A LECTURE NOTE ON:

Introductory Resistance
Breeding
B.Sc.Ag. 6th Semester

Prepared by:
Surya Dhungana
Assistance Professor
Department of Genetics & Plant Breeding
AFU, Rampur, Chitwan

Course Code : PLB 304

Course Title : Introductory Resistance Breeding
Credit Hours : 2 (2 + 0) Full Marks: 50 Theory: 50 Practical: 0

OBJECTIVES
Upon the completion of this course the students will understand Principles and Practices of
Resistance Breeding

I. SYLLABUS
Introduction to resistance breeding; natural enemies and their types; defence mechanisms against
pathogens, parasites, insects; a great diversity in mechanisms for resistance; source of resistance; test
of resistance; stage of development, application of natural enemies, composition of inoculums,
evaluation aspects; selection procedures; durability of resistance and application of non durable
resistance; development of resistant varieties in Nepal.

II. COURSE OUTLINE

A. Lecture
S.N. Topics No. of Lectures
1. Introduction to resistance breeding 1
2. Natural enemies and their types 2
2.1 Natural enemies
2.2 Types of natural enemies
3. Defence mechanisms against pathogens, parasites, insects 2
3.1 Defence mechanisms against pathogens, parasites
3.2 Defence mechanisms against insects
4. A great diversity in mechanisms for resistance 6
4.1 Broad resistance
 4.2 Non host resistance
4.3 Host range
4.4 Hypersensitivity resistance
4.5 Partial resistance
4.6 Supressors
5. Sources of resistance 3
5.1 Non host
5.2 Mutations
5.3 Genetic modification
6. Test of resistance 2
6.1 Field test
6.2 Invitro test
7. Stage of development, Application of natural enemies, Composition of inoculums 5
and Evaluation aspects
7.1 Stage of development
7.2 Application of natural enemies
7.3 Composition of inoculums
7.4 Evaluation aspects
7.4.1 Quantitative aspects
7.4.2 Qualitative aspects
8. Selection procedures 3
8.1 Back crossing and recurrent selection
8.2 Molecular markers
8.3 Marker assisted selection
9. Durability of resistance and application of non durable resistance 2
9.1 Durability of resistance
9.2 Application of non durable resistance (gene pyramiding, multilines, cultivar
mixtures, integrated control
Development of resistant varieties in Nepal 4
10.1 Cereal crops
10.2 Vegetable crops
10.3 Legumes
10.4 Oil seed crops
Total 30

