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As resistance to disease in plants is genetically controlled, molecular tools like breeding resistant cultivars has been an intensively used approach for crop protection since near beginning of human civilization, the time when we did not know its molecular aspects. Even today, molecular biology is applied in multiple ways to control plant diseases. Some of which are breeding, tissue culture, marker assisted breeding, QTL- mapping, identification of novel resistance genes etc. With the commencement of advanced technologies in the recent past, we are now able to genetically modify a plant without wasting a lot of time and avoiding problems of sexual incompatibility which we encounter in breeding programs.
Citation preview
1
Application of molecular
biology to conventional disease
strategies
Paper : Assignment
Supervisor:- Dr. Roopam Kapoor
Submitted by:- Satya Prakash
M.Phil. (Botany), 2013-14
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CONTENTS: Pg. no.
1. Introduction 3
2. Resistance breeding 3
2.1 Artificial selection 4
2.2 Hybridization 4
2.3 Polyploidy 4
2.4 Induced Mutations 4
3. Solutions to problems associated with plant breeding: 5
3.1 Tissue Culture 5
3.2 Marker- Assisted Breeding 6
3.2.1 Advancement: QTL-Mapping 9
3.2.2 Identification and application of resistance genes 10
4. Use of chemicals for disease control 11
4.1 Fungicides 12
a) Negative impacts of fungicide on the membrane 12
b) Effects on amino acid and protein synthesis 13
c) Effects on signal transduction 13
d) Effects on respiration 13
e) Effects on mitosis and cell division 13
f) Effects on Nucleic Acids Synthesis 13
4.2 Antiboitics 14
4.3 Nematicides 14
4.4 Implementation of molecular biology to design chemical structures 15
(agrochemicals) with increased effectiveness
Use of elicitors 15
Induction of systemic defense 15
4.5 Chemical immunization of plants against diseases 16
5. Summary 17
6. References 14
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Application of molecular biology to conventional disease strategies
1) Introduction:
As resistance to disease in plants is genetically controlled, molecular tools like breeding
resistant cultivars has been an intensively used approach for crop protection since near beginning
of human civilization, the time when we did not know its molecular aspects. Even today,
molecular biology is applied in multiple ways to control plant diseases. Some of which are
breeding, tissue culture, marker assisted breeding, QTL- mapping, identification of novel
resistance genes etc. With the commencement of advanced technologies in the recent past, we
are now able to genetically modify a plant without wasting a lot of time and avoiding problems
of sexual incompatibility which we encounter in breeding programs.
Any problem with a plant that causes a reduction in yield or appearance is called plant disease.
Plant diseases caused by biotic agents like viruses and bacteria are a threat to the food security of
developing countries, causing serious crop and income losses for people whose livelihoods
depend on farming. Demand for food is influenced by a number of forces, including population
growth, income levels, urbanization, lifestyles, and preferences. Almost 80 million people are
likely to be added to the world's population each year during the next quarter century, increasing
world population by 35 percent from 5.7 billion in 1995 to 7.7 billion by 2020 ( UN 1996). More
than 95 percent of the population increase is expected in developing countries, whose share of
global population is projected to increase from 79 percent in 1995 to 84 percent in 2020. Over
this period, the absolute population increase will be highest in Asia. With such rapidly increasing
human population and simultaneous rise in food requirement, scientists across the world are
aimed to increase yield percentage by several ways including reduction of loss in net output due
to plant diseases.
Modern science offers humankind a powerful instrument to assure food security for all. Through
enhanced knowledge and better technologies for food and agriculture, science has contributed to
astonishing advances in feeding the world in recent decades. If we are to produce enough food to
meet increasing and changing food needs, we must put all tools of modern science to work.
In this assignment, some of these tools of molecular biology are discussed with examples. The
cost associated with technologies and the possible solutions have also been discussed in brief.
2) Resistance breeding:
Plant breeding is the science of adapting the genetics of plants for the benefit of humankind. The
overall aim of plant breeding is to improve the quality, performance diversity of crops with the
objective of developing plants better adapted to human needs. This can be accomplished in
several ways including selection and propagation of superior individuals from the field, crossing
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different varieties to get improved one, use of tissue culture to get superior varieties, introduction
or modification of resistance gene/s in plant concerned etc.
