamin C. Cell membranes hold the majority of the lipid-soluble
antioxidants, such as vitamin E, carotenoids, and lipoic acid.
Small-molecule antioxidants and large-molecule antioxidants are two
further categories for the antioxidants. The ROS are neutralised and removed
by the small-molecule antioxidants through a process known as radical
scavenging. Vitamin C, vitamin E, carotenoids, and glutathione (GSH) are
the primary antioxidants in this group. The enzymes (SOD, CAT, and
GSHPx) and sacrificial proteins (albumin), which absorb ROS and stop them
from damaging other crucial proteins, are examples of large-molecule
antioxidants.

Page | 17
Mode of action of antioxidants
Antioxidants can reduce oxidative damage indirectly by increasing the
activity or expression of intracellular antioxidant enzymes or directly by
interacting with free radicals and reducing oxidative damage.
Antioxidants may act on 2 folds:
1. Primary or chain breaking antioxidants: break chain reaction and
resulting radical is less reactive
ROO + AH→ ROOH + A
2. Secondary or Preventive antioxidants They may act either by
 Chelators/Deactivate metals.
 Scavenge singlet oxygen (highly toxic).
 Remove ROS.
Factors affecting the efficiency of antioxidants
 Activation energy of antioxidants.
 Oxidation/reduction potential.
 Solubility.
Enzymatic antioxidant
Antioxidants that use enzymes neutralise and eliminate free radicals to
operate. In a multi-step process, the antioxidant enzymes convert harmful

Page | 18
oxidative products to hydrogen peroxide (H2O2) and then to water when
cofactors like copper, zinc, manganese, and iron are present.
Superoxide dismutase (SOD)
SOD is a member of the metalloenzyme family, which is ubiquitous in
aerobic species. SOD serves as the first line of defence against ROS-caused
harm when the environment is stressed. The SOD dismutates O•2 into O2 and
H2O in order to catalyse its elimination. As a result, the Haber-Weiss
reaction can no longer produce OH•. Based on the metal ion it binds, SODs
are divided into three isozymes: Mn-SOD (found in mitochondria), Fe-SOD
(found in chloroplasts), and Cu/Zn-SOD (found in cytosol, peroxisomes, and
chloroplasts). Abiotic stress conditions have been shown to increase SOD
regulation (Boguszewska et al., 2010).
2O2·⁻+ 2H+ H2O2 + O2

SOD is present in essentially every cell in the body which actually

represented by a group of metalloenzymes with various prosthetic groups.
SOD appears in three forms: based on metal cofactors
a) Cu-Zn SOD: in the cytoplasm
b) Mn-SOD: in the mitochondria
c) Fe -SOD: in chloroplast
 The Cu/Zn-SOD has a central role in scavenging toxic oxygen
radicals
 It is the major form in the leaves and is responsible for 65-80%
of the total activity
This is the first line of defence to protect cells from the injurious effects
of superoxide.

Page | 19
Catalase (CAT)
The enzyme CAT, which is tetrameric and contains heme, catalyses the
dismutation of H2O2 into H2O and O2. Although it is less selective for
organic peroxides (R-O-O-R), it has a high affinity for H2O2. It is
exceptional among antioxidant enzymes in that it does not require a reducing
equivalent and has a very fast turnover rate (6 106 molecules of H2O2 to H2O
and O2 min1). Due to oxidative stress, purine catabolism, photorespiration,
and fatty acid -oxidation, peroxisomes are the hotspots for H2O2 generation.
Though considerable CAT activity has not yet been observed, new reports
reveal that CAT is also present in other subcellular compartments such as the
cytosol, chloroplast, and mitochondria. Three CAT genes have reportedly
been found in angiosperms. It has been discovered that CAT3 is expressed in
leaves and vascular tissues (localised in the mitochondria), CAT1 is
expressed in pollens and seeds (localised in peroxisomes and cytosol), CAT2
is primarily expressed in photosynthetic tissues but is also expressed in roots
and seeds (localised in peroxisomes and cytosol), and CAT3 is expressed in
seeds. Stressful situations need the cell to produce and use more energy.
Increased catabolism, which produces H2O2, satisfies this. CAT uses less
energy to extract the H2O2.
 Catalase is a common enzyme found in nearly all living organisms
exposed to oxygen (such as bacteria, plants, and animals) which
catalyzes the decomposition of hydrogen peroxide to water and
oxygen.
 Catalase is a tetramer of four polypeptide chains, each over 500
amino acids long.

Page | 20
Ascorbate peroxidase (APX)
The Ascorbate-Glutathione (ASC-GSH) cycle requires APX to function
properly. While CAT primarily scavenges H2O2 in the peroxisomes, APX
does the same in the cytosol and chloroplast. The APX uses Ascorbic acid
(AA) as a reducing agent to convert H2O2 to H2O and DHA. The APX
family is divided into five isoforms depending on amino acid sequences and
location, including cytosolic, mitochondrial, peroxisomal, and chloroplastid
(stromal and thylakoidal) (Sharma and Dubey, 2004). Because APX is more
extensively distributed and has a higher affinity for H2O2 than CAT, it is a
more efficient H2O2 scavenger during times of stress.
 Ascorbate peroxidase that uses ascorbate (AsA) as its reducing
substrate.
 It is an enzyme that catalyzes the chemical reaction
 L-ascorbate + H2O2 dehydroascorbate + 2 H2O

Page | 21
Glutathione peroxidase (GPx)
A heme-containing enzyme called GPX, which has monomers between
40 and 50 kDa, removes extra H2O2 both during regular metabolism and
during stress. By destroying indole acetic acid (IAA) and using H2O2 in the
process, it protects against biotic stress and is essential for the formation of
lignin. As electron donors, GPX favours aromatic substances like guaiacol
and pyragallol (Asada, 1999). GPX is viewed as the major enzyme in the
elimination of H2O2 since it is active intracellularly (cytosol, vacuole), in the
cell wall, and extracellularly.
H2O2+GSH→H2O+GSSG
 The biochemical function of glutathione peroxidase is to
reduce lipid hydroperoxides to their corresponding alcohols and to
reduce free hydrogen peroxide to water.
 GPx is a selenium-dependent enzyme

Glutathione reductase (GR)

GR is a flavoprotein oxidoreductase that converts GSSG to GSH by
using NADPH as a reductant. In order to regenerate AA from MDHA and
DHA, reduced glutathione (GSH) must be used up, and as a result, it is
transformed into its oxidised form (GSSG). To maintain a high cellular
GSH/GSSG ratio, GR, an essential enzyme of the ASC-GSH cycle, catalyses
the creation of a disulfide bond in glutathione disulfide. Small amounts are
also present in the mitochondria and cytoplasm, but its concentration is
mostly found in chloroplasts. GSH, a substance with a low molecular weight,
functions as a reductant to stop the oxidation of thiol groups and interact
with harmful ROS members like 1O2 and OH•.
GSSG+NADPH→2GSH+NADP+

Page | 22
  Glutathione-disulfide reductase (GSR), often referred to as
glutathione reductase (GR).
 Glutathione reductase is an enzyme that catalyses the conversion of
glutathione disulfide (GSSG) to glutathione sulfhydryl form (GSH),
which is essential for protecting cells from oxidative stress and
preserving the reducing environment.

Monodehydroascorbate reductase (MDHAR)

Using NADPH as a reducing agent, MDHAR is in charge of rebuilding
AA from the transitory MDHA, ultimately replenishing the cellular AA pool.
It co-localizes with the APX in the peroxisomes and mitochondria because it
regenerates AA, and APX scavenges H2O2 while oxidising AA (Mittler,
2002). MDHAR has a number of isozymes that are restricted to the cytosol,
chloroplast, mitochondria, peroxisomes, and glyoxysomes.
MDHA+NADPH→AA+NADP+

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Dehydroascorbate reductase (DHAR)
Using Reduced Glutathione (GSH) as an electron donor, DHAR
converts dehydroascorbate (DHA) to AA (Eltayeb et al., 2007). This makes
it an additional agent that regenerates the cellular AA pool in addition to
MDHAR. It is essential for maintaining the redox status of the plant cell by
controlling the size of the AA pool in both the symplast and the apoplast
(Chen and Gallie, 2006). Seeds, roots, and both green and etiolated shoots
are rich sources of DHAR.

Non – enzymatic anti-oxidants

Non-enzymatic antioxidants function by halting the chain reactions of
free radicals. The non-enzymatic antioxidants, which include AA, GSH, -
tocopherol, carotenoids, phenolics, flavonoids, and the amino acid cum
osmolyte proline, make up the other half of the antioxidant machinery. They
play a crucial function in plant growth and development by adjusting cellular
processes like mitosis, cell elongation, senescence, and cell death. They not
only serve to protect various components of the cell from injury.

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Ascorbic acid (vitamin C)
AA is the most abundant and the most extensively studied antioxidant
compound. It is considered powerful as it can donate electrons to a wide
range of enzymatic and non-enzymatic reactions. Majority of AA in plant
cells is the result of Smirnoff-Wheeler pathway, catalyzed by L-galactano-γ-
lactone dehydrogenase in the plant mitochondria, with the remaining being
generated from D-galacturonic acid. 90% of the AA pool is concentrated not
only in the cytosol, but also substantially in apoplast, thus making it the first
line of defense against ROS attack (Barnes et al., 2002). AA is oxidized in
two successive steps, starting with oxidation into MDHA, which if not
reduced immediately to ascorbate, disproportionates to AA and DHA. It
reacts with H2O2, OH•, O•−2, and regenerates α-tocopherol from tocopheroxyl
radical, thereby protecting the membranes from oxidative damage (Shao et
al., 2005). It also protects and preserves the activities of metal-binding
enzymes. AA in its reduced state acts as the cofactor of violaxanthine de-
epoxidase and maintains the dissipation of the excess excitation energy
(Smirnoff, 2000). AA has also been reported to be involved in preventing
photo-oxidation by pH-mediated modulation of PSII activity and its down
regulation, associated with zeaxanthine formation.
 Ascorbic acid (vitamin C) is to act as an antioxidant to protect
cellular components from free radical damage.
 The antioxidant effect of vitamin C is due to its ability to donate
electrons from both the second and third carbon.

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Glutathione (GSH)
Low molecular weight glutathione (-glutamyl-cysteinyl-glycine)
tripeptide is widely distributed in nearly all cellular compartments, including
the cytosol, ER, mitochondria, chloroplasts, vacuoles, peroxisomes, and even
the apoplast. It is involved in a variety of processes, including cell
differentiation, growth and division, cell death, and senescence, as well as
the control of sulphate transport, detoxification of xenobiotics, conjugation
of metabolites, regulation of enzymatic activity, synthesis of proteins and
nucleotides, synthesis of phytochelatins, and expression of stress-responsive
genes (Mullineaux and Rausch, 2005). The strong reductive potential of
GSH is entirely responsible for its adaptability. The source of its reducing
power is a core nucleophilic cysteine residue. By creating adducts
(glutathiolated) or by reducing the various biomolecules in the presence of
ROS or organic free radicals and producing GSSG as a by-product, GSH
scavenges H2O2, 1O2, OH•, and O•2 and shields the various biomolecules.
Additionally essential to the process of regenerating AA into GSSG is GSH.
The GSSG therefore produced is then changed back to GSH by GR either
through de novo synthesis or enzymatically. In the end, this replaces the
cellular GSH supply. Through phytochelatin synthase, GSH also participates

Page | 26
in the production of phytochelatins, which scavenges another possible source
of ROS generation in plants by chelating heavy metal ions. Thus, it is
essential for preserving the redox state of the cell to maintain the delicate
balance between GSH and GSSG.
Glutathione (GSH; γ-glutamyl-cysteinyl-glycine) is a small intracellular
thiol molecule which is considered as a strong non-enzymatic antioxidant.
 It plays the role of an antioxidant as a scavenger
1. It directly quenches reactive hydroxyl free radicals or other
oxygen-centered free radicals.
2. GSH is the co-substrate of glutathione peroxidase.

α-Tocopherols (Vitamin E)
The -tocopherol is a member of a family of lipophilic antioxidants that
are effective scavengers of ROS and lipid radicals, making them vital
components of biological membranes and crucial protectors (Kiffin et al.,
2006). Of the four isomers (-, -, -, -), -tocopherol has the strongest
antioxidant properties. exclusively photosynthetic organisms can produce
tocopherols, which is why they are exclusively found in the green tissues of
plants. Tocopherol-methyl-transferase (-TMT; encoded by VTE4) converts -
tocopherol from -tocopherol. By interacting with O2 and quenching its

Page | 27
surplus energy, tocopherols are renowned for their ability to protect lipids
and other membrane components of the chloroplasts, thereby safeguarding
the PSII both structurally and functionally. Through its ability to stop the
LPO cycle's stage of chain propagation, tocopherol also functions as a potent
free radical trap. At the membrane-water interface, it interacts with the lipid
radicals RO•, ROO•, and RO*, reducing them and transforming itself into
TOH• in the process. By interacting with GSH and AA, the TOH• radical
proceeds through recycling to become its reduced form (Igamberdiev et al.,
2004).
 Tocopherols belong to a class of phenolic antioxidants
 The scavenging of singlet oxygen by α-tocopherol in chloroplasts
results in the formation of other products α -tocopherol quinone a
known contributor to cyclic electron transport in thylakoid
membranes, therefore providing photoprotection for chloroplasts.
 Isomers of tocopherols (α-, β-, γ) found in plants, among them α-
tocopherol has the highest antioxidative activity.

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Flavonoids
In the plant kingdom, flavonoids are widely distributed and are
frequently present in the leaves, floral organs, and pollen grains. Based on
their structural similarities, flavonoids can be divided into four groups:
flavonols, flavones, isoflavones, and anthocyanins. They provide a variety of
roles in the production of pigment in flowers, fruits, and seeds that are
important for plant fertility, pollen germination, and defence against plant
infections. Due to the extra excitation energy, flavonoids have been thought
of as a secondary ROS scavenging system in plants that are experiencing
damage to the photosynthetic apparatus (Fini et al., 2011). Additionally, they
help to scavenge 1O2 and lessen harm done to the chloroplastic membrane's
outer envelope (Agati et al., 2012).

 Flavinoids serve as ROS scavengers by locating and neutralizing

radicals before they damage the cell thus important for plants under
adverse environmental conditions.
 Their ability to act as antioxidants depends on the reduction
potentials of their radicals and accessibility of the radicals.
Carotenoids
The plastids of both photosynthetic and non-photosynthetic plant tissues
include the carotenoids, a family of lipophilic antioxidants. They can be
found in microorganisms as well as plants. They are a member of a class of
molecules called antennae that absorb light in the 450-570 nm range and
pass the energy on to the chlorophyll molecule.

Page | 29
 Carotenoids exhibit their antioxidative activity by protecting the
photosynthetic machinery in four ways,
a) Reacting with LPO products to end the chain reactions
b) Scavenging 1O2 and generating heat as a by-product
c) Preventing the formation of 1O2 by reacting with 3Chl* and excited
chlorophyll (Chl*)
d) Dissipating the excess excitation energy, via the xanthophyll cycle.
 Carotenoids are organized in pigment complexes in thylakoid
membrane of plants where they function as light harvesting
pigments and as antioxidants.
 They are efficient antioxidants scavenging singlet molecular
oxygen and peroxyl radicals.