REFERENCES
Jacobsen, E. 2010. Genetic Variation and Genetic Modification In Vitro. Wagenigen University and
Research Centre. Wageningen. The Netherlands.
Johnsen, R., 1984. A critical analysis of durable resistance. Phytopath. 22: 309-330.
Knott, D. R., 1989. The effect of transfers of alien genes for leaf rust resistance on the agronomic
and quality characteristics of wheat. Euphytica. 44: 65-72.
Niks, R. E. and W. H. Lindhout, 2010. Breeding for Resistance against diseases and pests.
Wagenigen University and Research Centre. Wageningen. The Netherlands.
Singh, B.D., 2005. Plant Breeding: Principles and Methods (7th Ed.). Kalyani Publishers. New Delhi.
India.
 Contents
1.1 Introduction ....................................................................................................................................................1
1.1.2 Abiotic Stress: ..............................................................................................................................................1
1.1.3 Biotic stress:.................................................................................................................................................2
1.1.4 Disease:........................................................................................................................................................3
1.1.5 Resistance: ...................................................................................................................................................3
1.2 Genetics of disease resistance: ......................................................................................................................4
1.3 Methods of breeding for disease resistance ..................................................................................................5
1.4 Mechanisms of plant resistance .....................................................................................................................6
1.5 Types of Plant Pathogens ...............................................................................................................................6
2.1 Introduction ....................................................................................................................................................7
2.1.1. Types of Natural Enemies: ..........................................................................................................................7
2.1.2 Establishment of Natural Enemies: .......................................................................................................... 11
2.1.3 Using Natural Enemies in the field: .......................................................................................................... 11
2.2 Advantages of using natural enemies in pest management: .................................................................. 13
2.3 Limitations of biological control in pest management ........................................................................... 14
2.4 Characteristics of effective natural enemies: .......................................................................................... 14
2.5 Examples of successful use of natural enemies: ...................................................................................... 15
2.6 Beneficial Organisms Commercially Available for Pest Management ........................................... 15
3.1 Introduction: ................................................................................................................................................ 16
3.2. Pathogen and detection of pathogen: ....................................................................................................... 17
3.2.1 Detection of microbial pathogens ............................................................................................................ 17
3.1.2 Detection of insects ................................................................................................................................. 18
3.3 Structural Defenses ..................................................................................................................................... 19
3.3.4 Proteins and Enzymes ............................................................................................................................ 23
4.1.1 Broad Resistance: ..................................................................................................................................... 25
4.1.2 Non host resistance: ................................................................................................................................. 25
4.1.3 Host range: ............................................................................................................................................... 27
4.2.1 Hypersensitivity Resistance ...................................................................................................................... 27
4.2.2 Partial Resistance ................................................................................................................................... 28
4.3 Ecological Resistance or Pseudo resistance: ............................................................................................... 29
Sources: ............................................................................................................................................................. 30
Test of resistance:.............................................................................................................................................. 31
Screening Techniques for Diseases Resistance ................................................................................................. 33
Measuring Resistance ...................................................................................................................................... 35
4.3.2 Inoculum Density: .................................................................................................................................. 36
4.4.3 Earliness: ................................................................................................................................................. 36
4.4.4 Plant Habitat:.......................................................................................................................................... 36
7.1 Mechanism of Disease resistance:- ............................................................................................................. 37
7.2 Sources of Disease Resistance: .................................................................................................................... 37
7.3 Methods of Breeding for Disease Resistance:-............................................................................................ 38
7.4 Mechanism of Insect- Pest resistance: ........................................................................................................ 38
8.1 Introduction ................................................................................................................................................. 39
8.2 Development of Resistance varieties: ......................................................................................................... 40
8.3 Source of Drought Resistance in Plant Breeding: ........................................................................................ 40
8.4 Mechanisms of Drought Resistance: ........................................................................................................... 41
8.5 Selection Criteria: ........................................................................................................................................ 43
8.6 Water use efficiency (WUE)......................................................................................................................... 44
9.1 Introduction: .............................................................................................................................................. 47
9.2 Heat-stress threshold: ............................................................................................................................... 48
9.3 Plant responses to heat stress ................................................................................................................... 48
9.3.1 Morphological symptoms: .................................................................................................................... 48
9.3.2 Anatomical changes.............................................................................................................................. 48
9.3.3 Phenological changes ........................................................................................................................... 49
9.4 Physiological responses ............................................................................................................................... 49
9.4.1 Waters relations: ................................................................................................................................... 49
9.4.2 Accumulation of compatible osmolytes: .............................................................................................. 49
9.4.3 Photosynthesis: ..................................................................................................................................... 50
9.4.4 Assimilate partitioning: ........................................................................................................................ 50
9.4.5 Cell membrane thermo-stability: .......................................................................................................... 50
9.4.6 Hormonal changes: ............................................................................................................................... 51
9.4.7 Secondary metabolites: ......................................................................................................................... 51
9.5 Molecular responses .................................................................................................................................. 52
9.5.1 Oxidative stress and antioxidants: ........................................................................................................ 52
9.5.2 Stress proteins:...................................................................................................................................... 52
9.5.2.1 Heat shock proteins: .......................................................................................................................... 52
9.5.2.2 Other heat induced proteins: .............................................................................................................. 52
9.6 Mechanism of heat tolerance ....................................................................................................................... 52
9.7 Acquired thermo-tolerance: ..................................................................................................................... 53
9.8 Temperature sensing and signaling: ........................................................................................................ 53
9.9 Genetic improvement for heat-stress tolerance ...................................................................................... 53
 9.9.1 Conventional breeding strategies.......................................................................................................... 54
9.10 Selection criteria for heat resistance:..................................................................................................... 55
10.1 Salt affected soils:...................................................................................................................................... 57
10.2 Genetics of salinity resistance ................................................................................................................... 58
10.3 Measurement of Salinity resistance .......................................................................................................... 59
Mineral Stress Resistance:..................................................................................... Error! Bookmark not defined.
Resistance to mineral deficiency stress:................................................................ Error! Bookmark not defined.
Mechanism of mineral deficiency resistance: ................................................................................................... 61
Genetics of mineral deficiency: ......................................................................................................................... 61
Sources: ............................................................................................................................................................. 61
Selection criteria:............................................................................................................................................... 61
Creation of environment: .................................................................................................................................. 62
Mineral Toxicity Resistance: .............................................................................................................................. 62
11.1 Chilling stress at plants level ..................................................................................................................... 65
11.2 Chilling stress at sub-cellular level............................................................................................................. 65
11.3 Chilling tolerance ....................................................................................................................................... 65
11.4 Genetics of chilling resistance ................................................................................................................... 66
11.5 Source of chilling resistance ...................................................................................................................... 66
11.6 Selection criteria ........................................................................................................................................ 66
11.7 Freezing stress ........................................................................................................................................... 67
11.8 Freezing resistance .................................................................................................................................... 68
11.9 Genetic resources: ..................................................................................................................................... 69
11.10 Selection environment: ........................................................................................................................... 69
11.11 Selection criteria: ..................................................................................................................................... 69
12.1 Back cross: ................................................................................................................................................. 71
12.2 Recurrent selection: .................................................................................................................................. 75
12.3 Molecular markers: ................................................................................................................................... 79
Mechanisms by which cultivar mixtures suppress disease ...................................................................... 91
Effect of cultivar mixtures on epidemic development ................................................................................... 92
Effect of cultivar mixtures on the evolution of pathogen races ..................................................................... 93
1.0 Introduction to resistance breeding
1.1 Introduction
With the prevalence of climate change scenarios, crops are exposed more frequently to episodes of
abiotic stresses such as drought, salinity, elevated temperature, and submergence and nutrient
deficiencies along with biotic stress. These stresses limit crop production.