2.1) Artificial selection:
As mentioned above, this kind of breeding approach is been in use since the time human started
agriculture. Many elite crop varieties that we see today are the result of thousands of years`
breeding practices. The art of recognizing desirable traits and incorporating them into future
generations is very important in plant breeding. Breeders scrutinize their fields and travel long
distances in search of individual plants that exhibit desirable traits. It is regarded as environment
friendly crop management.
Advantages:
a) We get cultivar with improved desirable trait. b) Resultant varieties are stable and uniform. c) No or reduced use of agrochemicals. d) Require no expertise.
e) Advanced technologies not needed.
Disadvantages:
a) It narrows genetic diversity.
b) Lack of genetic diversity favors spread of new pathogens and epidemic development.
c) Since focus stays on specific gene/s, other important gene/s may be lost in the process.
d) Relatively complex and time consuming method.
2.2) Hybridization:
The most frequently employed plant breeding technique is hybridization. The aim of
hybridization is to bring together desired traits found in different plant lines into one plant line
via cross- pollination.
Steps:
a) Generation of homozygous inbred lines.
b) Outcrossing
c) Progeny selection
d) Backcrossing
A homozygous inbred line is generated by continuous episodes of self-pollination until a pure
line is achieved. This step is followed by crossing of this pure line with another such kind of
inbred line. Thus, traits of two inbred lines are combined in one. However, not all the progenies
carry desired combinations of traits and hence, a selection procedure is required.
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Demerits:
a) If a trait from a wild relative of a crop species, e.g. resistance against a disease is to be
brought into the genome of the crop, a large quantity of undesired traits (like low yield, bad
taste, low nutritional value) are transferred to the crop as well. These unfavorable traits must
be removed by time-consuming back-crossing, i. e. repeated crossing with the crop parent.
b) Potential of hybridization is limited by sexual incompatibility between distant groups of
plants.
Merit:
a) Combining two highly inbred lines often result in heterosis i.e improved or increased
function of any biological quality in a hybrid offspring.
2.3) Polyploidy:
Ploidy is the number of sets of chromosomes in the nucleus of a biological cell. Most plants are
diploid. Plants with three or more complete sets of chromosomes are common and are referred to
as polyploids. The increase of chromosomes sets per cell can be artificially induced by applying
the chemical colchicine, which leads to a doubling of the chromosome number. Generally, the
main effect of polyploidy is increase in size and genetic variability.
Demerit: Polyploids often have lower fertility and grow more slowly.
2.4) Induced Mutations:
Mutations can be induced with the application of mutagens like radiation and mutagenic
chemicals. Instead of relying on wild relatives, we can induce mutations and look for desired
traits. This saves time and intensive work of backcrossing.
Demerits:
a) Mutagens cannot induce mutation at specific sites only.
b) Mutants often carry undesirable traits.
3) Solutions to problems associated with plant breeding:
3.1) Tissue culture:
As mentioned above, a very strong tool of plant breeding, hybridization is limited by phenomena
of sexual incompatibility making it very difficult to transfer genes from an incompatible plant to
commercial species. Tissue culture has opened up ways to alleviate such problems with
conventional breeding programmes. Protoplast fusion can be used for plants that might be
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impossible to cross by classical breeding methods and can be a way to incorporate resistance
genes. These plants then can be regenerated and taken through backcrossing programmes.
Added advantage: Tissue culturing has been shown to result in greater variability in progeny
plants, a phenomenon known as somaclonal variation. These variants often show more resistance
than their progenitors. For example, in experiments where plants were generated in tissue culture
from a line of potatoes susceptible to phytophthora infestans and Alternaria solani, 2.5 % were
found to be resistant to P. infestans , and 1% to A. solani .
3.2) Marker-assisted breeding:
Major problems with conventional breeding programmes include the time taken between
generations and the need to test with pathogens to select for the resistant progenies after each
cross. It becomes more problematic when pathogen only infects mature plants, or is difficult to
work with and maintain, perhaps because it is not an indigeneous organism and needs to be
maintained under strict license constraints.
This problem led to implementation of marker-assisted breeding strategy. Marker-assisted
breeding combines classical plant breeding with the tools and discoveries of molecular biology
and genetics, most specifically the use of molecular markers. A marker, in this context, is an
identifier (sometimes called a “tag”) of a particular aspect of phenotype and/or genotype; its
inheritance can easily be followed from generation to generation. Thus, marker used in a
breeding programme may be morphological (e.g. flowering time), biochemical (e.g. isozymes) or
molecular (e.g. microsatellites).