Page | 30
ASC-GSH cycle

Hydrogen peroxide (H2O2) is a reactive oxygen species that is created as

a waste product in metabolism and is detoxified via the glutathione-ascorbate
cycle. The cycle involves the enzymes that link the antioxidant metabolites
ascorbate, glutathione, and NADPH. Ascorbate peroxidase (APX) reduces
H2O2 to water in the first stage of this process while employing ascorbate
(ASC) as the electron donor. Monodehydroascorbate reductase (MDAR)
regenerates the oxidised ascorbate (monodehydroascorbate, MDA).
Monodehydroascorbate, on the other hand, is a radical and, if not reduced
quickly, disproportionally transforms into ascorbate and dehydroascorbate
(DHA). Dehydroascorbate reductase (DHAR) converts dehydroascorbate to
ascorbate at the expense of glutathione (GSH), resulting in oxidised
glutathione (GSSG). Finally, glutathione reductase (GR) uses NADPH as the
electron donor to decrease GSSG. Thus, ascorbate and glutathione are not
used up; instead, NADPH and H2O2 receive the majority of the electron
flow. Proteins with dehydroascorbate reductase activity, such as glutathione
S-transferase omega 1 or glutaredoxins, may catalyse the reduction of
dehydroascorbate or it may occur non-enzymatically.
In plants, the cytosol, mitochondria, plastids, and peroxisomes all
participate in the glutathione-ascorbate cycle. It is believed that the
glutathione-ascorbate cycle is essential for the detoxification of H2O2 since

Page | 31
glutathione, ascorbate, and NADPH are all found in significant amounts in
plant cells. However, additional enzymes (peroxidases), such as glutathione
peroxidases and peroxiredoxins, which utilise thioredoxins or glutaredoxins
as reducing substrates, also assist in the elimination of H2O2 in plants.
List of all the enzymatic and non-enzymatic antioxidants along with
their functions and cellular localization

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18. Mullineaux PM, Rausch T. Glutathione, photosynthesis and the redox
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 Chapter - 2
Synthetic Promoters and Plant Development

Author
Kaveri Chawan
University of Agricultural Sciences, Bengaluru, Karnataka,
India

Page | 35
Page | 36
 Chapter - 1
Synthetic Promoters and Plant Development
Kaveri Chawan

Abstract
For the advancement and application of transgenic techniques in research
and agricultural production, promoters are increasingly in demand. Plant
promoters are modular and composed of a variety of distinct domains arranged
in a certain orientation. This allows for the redesign of the 'cis architecture' of
a promoter DNA across the sequence backbone. Particularly upstream of the
TATA box, it is possible to swap or move the position of a crucial functional
domain between two distinct promoters to produce a special transcriptional
module that serves as both a donor and an acceptor. Light-responsive,
chemical-inducible, hormone-inducible, and biotic stress-inducible functional
regimes are divisions of synthetic promoters. Because they are flexible tools
for gene-based plant modification and could significantly aid in the creation
of a wide variety of value-added plants, synthetic promoters have a bright
future in translational research and plant biotechnology.
Keywords: CIS-engineering, gene expression, promoter structure, synthetic
promoter; transcription factors
Introduction
Plant promoters are made up of numerous discrete domains grouped in a
specific orientation and are modular, allowing for the redesign of a promoter
DNA's cis architecture across its sequence backbone. The position/s of critical
functional domains between two separate promoters can be shuffled/ swapped
(inter-/intra-molecularly), particularly upstream of the TATA box, to establish
a unique transcriptional module that performs both donor and acceptor
functions. The newly formed synthetic module's functionality is dependent on
the altered positioning of discrete cis-motif/s in terms of position, spacing,
orientation, and copy number/s. Approaches such as site-directed
mutagenesis, molecular evolution, linker-scanning hybridization, intron-
mediated mutagenesis, and bi-directional mutagenesis can specifically alter
the scattering of cis-motifs in the upstream/downstream of the native promoter

Page | 37
to generate a ''synthetic'' promoter. These newly designed synthetic promoters
are typically more efficient than native promoters because the spacing and
copy number/s of cis-motifs (either identical or different) in these synthetic
promoters promotes optimal interaction between cis-motifs and their
corresponding trans factors for maximum transcription.
Furthermore, synthetic promoters differ in terms of strength, inducibility,
and tissue-specificity; they are significant in planta for the following reasons:
1) Simultaneous co-expression of multiple genes in a plant cell is
required to develop a particular trait; in this gene-stacking approach,
the expression of each gene under a discrete promoter prevents
unwanted recombination or homology-dependent gene silencing.
2) Plant metabolic engineering requires different genes that can be
expressed at relatively higher or lower levels, necessitating the
development of synthetic promoters (having utmost sequence
diversity) with different levels of expression.
3) Use of correctly designed synthetic promoters is essential to address
the challenges of plant biotechnology under different environmental
conditions, e.g., biotic, abiotic, tissue-specific, hormonal or light-
stress conditions.
Promoter structure and scope of cis-engineering
Eukaryotic plant promoters have a distal (upstream activation sequence;
UAS) and proximal (core promoter) region, the latter of which contains the
TATA element. TFIID complex TATA-binding protein interacts to the TATA
region of the core promoter to activate transcription. Other DNA elements,
including enhancers, silencers, and insulators, influence the activity of a
promoter. Enhancers are DNA sequence segments that include motifs that
interact with DNA-binding proteins (activators). They promote gene
expression in a location and orientation-independent way. Silencers are DNA
elements that activate the cellular machinery to suppress gene expression.
Insulators are DNA sequences that prevent interference from an unrelated
enhancer or silencer in a neighboring domain. Insulators, enhancers, and
silencers are thus direct promoter activity regulators.
Several short sequence motifs (cis elements) are found in both distal and
proximal promoter regions, and their interaction with trans-factors in
conjugation with insulators, enhancers, and silencers affects promoter activity
under diverse environmental situations. As a result, rearranging the cis
elements in relation to the native promoter results in a distinct synthetic
module with a different function. Cis engineering is thus an important method

Page | 38
for generating many synthetic promoters from a single native promoter
sequence.
Modeling of synthetic gene switches
Synthetic promoters are usually designed by molding and adjusting the
cis-motifs orientation, spacing, and copy number. A synthetic promoter is
simply a systematic combination of a minimal promoter (CaMV35s mostly)
with cis elements. The minimal promoter has sequences for binding to the
transcriptional factors including the TATA box. The cis elements are linked
upstream to the minimal promoter. cis elements determine the expression of
the target gene thus they are selected looking at the characteristics of
transgene. Cis-acting elements contain variable number of individual cis-
motifs in various combinations.

Fig 1: A pre-initiation complex on a core promoter containing TATA, initiator (Inr)

and downstream promoter element (DPE) at the respective positions.
The core promoter contains many regulatory sequences such as GA
elements or a coreless sequence as well as some core elements such as CA
elements, Inr elements, CCAAT elements, Y patches, and so on. UBI relative
to the transcriptional start site, the position of the core promoter elements (50

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bp away from the transcription start site) along the length of a gene is
conserved in plants. The CaMV 35S promoter, which is around 54 bp long
and has a relatively low baseline expression level in monocots and dicots, is
the most efficient promoter for transcription start. The sequential and spatial
expression effectiveness of synthetic promoters is determined by the cis
element arrangement, type, and copy number. The synthetic motif library is
screened in-silico using a trial-and-error method, and cis-elements of interest
are selected and tested experimentally. The motifs are shuffled, added, or
eliminated as needed, and fresh motifs can be described through experimental
validation. To identify cis-regulatory elements, resources such as PLACE
(Plant cis-acting regulatory DNA elements database), TRANSFEC
(Transcription factor database), and Plant CARE (Plant cis acting regulatory
elements) can be employed. The spacing and copy number of motifs can be
optimized in designing synthetic promoter once the desired motifs are
identified. As the transcription factors attach to their corresponding cis
elements in an ordered fashion, the cis motifs in a synthetic promoter should
be properly spaced to get a perfect synergistic effect.
Table 1: Bioinformatics tools used for the identification of cis regulatory elements
and promoter prediction.

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 Fig 2: A typical synthetic promoter and its various elements are shown.
Strategies for designing synthetic promoters
Several methods have been employed to create synthetic promoters. Cis-
motif engineering has been utilized to change the position, spacing, and
number of important cis-elements. Recent improvements in bioinformatics
technologies have permitted the building of highly specialized synthetic
promoters capable of directing tightly regulated gene expression. Some of the
most essential ways to build synthetic promoters are hybridization, site-
directed mutagenesis, shuffling, bidirectionalities, and linker scanning.
Hybridization
This method entails joining two or more important promoter
segments/domains, such as the core promoter (CP) and upstream activation
sequences (UAS), from one (intramolecular hybridization) or more
(intermolecular hybridization) promoters in the same orientation as the native
promoter to produce a new promoter with specific activity. A promoter's
upstream activation (distal) fragment and another promoter's TATA box-
containing (proximal/core) domain are amplified by PCR using synthetic
primers to generate appropriate overhangs at the 5' end 3' end; amplified
fragments are digested with restriction enzymes and cloned into individual
cloning vectors. To construct a chimeric promoter, the TATA box-containing
core promoter is cloned into the vector containing the upstream activation
region.

Fig 3: A. Intra-molecular hybridization: taking the cis motifs from the same promoter
and putting them in a new combination.

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Fig 4: B. Inter-molecular hybridization: combining the cis regulatory elements of two
or more (different) promoters. P1 promoter 1, P2 promoter 2, DP distal promoter, PP
Proximal promoter.
Site-directed mutagenesis
One of the most frequent ways for cis-engineering is PCR-based site-
directed mutagenesis. It is typically used to insert mutations into the native
promoter segment to create/delete target CIS elements. In brief, PCR is used
to anneal a set of sense and antisense primers to a double-stranded starting
plasmid, which is then digested by the restriction enzyme DpnI. Following
digestion, the product is annealed again to produce a template with the desired
mutation. Similarly, synthetic oligonucleotides containing the cis-element/s
are frequently utilized to restructure the native promoter's cis-architecture by
adding or removing copies of one or more targeted cis-element/s.

Fig 5: PCR based site-directed mutagenesis involves the annealing of a (mutated)

sense and antisense primer pair by PCR to create or delete the target cis element
inside the native promoter.

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Shuffling
Shuffling is an in vitro DNA recombination process in which a mixture
of two or more promoter DNA pieces is digested by DNaseI into smaller
fragments, which are then allowed to reassemble randomly in the presence of
a DNA polymerase. Each of the reassembled promoter fragments, which now
contain multiple copies of distinct promoters, is amplified specifically in the
presence of 50 and 30 oligonucleotides specific to the native promoter DNA
sequence. Finally, the amplified recombinant (reassembled) promoter
segments are cloned into cloning vectors to form a library of synthetic
promoters.

Fig 6: Shuffling/recombination
Bidirectional promoter
Bidirectional promoters induce the transcription of the two genes flanking
the promoter. These promoters are important in the transcription of
bidirectional gene pairs, which are two genes placed head-to-head on opposite
strands of DNA. A bidirectional promoter module can be created by ligating
two unidirectional minimum promoters in opposing directions, with the
promoter cluster controlled by a common set of cis motifs. Two distinct
transgenes (or reporter genes) are joined at the 3' end of the two promoters in
opposite directions. The bidirectional promoter can effectively drive two
transgenes or reporter genes at the same time. The efficacy of bidirectional
promoters in driving both transgenes at the same time can be tested in a variety
of model-plant systems.

Fig 7: Bidirectional synthetic promoter designed by placing two core promoters at

the ends of the cis motif assembly.
Linker-scanning mutagenesis
Linker-scanning mutagenesis is a strong technique for re-structuring the
native promoter sequence. In this approach, a succession of deletion mutations

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is created in a specific region of interest. Appropriate oligonucleotides (also
known as linkers) are created with a restriction enzyme site that ligates with
the parent strand to produce a mutation. As the linker sequence passes over
the area, it causes the required point mutation/s to occur at various points.
Linker-scanning mutagenesis is useful for analyzing the contribution of
individual DNA sequence elements within a transcriptional regulatory region
in which discrete segments of native DNA are systematically replaced by
heterologous segments of the same length.

Fig 8: Linker-scanning mutagenesis restructures the sequence of native promoter.

Synthetic promoters in planta
Synthetic promoters are categorized into seven broad functional
categories: biotic stress-inducible, abiotic stress-inducible, light-responsive,
chemical-inducible, hormone-inducible, constitutively expressed, and tissue-
specific.
Biotic stress-inducible synthetic promoters
To reduce the difficulty in promoter expression, synthetic promoters
provide an effective strategy to controlling transgene expression in a precise
way at the time and place of pathogen plant contact. It has been reported that
a minimal promoter in conjunction with a cis-acting region directed reporter
gene production in response to pathogen. EL17, a W-box cis acting element,
was discovered in the promoter of the pathogen responsive gene ELI17
associated to parsley. Another cis-acting element F was discovered to be
effective against fungal infections, with 9 bp more motifs than EL17. Both the
EL17 and F elements contain functionally significant cis motifs. Pathogen
inducible promoters have been examined in various genes associated with
plant defense mechanisms.
Injury causes a variety of reactions in the plant, some of which are
involved in pathogen defense. The WUN-box is involved in the modulation of
the signaling pathway following damage. Because physical harm frequently

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mimics the effect of pest activity, wound-responsive promoters may also drive
some protective genes. Typically, wound-responsive promoters have only
been discovered in pathogenesis-related genes. Other gene families, such as
MYB, bZIP, ERF, WRKY, and MYC TFs, can be utilized to identify wound-
inducible promoters. Such promoters may be more effective in providing
protection during the early phases of pathogen colonization or shortly after
insect invasion.
Abiotic stress inducible synthetic promoters
Phytohormones aid plant adaptation to environmental changes by
adjusting growth and development as well as other metabolic activities. The
underlying molecular processes of plant hormone regulation and signaling can
be utilized to improve abiotic stress tolerance in transgenic crops. Amy 1x, rab
6x, rab 2x, and amy 6x were the first hormone-responsive synthetic promoters
to be identified. Higher plants have a sophisticated biochemical system that
detects and responds to light stimuli via photoreceptor proteins. Through a
complex signal transduction process, these photoreceptors respond to light by
regulating the expression of many genes that code for proteins that work in
photosynthesis and other metabolic pathways. Several transcription factors
and LREs (light-responsive elements) are activated by various pathways,
although they can also control common LREs. Antibiotics, herbicide safeners,
insecticide (methoxyfenozide), copper, benzothiadiazol (inducer of pathogen-
related proteins), and ethanol, steroids are chemical promoters of transgenic
expression.
Cold stress inducible promoters are described to reduce unfavorable
effects under various environmental situations in order to boost cold stress
tolerance and gene regulation at low temperatures. The stress inducible
promoter rd29A is frequently utilized in plants to improve stress tolerance and
lessen the negative impacts on plant growth and development. Cold stress
resistance has been reported in Arabidopsis COR gene promoters such as
cor47, cor6.6, and core 15a. Wsi18 and OsNCED3 gene promoters, which are
involved in abscisic acid production and signaling, were strongly inducible by
ABA, drought, and high salt treatments in transgenic rice. The Arabidopsis
Rd29A gene promoter successfully drove DREB1A gene expression in
transgenic wheat during drought stress.
Tissue-specific promoters have an advantage over constitutive promoters
in that they can drive transgene expression in a single cell type while leaving
the other tissues alone. Several tissue-specific promoters have been found and
characterized in planta over the last two decades. Insights into the fine

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architecture of cis regulatory elements in tissue specific promoters are critical
for leveraging these promoters' precise and targeted function in transgene
regulation. The database was used in silico approaches with plant gene
expression to develop the synthetic constitutive promoter Pmec. Pcec
expression cassette consists of two units: Pmec (138 bp expression cassette
with TATA box) and 312 bp transcription activation module for high level
GUS expression. Pcec predicts greater GUS expression, which was exploited
in the development of transgenic tomatoes resistant to lepidopteran insects
using the modified insecticidal cry1Ac gene from Bacillus thuringiensis.
Light-responsive synthetic promoters
Higher plants have evolved a complicated biochemical system that
detects and responds to light signals via photoreceptors, which are light-
sensitive proteins. Through a complicated signal-transduction cascade, light
received by these photoreceptors controls numerous genes that encode
proteins involved in photosynthesis and other developmental processes.
various light-activated pathways are thought to target various LREs (light-
responsive elements) and transcription factors inside the promoter region, but
they may also target similar LREs. Mutagenesis and deletion of the promoter
regions of light-induced genes were utilized to uncover various cis-acting
elements involved in the control of light-regulated transcription. The G-box,
GT-1 box, I-box, and H-box are the most important cis-sequences for light-
induced gene transcription.
The construction of a synthetic module with tetramer of box II-containing
GT-1 binding sites (bases 152-138 of the rbcS-3A promoter) and inserted
upstream of a -90 CaMV 35S promoter was described in preliminary research
of light-responsive synthetic promoter (Lam and Chua 1990). Under
illumination, this synthesized promoter complex induced chloroplast
expression. The as-1 tetramer fusion complex with the -90 CaMV 35S
promoter worked as a molecular switch, modulating promoter activity under
light. When coupled to the minimal -90 CaMV 35S promoter with the
activator sequence-1 (as-1) in the presence of light, the synthetic promoter
consisting of tetramer GT-1 regulated transcription but showed no activity
when coupled to the -46 CaMV35S with no as-1 (Gilmartin and Chua 1990).
Puente et al. (1996) developed synthetic promoters by merging four
LREs: GT-1, Z-box, G-box, and NOS101. This group also investigated the
potential of these four LREs, alone or in various combinations, to modulate
the light responsiveness of newly synthesized promoters. Individual motifs
(GT-1, Z-box, G-box, and NOS101) were unable to generate light

Page | 46
responsiveness, however pairwise combinations (4xG4xGATA, 4xGT1-
4xGATA, or 2xZ- 4xGATA) did. Under light, the 4xG4xGATA and 2xZ-
4xGATA pairs showed 4- to 8-fold higher GUS activity, but the 4xGT1-
4xGATA combination showed 8- to 15-fold higher GUS activity. These
synthetic promoters outperformed their native promoter Arabidopsis cab1.
Chemical-responsive synthetic promoters
Antibiotics, hormones, ethanol, benzothiadiazole (a pathogen-related
protein inducer), herbicide safeners, and insecticides are all known to affect
transgenic expression. Chemically triggered synthetic promoters are
composed of a dense array of particular cis-motif repeats (varying from two
to ten) that increase transgene transcription.
Gatz et al. (1992) created a triple-operator synthetic promoter with three
copies of an operator both upstream and downstream of the TATA-box-
containing CaMV 35S promoter and created a transgenic plant that can
express GUS up to 500-fold higher than the wild type in the presence of
tetracycline. By efficiently de-repressing its activity in the presence of certain
inducers such as tetracycline, this synthetic promoter proved capable of
recovering transgenic plants containing a repressed transgene.
Konishi and Yanagisawa (2010) created a synthetic promoter (4x NRE-
min synthetic promoter) that fused a tetramerized 43-bp enhancer fragment in
the NIR promoter (which imparted a high nitrate-enhancer function) to the
CaMV 35S minimal promoter. This construct was expressed in transgenic
Arabidopsis plants, which demonstrated increased nitrate inducibility.
Nitrogen-depleted seedlings of these transgenic Arabidopsis responded to
nitrate treatment with enhanced GUS activity. When seedlings were cultivated
in half MS with enough nitrogen, the synthetic promoter produced
significantly more GUS than the native NIR promoter.
Zhu et al. (2015) created artificially synthetic promoters with tandem
copies of cis-elements (SARE, JERE, GCC, GST1, HSRE, and W-box) and
used the chemical inducer probenazole (PBZ), which has been shown to
induce systemic acquired resistance (SAR) in plants by inducing salicylic acid
(SA) biosynthesis. Tetramers of the following cis-elements, SARE, JERE,
GCC, GST1, HSRE, and W-box, increased GUS activity in transgenic
Arabidopsis by twofold, ninefold, fourfold, twice, and twofold, respectively,
when treated with 0.5 mmol/L PBZ. They also created a PBZ-inducible
chimeric construct GWH including 29 GST1, 69 W box, and 29 HSRE, which
demonstrated threefold stronger GUS activity in transgenic lines than single
cis elements after PBZ treatment.