The performance of a plant is determined by its genotype along with the environment on which it
thrives and the interaction of its genotype with the particular growing environment. The environment
on which it thrives is the sum totals of all the factors other than its individual characters.
Environment consists of two major factors named as biotic and abiotic based upon their biological
and non-biological nature. If there is not interference by any one the environmental factors then it is
known as optimal environment where there is stress free environment but if there is any type of
interference in the complete expression of genotypic potential by any of the environmental factors
then it is known as stress environment. Such stress may be created due to biotic factors like presence
of pathogens, pests, weeds etc and abiotic factors like drought, heat, cold etc depending on their
biological nature.

Physical terms: Stress is defined as the force per unit area acting upon a material, inducing strain and
leading to dimensional change. More generally, it is used to describe the impact of adverse forces,
and this is how it is usually applied to biological systems.

Biological terms: In the widest biological sense, stress can be any factor that may produce an adverse
effect in individual organisms, populations or communities. Stress is also defined as the
overpowering pressure that affects the normal functions of individual life or the conditions in which
plants are prevented from fully expressing their genetic potential for growth, development and
reproduction (Levitt, 1980; Ernst, 1993).

Agricultural terms: Stress is defined as a phenomenon that limits crop productivity or destroys
biomass (Grime, 1979).

Classification of Stress:

In normal conditions or field conditions it is rarely possible to maintain the ideal conditions as plants
will face sometime of stress either by biotic or abiotic factors.

1.1.2 Abiotic Stress:

Abiotic stress corresponds to any environmental factor that can limit plant growth and productivity.
Plants, due to their sessile nature, face several environmental adversities. Abiotic stresses such as
heat, cold, drought, heavy metals, and salinity are serious threats to plant production and yield.

1
Abiotic stress management is one of the most important challenges facing agriculture. Abiotic stress
can persistently limit choice of crops and agricultural production over large areas and extreme events
can lead to total crop failures. Abiotic stresses adversely affect the livelihoods of individual farmers
and their families as well as national economies and food security. It includes potentially adverse
effects of Salinity, Drought, Flooding, Metal toxicity, Nutrient deficiency, High temperature and
Low temperature. In addition, abiotic stresses can include Shade, UV exposure Photo inhibition, and
Air pollution, Wind, Hail and Gaseous deficiency which are often sporadic and highly localized in
occurrence.

Abiotic stress helps to determine the potential cultivable land which can be used for agriculture. It
has been estimated that around 24.2% of the totals world’s geographical area is potentially arable
whereas only 10.6% of the geographical area is under cultivation as rest in not accessible for
cultivation due to prevalence of one or more abiotic stresses. Abiotic stresses are the major source of
yield losses as they limit the production to mere 20% or less of the total crop’s potential yield.