The following four stages should be followed carefully during MAB programmes aiming at the
introgression of stress resistance:
I. Identification of marker trait Association for stress resistance using the concept of
linkage mapping
II. Validation of marker trait association in different genetic background using cross
breeding population
III. Test the usefulness of marker in characterization core germplasm
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IV. Implementation of the validated marker in the breeding programmeme
Prepare simple genotyping protocol (marker assay) for the
marker detection and its allele size
↓
Provide the marker data along with allelic information to the
molecular breeders for the MAB for stress resistance
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Strategy:
Figure 1
The rationale behind using molecular markers in breeding programmes. M is a molecular marker tightly linked to
the resistance gene (see text for details). Following each cross, progeny containing the resistance gene are selected
by pathogen screens or by detection of the tightly linked marker (M) if using marker-assisted breeding. ( Source:
M.Dickinson (2005) Molecular Plant Pathology. BIOS Scientific Publishers, New York, USA).
Gao et.al, 2013, did Marker-assisted breeding for rf1, a nuclear gene controlling A1 CMS in
sorghum (Sorghum bicolor L. Moench). MAS study was conducted on the offspring population of
two crosses between a maintainer line, BTx-622, and two sweet sorghum lines, BJ-299 and Lunen-2, to
test the effectiveness of the MAS method and develop maintainer lines with sweet and juicy stalks and
corresponding cytoplasmic nuclear male sterility (CMS) lines. The simple sequence repeat marker
Xtxp18 exhibited a high accuracy (95.098 %) for selecting recessive homozygotes for the rf1 gene. The
segregation ratio matched the expected ratio calculated according to the reported genetic distance in
the F2 population of the two crosses used. Finally, four excellent maintainer lines/CMS line pairs
(F5/BC3) with high stalk juice and stalk juice sugar contents were developed. The MAS method based on
Xtxp18 for the sorghum rf1 gene could be used for hybrid breeding programs at a low cost in the future.
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3.2.1) Advancement: QTL-Mapping
Quantitative trait locus (QTL)-Mapping is one of the new techniques adopted to improve plant
breeding methodologies. Many important traits for crop improvement are quantitative (e.g.
yield). This technique allows us to monitor multiple locus instead of relying on single gene
breeding. This mapping approach is a highly effective approach for studying genetically complex
forms of plant disease resistance. With QTL mapping, the roles of specific resistance loci can be
described, race-specificity of partial resistance genes can be assessed, and interactions between
resistance genes, plant development, and the environment can be analyzed. Outstanding
examples include: quantitative resistance to the rice blast fungus, late blight of potato, gray leaf
spot of maize, bacterial wilt of tomato, and the soybean cyst nematode. These studies provide
insights into the number of quantitative resistance loci involved in complex disease resistance,
epistatic and environmental interactions, race-specificity of partial resistance loci, interactions
between pathogen biology, plant development and biochemistry, and the relationship between
qualitative and quantitative loci. QTL mapping also provides a framework for marker-assisted
selection of complex disease resistance characters and the positional cloning of partial resistance
genes.
Hatakeyama et,al ,2012, Identified and Characterized Crr1a, a Gene for Resistance to Clubroot
Disease (Plasmodiophora brassicae Woronin) in Brassica rapa L. Clubroot disease, caused by the
obligate biotrophic protist Plasmodiophora brassicae Woronin, is one of the most economically
important diseases of Brassica crops in the world. Although many clubroot resistance (CR) loci
have been identified through genetic analysis and QTL mapping, the molecular mechanisms of
defense responses against P. brassicae remain unknown. Fine mapping of the Crr1 locus, which
was originally identified as a single locus, revealed that it comprises two gene loci, Crr1a and
Crr1b.
Q. How Is QTL Analysis Conducted?
o The very first requirements are two or more varieties of a plant that differ genetically with
regard to trait of interest (e.g. plant height). These are termed parental lines.
o Second, researchers also require genetic markers that distinguish between these parental
lines. Common markers used are single nucleotide polymorphisms (SNPs), simple
sequence repeats (SSRs, or microsatellites), restriction fragment length polymorphisms
(RFLPs), and transposable element positions.
o Cross between parental lines gives heterozygous individuals (F1).