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Hormonal responsive synthetic promoters
Through site-directed mutagenesis in the AuxRE region of the soybean
GH3 promoter, an auxin-inducible synthetic promoter DR5 was created
(Ulmasov 1997). This promoter was more auxin responsive than the GH3
promoter and a natural variant AuxRE. A single copy of DR5 demonstrated
activity comparable to the CaMV 35S minimum promoter, whereas a two-
copy construct had threefold enhanced activity after auxin induction. Auxin
induction was increased by increasing the copy number to 4, 6, and 7, but an
8-copy construct DR5 (8X) was induced 25 to 50-fold by 1-NAA; in contrast,
the natural D1-4 (8X) containing eight tandem copies of the 11-bp natural D1-
4 AuxRE from the GH3 promoter showed a fivefold induction in response to
1-NAA.
Ganguly et al. (2011) investigated two abscisic acid-inducible promoters,
4X ABRE and 2X ABRC, connected to the GUS reporter gene in transgenic
rice plants. When exposed to 150 mM NaCl and 100 lM ABA, the 4X ABRE
promoter showed roughly twofold greater GUS expression, while the 2X
ABRC promoter demonstrated highest promoter induction (nearly thrice).
Synthetic promoters directing multiple types of expression in planta
Under pathogen and chemical (probenazole) treatment, the synthetic
promoters 4x SARE, 4x JERE, 4x GCC, 4x Gst1, 2x W box were inducible
(Rushton et al. 2002, Zhu et al. 2015). Both UV light and pep25 (synthetic
peptide elicitor) generated from Phytophthora sojae were responsive to the
synthetic promoters PcCHS-m1, PcCHS-m2, PcH3-7, and PcH3-7 m with
ACGT-containing elements (Logemann and Hahlbrock 2002).
Three SA- and JA-inducible synthetic promoters, EFCFS-HS-1, EFCFS-
HS-2, and EFCFS-HS-3, have demonstrated improved activity in transgenic
tobacco vascular tissue. (2012) (Ranjan and Dey). The modular character of
various synthetic promoters (C18, C19, C20, C21, C22, C23, C24, C25, C26,
C27, C28, C29) was established by their reactivity to abscisic acid and
environmental stress (including salt, cold, and drought) (Shen et al. 1996).
ABA and osmotic stress promoted the expression of two synthetic
promoters, RB70 and RB40, in diverse tissues of vegetative and floral organs.
The synthetic bi-directional constructs P1301A and P1301B showed increased
activity when exposed to SA, NaCl, and IAA (Indole-3-acetic acid) in both
transiently and stably transformed tobacco leaves (Chaturvedi et al. 2006).
Furthermore, two synthetically created promoters (4X ABRE and 2X ABRC)
were activated by NaCl and ABA stress (Ganguly et al. 2011). 4H1-46 and
4H3-46 synthetic promoters with three roles provided seed-specific

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expression and transcriptional increase in response to desiccation, salt, and
ABA (Lam and Chua 1991). Xie et al. (2001) created a set of bidirectional
synthetic promoters (pGLbd1-pGLbd 6) that demonstrated constitutively
elevated expression (GUS and GFP) as well as induction by wound, jasmonic
acid, and leaf senescence.

Fig 9: Venn-diagram depicting the total number of synthetic promoters developed so

far under the seven functional groups and also showing the overlap among the
promoters exhibiting bipartite or tripartite functions.

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 Table 2: Combination and dosage of cis regulatory elements used in synthetic
promoters against different stresses

Conclusion and future perspectives

Synthetic promoters have a promising future in translational research and
plant biotechnology. Synthetic promoters are versatile tools for gene-based
modification of plants and have the potential to contribute significantly to the
development of a wide spectrum of value-added plants. Plants belonging to
one end of the spectrum can grow in marginal lands to boost agricultural
productivity, while plants at the other end can efficiently produce essential
commodities and pharmaceuticals. Recombinant promoters can be used to
elucidate the basal mechanism of underlying genes and their interactions with
proteins (transcription factors) and better understand the regulation of gene
expression in eukaryote systems. Synthetic promoters are thus of immense
value in plant molecular biology at recent times.
Synthetic promoters with bipartite (active under two different
environmental stimuli/ stresses) and tripartite functions (active under three
different environmental stimuli/stresses). Synthetic promoters with
bipartite/tripartite functions will be of great importance in the development of

Page | 50
transgenic plants capable of withstanding multiple simultaneous stresses.
Based on current global climatic and social conditions, the development of
multi-stress tolerant plants is critical, especially in developing countries. To
develop synthetic promoters, specific cis elements with discrete functions are
required. However, the use of integrated strategies, including bioinformatics
and biotechnological tools, for the development and testing of synthetic
promoters has been limited. Thus, construction of synthetic promoters with
suitable specificity and efficiency, as we assume, is still in its infancy.
In recent years, advancements in bioinformatics and plant genomics
techniques have facilitated the whole genome sequencing of several plants,
while sophisticated tools such as RNAi, zinc finger nucleases (ZFNs),
TALEN, and CRISPR-Cas9 are being used for targeted genome editing to
develop plants with desired trait. These cutting-edge tools are being used to
modify transcription factors/effectors responsible for plant gene expression
under different climatic conditions. A combinatorial approach including
designed or custom-made synthetic promoters in conjugation with engineered
transcription factors has the potential to revolutionize gene-technology-based
plant modification in the near future.
References
1. Chaturvedi CP, Sawant SV, Kiran K, Mehrotra R, Lodhi N, Ansari SA,
Tuli R (2006) Analysis of
2. Chaturvedi CP, Sawant SV, Kiran K, Mehrotra R, Lodhi N, Ansari SA
et al. Analysis of polarity in the expression from a multifactorial
bidirectional promoter designed for high-level expression of
transgenes in plants. J Biotechnol. 2006;123:1-12.
3. Ganguly M, Singh SK, Sharma I, Kumar R, Srinivasan R. Inducibility
of three salinity/abscisic acid-regulated promoters in transgenic rice
with gusA reporter gene. Plant Cell Reports. 2011;30:1617-1625.
4. Gatz C, Frohberg C, Wendenburg R. Stringent repression and
homogeneous de-repression by tetracycline of a modified CaMV 35S
promoter in intact transgenic tobacco plants. Plant J. 1992;2:397-404.
5. Gilmartin PM, Chua NH. Localization of a phytochrome-responsive
element within the upstream region of pea rbcS-3A. Mol Cell Biol.
1990;10(10):5565-5568.
6. Konishi M, Yanagisawa S. Identification of a nitrate-responsive cis-
element in the Arabidopsis NIR1 promoter defines the presence of
multiple cis-regulatory elements for nitrogen response. Plant J.
2010;62:269-282.

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7. Lam E, Chua NH. Tetramer of a 21-base pair synthetic element confers
seed expression and transcriptional enhancement in response to water
stress and abscisic acid. J Biol Chem. 1991;266(26):17131-17135.
8. Logemann E, Hahlbrock K. Crosstalk among stress responses in plants:
Pathogen defense overrides UV protection through an inversely
regulated ACE/ACE type of light-responsive gene promoter unit. Proc
Natl Acad Sci USA. 2002;99:2428-2432.
9. Puente P, Wei N, Deng XW. Combinatorial interplay of promoter
elements constitutes the minimal determinants for light and
developmental control of gene expression in Arabidopsis. EMBO J.
1996;15:3732-3743.
10. Rushton PJ, Reinstädler A, Lipka V, Lippok B, Somssich IE. Synthetic
plant promoters containing defined regulatory elements provide novel
insights into pathogen- and wound-induced signaling. Plant Cell
Online. 2002;14:749-762.
11. Shen Q, Zhang P, Ho TH. Modular nature of abscisic acid (ABA)
response complexes: Composite promoter units that are necessary and
sufficient for ABA induction of gene expression in barley. Plant Cell.
1996;8:1107-1119.
12. Zhu Z, Gao J, Yang JX, Wang XY, Ren GD, Ding YL, et al. Synthetic
promoters consisting of defined cis-acting elements link multiple
signaling pathways to probenazole-inducible system. J Zhejiang Univ.-
Sci. B. 2015;16(4):253-263.
13. Xie M, He Y, Gan S. Bidirectionalization of polar promoters in plants.
Nature Biotechnol. 2001;19:677-679.

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 Chapter - 3
Genetic Enhancement in Pre-Mendelian Era and
21st Century

Authors
A. Krishnamoorthi
Ph.D. Research Scholar, Division of Plant Genetic Resources,
NBPGR, ICAR-Indian Agricultural Research Institute, New
Delhi, India
V. Lakshmi Prasanna Kumari
Assistant Professor, (Dept. of Floriculture and Landscaping),
Faculty of Agricultural Sciences, Siksha “O” Anusandhan
(Deemed to be university), Bhubaneshwar, Odisha, India

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Page | 54
 Chapter - 3
Genetic Enhancement in Pre-Mendelian Era and 21st
Century
A. Krishnamoorthi and V. Lakshmi Prasanna Kumari

Abstract
Rick used the term pre-breeding to describe the same activity. Now terms
genetic enhancement and pre-breeding are used as synonyms and
interchangeable. However, the term genetic enhance is in more usage.
Keywords: Domestication, domestication syndrome, trait.
Introduction
The term genetic enhancement was first used by Jones in 1983. It refers
to transfer of useful genes from exotic or wild types into agronomically
acceptable background. In 1984,
Genetic enhancement is carried out for broadening the genetic base of the
population. It increases genetic diversity in the population. In other words, it
helps in developing population with more genetic diversity. (Allen et al 2002)
GE consists of three main steps, viz.
a) Identifying a useful character
b) Capturing its genetic diversity, and
c) Putting those genes into usable form.
Base material
For launching genetic enhancement programme, three types of material
viz. agronomic base, exotic germplasm and wild species are required. The
former is used as the recipient parent and last two as donor parents. (Newman
et al 2005)
Breeding methods used
The backcross method is widely used for genetic enhancement, which
helps in transferring useful genes from exotic germplasm and wild species into
acceptable agronomic base. Now biotechnological approaches are also used
for genetic enhancement. (Morrison et al (1967)

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Impact
The major impact of genetic enhancement is value addition in the
germplasm. In other words it leads to genetic improvement of germplasm for
various economic characters. It also leads to creation of new variability. It
improves adaptation of the population.
End product
The end products of genetic enhancement programme are improved
germplasm lines which can be used as parents for developing productive
cultivars/hybrids in traditional breeding.
Domestication
It is the most important technological advance in our history. It allowed
us to develop into the highly complex civilization we have become. Traits that
made domestication possible were controlled by few genes and were fixed
quickly. Example: shattering system in wheat and barley - controlled by a
single gene.
Domestication syndrome
Among the cultivated species, a certain set of traits exist that are common
to nearly all of them. The differences seen in cultivated plants from their wild
relatives was due to selection pressures by early man. These are the traits that
make the plants productive and beneficial to human society. These traits
imbued the crop plants with uniformity, predictability, and high productivity.
It was originally postulated by Charles Darwin.
Traits associated with plant domestication

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The bottleneck concept
The genetic diversity of our crops is much lower than that of their wild
relatives. Early farmers noticed beneficial few mutants, kept them and planted
them over and over again. The bottleneck effect of domestication was due to
small founder populations of the plants and strong selection pressures imposed
upon these populations.
Plant breeding before Mendel
17th century – anatomical investigations
In the early part of the 17th century, pilgrims from Europe to the New
World discovered that their traditional crop varieties were eminently unsuited
to their new home. Two of the leading plant anatomists, Marcello Malpighi
and Nehemiah Grew, each made substantial contributions to early
understandings of plant anatomy and reproduction. Malpighi described plant
structures and the generation of seeds in 1675 and germination and growth of
young plants in 1679. Flower sexuality was still an unknown concept at that
time. He used his considerable background in medicine and animal anatomy
to give names to some of the reproductive parts of the plant. Grew (1682) also
made comparisons between animal and plant structure and development. He
recognized that plants have two sexes but misidentified the mega gametophyte
as male instead of female. In 1694 Rudolph Camerarius demonstrated the
necessity of pollen through a series of systematic experiments. He
emasculated flowers from a variety of species and hand-pollinated some of
them. Only the pollinated flowers produced seed. He concluded that the
anthers, which produce the pollen, must be male, and the ovaries, style, and
stigmas that had received the pollen must be female.
18th century - new innovation in agriculture
During 1717, Thomas Fairchild made a cross between a carnation and a
sweet william that resulted in a (sterile) flower - ‘Fairchild’s Mule’.
Koelreuter demonstrated that hybrid offspring display characters from both
parents in an intermediate form, and that these hybrids only produced seed
when their parents were of closely related species. Additionally, by
performing crosses from hybrid tobacco and dianthus plants back to their
parents over many generations the progeny resumed the phenotype of the
recurrent parent. This process is the foundation of the backcrossing method,
which is still extensively used today. Thomas Andrew Knight, gentleman
farmer performed substantial amounts of plant breeding during the latter part
of the 18th century. Knight performed hybridization of various fruits and
vegetables in order to generate improved cultivars. During 1787, he created

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two improved hybrid strawberry varieties. He began experimenting with
crossing inbred lines of sweet pea plants. Although he could not explain the
performance of what we now know as dominant and recessive alleles, Knight
(1809) was able to produce practical breeding results, even without having a
scientific background himself. Franz Achard – 1786 to 1830 - initiated a
breeding program for sugar beets by performing mass selection on fodder
beets for increased sucrose content.
19th century - plant breeding by various groups and organizations
Luther Burbank – created approximately 800 varieties of a wide range of
plants, including a (now extinct) spineless cactus intended for livestock
fodder, the Santa Rosa plum, the Freestone peach and the Burbank and Russet
Burbank potatoes.
1862 - USDA - “the largest organized governmental plant-breeding
enterprise in the world” - continued the American government’s tradition of
collecting and disseminating germplasm to growers.
1840 - French Vilmorin Company, founded by the de Vilmorin family
focused on an original method of selection based on plant lineages, rather than
individuals, known as “genealogical selection”. 1870s followed methodical
combination of hybridization and selection wheat and other crops.
Plant breeding in 20th century
The Rockefeller foundation
Between 1920 and 1940, The Rockefeller Foundation started supporting
hybridizing efforts in corn to produce improved crop for industrial agriculture.
As a result there was a boost in hybrid corn seed sales around the 1930s and
corn yield increased significantly after introduction of double cross hybrids.
During 1943 it launched its Mexican Agricultural Program to develop HYVs
having higher response to agrochemicals. It established an independent
institute known as CIMMYT in Mexico (1963). It spread its hybrid corn
production program to Brazil in 1946, Argentina in 1947 and Kenya in 1956.
Rockefeller and Ford foundations joined hands with the Philippines
government to establish the International Rice Research Institute (IRRI) near
Manila in 1960. At IRRI, the focus was totally on research and breeding of
rice which is the staple food of over one billion poor people across the world.
Legacy of Norman E. Borlaug
Norman E. Borlaug is called as the Father of the Green Revolution
because of the credit for success of the Green Revolution. He spent almost his

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entire life working to alleviate food scarcity from the world. In 1970 he was
awarded Nobel Peace Prize. During 1963 – 1979, he was the leader of the
Wheat Program in CIMMYT. The new varieties developed at CIMMYT,
along with improved management practices, revolutionized the wheat
production in Mexico in the 1950s. He spread this successful model of wheat
production technology to other developing nations like India and Pakistan in
the mid-60s. As a result, between 1964 and 2001, the wheat production in
India increased from 12 to 75 million tonnes while in Pakistan an increase
from 4.5 to 22 million tonnes was achieved. Thus, the work of Dr. Borlaug
revolutionized agriculture in the developing countries and saved millions of
people from starvation.
Emergence of CGIAR
Rockefeller and Ford foundation, with support from the World Bank and
the UN Food and Agriculture Organization, established the CGIAR
(Consultative Group on International Agricultural Research) in 1971. An
organization which coordinates agricultural research in developing countries
worldwide with support from the World Bank and various Governments. At
present, CGIAR has about 15 international agricultural research centers.
New facets of 21st century plant breeding
Synergy of plant breeding and biotechnologies
Genetic modification has been the basis for betterment of crops in modern
agriculture. Biotechnology tools become a necessary component for the
potential use of information and products derived from the molecular genetics.
Tissue culture, micro-propagation, haploids, embryo rescue and protoplast
fusion greatly facilitated rapid generation of uniform plants and obtaining
hybrids between incompatible species. Further innovations in plant breeding
came from the introduction of DNA based technologies and insight into the
genome - sequence information and its functional relatedness.
Two such important technologies widely used are:
• DNA markers for identification, tracking of targeted gene or
selection of favourable combination of genes (DNA marker
technology)
• Introgression of alien genes through genetic engineering
(Recombinant DNA technology).
The capability to transport the genes from secondary, tertiary and
quaternary gene pools via transgenesis has brought a new dawn in plant

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breeding, enabling breeders’ access to here-though inaccessible genes/ gene-
pools. Marker technology facilitated breeders to exploit the genetic variation
within the crop gene pool while genetic engineering provided access to
biodiversity as a whole.
New generation plant breeding techniques
Innovation in plant breeding is necessary to meet the challenges of
population growth and climate change. Agriculture has been able to cope with
these challenges until now. However, further efforts are needed and therefore
plant breeder’s quest for new plant breeding techniques
• Cisgenesis and Intragenesis
• Zinc finger nuclease (ZFN) technology
• Oligonucleotide directed mutagenesis (ODM)
• RNA-dependent DNA methylation (RdDM)
• Grafting on GM rootstock
• Reverse breeding
• Agro-infiltration
• Synthetic chromosomes
Cisgenesis and intragenesis
When applying the cisgenesis/intragenesis technology a DNA fragment
from the plant species itself or from a cross-compatible plant species is
inserted into the plant genome (Figure 8). In the case of cisgenesis, the inserted
gene is unchanged and includes its own introns and regulatory sequences. In
the case of intragenesis, the inserted DNA can be a new combination of DNA
fragments from the species itself or from a cross-compatible species. Cisgenic
and intragenic plants are produced by the same transformation techniques as
transgenic plants, e.g. Agrobacterium-mediated transformation, following the
isolation of genes from the host. Biolistics could also be used. The changes
intended when applying this technique relate to modifying the expression of
target genes through stable integration in the host genome, as is the case for
transgenesis.
Zinc finger nuclease (ZFN) technology
ZFNs are proteins custom-designed to cut at specific DNA sequences.
They consist of a “zinc finger” domain (recognizing specific DNA sequences
in the genome of the plant) and a nuclease that cuts double stranded DNA.