Abiotic or non-infectious diseases:

These diseases are caused by conditions external to the plant, not living agents. They cannot spread
from plant to plant, but are very common and should be considered when assessing the health of any
plant. Examples of abiotic diseases include nutritional deficiencies, soil compaction, salt injury, ice,
and sun scorch

Characteristics of abiotic stress:

 They vary considerably depending on the location.

 Relative importance may be different based on locations
 Occurrence and degree of stress may be unpredictable
 Degree of stresses may vary during the crop season.
 Some types of stresses can be relieved by using management practices where others may be
impossible to manage
 Different crops may show different behavior based on their abilities to withstand the stress
 Stress during the reproductive phase can produce more significant effect than those prevailed
before.

1.1.3 Biotic stress:

It includes attack by various pathogens such as fungi, bacteria, oomycetes, nematodes and
herbivores. Diseases caused by these pathogens accounts for major yield loss worldwide. Being
sessile plants have no choice to escape from these environmental cues. Expertise in tolerating these
stresses is crucial for completing the lifecycle successfully.

2
Biotic or infectious diseases:

These diseases are caused by living organisms. They are called plant pathogens when they infect
plants. For the purposes of discussing plant pathology, only plant disease pathogens will be
discussed. Pathogens can spread from plant to plant and may infect all types of plant tissue including
leaves, shoots, stems, crowns, roots, tubers, fruit, seeds and vascular tissues

1.1.4 Disease:
Any physiological deviation from the normal functioning of the organism (i.e., the crop plant) caused
by pathogenic organisms (fungi, bacteria or viruses) is known as disease. The plant affected by the
disease is known as host while the organism that produces the disease is known as pathogen.
Therefore, disease is an abnormal condition in the plant produced by an organism/pathogen. Thus,
the abnormalities produced by the non-biological environment or by the genetic factors present in the
host are not diseases.

Effect of disease:

 Killing of plants
 Killing of branches
 General stunting
 Damage of leaf tissues
 Damage to the reproductive organs including fruits and seeds

1.1.5 Resistance:
Resistance refers to the ability of the host to interfere with the normal growth and for development of
the pathogen. The earliest demonstration of the behavior of “disease-resistance” as a character
transmissible from parent to off-spring in the “Mendelian” fashion was given by Biffen (1905) in his
work on yellow rust of wheat. Pathogenicity is the ability of a pathogen to attack a host which
includes virulence and aggressiveness. Virulent strains of pathogen cause much severe symptoms of
the disease and they carry the virulence gene that enables it to attack a particular host genotype.
Virulence is due to the action of one or a few genes. An aggressive strain of a pathogen causes severe
disease on all the host genotypes which they are able to attack and aggressiveness is polygenically
inherited. Physiological races are strains of a single pathogen species differing in their ability to
attack different varieties of same host species. The varieties of a host species used to identify
physiological races of a pathogen are known as ‘differential hosts’ or ‘host testers. The differential
hosts are chosen on the basis of differences in their resistance to the pathogen. Each of the
differential hosts should possess a single resistance gene which is different from those present in the
others.

3
  Susceptible Reaction: In case of susceptible reaction, the disease development is profuse and
is not checked by the genotype.
 Resistance Reaction: Resistance denotes less disease development than the susceptible
variety and is a relative attribute. Infection and establishment to take place but the growth of
the pathogen in the host tissue is restricted. This results in smaller spots or pustules than in
susceptible variety.
 Tolerance reaction: Tolerance implies that the host is attacked by the pathogen but there is no
less in biomass production or yield. It is desirable not to use the word ‘tolerance’ unless it has
been clearly shown to be the case.
 Vertical Resistance: It is also known as race-specific, pathotype specific or simply specific
resistance. Vertical resistance is determined by the major genes and is characterized by
pathotype specificity. Pathotype specificity denotes that the host carrying a gene for vertical
resistance is attacked by only that pathotype which is virulent towards that resistant gene. To
all other Pathotype, the host will be resistant.
 Horizontal Resistance: It has many synonyms e.g., race- nonspecific, partial, general and
field resistance. Horizontal resistance is generally controlled by polygenes i.e., many genes
with small individual effects and it is pathotype nonspecific. Thus, it is also known as general
resistance. Horizontal resistance does not prevent the development of symptoms of the
disease, but it slows down the rate of spread of disease in the population.