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o F1 individuals are then crossed using one of the different schemes.
o Finally, the phenotypes and genotypes of the derived (F2) population are scored. Markers
that are genetically linked to a QTL influencing the trait of interest will segregate more
frequently with trait values, whereas unlinked markers will not show significant association
with phenotype.
3.2.2) Identification and application of resistance genes:
Molecular genetics is the boon helping with the identification of novel resistance genes and
aiding in breeding of resistant cultivars.
Strategies:
a) Cloned resistance genes and primers to the conserved regions of resistance genes can be
used in PCR to identify similar genes in wild relatives that might be potentially useful.
Homologues of mlo resistance genes have been identified in a number of plant
species.
b) Identification of proteins required for full pathogenicity and then their transient
expression in a wild crop to see hypersensitive response . The gene(s) responsible for
this response can then be transferred to commercial cultivars.
Transient expression of extracellular fungal protein EPC2, required for full
pathogenicity in tomato genotype. A dominant gene, the Cf-ECP2 gene was
identified and transferred to commercial cultivars. ( EPC2 expressing PVX system
can be used at each step of breeding and backcrossing programme)
c) DNA shuffling techniques can also be used. This involves random generation and testing
of mixed sequences from resistant genes and sequences from such genes and
identification of combination that gives best results.
There is therefore, enormous potential for molecular biology to be used in improvements to crop
protection methods based on resistance mechanisms with or without the use of transgenic plants.
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Figure 2
The use of the PVX transient expression system to isolate a resistance gene that recognises the ECP2 protein from
Cladosporium fulvum. ECP2 cDNA has been fused to the PVX coat protein gene so that a fusion protein is
expressed in the PVX system.
4) Use of chemicals for disease control:
Chemical disease control employs the use of chemicals that are either generally toxic and used as
disinfectants or fumigants or chemicals that target specific kinds of pathogens, as in the case of
fungicides, bactericides (or antibiotics) and nematicides. These chemical agents can be sold as
dusts, concentrated solutions, wettable powders, granules or emulsions. India ranks four in
agrochemical production in the world. This industry is important for Indian economy. In India,
there are about 125 technical grade manufacturers (10 multinationals), 800 formulators, over
145,000 distributors. 60 technical grade pesticides are being manufactured indigenously.
Properties of Ideal chemical agent:
a) It should be effective at concentrations that will not harm the plant.
b) It should have low risk to humans and animals.
c) It should have minimal effect on the normal microflora on the plants and in the soil.
d) There should be little chance of the pathogen quickly developing resistance to it.
e) It should be suitable for long periods of storage in ambient conditions.
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4.1) Fungicides:
Fungicides are chemicals used in the control of fungal diseases. (Whole live or dead organisms
that are efficient at killing or inhibiting fungi can sometimes be used as fungicides)
Examples: - Neem oil, tea tree oil, copper sulfate pentahydrate, hexachlorobenzene etc.
Types:
Contact – Fungicides that are not taken up into the plant tissue.
Translaminar – These fungicides redistribute the fungicide from the upper, sprayed leaf surface
to the lower, unsprayed surface.
Systemic – Fungicides that are taken up and redistributed through the xylem vessels.
Mode of action:
a) Negative impacts of fungicide on the membrane of microorganisms were found to alter
the structure and function of soil microbial communities.
o For example, fungicides of the Aromatic Hydrocarbons (AH) group can modify
the lipid structure.
i. Dicloran (2,6-dichloro-4-nitroaniline) treatment results in increased
sensitivity of treated fungi to solar radiation , which then destroys the
structure of Linoleic acid.
ii. Etridiazole (5-ethoxy-3(trichloromethyl)-1,2,4-thiadiazole), causes the
hydrolysis of cell membrane phospholipids into free fatty acids and
lysophosphatides , leading to the lysis of membranes, in fungi.
o Sterols are another important component of cell membrane in fungi.