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Oligonucleotide directed mutagenesis (ODM)
ODM employs oligonucleotides for the induction of targeted mutations in
the plant genome. They target homologous DNA and induce site-specific
nucleotide substitutions, insertions or deletions through repair mechanisms.
RNA-dependent DNA methylation (RdDM)
When applying the RdDM technique, genes encoding RNAs which are
homologous to plant sequences, like promoter regions, are delivered to the
plant cells. These genes, once transcribed, give rise to the formation of small
dsRNAs. They induce methylation of the homologous sequences and
consequently inhibit their transcription. The efficiency of silencing can be up
to 90% and is dependent on the active transcription of the promoter. Generally,
the degree of silencing is related to the degree of methylation, but this is not
always the case. Silencing and differences in silencing have been observed to
be transmitted to at least the F3 generation. Methylation is restricted to the
region of sequence homology with the dsRNA.
Grafting on GM rootstock
It is conceivable that there might be an intention to transform only the
rootstock with a view to changing protein or gene expression in the scion due
to the movement of specific proteins and/or RNA from the roots to the scion.
In this way a GM rootstock could be used to introduce new traits into a range
of genetically distinct scions.
Reverse Breeding
The intended goal of the reverse breeding technique is to generate
perfectly complementing homozygous parental lines through a suppression of
meiotic crossovers and the subsequent fixation of non-recombinant
chromosomes in homozygous doubled haploid (DH) lines.
Agro-infiltration
Depending on the tissues and the type of constructs infiltrated, three types
of agro-infiltration can be distinguished:
1. “Agro-infiltration sensu stricto”: Non-germ line tissues are
infiltrated with a liquid suspension of Agrobacterium sp. containing
a genetic construct in order to obtain localized expression in the
infiltrated area.
2. “Agro-inoculation” or “agro-infection”: Non-germ line tissues
(typically leaf tissues) are infiltrated with a construct containing the
foreign gene in a full length virus vector in order to obtain expression
in the entire plant.

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 3. “Floral dip”: Germ line tissues (typically flowers) are infiltrated with
a DNA construct in order to obtain transformation of some embryos
that can be selected at the germination stage.
Synthetic chromosomes
Synthetic chromosomes provide the means to stack transgenes
independently of the remainder of the genome. Telomere-mediated truncation
coupled with the introduction of site-specific recombination cassettes has been
used to produce mini chromosomes.
Conclusion
With the vast panorama of possibilities available and emerging to
manipulate the plant’s genetic architecture dealt above, it is clear that the
process of plant breeding continues to evolve from a skillful art to more and
more molecular biology dependent
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Page | 64
 Chapter - 4
Fertility Variation in Forest Tree Seed Orchards:
Implications for Genetic Diversity and
Conservation

Author
Rakesh M. Jaliya
Ph.D. Scholar, Department of Forest Biology and Tree
Improvement, College of Forestry, Navsari Agricultural
University, Navsari, Gujarat, India

Page | 65
Page | 66
 Chapter - 4
Fertility Variation in Forest Tree Seed Orchards:
Implications for Genetic Diversity and Conservation
Rakesh M. Jaliya

Abstract
Fertility variation in seed orchards of forest trees and its far-reaching
implications. Seed orchards, designed for the mass production of genetically
modified seeds, play a vital role in ensuring controlled genetic quality.
Understanding the dynamics of fertility variation within these populations is
central to assessing genetic gain, diversity, and overall commercial value. The
chapter delves into key factors such as reproductive energy, reproductive
success, and the parental balance curve, shedding light on the variability
observed among seed orchard parents. Uneven contributions from highly and
lowly productive genotypes pose challenges, leading to relatedness,
inbreeding, and a reduction in genetic diversity. Recognizing the
consequences of fertility variation is crucial for effective tree breeding and
gene conservation programs, necessitating the quantification, evaluation, and
mitigation of these variations to preserve the genetic diversity of forest tree
populations.
Keywords: Fertility variation, sibling coefficient, status number, group
coancestry
Introduction
Forest tree species are usually propagated through sexual reproduction. In
managed forests, seed trees left over after harvesting ensure that fresh forests
will naturally regenerate. Seeds for planting and sowing are usually collected
from seed orchards, good stands, and seed production areas. (Zobel and
Talbert, 1984; Eldridge et al., 1993).
A population where genetically modified seeds are economically mass-
produced is called a seed orchard (Zobel et al., 1958). The genotypes in seed
orchard populations are carefully chosen and isolated or manipulated to
minimise external pollination (gene flow) and produce frequent, abundant, and
easily harvested seed crops (Feilberg and Soegaard, 1975). Genetic gain and

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diversity estimate which are assessed based on parental breeding values,
gametic contribution, and the extent of parental genetic representation to the
seed crop, are frequently used to assess the genetic quality of crops grown in
seed orchards (Funda and El-Kassaby, 2012). Depending on the number of
improvement cycles, seed orchards are typically divided into three
generational levels (i.e., 1st, 2nd, or advanced generation). First generation
orchards are usually established by parents whose genetic value is unknown,
and the trees are typically closely spaced to allow for the rouging of poor
genotypes while maintaining a fully operational seed orchard (Zobel and
Talbert, 2003).
A seed orchards number of female and male strobili (reproductive energy)
and the conversion rate of female strobili to seed-cones (reproductive success)
are important pieces of information for determining the genetic gain and
diversity of its resultant seed crops. Considering the genetic gain and diversity
of seed orchard crops reflect their commercial values, parameters such as
genetic relatedness, inbreeding, and genetic diversity should be measured for
each seed crop. (Kang and Lindgren, 1998; Kang, 2000; Gomory et al., 2000;
Ertekin, 2010). It is commonly observed that seed orchard parents vary in both
reproductive energy and success with consistently high and low producers
resulting in disproportionate contribution from a reduced subset of parents (El-
Kassaby et al. 1989). A cumulative contribution curve, known as parental
balance curve, is often used to quantify fertility variation in seed orchards
(Griffin, 1982; Adams and Kunze, 1996).
Variation in fertility has consequences in both tree breeding and gene
conservation programmes. By over representing the most fertile genotypes,
uneven contribution of gametes among trees influences the genetic makeup of
the offspring. (Bilir et al., 2002). This leads to the accumulation of relatedness
and inbreeding and a reduction in diversity. Differences in fertility within
populations are important elements to consider when managing forest genetic
resources; they should be quantified and their impacts in the population should
also be evaluated and mitigated to retain genetic diversity (Varghese et al.,
2006). Predictions of conservation and breeding operations (e.g., germplasm
collection, establishment and utilization of seed stands and seed orchards)
require information on fertility variation. In most cases, however, the
information does not exist or cannot be collected due to the young age of the
populations concerned. Information from seed orchards and mature stands can
be utilized for predicting fertility variation. For the present chapter, we
compiled published data on fertility variation in seed orchards and its
implications. For the chapter, we gathered and estimated previously published
data on fertility variation within forest tree populations.

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Fertility Estimation
The numbers of female and male gametes produced by an individual are
considered to indicate maternal and paternal fertility, respectively. Expected
total fertility can be estimated as the average of maternal and paternal fertility.
Fertility variation can be described by sibling coefficient (Kang and Lindgren,
1999) that is the probability of two genes taken from the gamete gene pool to
originate from the same parent. This parameter was compared under the
assumption that all clones contribute as parents to the same number of
offspring.

Sibling coefficient (Ψ)

The sibling coefficient (Ψ) is related to the coefficient of variation (CV)
in parental fertility (Kang and Lindgren, 1999). The sibling coefficient for the
parental fertility can be divided into maternal sibling coefficient (Ψm) and
paternal sibling coefficient (Ψp) as,
Ψm = 𝑁 ∑𝑛𝑖=1 𝑚𝑖2 = 𝐶𝑉 2 𝑚 + 1, Ψp = 𝑁 ∑𝑛𝑖=1 𝑝𝑖2 = 𝐶𝑉 2 𝑝 + 1
Where, CVm and CVp are the coefficients of variation in maternal and
paternal fertility, Nm and Np are the numbers of female and male parents, and
mi and pi are the maternal and paternal contributions of the i-th clone,
respectively.
Total fertility variation (Ψ) was estimated as (Kang, 2001):
𝑓𝑖+𝑚𝑖 2
Ψ = 𝑁 ∑𝑛𝑖=1 𝑝𝑖2 = 𝑁 ∑𝑛𝑖=1 ( )
2

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 Where N is the census number, fi and mi are female and male fertility,
respectively.
Group coancestry (Θ)
Group coancestry (Θ) is the probability that two genes taken at random
from the gene pool of the expected seed orchard crop will be identical by
descent. It is applied for a diploid population is the average of all coancestry
values between population members (including self-coancestry), but as group
coancestry depends only on the gene pool and not how the genes are organized
into individuals, the group coancestry concept can as well be applied to
successful gametes before they form diploid zygotes (Lindgren and Mullin,
1998).
As an expression of the accumulated loss of gene diversity, group
coancestry can be estimated relative to the reference population. The reference
population is defined as having an infinite number of unrelated individuals,
and thus a group coancestry of 0 and gene diversity of 1 (Kang and Lindgren,
1999). In a seed orchard with finite number of related individuals, as described
by (Lindgren and Mullin, 1998) the group coancestry of seed orchard crops
(Θ) is given by,
Θ = ∑ 𝑝𝑖 𝑝𝑗 𝜃𝑖𝑗
Where, pi (or pj) is proportion of tree i (or j), and θij is coancestry between
tree i and j.
Gene diversity (GD)
Gene diversity Expected gene diversity (GD) is a function of the group
coancestry and can be calculated in the orchard relative to a reference
population. The reference population, which is the natural forest, has zero
group coancestry as it is considered to have infinite number of unrelated
individuals.
GD = 1 - Θ
Thus, group coancestry can be regarded as the fraction of gene diversity
lost.
Status number effective population size
It is convenient and understandable to characterize a seed orchard by an
“effective number”. Status number is such a number. For an ideal seed orchard
where the fertility of the entries is constant and there is no inbreeding or
relatedness or migration, status number of the seed orchard crop and census
number is the same.

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 Status number is half the inverse of group coancestry.
0.5
Ns =
Θ

When the seed orchard trees are considered non inbred and non-related,
status number can be calculated as,
1
N s = ∑𝑛 2
𝑖=0 𝑝𝑖

where pi is the contribution from individual genotype i to the gamete pool

and N the census number of trees in the orchard.
Relative status number (Nr) was used to compare the effective number of
trees contributing to random mating with the actual number of trees retained
in the orchard.
𝑁𝑠
Nr =
𝑁

Variance effective population size (Ne(v))

It describes the size of the population that would give the same drift in
gene frequencies as that which occurs between the seed orchard genotypes and
their seed crop. It can be calculated from sibling coefficient and coancestry
values (Kang et al., 2001).
(𝑣) Ψ
𝑁𝑒 =
2Θ(Ψ − 1)
Summary of the Published Experimental Studies on Fertility Variation in
Forest Trees
Varghese et al. (2006) compared two 25-year-old Teak clonal seed
orchards comprising 15 (CSO-I) and 20 clones (CSO-II) for fertility variation.
Out of the 15 clones, 14 were fertile in the Top slip orchard (CSO-I) whereas
in CSO-II (Walayar), 14 out of the 20 clones were fertile. Group coancestry
was high in CSO-II compared to CSO-I which had a higher effective number
of clones (Ns = 8.7) and gene diversity (GD = 0.943). The relative population
size was almost 5 times higher in CSO-I than in CSO-II. Fertility variation
differed considerably between the orchards as indicated by sibling coefficient
(Ψ) values. CSO-II had a high average Ψ value of (8.33) and CSO-I (1.71),
respectively.
Kamalakannan et al. (2007) seedling seed orchards of Eucalyptus
tereticornis (N=192 & 505) and Eucalyptus camaldulensis (N=182 & 525)
were established at two sites (one moist and one dry) in southern India. The
fertility (based on the number of flowers and fruits) was registered for each
tree at age eight and nine years. E. camaldulensis on the moist location had

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73% fertile trees and low fertility difference (sibling coefficient, Ψ, was 2.27)
at eight years. Whereas, only 23% trees were fertile in the E. tereticornis
orchard at the same site and the fertility variation was high (Ψ =11.71). In the
dry location, fertility was almost same in both the species at nine years, with
45 & 51% fertile trees in E. camaldulensis (Ψ = 5.4) and E. tereticornis (Ψ =
5.2), respectively. The coancestry values were low for all the orchards except
the E. terticornis orchard at Panampally. Gene diversity values of the seed
crop estimated for two consecutive years were fairly high except for the E.
tereticornis (GD = 0.9650 and 0.9690) orchard located in the moist site.
Nicodemus et al. (2009) observed two clonal seed orchards (CSO) of
Teak for four consecutive years (2003–2006). In CSO II, the male fertility
variation (Ψm = 2.5–6.79) was higher than the female fertility variation (Ψf =
2.09–5.68) whereas in CSO I, the trend was opposite (Ψm = 1.7 to 2.37 and
Ψf = 2.51 to 3.47) except in 2005. The smallest fertility variation (Ψ =1.49)
was observed in CSO I at the time of abundant flowering in the year of 2005.
Status number, relative status number and gene diversity were higher in CSO
I compared to CSO II.
Sumardi (2011) studied 27-year-old clonal seed orchard of Teak in
Padangan, East Java comprising 24 clones for fertility. The number of
inflorescences, flowers and fruits produced varied among clones. The average
inflorescences per clone varied from 10 (clone 7) to 53 (clone 2). The number
of flowers per clone varied from 17.725 (clone 7) to 165.053 (clone 2). The
number of fruits produced per clone varied from 4 (clone 5) to 783 (clone 3).
Fertility variation of the teak CSOs as indicated by the sibling coefficient
value (Ψ) was 1.62. Male fertility variation (Ψm) was 1.43 and female fertility
variation (Ψf) was 1.54. Coancestry (Θ), effective population size (Np), and
genetic diversity (GD) of teak clonal seed orchard in Padangan were 0.03, 15
and 0.97 respectively.
Varghese et al. (2012) compared two seed orchards each of Casuarina
equisetifolia and Casuarina junghuhniana established by selectively thinning
provenance trials located in coastal and inland regions in southern India for
fertility. There were around 50% female and 25% male trees in both sites and
13–15% trees were either monoecious or non-flowering. Fertility variation in
C. junghuhniana was very high in the inland seedling seed orchard (as
indicated by Ψ = 7.06), a contributing reason may be that many trees were not
flowering at all.
Suraj et al. (2019) compiled fertility in three second generation (F2) seed
stands (SPA 1-3) and two clone trials (CSO 1&2) of Eucalyptus camaldulensis
to assess the impact on seed crop. Fertility variation, as indicated by sibling

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coefficient, was high (Ψ, 9-14) in the SPAs as only about 26% trees were
fertile compared to 81% trees in CSOs. Effective population size was higher
in SPA 1 and 2 (Ns, 95 and 74, respectively) than SPA 3 (Ns = 39). Fertility
was highly skewed in CSO 2 resulting in low effective population size (Ns =
2) compared to CSO 1 (Ns = 11).
Bilir et al. (2002) studied the clonal variation in the production of female
and male strobili in seed orchards of Pinus brutia in southern, and Pinus nigra
and Pinus sylvestris in northern Turkey. The fertility variation (CV) in P.
brutia was higher than the others. Large differences in female and male
fertility among clones were found. Male fertility variation was larger than
female fertility variation in P. brutia and P. nigra seed orchards, while it was
an opposite situation in the P. sylvestris seed orchard. The status number and
relative status number of female parents were higher than those of male
parents in most seed orchards, except in P. sylvestris.
Kang et al. (2005) recorded clonal differences in fertility for three
consecutive years (2002–2004) in a clonal seed orchard of Cryptomeria
japonica in Korea. Average female and male strobilus production reached its
peak in 2003 where the coefficient of variation (CV) in strobilus production
among clones was lower. The CV of female strobilus production was lowest
in 2003 while the male strobilus production was lowest in 2004.Variation in
female fertility was higher than male fertility, and this variation was reflected
on female and male parents’ status numbers.
Bilir et al. (2006) studied clonal differences in fertility for three years
(2001, 2005 and 2006) in a clonal seed orchard of Scots pine (Pinus sylvestris).
Variation in female fertility was higher than that in male fertility in 2001, but
it was opposite situation in 2006. Such variation was reflected on female and
male parents’ status numbers. Clonal fertility variation measured as the sibling
coefficient was 1.10, 1.01 and 1.04 for the three years. The status numbers
varied from 22.95 to 24.69 among the three years of study.
Ertekin (2010) recorded clonal variation in the number of female and
male strobili for three consecutive years (2002–2004) in a clonal black pine
seed orchard in Turkey. The correlation between female and male strobilus
production was negative and statistically significant in 2004, a good-flowering
year. The CV for female flowering was lower than the CV for male flowering.
Male fertility variation was higher than female fertility variation in the three
years. The status number, a measure of genetic diversity, was calculated as
26.4 (2002), 23.9 (2003), and 24.0 (2004).