1.2 Genetics of disease resistance:

 Oligogenic inheritance: In such cases, disease resistance is governed by one or few major
genes and resistance is generally dominant to the susceptible reaction. In many cases, the
action of major resistant genes may be altered by modifying the genes e.g., Bunt resistance in
wheat. But, in some other cases, modifying genes are not known e.g., Resistance to X and Y
viruses in potato
 Polygenic Inheritance: In this case, disease resistance is governed by many genes with small
effects, and a continuous variation for disease reaction is produced, ranging from low
resistance (extreme susceptibility) to good resistance (low susceptibility). In case of
polygenic resistance, disease resistance is not classifiable into clear cut resistant and
susceptible class as is the case with Oligogenes. The polygenes, show both additive and non-
additive effects and there is a large environmental effect as is the case with most of the
quantitative traits. Polygenic inheritance is quantitatively inherited and visually has a large
SCA component.
 Cytoplasmic Inheritance: In some cases, resistance is determined by cytoplasmic gene(s) or
plasma gene(s). For example, maize strain having ‘T’ male sterile cytoplasm (CMS-T) are
extremely susceptible to Helimenthosporium leaf blight, while those having the normal or

4
 non-T cytoplasm are resistant to this disease. Cases of cytoplasmic inheritance of disease
resistance are rare.

1.3 Methods of breeding for disease resistance

 Selection: Selection of resistant plants from a commercial variety is the cheapest and quickest
method of developing a resistant variety.
 Introduction: Resistant variety may be introduced for cultivation in a new area. This offers a
relatively simple and quick means of obtaining resistant varieties
 Mutation: Selection of spontaneous and induced mutant plants with resistant to diseases
through the use of mutagenesis.
 Hybridization: It is the most common method of breeding for disease resistance.
Hybridization serves two chief purposes:
 Transfer of disease resistance from an agronomically undesirable variety to a susceptible but
otherwise a desirable variety (by backcross method).
 Backcross method: This is useful for transferring genes for resistance from a variety that is
undesirable in agronomic characteristics to a susceptible variety which is widely adapted and
is agronomically highly desirable. The resistant parent variety is the donor of the resistance
gene and thus is known as the donor parent or non-recurrent parent. The susceptible variety to
which the resistance gene is transferred is used as a parent in the recessive backcrosses, hence
it is known as the recurrent parent. The backcross program would differ depending upon the
allelic relationship of the resistance genes i.e., whether it is resistance or dominant to the
allele for susceptibility. Generally, backcrosses are made for the recovery of recurrent parent
phenotype along with transfer of disease resistance.
 Combining disease resistance and some desirable characters of one variety with superior
characteristics of another variety (by pedigree method)
 Pedigree method: It is quite suited for breeding for horizontal or polygenic resistance. In
breeding for disease resistance, artificial disease epidemics are generally produced to help in
selection for disease resistance.

Ecological resistance is resistance related to favorable environmental conditions at a given location

at a particular time. There are three forms of ecological resistance:

 Host evasion – occurs when a host passes through the susceptible stages very quickly or
during a period when pests are fewer. This type of resistance applies to an entire species
population.
 Induced resistance – resistance stemming from some type of changed condition for the plant,
such as an increase in available nutrients or water

5
  Escape – this is more or less luck as there is an absence of infestation or injury to a host plant
as a result of incomplete infestation.

1.4 Mechanisms of plant resistance

 Antixenosis (non-preference) resistance mechanisms are those by which host plant has
characteristics that result in non-preference for insects in terms of shelter, oviposition,
feeding, etc.
 Morphological or chemical factors that influence insect behavior and results in the poor
establishment of insects. Plant shape and color can also be an important influencing factor.
 Antibiosis, i.e. the negative effect of a host plant on the biology of an insect. This can include
decreased rates of survival, development and/or reproduction and is a result of biochemical
and biophysical factors. Antibiosis may be a result of the presence of toxic substances, the
absence of essential nutrients or a nutrient imbalance. Physical factors, such as thick cuticles,
glandular hairs and silica deposits, also contribute to antibiosis.