Demethylation-inhibiting (DMI) fungicides inhibit sterol biosynthesis in
fungal cells. Triadimefon ((RS)-1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-
1,2,4-triazol-1-yl)butan-2-one) demethylated at C-14, introduced a double
bond at C-22, and reduced a double bond at C-24 in the carbon skeleton of
sterols in a fungal membrane, causing disfunction and cell lysis.
o Some fungicides target fungal intracellular membrane systems and their
biological functions. A widely used fungicidal compound, acriflavine (3,6-
diamino-10-methylacridin-10-ium chloride), increases mitochondrial
permeability and releases cytochrome c in fungal cells, repressing plasma
membrane receptor activation, disordering proton stream and collapsing the
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electrochemical proton gradient across mitochondrial membranes. As a
consequence, ATP synthesis is decreased leading to cell death.
b) Effects on amino acid and protein synthesis:
o It can lead to misincorporation of amino acid ( e.g streptomycin in E. coli,
caused misincorporation of an isoleucine molecule in the phenylalanine
polypeptide chain associated with 70S ribosomes) .
o Inhibiting protein synthesis by interfering the binding of RNA complex with
accepter site of ribosome (e.g. Oxytetracycline).
c) Effects on signal transduction:
o Affecting the genes involved in two-component signal transduction system
(e.g. fludioxonil).
d) Effects on respiration: Several fungicides with different modes of action were reported
to inhibit microbial respiration.
o By inhibiting NADH oxidoreductase, Complex I (e.g. Diflumetorim), succinate-
dehydrogenase, Complex II (e.g. boscalid (2-chloro-N-(4′-chlorobiphenyl-2-yl)
nicotinamide), carboxin (5,6-dihydro-2-methyl-1,4-oxathiine-3-carboxanilide),
and flutolanil(α,α,α-trifluoro-3′-isopropoxy-o-toluanilide) and cytochrome bc1 ,
Complex III .
o By uncoupling oxidative phosphorylation.
i. The metabolic state of mitochondria in target cell was found to be
inhibited after exposure to fluazinam, which may be caused by the
conjugation of the chemical with glutathione, in mitochondria.
e) Effects on Mitosis and Cell Division: The methyl benzimidazole carbamate (MBC)
fungicides are known to impact mitosis and cell division in target fungi.
o Inhibiting polymerization of tubulin into microtubules.
f) Effects on Nucleic Acids Synthesis :
o Inhibiting the activity of the RNA polymerase I system.
i. For example, metalaxyl (methyl N-(methoxyacetyl)-N-(2,6-xylyl)-DL-
alaninate), a widely used PA fungicide, inhibits uridine incorporation into
the RNA chain.
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Picture of some commercially available fungicides in India.
4.2) Antibiotics:
Relatively few antibiotics are routinely used to control plant diseases. Antibiotics are chemical
produced by micro-organisms, which destroy or injure living organisms, in particular, bacteria.
Streptomycin is effective against a few fruit pathogens, such as blights and cankers, and
cyclohexamine can be used to control some fungal pathogens of crops, particularly powdery
mildews and rusts.
Disadvatage: Bacteria, as well as fungi, have the ability to develop resistance to antibiotics.
Examples: streptomycin, Tetracyclines , cyclohexamide etc.
4.3) Nematicides:
A nematicide is a type of chemical pesticide used to kill plant-parasitic nematodes. The use of
nematicides is confined largely to high-return horticultural crops, because they are expensive.
Additionally, they are all highly toxic, and alternative measures for controlling nematodes are
being investigated.
Example: Aldicarb, polysulfide etc.
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4.4) Implementation of molecular biology to design chemical structures (agrochemicals) with
increased effectiveness:
To make use of chemicals for plant disease control more effective, we can identify metabolic
pathways & biochemical processes that are essential for pathogenicity. One potential approach is
insertion mutagenesis of pathogens. Essential factors thus identified can be targeted selectively
to control plant diseases. For example there are chemical agents that can be used to attack cell
wall component, chitin in fungi.
Important: the processes which are targeted must be non-essential in host plant and animals, to
ensure that any agrochemical thus obtained is specific to pathogen and is not harmful to host
plant and consumers.
Some examples of pathways and processes that can be targeted are:
a) Important steps in fungal penetration (e.g. formation of appressoria, melanisation).
b) Genes involved in toxin production.
c) Biosynthesis of specific cell wall components ( e.g. chitin in fungi). Etc.