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Conclusion
Loss of diversity resulting from over representation of highly fertile
clones in seed orchard is a serious concern. Fertility variation also leads to
poor fruit set due to inadequate pollen supply, sexual asymmetry and
unbalanced parental contribution. The orchard manager can adopt corrective
measures to reduce the magnitude of undesirable effects of fertility variation.
Making collections with ramet and clone identity and mixing them in equal
proportions would keep the coancestry levels low. It could be achieved either
by total removal of some ramets of the clone if the fertility status is highly
skewed or by just retaining them as pollen producers. Another measure that
can be adopted to increase gene diversity is to mix seeds from different seed
orchards. These measures can help to reduce the loss of gene diversity during
domestication. As fertility variation can be high in poor flowering years, seed
collection may even be avoided during those years to avoid genetic drift.
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 Chapter - 5
Molecular Characterization of Phytoplasma: An
Overview

Authors
Shoeb Ahmad
Department of Botany, Gandhi Faiz-e-Aam College,
Shahjahanpur, Affiliated to M. J. P. Rohilkhand University,
Bareilly, Uttar Pradesh, India
Shveta Malhotra
Department of Botany, Gandhi Faiz-e-Aam College,
Shahjahanpur, Affiliated to M. J. P. Rohilkhand University,
Bareilly, Uttar Pradesh, India
Syed Saad
Department of Botany, Gandhi Faiz-e-Aam College,
Shahjahanpur, Affiliated to M. J. P. Rohilkhand University,
Bareilly, Uttar Pradesh, India
Akil Ahmad Khan
Department of Botany, Gandhi Faiz-e-Aam College,
Shahjahanpur, Affiliated to M. J. P. Rohilkhand University,
Bareilly, Uttar Pradesh, India
Azahar Sajjad
Department of Botany, Gandhi Faiz-e-Aam College,
Shahjahanpur, Affiliated to M. J. P. Rohilkhand University,
Bareilly, Uttar Pradesh, India

Page | 77
Page | 78
 Chapter - 5
Molecular Characterization of Phytoplasma: An Overview
Shoeb Ahmad, Shveta Malhotra, Syed Saad, Akil Ahmad Khan and Azahar Sajjad

Abstract
Over the past four decades, a variety of plant diseases characterized by
yellowing have been linked to phytoplasmas, which are prokaryotic
pathogens lacking a cell wall. As of yet, these viruses have not been
cultivated in a pure culture; therefore, advancements in their study primarily
rely on molecular approaches. Global occurrences of severe disease
outbreaks linked to the presence of phytoplasma have been documented.
Molecular approaches can now be used to detect and characterize
phytoplasmas that infect various plant species. These methods rely on the
analysis of 16SrDNA polymorphisms. Examining genes that encode
ribosomal proteins like S3, tuf, SecY, amp, imp, and other genes allows one
to observe the molecular diversity of phytoplasmas. Phytoplasmas possess
abnormally compact genomes, and these repetitions may be associated with
their ability to thrive across many kingdoms and their capacity to cause
diseases.
Keywords: Phytoplasma, genome sequence, membrane protein.
Introduction
Phytoplasmas are phytopathogenic microorganisms that are exclusive to
elements of the phloem sieve. Spread of these pathogens to many species is
done by phloem-feeding insects like leafhoppers, plant hoppers, and psyllids
(Trivellone, V., & Dietrich, C. H., 2021). Phytoplasma infections cause
plants to grow more slowly, get a purple top (because anthocyanin builds
up), get dwarfed, wilt, sprout early, turn yellow, make witches' broom (many
shoots with small leaves), show virescence (abnormal green coloration),
phyllody (the growth of leaf-like structures instead of regular flowers), and
generally decline. Agricultural production and quality are notably affected
by these symptoms (Namba, S., 2019). According to Chiu et al. (2023), more
phytoplasma strains from the 16SrII-V subgroup were found in Taiwan.
They are very closely related to the phytoplasma strains found in this study.
These results not only indicate the local distribution of phytoplasma strains

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belonging to the 16SrII-V subgroup, but they also serve as a cautionary tale
about the importance of preventing the spread of this threat before it
becomes pervasive.
An analysis of the infection system or virulence mechanism of
phytoplasmas is difficult due to the impossibility of cultivating them in a
laboratory environment. Because of recent developments in sequencing
technology, it is now possible to ascertain the complete genomic sequences
of non-cultivable bacteria. As of now, the complete genome sequences of
four phytoplasmas have been effectively deduced by scientists (Oshima et
al., 2004; Bai et al., 2006; Kube et al., 2008; Tran-Nguyen et al., 2008). The
genomes of phytoplasmas contain an estimated 500-840 genes, of which 40-
50% encode putative proteins, the exact function of which is unknown.
Plant-pathogenic bacteria typically contain an assortment of pathogenicity
genes (Abramovitch et al., 2006; Jones and Dang et al., 2006). The
clustering of these genes on a chromosome is referred to as a "pathogenicity
island." A type-III secretion system is crucial for numerous phytopathogenic
bacteria as it facilitates the entry of effector proteins into host cells (Grant et
al., 2006). There are no genes in the genomes of phytoplasma that resemble
any of the identified pathogenicity genes. This implies that the interaction
between phytoplasmas and their hosts involves novel mechanisms (Oshima
et al., 2002, 2004, 2007). Without cell walls, phytoplasmas are
microorganisms that reside within host cells. Phytoplasma-produced or
membrane-localized proteins appear to exert a direct influence on the
cytoplasm of insect and host plant cells (Hogenhout et al., 2008). In order to
comprehend the interaction between phytoplasma and its host, it is crucial to
possess knowledge regarding the functions of membrane proteins or secreted
proteins whose names are encoded in phytoplasma genomes. This segment
presents a comprehensive summary of recent publications pertaining to the
secretion system and membrane proteins of phytoplasmas.
The phytoplasma secretory system is functional
The phytoplasma found on parthenium vegetation and green peas is the
initial documentation of such organisms in Taiwan. The in-depth molecular
analysis showed that green legumes and parthenium weed were infected by
the 16SrII-V subgroup phytoplasma strains, which are biologically very
similar to the peanut-associated phytoplasma strain. As symptomatic
parthenium weeds and green peas were detected during field surveys
conducted in close proximity to or adjacent to peanut fields, prevention and
treatment strategies for this disease must be carefully considered.
Epidemiological cycles of phytoplasma encompass both vegetation and

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invertebrates. Some invasive weeds in Mailiao, Yunlin, Taiwan were found
to have 16SrII-V subgroup phytoplasma strains on them. These weeds are
Nicotiana plumbaginifolia, Eclipta prostrata, Ixeris chinensis, Digera
muricata, and Eclipta prostrata (Mejia, H. M., et al., 2022). Therefore, it is
critical to identify insect vectors in order to make well-informed decisions
regarding the management of phytoplasma diseases, which threaten the
agricultural sector and food security of Taiwan. Phytoplasma is responsible
for substantial agricultural losses and poses health risks to both humans and
livestock. In extreme circumstances, it can induce cell mutagenicity and
contact dermatitis (Ojija, F., 2022). On the contrary, certain communities
employ P. hysterophorus medicinally to treat a range of ailments such as
inflammation, skin disorders, colds, and heart problems (Kaur, L., et al.,
2021). Notwithstanding its limited economic utility, parthenium weed
exhibits the potential to serve as an alternative plant host for disease
transmission, owing to its remarkable adaptability to a wide range of
environmental conditions. Huang et al. (2022) have established a connection
between several agricultural diseases in Taiwan and plant pathogens
belonging to the 16SrII, 16SrVIII, 16SrX, 16SrXI, and 16SrXII groups. Pear
decline, rice yellow dwarf, papaya yellows, and peanut witches' broom are a
few examples. It has been shown that pea plants in Europe, Asia, and North
America have been infected with phytoplasmas from different subgroups and
groups (16SrI, 16SrII-C, 16SrII-D, and 16SrXII-A). Some phytoplasma
infections, like those linked to 16SrII-A, 16SrII-C, and 16SrII-D, have been
found in Parthenium plants in Asia and Africa (Huang, C.-T., et al., 2022).
According to this investigation, the 16SrII-V subgroup phytoplasma strain,
which was first identified in Taiwan, is associated with green peas and
parthenium vegetation. Finding the SAP11 and PHYL1 effectors helps us
understand more about the bad things that happen in green peas and
parthenium weed, which are both infected with phytoplasma.
Bacteria possess a minimum of five distinct protein export mechanisms
(Economou, 1999). Escherichia coli uses these systems to eliminate a wide
range of proteins, including adhesins, toxins, and hydrolytic enzymes.
Among these systems, only the Sec system is of critical importance for
cellular survival. Among four distinct transport pathways, the Sec pathway is
regarded as the most significant in Bacillus subtilis (Tjalsma et al., 2000).
The Sec protein translocation system, which comprises a minimum of eleven
proteins and one RNA species, has been the subject of extensive research in
E. coli (Economou, 1999). A translocase complex composed of the proteins
SecY, SecE, SecG, and SecA serves as an export mechanism across the

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cytoplasmic membrane. In E. coli, protein translocation and cell survival are
dependent on SecA, SecY, and SecE (Economou, 1999). Protein
translocation can be effectively reinstated using only these three proteins,
according to in vitro experiments (Akimaru et al., 1991); however, SecG is
not regarded as an essential component of this process. SecYEG hetero
tritrimers create a pore to pass through the membrane. The N-terminal region
of secretory proteins, which are targets of the Sec system, contains the signal
peptide. The signal recognition particle (SRP) identifies the signal peptide
during the transcription of a secretory protein. The signal recognition particle
(SRP) in E. coli consists of two components: a protein called Ffh and a
molecule called 4.5SRNA. The Signal Recognition Particle (SRP)
specifically directs particular proteins to the cellular membrane, with FtsY
being widely believed to function as the receptor for SRP. SecB is a
chaperone that is particular to the Sec system. By identifying specific
sequence patterns in the mature region of a secretory protein, it inhibits the
folding process of the protein. To facilitate the transport of a secretory
protein to the SecYEG pore, SecA assembles with a chaperone. SecA drives
the sequential secretion of proteins by utilizing its ATPase function.
Scientists have discovered the SecA, SecY, and SecE proteins' genes in the
"Candidatus Phytoplasma asteris" OY strain (Kakizawa et al., 2001, 2004).
Significant components of the Sec system are these proteins (Economou,
1999). Furthermore, previous studies have confirmed the existence of SecA
protein in phytoplasma-infected plants (Kakizawa et al., 2001; Wei et al.,
2004). The AY-WB phytoplasma genome investigation, which Bai et al.
conducted in 2006, also identified the aforementioned three genes.
Furthermore, Lee et al. (2006) documented the cloning of SecY genes from
additional phytoplasmas. The findings presented persuasive proof that the
Sec system is prevalent in phytoplasmas. Antigenic membrane proteins
(Amps) are a distinct category of membrane proteins that are present in
phytoplasmas and play a role in eliciting immune responses. At its N-
terminus is a signal sequence referred to as the Sec system. The fact that the
Amp signal sequence is cut in OY phytoplasma shows that the Sec system is
working in phytoplasma.
Prediction of phytoplasma secretory proteins
It is anticipated that the cell-wall-less phytoplasmas' membrane proteins,
or secreted proteins, play a direct functional role in the cytoplasm of both the
insect and host plant cells. So, it is thought that proteins like adhesins,
proteases, and hydrolytic enzymes might be sent from the phytoplasma
cytoplasm to either the phytoplasma cell surface or the host cytoplasm

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through the Sec pathway. The transfer of these proteins could potentially
affect pathogenicity. Therefore, it is imperative to identify secreted proteins
within the phytoplasma genome in order to fully comprehend the host-
phytoplasma interactions. Generally, secretory proteins that employ the Sec
system are equipped with a signal peptide situated at the N-terminus. The
consensus signal sequence is made up of three parts: an N-terminal domain
that is positively charged and has at least one arginine or lysine residue; a
hydrophobic core domain that goes into the inner membrane as an alpha-
helix; and an A-X-Z domain. The consensus sequence functions as the signal
peptidase I (SPaseI) cleavage site. The two alanine residues in this consensus
sequence can be switched out for other minor and uncharged residues, such
as valine, leucine, isoleucine, and others (Tjalsma et al., 2000). The expected
export signal sequence of OY Amp exhibits a high degree of similarity to the
customary signal peptides found in the Sec system. It appears that the Sec
systems of both E. coli and phytoplasma export OY Amp along with the
processing of its signal sequence. Kakizawa et al. (2004) state that this
illustrates the similarity between the signal sequence identification methods
utilized by E. coli and phytoplasma. Prediction programs such as PSORT
(Nakai and Kanehisa, 1991) or SignalP (Nielsen et al., 1997) could therefore
be utilized to identify signal sequences in phytoplasma proteins. This would
enable the identification of phytoplasmic secretory proteins. The secretory
proteins of phytoplasmas, including both membrane-embedded and
cytoplasmic secreted proteins, are anticipated to have direct interactions with
host plant and insect cells and are believed to have important functions in the
interactions between the host and phytoplasmas. As a result, it is crucial to
investigate the proteins that phytoplasmas transport and research the
functions of these exported proteins in order to comprehend how the host
and the phytoplasma interact. Recent reports indicate that the AY-WB strain
of 'Ca. Phytoplasma asteris' produces a protein that specifically targets the
nuclei of plant cells. According to Bai et al. (2009), this protein is one of the
potential factors influencing the virulence of phytoplasma.
Other protein secretion systems in phytoplasma
Gram-negative pathogens are capable of infecting animals and
vegetation. Virulence-enhancing proteins can enter host cells via type III
secretion systems (T3SS) (Cornelis & van Gijsegem, 2000). The
evolutionary origins of the Type III Secretion System (T3SS) and flagella
are intricately intertwined, and their foundational structures are remarkably
similar. However, it is worth noting that flagella and the Type III Secretion
System (T3SS) are exclusively observed in gram-negative bacteria.
Phytoplasmas, being categorized as Gram-positive bacteria, do not possess

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any identified Type III Secretion Systems (T3SS). The type IV secretion
system (T4SS) is an important secretion system that is present in diseases of
both plants and animals. A heterogeneous collection of translocation
systems, the Type IV Secretion System (T4SS) operates as a pilus-like
structure. It enables the translocation of protein and DNA substrates to
bacterial or eukaryotic cells via the cell envelope. As described by
Grohmannet et al. in 2003, this transfer frequently happens through direct
cell-to-cell contact. Both Gram-positive and Gram-negative bacteria have
proteins that are part of the conjugative transfer system. These proteins have
sequences that are similar to those of the T4SS. As a result, it is believed that
the conjugative transfer system and the T4SS have a common ancestral
relationship (Grohmann et al., 2003). Hence, Christie and Cascales (2005)
proposed that the Type IV Secretion System (T4SS) is extensively present in
both Gram-negative and Gram-positive bacteria. Phytoplasmas lack both pili
and T4SS. Phytoplasma genomes lack genes that show similarity to the
proteins found in pili or T4SS. In addition, electron microscopy analysis has
not uncovered any indications of pililike structures. On the other hand,
spiroplasmas exhibit structures resembling pili (Ammar et al., 2004). The
process of incorporating newly synthesized membrane proteins into the cell
membrane is facilitated by YidC. It was observed that YidC exhibited co-
purification with components of the Sec system (Scotti et al., 2000). Urbanus
et al. (2001) hypothesized that YidC collaborates with the Sec translocase to
facilitate the passage of Sec-dependent substrate proteins across the
membrane and into the hydrophobic bilayer. Recent studies have
demonstrated that YidC in isolation can facilitate the passage of a membrane
protein (Pf3 coat protein) across a laboratory membrane. This illustrates the
capability of YidC to function autonomously from the Sec system. (Dalbey
and Kuhn, 2000; Samuelsone et al., 2000) YidC is utilized exclusively for
the incorporation of membrane proteins and is not implicated in the
translocation of exported proteins. The probable function of YidC is to
identify the water-averse regions of a membrane protein and facilitate their
trans membrane orientation when joining with the membrane bilayer (Serek
et al., 2004). YidC is encoded by a single gene in both the OY and AYWB
phytoplasma genomes (Oshima et al., 2004; Bai et al., 2006). The
phytoplasma therefore possesses this system for integrating YidC. It is
conceivable that YidC, an essential protein in E. coli (Samuelson et al.,
2000), exerts a comparable influence in phytoplasmas.
The major membrane protein of phytoplasmas
In earlier research, it was shown that immuno dominant membrane
proteins (IDPs), a group of proteins found in the cell membrane, make up a