1.5 Types of Plant Pathogens

Plant pathogens are very similar to those that cause disease in humans and animals. Fungi, fungal-
like organisms, bacteria, phytoplasmas, viruses, viroids, nematodes and parasitic higher plants are all
plant pathogens.
Fungi and Fungal-like Organisms (FLOs): fungi and FLOs cause the most plant disease than any
other group of plant pathogens. These organisms cannot make their own food, lack chlorophyll, have
filamentous growth, and may or may not reproduce by spores. Fungi and FLOs are able to
overwinter in soil or on plant debris. However, some fungi and FLOs cannot overwinter in northern
climates because of low winter temperatures. These pathogens overwinter in southern climates and
then are transported by air currents back to northern climates. Disease movement from southern to
northern climates can be monitored during the growing season
Bacteria: Bacteria are single-celled microscopic organisms with cell walls that reproduce by binary
fission (one cell splits into two). Introduction to the plant must occur through natural openings or
wounds in the plant. Bacteria overwinter primarily in soil and in or on plant material that does not
decompose, but some survive inside insect vectors
Phytoplasmas: Phytoplasmas are microscopic, bacteria-like organisms that lack cell walls and thus
appear filamentous
Viruses and viroid: Viruses are intracellular (live inside the cell) nucleic acid particles with a protein
coat that infect other living organisms and replicate in the hosts they infect. Viroids are virus-like
particles but lack a protein coat. Viruses and viroids are primarily transmitted by vectors including
insects, nematodes, and fungi, which introduce the virus or viroid during feeding. Viruses and viroids
can also be transmitted through seed, vegetative propagation and pruning

6
Nematodes: Nematodes are microscopic worm-like animals. The majority of nematodes are soil
dwelling animals and move with soil. However, there are some nematodes that are transmitted
through insects and infect above ground plant parts

2.0 Natural enemies and their types:

2.1 Introduction
Insects are harmful to man as pests of cultivated crops, animals, stored products, carries of
human diseases and pests of household and industrial articles. They are also helpful as producers of
honey, lac, silk, dyes, etc., pollinators of crops and as natural enemies of crop pests. They also serve
as important link in the food web of biological cycle in ecosystem. Pests are those species that attack
some resource we human beings want to protect, and do it successfully enough to become either
economically important or just a major annoyance. They are only a tiny fraction of the insect species
around us.

Natural enemies are insects that control other insect pests, and thus help regulate pest
densities in nature, making cropping systems sustainable. Natural enemies of insects play an
important part in limiting the densities of potential pests. This has been demonstrated repeatedly
when pesticides have devastated the natural enemies of potential pests. Insects which were
previously of little economic importance often become damaging pests when released from the
control of their natural enemies. Conversely, when a non-toxic method is found to control a key pest,
the reduced use of pesticides and increased survival of natural enemies frequently reduces the
numbers and damage of formerly important secondary pest species. Biological control is the use of
living organisms to suppress pest populations, making them less damaging than they would
otherwise be. Biological control can be used against all types of pests, including vertebrates, plant
pathogens, and weeds as well as insects, but the methods and agents used are different each type of
pest. Biological control agents such as these include predators, parasitoids, pathogens,
and competitors. Biological control agents of plant diseases are most often referred to as antagonists.
Biological control agents of weeds include seed predators, herbivores, and plant pathogens.

2.1.1. Types of Natural Enemies:

The three categories of natural enemies of insect pests are: predators, parasitoids, and pathogens.

Predators: Predators simply eat other insects during one or more life stages of their life cycle, e.g. a
ladybug or lady beetle eats aphids, caterpillar eggs, and small caterpillar larvae in its adult and larval
stage. Normally, predators are not as specific as parasitoids in their host range, and may feed on a
number of different insects. Many different kinds of predators feed on insects. Insects are an

7
important part of the diet of many vertebrates, including birds, amphibians, reptiles, fish, and
mammals. These insectivorous vertebrates usually feed on many insect species, and rarely focus on
pests unless they are very abundant. Insect and other arthropod predators are more often used in
biological control because they feed on a smaller range of prey species, and because arthropod
predators, with their shorter life cycles, may fluctuate in population density in response to changes in
the density of their prey. Important insect predators include lady beetles, ground beetles, rove
beetles, flower bugs and other predatory true bugs, lacewings, and hover flies. Spiders and some
families of mites are also predators of insects, pest species of mites, and other arthropods.