Thus, after identification of gene(s), the corresponding protein`s/ proteins` structure can be
analyzed. Here, use of computer programs and software is required. Three dimensional
modelling of the encoded protein(s) can be undertaken to help with the identification of active
site and domains that are significant for the activity of protein(s) concerned. These significant
portions of protein can be utilized generate and test the chemical structures made to disrupt these
active sites or domains.
The different potential approaches we can use to target significant locations on proteins are:
a) Chemical structures can be designed that disrupt target domain or active site.
b) Recombinant DNA technology (e.g. Phage display technique and Yeast two hybrid
methods) can be employed to identify structures that interact and inhibit the target
protein.
4.5) Chemical immunization of plants against diseases:
Plant immunization is the process of activating natural defense system present in plant induced
by biotic or abiotic factors. As pesticides contribute to the problem of environmental
deterioration which in turn has a marked influence on the economy, health, and the quality of
life, immunizing plants chemically against plant diseases is now popular. Although, the use of
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agrochemicals is still continued and growing, but making use of natural defense mechanism has
been in practice for many years. A popular example is discovery of phytoalexins resulted in
strategies in which these were sprayed onto plant to control diseases.
Examples: Rishitin and Phytuberin against potato blight and isoflavonoid phytoalexins against
rusts and mildews.
Disadvantages:
Fungicides are more effective.
Phytoalexins are ineffective as eradicants.
Economically not viable.
Suffer from problems of reliability.
Use of elicitors:
Rather than using a single component (e.g. phytoalexins) of defense pathway, we can induce the
battery of defense mechanism by employing the use of elicitors.
Examples:
Oligogalacturonides like Chitins, chitosan, etc.
Mechanism: rapid and transient membrane depolarization (e.g. harpin produced by Erwinia
amylovora has been developed into commercial product in the USA).
Induction of systemic defense:
Chemicals like salicyclic acid, jasmonic acid and ethylene can induce systemic defence in plants.
Problem:
These chemicals are effective in laboratory study but in many cases commercial
applications have not been feasible.
Diversion of biochemical pathways into defense sometimes result in low yield.
Technical problems are also there.
However, a benzathiodioazole (BTH) compound has been developed which mimics the effects of
salicylic acid as an inducer of systemic acquired resistance, and this has been marketed under the
tradename Bion as an effective chemical for the control of certain diseases in tobacco, tomato,
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Arabidopsis, wheat, rice and cucumber, and when appropriate concentrations are used no
harmful effects on the health/productivity of crop is observed.
5) Summary:
Crop improvement for diseases resistant is necessary to increase food production to feed rapidly
growing world population. Breeding for disease resistance has been practiced by humans since
the time human civilization evolved. Many problems associated with conventional plant breeding
strategies have been omitted with the introduction and development of molecular biology. Use of
molecular markers has made breeding much more efficient and easy as it reduces the number of
crosses required. With the introduction of tissue culture, now people are able to cross even the
incompatible plants which were earlier impossible to cross with the aided advantage of increases
variation due to somaclonal variation in some cases. Beside breeding of resistance, a wide array
of chemicals, both inorganic and organic are currently used in crop protection, and new
agrochemicals are constantly being produced and evaluated. These chemicals include fungicides,
bactericides, nematocides and virocides as well as insecticides that can be used to control vectors
for viral and bacterial diseases and herbicides that can be used on parasitic plants and weeds.
As with the use of resistance breeding, persistent use of chemicals can influence the way the
pathogen populations evolve and result in resistance developing or increasing in pathogen
populations against the chemical, compromising the effectiveness of the control. Strategies for
using mixtures of chemicals with differing modes of action, and combining the use of chemicals
with methods such as disease-resistant cultivars into integrated control programmes, can increase
the long-term effectiveness of chemicals and also reduce the quantities required.
Rather than the somewhat hit-and-miss approaches that have been used in the past for
agrochemical identification, molecular biology has the potential to be used in a more targeted
approach. The aim would be to identify, potentially through insertion mutagenesis of pathogens,
metabolic pathways and biochemical processes that are essential for pathogenicity. Once a
pathway and/or gene has been identified, three-dimensional modelling of the encoded protein(s)
can be undertaken to help with identification of the active site and other domains that are
significant. Recently, the use of chemicals like salicylic acid , jasmonic acid and elicitors of plant
defense response has opened up new ways of increasing plant productity.
All these approaches in an integrated manner has led to revolution resulted in development of
new strategies for crop improvement.
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