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big part of the total cellular membrane proteins in most phytoplasmas (Shen
and Lin, 1993). Electron microscopy and immuno gold labeling experiments
showed that IDP is on the cell membrane's edge (Milne et al., 1995).
Because mollicute membrane proteins probably help bacteria stick to the
surface of the host cell, it is possible that the IDP changes how the host and
phytoplasma interact with each other. Several phytoplasmas have yielded
genes that encode IDP, which have been identified and isolated (Berg et al.,
1999; Blomquist et al., 2001; Barbara et al., 2002; Morton et al., 2003;
Kakizawa et al., 2004, 2006a). The proteins demonstrate considerable
heterogeneity in terms of their antigenic properties and amino acid
composition. In addition to a primary hydrophilic region, which may be
situated on the outer surface of the phytoplasmal cell, every protein
comprises one to two trans membrane domains. During protein localization,
immuno dominant membrane proteins are likely secreted through the
phytoplasmal cell membrane. Antigenic membrane protein (Amp), immuno
dominant membrane protein A (IdpA), and immuno dominant membrane
protein (Imp) comprised the three distinct forms of IDPs. The amino acid
composition of these internally displaced persons (IDPs) is completely
dissimilar, and they are located in discrete regions of the genome. An
external location is not a prerequisite for the presence of a central
hydrophilic region (CPR) in internally displaced persons (IDPs).
Nevertheless, there is variation in the configuration of the hydrophobic trans
membrane anchor (Kakizawa et al., 2006b). These three distinct categories
of IDPs are therefore not homologous. Stabilization of the initial type, Imp,
is attributed exclusively to N-terminal trans membrane regions. The IdpA
subtype, which is the second in nature, comprises trans membrane regions at
both its N- and C-termini, neither of which is subject to cleavage. Barbara et
al. (2002) describes the third type, Amp, which also comprises two trans
membrane regions; however, it is noteworthy that only the C-terminal region
serves as an anchor since the N-terminal region is cleaved. These two
phytoplasmas the Western X-disease phytoplasma (WX) and the OY
phytoplasma both had the imp gene found in their genomes, along with their
original IDP genes in 2003 and 2009. Despite the relatively low sequence
similarity of imp between OY and WX, the gene arrangement encompassing
imp is exceptionally conserved. It was found by Oshima et al. (2004), Bai et
al. (2006), Kube et al. (2008), Tran-Nguyen et al. (2008), and Kakizawa et
al. (2009) that the WX IDP was missing from the full genome sequences of
other phytoplasmas. This was done by using comparison-based search or
genomic structure-based search. As an additional point, the complete
genomic sequence of 'Ca. Phytoplasma mali' lacked the homologous

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counterpart of amp. Based on the available data, it is probable that the
phytoplasma ancestor possessed imp. Subsequently, the AY or WX groups
may have acquired Amp or IdpA, respectively, through evolution.
The variability and positive selection of IDPs
In earlier research, it was shown that immuno dominant membrane
proteins (IDPs), a group of proteins found in the cell membrane, make up a
big part of the total cellular membrane proteins in most phytoplasmas (Shen
and Lin, 1993). Electron microscopy and immuno gold labelling experiments
showed that IDP is on the cell membrane's edge (Milne et al., 1995).
Because mollicute membrane proteins probably help bacteria stick to the
surface of the host cell, it is possible that the IDP changes how the host and
phytoplasma interact with each other. Several phytoplasmas have yielded
genes that encode IDP, which have been identified and isolated (Berg et al.,
1999; Blomquist et al., 2001; Barbara et al., 2002; Morton et al., 2003;
Kakizawa et al., 2004, 2006a). The proteins demonstrate considerable
heterogeneity in terms of their antigenic properties and amino acid
composition. In addition to a primary hydrophilic region, which may be
situated on the outer surface of the phytoplasmal cell, every protein
comprises one to two trans membrane domains. During protein localization,
immuno dominant membrane proteins are likely secreted through the
phytoplasmal cell membrane. Antigenic membrane protein (Amp), immuno
dominant membrane protein A (IdpA), and immuno dominant membrane
protein (Imp) comprised the three distinct forms of IDPs. The amino acid
composition of these internally displaced persons (IDPs) is completely
dissimilar, and they are located in discrete regions of the genome. An
external location is not a prerequisite for the presence of a central
hydrophilic region (CPR) in internally displaced persons (IDPs).
Nevertheless, there is variation in the configuration of the hydrophobic trans
membrane anchor (Kakizawa et al., 2006b). These three distinct categories
of IDPs are therefore not homologous. Stabilization of the initial type, Imp,
is attributed exclusively to N-terminal trans membrane regions. The IdpA
subtype, which is the second in nature, comprises trans membrane regions at
both its N- and C-termini, neither of which is subject to cleavage. Barbara et
al. (2002) describes the third type, Amp, which also comprises two trans
membrane regions; however, it is noteworthy that only the C-terminal region
serves as an anchor since the N-terminal region is cleaved. Alongside their
original IDP genes, the imp gene was discovered in the genomes of the
Western X-disease phytoplasma (WX) and the OY phytoplasma (Kakizawa
et al., 2009) (Liefting and Kirkpatrick, 2003). Despite the relatively low

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sequence similarity of imp between OY and WX, the gene arrangement
encompassing imp is exceptionally conserved. Oshima et al. (2004), Bai et
al. (2006), Kube et al. (2008), Tran-Nguyen et al. (2008), and Kakizawa et
al. (2009) have all reported that the IDP of WX was absent from other
phytoplasmas' complete genome sequences. Using comparison-based search
or genomic structure-based search, this was discovered. As an additional
point, the complete genomic sequence of 'Ca. Phytoplasma mali' lacked the
homologous counterpart of amp. Based on the available data, it is probable
that the phytoplasma ancestor possessed imp. Subsequently, the AY or WX
groups may have acquired Amp or IdpA, respectively, through evolution.
Amp forms the complex with insect microfilaments
According to a study by Suzuki et al. (2006), the manner in which the
Amp protein (a membrane protein that is immunologically dominant) of OY
phytoplasma interacts with the insect microfilament complex determines the
specific insect vector. The aforementioned data is illustrated in Plate 1. The
OY phytoplasma was specifically detected in the microfilaments of the
visceral smooth muscle encircling the digestive system of the insect. In
addition, the Amp forms a complex with actin, myosin heavy chain, and
myosin light chain, three insect proteins, in both laboratory and living
organism environments. The ability of leaf hoppers to transmit phytoplasmas
was discovered to be associated with the formation of the Amp-
microfilament complex (AM complex). This indicates that the manner in
which Amp and insect microfilament complexes interact is a critical
determinant in determining the ease of phytoplasma transmission.
The interactions between the surface proteins of bacteria and host
microfilaments have been the subject of numerous reports. In the case of
bacteria that induce diseases in mammals, including Listeria, Salmonella,
and Shigella, interactions between the surface membrane proteins of the
bacteria and the microfilaments of host cells have been extensively
documented. A multitude of scholarly investigations have documented these
interactions, including those by Hayward and Koronakis (1999, 2002),
Gouin et al. (1999), van Nhieu et al. (1999), Zhou et al. (1999), Juris et al.
(2000), Pantaloni et al. (2001), Delahay and Frankel (2002), and Cossart et
al. (2003). The capacity to establish a complex with the microfilaments of
the host cell seems to be a critical factor in determining the host cell's fate.
Bacterial translocation across compromised epithelial cells is facilitated by
the actin polymerization apparatus. This facilitates the bacteria's migration
from the cytoplasm to adjacent epithelial cells (Goldberg, 2001). VirG, an
outer-membrane protein, is of paramount importance in facilitating the actin-

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based motility of Shigella. Critical to this process is the interaction between
VirG and its specific host ligand, neural. (Suzuki et al., 2002) The Wiskott-
Aldrich syndrome protein (N-WASP) regulates the actin cytoskeleton and
determines the type of host cell, thereby facilitating the proliferation of actin-
based structures.
The microfilament of the host cell interacts with a bacterial membrane
protein. This, in conjunction with the intricate interplay between the
phytoplasma Amp and the microfilament of the insect host, constitutes a
comprehensive system that is critical for the effective invasion of bacteria.
There are several phases involved in the infection of insect hosts by
phytoplasmas or spiroplasmas (Hogenhout et al., 2008). Initially, the insect
consumes mollicutes from plant phloem elements using its stylet. Ultimately,
the microbes adhere to the insect host's gastric epithelial cells. Following
this, the parasites proceed to invade the cells of the gastrointestinal tract,
reproduce, pass through the intestinal barrier, and then infiltrate the
hemolymph. There, they conduct additional replication and spread to various
parts of the body. In the end, the parasites successfully invade the salivary
gland, proliferate, and become introduced into the phloem of the plant
through the insect's ingestion of sustenance, thereby facilitating transmission
to an alternative plant host. In order to investigate the mode of transportation
employed by phytoplasma, scientists examined its ability to traverse the
intestinal tract of insects, which consisted of the salivary gland, connective
tissue, epithelial cells, and visceral muscle (Purcell et al., 1981). 1979 saw
the discovery by Markham and Townsend that spiroplasmas cannot spread
without effectively traversing the salivary gland. It is critical to establish the
AM complex in order to surmount the obstacles presented by the host.
Spiroplasma citri is hypothesized to enter the intestinal epithelium of its host
insect, Circulifer tenellus (Baker), via endocytosis mediated by receptors
(Kwon et al., 1999). It is probable that the epithelial cells of leaf hopper
intestines contain receptors that recognize particular proteins present in the
spiroplasma membrane. Numerous prospective genes responsible for
encoding attachment proteins of S. citri have been discovered, including P58
(Ye et al., 1997), SARP1 (Berg et al., 2001), the immunological dominant
membrane protein (spiralin) (Foissac et al., 1997; Duret et al., 2003), and
P32 of the pSci6 plasmid (Berho et al., 2006). Reports indicate that the
efficacy of spiralin in facilitating its dissemination was diminished due to a
defective variant (Fletcher et al., 1996). Furthermore, spiralin has been found
to bind to the glycoproteins of Circulifer haematoceps (Mulsant & Rey), the
insect carrier of spiralin (Killiny et al., 2005). Although no discernible

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resemblance has been found between spiralin and Amp, it is widely
recognized that the immunologically dominant membrane proteins of
spiroplasmas and phytoplasmas exert a substantial influence on insect vector
transmission.
According to a recent study by Berho et al. (2006), the plasmid pSci6
confers the capacity to transmit insects to a strain of S. citri that lacks this
capability by nature. The pSci6 plasmid harbors the genetic material
necessary to encode the P32 protein, a protein that was formerly
hypothesized to be associated with insect disease transmission. When the
non-transmissible strain of S. citri was introduced with the p32 gene alone,
however, no restoration of transmissibility was observed. Hence, it is
conceivable that there exist additional pivotal pSci6-encoded factors that are
indispensable for insect transmission, in addition to P32. As a result, a
comprehensive analysis of pSci6 is expected. The AM complex is essential
for insect transmissibility in phytoplasma; however, it does not adequately
characterize the entirety of the insect transmission process, which involves
two barriers to the insect host being overcome. As a result, further
components would also be required. Previous research (Oshima et al., 2001a,
b; Nishigawa et al., 2002) has established that the ORF3 gene, which is
encoded on plasmids, is heavily involved in insect transmission.
Furthermore, it is imperative to undertake a more thorough investigation of
ORF3 so as to clarify the exact mechanism through which insect
transmission takes place.
Amp-microfilament complex determines insect vector specificity
Insect-borne infections have the potential to inflict severe harm on
humans, animals, and plants due to their ability to spread quickly across a
large geographical area. Even if they are closely related, specific insect
vectors typically spread insect-transmissible diseases like phytoplasmas
rather than other insects (Lee et al., 2000; Alavi et al., 2003). As a result, the
quantity of insect vector species with the capacity to spread the infection
heavily influences the degree of harm that a virus causes (Lee et al., 2000).
Understanding the mechanisms that determine the specificity of insects as
vectors can help distinguish between insects that are vectors and those that
are not. This knowledge can also aid in monitoring and predicting the spread
and infection route of the pathogen. The plant-host ranges are diverse, and
each phytoplasma species typically has a unique vector insect. According to
research by Lee et al. (2000) and Hogenhout et al. (2008), phytoplasmas are
capable of infecting a minimum of 700 plant species from 98 families.
Additionally, McCoy et al. (1989) reported that AY has the capacity to infect

Page | 89
about 161 plant species across 120 genera and 39 families. Most
phytoplasmas have the ability to infect the periwinkle plant (Lee et al.,
1998). Conversely, the level of specificity for the insect host is typically
more stringent. Macro stelesstriifrons (Fallen) is capable of transmitting OY
phytoplasma; however, it is unable to propagate rice yellow dwarf (RYD)
phytoplasma, specifically the strain known as 'Ca. Phytoplasma oryzae'. On
the contrary, Nephotettix cincticeps Uhler is capable of transmitting RYD
phytoplasma, but it is not susceptible to infection by OY phytoplasma.
Hence, the identification of insect vectors plays a crucial role in determining
the complete range of hosts that phytoplasmas can infect in their natural
environment. The interactions between the Amp of OY phytoplasma and the
insect microfilament complex. These interactions are linked to a certain type
of insect vector (Suzuki et al., 2006). As a result, the development of the AM
complex is crucial in determining the variety of hosts that phytoplasmas can
infect in their natural environment as well as the extent of agricultural
damage these phytoplasmas can cause. While the significance of the AM
complex in determining insect vector specificity is well recognized, there are
still numerous uncertainties surrounding this topic. It is uncertain if the AM
complex is linked to these three potential stages, and further investigation is
required to establish clarity. Furthermore, as previously stated, there are two
primary obstacles, namely the insect intestine and the salivary gland, that
must be overcome for a phytoplasma to successfully infect. The relationship
between the AM complex and specific obstacles remains uncertain.
Furthermore, which specific protein directly interacts with Amp? So far,
researchers have discovered three proteins that have the ability to bind Amp:
actin, myosin light chain, and myosin heavy chain (Suzuki et al., 2006). It
remains uncertain whether Amp has the ability to attach to any of these three
components or if there are more variables that play a role in the AM
complex. Furthermore, what causes the amp to exhibit such variability? As
previously stated, Amp exhibited positive selection, indicating that amino
acid changes in Amp confer fitness benefits to phytoplasmas. While Amp
does indeed have the potential to combine with the insect microfilament, this
particular characteristic does not account for the wide range of variations
observed in Amp. Prior research has indicated that interactions between
hosts and bacteria contribute to the diversity of bacterial membrane proteins
(Deitsch et al., 1997). In the case of the membrane protein OspC of
Borreliaburgdorferi, the bacterium responsible for causing Lyme disease, the
attachment protein plays a crucial role in infection. It is necessary for this
protein to undergo co-evolution with the host and potentially undergo
positive changes in order to adapt to a new host species. Based on these

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examples, we can guess that phytoplasmas may be subject to different types
of selective pressure, such as having to change in order to avoid being killed
by the immune systems of insects that carry them (this is similar to how
some membrane proteins in animal pathogens change to avoid being killed).
Another possibility is that they attach to host proteins, which is a crucial step
in establishing infection for various pathogens. Further analysis will be
needed to determine the cause of positive selection on Amp and the links
between the function and variability of Amp.
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Page | 100
 Chapter - 6
Ethnobotanical Importance and Pharmacological
Action of Moringa oleifera

Author
Vidyan Kumari
Assistant professor, University Department of Botany, Dr
Shyama Prasad Mukherjee University, Ranchi, Jharkhand,
India

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Page | 102
 Chapter - 6
Ethnobotanical Importance and Pharmacological Action of
Moringa oleifera
Vidyan Kumari

Abstract
Moringa oleifera, native to the Asian subcontinent, is cultivated across
Southeast Asia, where it is considered one of the indigenous species. The
pods and leaves of the plant species are valuable source of nutrition and
medicinal properties. It is also used as protein-rich animal fodder. The plant
is very common to use as a live fencing materials in backyard farming and
cultural sues in the tribal areas. The plant is also applied as a water purifier.
This chapter reviews the ethnobotanical importance and pharmacological
action of the M. oleifera as there is potential to improve its medicinal and
nutritional value for the benefit of tribal and poor people. Rural people could
also be benefited by further adoption of the species in agroforestry systems.
Keywords: Ethnobotanical, Moringa oleifera, non-timber forest product,
nutritional & medicinal value, traditional knowledge.
Introduction
Moringa oleifera Lam, belongs to family Moringaceae is commonly
known as drumstick is a multi-functional non-timber plant species mostly
used by poor and tribal people as food, nutritional source, medicinal use,
fodder and as a biological fencing for small backyard farm. It is a medium
height, evergreen, fast growing; deciduous tree which is generally grows up
to 12 or 15 m in height. The plant species is found in Sub Himalayan Tracts,
Peninsular India, Bengal and Assam (Gupta 2010).
Recently, M. oleifera has gained attention especially because of its
medicinal and nutritional importance. Root, bark, leaf, flower, pod and seeds
of the plant are considered as important sources of essential minerals,
proteins, lipids and vitamins for human as well as livestock health.
Therefore, the plant species is integrated into different programs of food aid
for preventing malnutrition of adolescent, children and nursing mothers
(Sreelatha and Padma 2009). Every part of this plant has valuable medicinal
properties. The plant parts hold rich source of the vitamin A, vitamin C and

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protein. Various kinds of active phytoconstituents such as protein, alkaloids,
flavonoids, steroids, glycosides, quinine, saponins, tannin, fats and fixed oil
are found in the plant. Some other phyto-constituents present in the plant
parts are niazimicin A, niaziminin B, niazinin A and niazinin B. The seeds of
M. oleifera have antioxidant properties and thus acquire potential anticancer
effect (Kumar et al. 2007). Hypotensive elements and antispasmodic activity
of moringa give scientific basis for traditional uses in the control of gastro-
intestinal problems (Kalogo et al. 2000). The present review discusses the
Ethnobotanical importance and pharmacological properties of the plant
species.