Examples:

 Coccinellid beetles are commonly called as ladybirds. These feed on aphids, scale insects,
mealy bugs and mites. Both grubs and adults are voracious feeders.
 Vadalia beetle, Rodolia cardinalis is used for the control of cottony cushion scale on citrus.
Australian lady bird beetle, Cryptolaemus montrouzieri is commonly used for the
management of mealy bugs and scale insects.
 Coccinellids, Pharoscymnus horni and Chilocorus nigrita and Stichlotis madagassa are used
in sugarcane for the control of scale insect, Melanaspis glomerata. Lady bird beetle, Curinus
coerulens is used for the control of phyllid, Heteropsylla cubana on subabul plantations.
 Chrysoperla carnea and Mallada boninensis are used in cotton and citrus ecosystem for
protection from aphids and other soft bodied insects. C. carnea feed on aphids, red mites,
thrips, white and black flies, and eggs of leaf hoppers, moths, leaf miners and small
caterpillars.
 Anthocorid bugs are used in sunflower for the management of thrips, aphids, eggs and young
larvae of mouth. Syrphid or hover flies are important predators of aphids of several crops.
Syrphid, Ischiodon scutellaris is used in large scale in mustard and other crops.
 Crytorhinus livedipennis is the most promising predator for the control of brown plant
hopper, Nilaparvata lugens in paddy.
 Predatory mites, Phytoseiulus spp. Amblyseius spp. are important in controlling
phytophagous mites in several crop ecosystems.

Parasitoids: Parasitoids are free living wasps or flies that lay their eggs on a specific prey. Larvae
hatch inside that prey and feed on it internally, killing it, and giving birth to new parasitoid adults.
Wasp parasitoids are more common than fly parasitoids. Parasitoids are very susceptible to pesticides
because they feed on flower nectars which contain no natural poisons, and are normally exposed to
pesticides because they are active searchers. Parasitoids are targeted and attack only one stage of an
insect life cycle. They are also specific in the number of different organisms that they parasitize.
Normally a parasitoid only parasitizes one insect species, or at times the different species of one

8
genus. In nature it is common to find egg parasitoids, larval parasitoids, and pupal parasitoids.
Parasitoids of nymphs or adults are less common because the insects are much more mobile during
these life cycle stages. Parasitoids are insects with an immature stage that develops on or in a single
insect host, and ultimately kills the host. The adults are typically free-living, and may be predators.
They may also feed on other resources, such as honeydew, plant nectar or pollen. Because
parasitoids must be adapted to the life cycle, physiology and defenses of their hosts, they are limited
in their host range, and many are highly specialized. Thus, accurate identification of the host and
parasitoid species is critically important in using parasitoids for biological control.

Types of Parasitoids:

 Egg prasitoids: Trichogramma egg parasitoids are used the management of tissues borers in
sugarcane, stem borer in rice, boll worms in cotton and pests of several crops. Trichogramma
chilonis is used for the control of bollworms in cotton, intermodal borer in sugarcane and rice
leaf folder. Telenomus remus is used for the control of tobacco caterpillar.
 Egg larval parasitoids: Chelonus balackburni is used for the control of spotted boll worm.
 Larval parasitoids: Campolestis chloridae is used the control of Helicoverpa armigera. Bracon
hebetor and Bracon brevicornis for the control of coconut black headed caterpillar Platygaster
oryzae is used for the control of rice gal midge.
 Larval pupal parasitoids: Isotima javensis is used for the control of top shoot borer of
sugarcane.
 Pupal parasitods: Trichospilus pupivora, and Brachymeria nephantidis are used for the
control of coconut black headed caterpillar.
 Nymphal and adult parasitoids: Aphelinus mali is used for the control of apply wooly aphid.
Encarsia formosa for the control of whitefly. g) Ecto-parasitoid: Ecto-parasitoid, Epiricania
melanoleuca is used for the control of Pyrilla perpusilla on sugarcane

Entomopathogens/ pathogens: Entomopathogens are disease causing organisms such as bacteria,

viruses, fungi and protozoa in insect pest, which kill their host or debilitate the future generations.
The infected insects are unable to feed properly, remain stunted, lose their body color and gets
immobile or paralyzed. Under certain conditions they cause disease epizootics in the field. Insects,
like other animals and plants, are infected by bacteria, fungi, protozoans and viruses that cause
disease. These diseases may reduce the rate of feeding and growth of insect pests, slow or prevent
their reproduction, or kill them. In addition, insects are also attacked by some species of nematodes
that, with their bacterial symbionts, cause disease or death. Under certain environmental conditions,
diseases can multiply and spread naturally through an insect population, particularly when the
density of the insects is high.