Traditional uses
M. oleifera or drumstick also known as sahajan or munga is traditionally
used as antispasmodic, stimulant, antilithic, antiseptic, expectorant and
diuretic. The plant is also used as cardiac tonic. Stem-bark and flowers have

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hypoglyceamic properties. Leaf juice is given in hiccough; cooked leaves are
given in influenza and catarrhal affections. Bark is emmenogogue and even
abortifacient, antifungal and antibacterial. Flowers are cholagogue,
stimulant, tonic and diuretic and useful to increase the flow of bile. Pods are
antipyretic, anthelmintic and fried pods are used in diabetes. Gum is bland
and mucilaginous. Seeds are acrid and stimulant. Infusion of seed has anti-
inflammatory, antispasmodic and diuretic properties. It is also given in
venereal diseases. Fresh root is acrid and vesicant. Root juice is given in
cardiac tonic, antiviral, anti-inflammatory, analgesic and antiepileptic. It is
also used for nervous debility, asthma, enlarged liver and spleen, deep seated
inflammation and as diuretic in calculus affection. Decoction of the root is
used as a gargle in hoarseness and sore throat. Root and fruit have
antiparalytic properties (Nadkarni 2009). The Ayurvedic Pharmacopoeia of
India reported the use of the leaf, seed, root bark and stem bark in internal
abscess and piles. The dried root barks are effective in lipid disorders, goiter
and glycosuria (Khare 2007). The poor rural and tribal people generally use
the plant parts as a medicine as well as vegetable and other meals. A review
of the documented traditional knowledge of M. oleifera in response to
medicinal properties is discussed below.
Traditional use of leaf
S. No. Ailments Method of use
Crushed fresh leaves of Moringa are mixed with water
1. Anemia along with two lumps of sugar and condensed milk. Given
to the anemic patient twice a day.
Fresh leaves are grinded along with potassium hydroxide
2. Abscess
and applied on the abscess and covered it with bandage.
It is a generalized physical weakness or lack of strength.
3. Asthenia Traditionally leaf powder is mixed in the meals and given
to the patient every morning.
The leaf is crushed with the leaves of Newbouldia laevis
4. Cough along with lemon in water. The mixture is given to the
patient every morning.
The fresh leaves are crushed and juice is extracted. Two
5. Eyesight
drops of fresh juice is dropped in the eyes.
Grounded fresh leaves juice are given to the patient three
6. Fever
teaspoons every day.
Fresh leaves are grinded along with seven hot peppers and
7. Gonorrhea salt. It is used by the patient one cup every morning and
evening for15 days.

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 Leaf powder are mixed with the meals and given it
8. Hemorrhoids
regularly.
9. High B.P. Dried leaf powder is used with meals every morning.
Headaches and Grinded fresh leaf juice is dropped in the eyes or massages
10.
Migraine the forehead in case of pain.
Immune Dried leaf powder is consumed with a cup of lukewarm
11.
deficiency milk every evening.
12. Infertility Dried leaf powder is use with meals twice a day.
Intestinal Dried leaf powder is mixed in water and given to the
13.
worms patient every evening for seven days.
Infusion of leaf along with the leaves of Spondias mombin
14. Malaria
and lemon juice is drink by the patient.

Traditional use of root

S. No. Ailments Method of use
Aqueous root extraction of Moringa is given to the patient
1. Dysentery
every day 5 times.
Two drops of extracted fresh root juice is dropped I the
2. Ear pain
ears every morning and evening.
Small pieces of roots of Moringa is mixed with Carica
papaya, orange peel, paste of white onion, seeds of Xylopia
3. Hernia
aethiopica and Monodora myristica. The drink is given to
the patient thrice every day.
Headaches, Fresh roots are crushed and put some drops in the eyes as
4.
Migraine well as massage on forehead every morning and evening.
Crushed root of Moringa is mixed with lemon juice and the
5. Joint pain
paste is applied to the joint part.
Dry root powder is mixed in water and given to the patient
6. Stomach pain
three times in a cup.
Fresh root is washed and cut into small pieces. The root is
7. Tooth ache
chewed two to three times every day.
8. Sinusitis Root juice is inhaled every evening.
Sexual Dry root powder of Moringa is mixed with hot pepper and
9.
weakness water. The preparation is used every evening.

Traditional use of bark

S. No. Ailments Method of use
1. Tooth decay Bark is cut into small pieces and chewed and holds the

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 juice. Use three times every day.
Bark is soaked into water for 5 hours. The water is used
2. Fever
during bath.
Bark powder is soaked into water; sugar is added and given
3. Malaria
to the patient two times every day.
Wash the bark, cut into small pieces and chew regularly.
4. Indigestion
Swallow the juice thrice a day.

Traditional use of pod and seed

S. No. Ailments Method of use
Remove the winged shell of the seed. Eat two kernels every
1. Diabetes
day.
2. Fever Pod is soaked into water and bathed every morning.
Dry pods are washed and soaked into water. The water is
3. Malaria
taken to the patient every morning and evening.
Sexual Two kernels of the seed is taken every morning and
4.
weakness evening.

Used as fodder
Fresh leaf of Moringa is fed to rabbits, sheeps, and pigs. Powder of the
leaf is dissolved into water and given to poultry. The leaf is rich source of
nutrition, minerals and vitamins which help in the growth and eases birth of
livestock as well as enhances milk production. Regular drink of water mixed
with Moringa leaf powder protects poultry from diseases and favors bigger
and more numerous eggs.
Pharmacological action
Moringa oleifera is a nutritional plant contains valuable
pharmacological action like anti- diabetic, anti-asthmatic, haepato-protective,
anti-inflammatory, anti- fertility, anti-cancer, anti- microbial, anti-oxidant,
cardiovascular, anti-ulcer, CNS activity, anti-allergic, wound healing,
antipyretic and analgesic properties. Review of the pharmacological actions
of the species is mentioned below.
The fresh leaf juice and aqueous extracts from the seeds inhibit the
growth of Pseudomonas aeruginosa and Staphylococcus aureus. No activity
was expressed against other pathogenic Gram-positive and Gram-negative
bacteria. Candida albicans was observed antimicrobial effect of Moringa
oleifera leaves, roots, bark and seeds in vitro against bacteria, yeast,
dermatophytes and helminthes by a disk-diffusion method (Caceres et al.

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1991). Antifungal action of Moringa oleifera was observed against seven
pathogenic fungi using the broth dilution and agar plate methods. Leaf
extracts of the plant were effective against T. mentagraphytes and
Trichophyton rubrum (Nwosu et al. 1995). It was found in the investigations
that antimicrobial effect was present in three fractions of Moringa oleifera
leaves which were found by Sephadex G-25 column chromatography. Single
bands of these fractions were detected on Polyacrylamide SDS gel
electrophoresis. An antibacterial action of small protein/peptide was tested
against E. coli Kl. aerogenes, Kl. pneumoniae, S. aureus, and B. subtilis.
Fractions 1 showed strong inhibitory activity against E. coli, S. aureus and B
subtilis but clear zone of inhibition was also noted against K1. aerogenes
with peptide 1. Fraction 2 showed significant zone of inhibition against
Aspergillus niger (Dahot 1998). Researchers investigated anti-inflammatory
activity from the ethanolic extract of seeds of Moringa oleifera. The extract
was pharmacologically evaluated against immune mediated inflammatory
responses in toluene di-isocyanate (TDI as antigen) induced asthma in
Wistar rats (Mahajan et al. 2007). Antioxidant potential of Moringa oleifera
was investigated on hepatic marker enzymes, lipid peroxidation, and
antioxidants. The antioxidant property was investigated during antitubercular
drug (isoniazid, rifampicin, and pyrazinamide) induced toxicity in rats.
Enhanced hepatic marker enzymes and lipid peroxidation of anti-tubercular
drug treatment was accompanied by a significant decrease in the levels of
vitamin C, reduced glutathione, superoxide dismutase, catalase, glutathione
peroxidase, and glutathione Stransferase. Application of Moringa oleifera
extract and silymarin significantly decreased hepatic marker enzymes and
lipid peroxidation with a simultaneous increase in the level of antioxidants
(Kumar and Pari 2003). The cytotoxic effect of various extracts of leaves of
Moringa oleifera was investigated. The cytotoxic efficacy was monitored on
human multiple myeloma cell lines. Of the organic extracts, methanolic
extracts of Moringa leaves showed least viability at highest dose (Parvathy
and Umamaheshwari 2007). The antifertility properties were observed from
the aqueous extract of Moringa oleifera roots. The effect of aqueous extract
has been studied on histoarchitecture of the uterus during pre and post-
implantation stages in rats (Prakash et al. 1987). A Hematological and
hepatorenal property of methanolic extract of M. oleifera roots was
investigated. Doses of the crude extract on liver and kidney functions and
hematological parameters in mice were reported. No alteration in
hematological and biochemical parameters at low and moderate dose level of
daily and low dose level of weekly treatment of the extract was observed.
However, the extract at moderate dose level in weekly treatment changed

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serum aminotransferase and plasma cholesterol levels significantly. High
dose in addition to the above parameters changed total bilirubin, non protein
nitrogen, and blood urea and plasma protein. High dose of daily treatment
and moderate and high dose of weekly treatment of CE increased WBC
count and decreased clotting time significantly (U.K. Mazumder et al. 1999).
Some of the researchers investigated cardioprotective effect of lyophilized
hydroalcoholic extract of M. oleifera in the isoproterenol induced model of
myocardial infarction in male wistar albino rats. Chronic treatment with the
species resulted in significant favorable modulation of the biochemical
enzymes (superoxide dismutase, catalase, glutathione peroxidase, lactate
dehydrogenase and creatine kinase) but failed to demonstrate any significant
effect on reduced glutathione compared to the ISP control group. Moringa
treatment significantly prevented the rise in lipid peroxidation in myocardial
tissue (Nandave et al. 2009). The wound healing property from the aqueous
extract of leaves of M. oleifera was evaluated in male Swiss albino mice.
Significant increase in wound closure rate, skin breaking strength,
granuloma breaking strength, hydroxyproline content, granuloma dry weight
and decrease in scar area was observed (Rathi et al. 2006). The anti-diabetic
activity of aqueous extract of M. oleifera leaves was observed on glycemic
control, haemoglobin, total protein, urine sugar, urine protein and body
weight (Jaiswal et al. 2009).
Conclusion
Moringa oleifera is an important plant species with high value of
nutrition as well as medicinal properties. All the parts of the plant including
flower, leaves, roots, pods, seeds and bark are great source of effective
phytochemicals. The leaves are used by various ethnic groups as a source of
food. The fresh leaves are consumed as a vegetable and dry leaf powder is
used as a source of nutrition. The plant is used to help to cure various
diseases such as digestive diseases, infertility disorders, venereal diseases,
cardiovascular diseases, infectious diseases, tropical dis- eases, inflammatory
complaints, otorhino-laryngologocal complaints, skin infections etc. The
leaves are also used as fodder, and they are included in compounds used to
increase the pro- duction milk and body mass. Bark of the plant is
emmenogogue and even abortifacient, antifungal, antibacterial. Flowers are
cholagogue, stimulant, tonic and diuretic. Root-bark is used as antiviral, anti-
inflammatory, analgesic. Pharmacologically reported activities includes
antimicrobial, anti-inflammatory, antioxidant, anticancer, antifertility,
hepatoprotective, cardiovascular, antiulcer, analgesic, wound healing,
anticonvulsant, antiallergic and anthelmintic activities. Phytochemically, the

Page | 109
plant is known as rich source of glycosides, phenols, sterols, flavanol
glycosides which is medicinally important and nutritionally valuable. The
modern research work validates the Ethnobotanical importance of the plant
species.
References
1. Caceres A, Cabrera O, Morales O, Mollinedo P, Mendia P. Journal of
Ethnopharmacology. 1991;33(3):213-216.
2. Jaiswal D, Kumar Rai P, Kumar A, Mehta S, Watal G. Journal of
Ethnopharmacology. 2009;123(3):392-396.
3. Dahot MU. Journal of Islamic Academy of Sciences. 1998;11(1):27-32.
4. Gupta RK. Medicinal & Aromatic Plants. CBS publishers &
distributors, 2010, 151-152.
5. Kalogo Y, Rosillon F, Hammes F, Verstraete W. Effect of a water
extract of Moringa oleifera seeds on the hydrolytic microbial species
diversity of a UASB reactor treating domestic wastewater. Letters in
Applied Microbiology. 2000;31(3):259-264.
6. Khare CP. Indian medicinal plants. Springer. 2007:422-423.
7. Kumar S, Kumar D, Singh N, Vashishta BD. In vitro free radicals
scavenging and antioxidant activity of Moringa oleifera pods. Journal of
Herbal Medicine and Toxicology. 2007;1:17-22.
8. Kumar NA, Pari L. J Med Food. 2003;6(3):255-259.
9. Mahajan SG, Mali RG, Mehta AA. Journal of Immunotoxicology.
2007;4(2):85-96.
10. Mazumder UK, Gupta M, Chakrabarti S, Pal D. Indian Journal of
Experimental Biology. 1999;37(6):612-614.
11. Nadkarni KM. Indian Materia Medica. Bombay Popular Prakashan.
2009;I:811-816.
12. Nandave M, Ojha SK, Joshi S, Kumari S, Arya DS. J Med Food.
2009;12(1):47-55.
13. Nwosu MO, Okafor JI. Mycoses. 1995;38(5-6):191-195.
14. Parvathy MVS, Umamaheshwari A. Trends in Medical Research.
2007;2(1):44-50.
15. Prakash AO, Pathak S, Shukla S, Mathur R. Acta Eur Fertil.
1987;18(2):129-135.

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16. Rathi BS, Bodhankar SL, Baheti AM. Indian Journal of Experimental
Biology. 2006;44:898-901.
17. Sreelatha S, Padma PR. Antioxidant activity and total phenolic content
of Moringa oleifera leaves in two stages of maturity. Plant Foods for
Human Nutrition (Dordrecht, Netherlands). 2009;64:303–311.

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Page | 112
 Chapter - 7
Diversity of Loranthaceae Juss. Members
Occurring in Chirang Reserve Forest, BTR,
Assam, India

Authors
Sanswrang Basumatary
Department of Botany, Kokrajhar Govt. College, Kokrajhar,
Assam, India
Sanjib Baruah
Department of Botany, Bodoland University, Kokrajhar,
Assam, India

Page | 113
Page | 114
 Chapter - 7
Diversity of Loranthaceae Juss. Members Occurring in
Chirang Reserve Forest, BTR, Assam, India
Sanswrang Basumatary and Sanjib Baruah

Abstract
The diversity of the Loranthaceae family has been studied and recorded
in a total of 7 species under 5 genera from Chirang Reserve Forest (CRF),
Assam. The species were enumerated taxonomically along with associated
information like flowering and fruting, distribution, host plant etc. Among
those species, Scurrula parasitica, Scurrula pulverulenta, and Tolypanthus
involucratus have been recorded for the first time in the flora of BTR. For
easy recognition of the species, the taxonomic keys of the genera and the
species along with, the photographs have been provided.
Keywords: BTR, Diversity, Manas Biosphere Reserve, Loranthaceae
Introduction
The members found in the Loranthaceae Juss. family are mistletoe,
which is a group of hemi-parasitic shrublets that grow attached to and within
the branches of trees and shrubs (Kulkarni and Kumbhojkar 2002, Macphail
et al. 2012). The family is very close to Viscaceae Batsch (now treated under
Santalaceae R.Br. as per APG IV 2016) but differs in many features of
embryology, floral anatomy and floral morphology (Barlow 1964). The
family is represented by 78 genera and about 900 species across the world.
However, in India represents 49 species under 8 genera (Rajasekaran 2012,
POWO 2023). The state Assam represents 7 genera and 13 species (Barooah
and Ahmed 2014). Bodoland Territorial Region (BTR) earlier known as
Bodoland Territorial Area District (BTAD) represented 5 species and 4
genera (Borthakur et al. 2018).
The study area, Chirang Reserve Forest (CRF), is located in the
westernmost part of state Assam and falls under Kokrajhar and Chirang
districts of Haltugaon and Chirang Division (Figure 1). The total area of the
forest is 592.54 sq. km. The area shows varied vegetation and diverse
habitat, which endow diverse groups of plants. In the entire forest area,

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Ultapani has one of the richest plant diversity with diverse habitats (swampy,
streams inside forest, highlands, semi-evergreen forest, climax vegetation,
etc.) and is regarded as ‘Orchid Valley’ (‘Daothu Bibarni Halam’ in Bodo),
as termed by local inhabitants, and this area represents over 100 species of
orchids, which is the richest area in the entire Bodoland Territorial Region of
Assam.

Fig 1: Location map of Chirang Reserve Forest (CRF), Assam

Materials and Methods

The extensive floristic surveys were conducted in Chirang Reserve
Forest under the Manas Biosphere Reserve, Bodoland Territorial Area
District, Assam. Collected species are prepared for herbarium specimens
following the standard field and herbarium techniques (Jain and Rao 1977).
Identification of the flora has been done with the help of several regional and
local floras (Kanjilal et al. 1940; Nath 1999; Bora and Kumar 2003; Begum
2008; Daimary 2011; Rajasekaran 2012, Boro 2016; Borthakur et al. 2018)
and online herbarium specimens deposited in K, L, AMES, NY, etc. For up-
to-date nomenclature of the taxa the International Plant Name Index (IPNI)

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has been consulted. The voucher specimens were deposited at Bodoland
University Botanical Herbaria (BUBH), Kokrajhar, Assam for future use.
Results
The present study deals with the diversity of Loranthaceae family
members in the CRF of BTR. A total of 7 species under 5 genera have been
enumerated taxonomically and discussed.
Taxonomic enumeration
Loranthaceae: Juss. in Ann. Mus. Natl. Hist. Nat. 12:292. 1808.
Key to the genera
1a. Bracts foliaceous……………………………………5. Tolypanthus
1b. Bracts not foliaceous………………………………………………2
2a. Each flower subtended by 3 bracts…………………..3. Macrosolen
2b. Each flower subtended by 1 bract…………………………………3
3a. Corolla lobes free…………………………………..2. Helixanthera
3b. Corolla lobes fused into tube………………………………………4
4a. Flowers 4-merous………………………………………..4. Scurrula
4b. Flowers 5-merous………………………………….1. Dendrophthoe
1. Dendrophthoe Mart. in Flora 13(1):109. 1830.
1. Dendrophthoe falcata (L.f.) Ettingsh. in Denkschr. Kaisel. Akad.
Wiss. Math. Naturwiss. Kl. 32:52. 1871; Kanjilal et al., Fl. Assam
4:123. 1940. Loranthus falcatus L.f., Suppl. Pl. 211. 1781. L.
longiflorus Hook.f., Fl. Brit. India 5:214. 1886. (Plate 2A).
Shrub, parasite, epiphytic, up to 2 m tall. Branches greenish brown,
brown dots when young. Leaves opposite or alternate, lanceolate to elliptic,
11-13 × 3-6 cm, coriaceous, glabrous both sides, margin yellow lined, entire,
base cuneate, rounded at apex; lateral nerves 2-4 pairs, inconspicuous.
Petiole short, 4-7 mm long. Inflorescence short racemes, 5-10 cm long,
solitary or 2 together, 15-22 flowered. Peduncle 3-5 mm in diameter, green,
glabrous. Bracts green, broadly ovate, 2 mm across, glabrous. Calyx
unlobed, 4 mm long, circular, green. Corolla tubular, 5 mm in diameter,
lower part pink, upper part yellowish-green, 5-lobed. Stamens 5, epipetalous,
1-1.2 cm long. Aanther yellow, 5 mm long, backside red; filament 7 mm
long, red. Ovary 1 mm across. Style 5-6.5 cm long. Stigma globose. Fruits
ellipsoid, glabrous, yellowish-orange.

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 Flowering and Fruiting: September-December.
Occurrence: Found in deciduous forest.
Distribution: INDIA (Throughout the country), BANGLADESH,
NEPAL, SRI LANKA.
Specimen examined: Kokrajhar, Ultapani, S. Basumatary 0061
(BUBH), 21.11.2018.
Host plants: Melia azedarach L., Syzygium formosum (Wall.) Mason.
Note: Found variable forms of leaves within this species.
2. Helixanthera Lour., Fl. Cochinch. 142. 1790.
Key to the species
1a. Flowers 5-merous, yellow…………………………2. H. parasitica.
1b. Flowers 4-merous, red……………………………...1. H. ligustrina.
1. Helixanthera ligustrina (Wall.) Danser in Bull. Jard. Bot.
Buitenzorg ser. 3, 10:317. 1929; Kanjilal et al., Fl. Assam 4:121.
1940. Loranthus ligustrinus Hook. f., Fl. Brit. India 5:207. 1890.
(Plate 2E).
Shrub, parasitic, up to 1 m tall, densely branched, crown at the base, old
stem rusty brown, young stem greenish-brown, terete. Leaves opposite,
sometimes sub-opposite or alternate, elliptic, sub-lanceolate, 2.5-4 × 6-8 cm,
glabrous both sides, base broadly acute, margin entire, apex acuminate,
lateral nerves 4-6 pairs. Petiole 5-10 mm long. Inflorescence axillary, short
raceme, up to 4 cm long, 3-6 flowered. Flowers 8-12 mm long. Pedicel 2 mm
long, bent at the attachment of tubular calyx. Bract 1, arising from the tip of
pedicel, 1-2 mm long, green, margin brown. Calyx tubular, green, brown
dotted, 2 mm long. Corolla 4, 6-8 mm long, red, yellow during immature,
bent downwards from the middle. Stamens 4, epipetalous, red, 5 mm long,
surrounds style. Gynoecium 1 cm long. Ovary 2 mm across. Style 6-8 mm
long, yellow. Stigma red.
Flowering and Fruiting: February-June.
Occurrence: Found in open forest or in deciduous forest.
Distribution: INDIA (NE India), BANGLADESH, MYANMAR,
NEPAL, PHILIPPINES, THAILAND, VIETNAM.
Specimen examined: Kokrajhar, Jharbari, S. Basumatary 0015 (BUBH),
24.03.2018.

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 Host plants: Ficus hispida L.f., Melia azedarach L.
2. Helixanthera parasitica Lour., Fl. Cochinch. 1:142. 1790; Kanjilal
et al., Fl. Assam 4:120. 1940; Deb, Fl. Tripura 1:400. 1981; Harid.
and Rao, Forest Fl. Meghalaya 2:757. 1987; Chowdhery et al. in
Giri et al., Materials for the Fl. Arunachal Pradesh 2:354. 2008.
Loranthus pentapetalus Hook. f., Fl. Brit. India 5:206. 1886. (Plate
2F).
Shrub, parasitic, up to 1-1.5 m tall. Leaves opposite, ovate-lanceolate, 6-
10 × 3-5 cm, coriaceous, margin entire, acuminate at apex, base broadly
acute or sub-rounded; lateral nerves obscure, 6-8 pairs. Petioles 1-2 cm long.
Inflorescence axillary, yellow or red, solitary or in pair, 7-15 cm long, 15-40-
fowered. Bracts ovate to ovate-triangular 1-1.5 mm long. Flowers yellow.
Pedicel 1-2 mm long. Calyx limb annular, 5-denticulate; Corolla 5-lobed,
yellow or red, 4-8 mm long; lobes obovate-lanceolate, reflexed. Stamens 5,
epipetalous. Filaments 1-2 mm long; Anther 1-1.5 mm long. Style, 3-5 mm
long, constricted at middle. Stigma capitate; Berry ellipsoid, 5-6 × 3-4 mm,
reddish.
Flowering and Fruiting: April-July.
Occurrence: Found in roadsides of forest.
Distribution: INDIA (NE India), BANGLADESH, BORNEO,
CAMBODIA, CHINA, LAOS, MYANMAR, NEPAL, PHILIPPINES,
THAILAND, VIETNAM.
Specimen examined: Kokrajhar, Diglipara, S. Basumatary 0380
(BUBH), 11.04.2021.
Host plant: Litsea monopetala (Roxb.) Pers.
3. Macrosolen (Blume) Rchb., Deut. Bot. herb.-Buch 73. 1841.
1. Macrosolen cochinchinensis (Lour.) Tiegh. in Bull. Soc. Bot. Fr.
41:122. 1895; Kanjilal et al., Fl. Assam 4:127. 1940; Deb, Fl.
Tripura 1:401. 1981. Loranthus ampullaceus Hook. f., Fl. Brit.
India 5:220. 1890. (Plate 2C).
Shrub, parasitic, epiphytic, up to 1 m tall. Branches greyish, terete,
scattered lenticellate. Leaves alternate, ovate-elliptic or lanceolate, 4-5 × 10-
12 cm, coriaceous, base broadly cuneate, margin entire, apex acuminate;
lateral nerves 4-6 pairs, inconspicuous, glabrous both sides. Petiole 1 cm
long. Inflorescence 2-3-fascicled, axillary, sometimes at older leafless nodes,
short raceme, 2-4 cm long, 4-8-flowered. Peduncle 1-2 cm long; bracts 3,

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broadly ovate, 1-2 mm across, green, persistant; Flowers 1.5–2 cm long.
Pedicel 2 mm long. Calyx tubular, 4 × 2 mm, green. Corolla 6-lobed, lobes
bent downwards from the middle, reddish-brown colouration at middle, base
connate. Stamens 6, epipetalous. Anther yellow, 1 mm long; filament 5 mm
long, green. Style 1-1.3 cm long, green; stigma pink. Ovary ellipsoid, 2 mm
in diameter. Berry ellipsoid to subglobose, 7 × 5 mm, orange to yellow.
Flowering and Fruiting: February-May.
Occurrence: Found in roadside or in deciduous forest.
Distribution: INDIA (NE India), BANGLADESH, BORNEO,
CAMBODIA, CHINA, MYANMAR, NEPAL, THAILAND, VIETNAM.
Specimen examined: Kokrajhar, Ultapani, S. Basumatary 0017
(BUBH), 27.03.2018.
Host plants: Artocarpus heterophyllus Lam., Careya arborea Roxb.,
Mangifera indica L., Melia azedarach L.
4. Scurrula L., Sp. Pl. 1:110. 1753.
Key to the species
1a. Inflorescence fascicled; leaves subsessile…………...1. S. parasitica
1b. Inflorescence pedunculate; leaves petiolate………2. S. pulverulenta
1. Scurrula parasitica L., Sp. Pl. 1:110. 1753; Kanjilal et al., Fl.
Assam 4:124. 1940. Loranthus scurrula Hook.f., Fl. Brit. India
5:208. 1886. (Plate 1).
Shrub, parasite, epiphytic, up to 1 m tall; branches and leaves covered
with rusty hairs when young, glabrous when old. Leaves opposite, ovate,
oblong-elliptic, 7-10 × 3-5.5 cm, glabrous both side, coriaceous, margin
entire, base cordate or shallowly cordate, apex tapering subacuminate; lateral
nerves 4-7 pairs; midrib distinct, extending to apex; petioles 1-3 mm long,
rusty. Inflorescence fascicle, 4-10-flowered, spreading 1-4 cm in diam.
Bracts ovate, 1 mm across, rusty above. Flowers 2-2.5 cm long. Pedicel 2
mm long. Calyx limb annular, rusty brown. Corolla 4-lobed, lobes
lanceolate, rusty brown ouside, dark purple inside, base tubular with median
split. Stamens 4, epipetalous. Anther 1 mm long, yellow; filament dark-pink.
Ovary 4 × 1-2 mm, rusty brown; style up to 1-1.5 cm long, dark pink or
purple, quadriangular, glabrous; stigma globose. Berry pyriform, 6-13 × 2-4
mm, pale green, covered with rusty brown hairs.
Flowering and Fruiting: June-December.

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 Occurrence: Found growing in semi-evergreen or in deciduous forest.
Distribution: INDIA (Throughout the country), BANGLADESH,
CHINA, MYANMAR, NEPAL, SRI LANKA, THAILAND, VIETNAM.
Specimen examined: Kokrajhar, Ultapani, S. Basumatary 0530
(BUBH), 12.12.2018.
Host plants: Bridelia tomentosa Blume, Ficus hispida L. f., Melastoma
malabathricum L., Mallotus tetracoccus (Roxb.) Kurz.
Note: Leaves are variable. Sometimes the leaves are sessile, petiolate, or
subsessile. The shape and colour of the leaves also vary, referring to the
habitat in which they grow.
2. Scurrula pulverulenta (Wall.) Don, Gen. Hist. 3:421. 1834;
Kanjilal et al., Fl. Assam 4:125. 1940. Loranthus pulverulentus
Hook. f., Fl. Brit. India 5:211. 1886. (Plate 2D).
Shrub, parasite, up to1.5 m tall. Branches greyish, lenticellate, glabrous,
immatured branchlets tomentose with stellate hairs. Leaves opposite, ovate-
lanceolate, 8-12 × 4-5 cm, coriaceous, both side pubescent during young,
glabrous when old, margin entire, tapering and rounded or bluntly acute at
apex, base rounded; lateral nerves 4-5 pairs. Petioles 1.5-2.5 cm long.
Inflorescence axillary, 1-3 fascicled, 3-5 cm long, 4-12-flowered. Both
peduncle and rachis with white stellate hairs. Bracts ovate, 1 mm across.
Flowers 3-4 cm long. Pedicel flower 4-6 mm long. Calyx limb annular,
woolly brown. Corolla 4-lobed; lobes oblong-lanceolate 6-10 mm long,
reflexed, base tubular, slightly bent, split up to below middle, woolly
pubescent. Stamens 4; filaments 2-3 mm long, blood-red. Anther 5 mm
across, pale brown, reddish-pink outside. Style red. Stigma capitate. Berry
pyriform, pubescent.
Flowering and Fruiting: June-December.
Occurrence: Found in the semi-evergreen or in deciduous forest.
Distribution: INDIA (Throughout the country), BANGLADESH,
BHUTAN, CHINA, MYANMAR, NEPAL, THAILAND.
Specimen examined: Kokrajhar, Ultapani, S. Basumatary 0247
(BUBH), 08.11.2020.
Host plants: Cordia dichotoma G. Forst.
5. Tolypanthus (Blume) Rchb., Deut. Bot. Herb.-Buch 73. 1841.
1. Tolypanthus involucratus (Roxb.) Tiegh. in Bill. Soc. Bot. France
42:248. 1895; Kanjilal et al., Fl. Assam 4:126. 1940; Deb, Fl.

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 Tripura 1:402. 1981; Haridasan and Rao, For. Fl. Meghalaya 2:761.
1987. Loranthus involucratus Hook. f., Fl. Brit. India 5:218. 1890.
(Plate 2B).
Shrub, parasitic, up to 1.5 m tall, semi-pendulous. Stem brownish,
lenticellate, terete, arising from a tuft of main stalk. Leaves opposite to sub-
opposite, rarely alternate, ovate or ovate-elliptic, 5–5.5 × 9–11 cm, margin
entire, apex tapering obtuse, upper surface sparsely pubescent, sometimes
with brown spots of fungal colony, lower surface pubescent, mid vein
brownish; lateral nerve 5–7 pairs. Petiole 1–1.5 cm long. inflorescence
cymose, axillary, crowded or solitary, 2–2.5 cm long, flowers enclosed by
involucral bracts, number of flower is equal in respect to number of
involucral bracts (generally 3–5-flowered). Involucral bracts ovate, 2–2.3 ×
1–1.3 cm, pubescent, apex acute, margin entire. Flowers 2–2.3 cm long.
Calyx tubular, densely pubescent, minutely 5-lobed. Corolla tubular, 5-
lobed, sparsely pubescent, 1.6–1.8 cm long, reddish lined. Stamens 5,
epipetalous. Anther 2 mm across, reddish with yellowish pollen grainsh;
filament 3 mm long. Gynoecium 2 cm long. Ovary 2 mm across. Stigma red.
Berry ellipsoid, densely pubescent, yellow when ripe.
Flowering and Fruiting: January-May.
Occurrence: Found in roadsides or in deciduous forest.
Distribution: INDIA (NE INDIA), BANGLADESH, MYANMAR,
NEPAL.
Specimen examined: Kokrajhar, Ultapani, S. Basumatary 0010
(BUBH), 22.03.2018.
Host plants: Lagerstroemia speciosa (L.) Pers., Syzygium formosum
(Wall.) Mason.

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Fig 1: Scurrula parasitica L.: A. Habit; B. Showing inflorescence; C. Leaf; D.
Bracts; E. Flower; F. Gynoecium; G. Berry.

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Fig 1: A. Dendrophthoe falcata (L.f.) Ettingsh.; B. Tolypanthus involucratus (Roxb.)
Tiegh.; C. Macrosolen cochinchinensis (Lour.) Tiegh.; D. Scurrula pulverulenta
(Wall.) Don; E. Helixanthera ligustrina (Wall.) Danser; F. Helixanthera parasitica
Lour.

Discussion
The study area CRF has a diverse habitat, which is home to 53.85%
Loranthaceae members compared to rest of the parts of Assam. The species
found within this family exhibit great variation in their leaf morphology and
colouration. The genus Scurulla and three species viz. Scurrula parasitica,
Scurrula pulverulenta, and Tolypanthus involucratus have been added as
new distributional records for the flora of Bodoland Territorial Region.
Along with the members of Loranthaceae, many important medicinal plant
species, threatened, endemic, and other flora and fauna prefer this area as
their suitable habitat. But these biodiversity rich and diverse habitats are now

Page | 124
under threat due to deforestation and encroachment. Several human activities
such as illegal tree felling, poaching, forest fire, use of chemicals fertilizers,
and cattle grazing etc. are involving in the destruction of these healthy
ecosystems of the forests. It will be difficult to sustain life on earth if we fail
to preserve such a healthy forest, which offers several ecological,
economical, and societal benefits. Protecting and conserving forests is
therefore paramount for current and future generations.
References
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form Western Maharashtra, India. Ancient Science of Life.
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10. Kanjilal UN, Kanjilal PC, Das A, De RN. Flora of Assam, Government
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