9
Types:

 Entomopathogenic bacteria: They are extra cellular associated with insects and enter the body
cavity through contaminated feed and multiply. Death of the insect may be either due to
intoxication, sudden lack of oxygen, chemical changes in the gut or by the toxins or crystal
produced in the bacterial cells. Several commercial products based on Bacillus thuringiensis
and its sub sp. is available and is widely used for controlling lepidopteran pests. B
thruingiesnsis is recommended for the suppression of Helicoverpa armigera on tobacco,
sunflower and pulses; Achaea janata on castor; Spodotera litura on tobacco and beet root and
Adisura atkinsoni on field beans.
 Baculoviruses Viruses are sub microscopic, obligate, intracellular pathogenic entities. Viruses
in the family of baculoviridae are the best known of all the insect viruses because the disease
symptoms are easily recognized and they have potential for the development as microbial
insecticides. Baculoviruses are double stranded DNA viruses having bacilliform or rod
shaped virions. Important sub groups within the family are Nuclear Polyhedrosis Viruses
(NPV) and the Granulosis Viruses (GV) which are widely used in pest control the NPVs are
specific to host species or genus. GVs are more specific than the NPVs and are reported from
Lepidoptera. Commercial formulation of NPVs is commonly used by the farmers for the
management to tobacco caterpillar and gram caterpillar.
 Entomo-fungi: Different enotmofungal pathogens such as white muscardine, green
muscardine and yellow muscardine are known to attack various pest. The spores of the
fungus directly penetrate integument of pest and body cavity is attack. The fungus with its
mycelium and spores cover the body of host. Beauveria basiana against rice hispa,
Spodoptera litura and lepidopteran tissue borers’ sugarcane.
 Protozoans: Protozo kill the insect either directly or by reducing the fecundity of the adult.
Their effect on host is chronic. They prolong the larval life in the field, thus exposing the
insect longer to predators and parasitoids. These are called debilitating infections. They are
always associated with other pathogens. Ex : Nosema melolonthae against chaffer beetles
 Entomo-pathogenic nematodes (EPN): Entomopathogenic nematodes (EPN)
(Steinernematids and Heterorhabdits) directly attack the insect pest. The invasive larvae of
EPNs carry the associated bacteria Xenorhabdus sp. or Photorhabdus sp. In their gut, which
not only kill the invaded host within 24-48 hr but also produce a biostatic substance that
retards the purification of the cadaver. The nematodes feed on these bacteria and are capable
of producing several generations in their dead hosts.

10
 2.1.2 Establishment of Natural Enemies:
Natural enemies have four basic needs: food, reproduction, a good living environment, and defense
against strong winds and climatic conditions. Plant biodiversity allows for the maintenance of
diverse prey, flower nectar, and pollen for natural enemy survival. Cucurbit, solanaceous and
malvaceaous crops (cotton, okra) bloom continuously and help maintain food sources for natural
enemies. At times, blooming plants that are attractive to wasps and flies are planted as reservoirs for
natural enemies. Ideally, these plants should also have commercial value. Ideal plants are those that
help maintain natural enemies and can either be sold as fresh flowers or spices, or have insecticide,
nematicide, fungicide or bactericide properties. Special attention should be given to the fact that
these reservoirs may maintain aphids, whiteflies and viruses at the same time. In such cases, the
reservoir plant should be destroyed. The use of live barriers of corn or sorghum also promotes the
establishment of natural enemies.

2.1.3 Using Natural Enemies in the field:

There are three primary methods of using biological control in the field:

1. Conservation of existing natural enemies:

Reducing pesticide use: Most natural enemies are highly susceptible to pesticides, and pesticide use
is a major limitation to their effectiveness in the field. The original idea that inspired integrated pest
management (IPM) was to combine biological and chemical control by reducing pesticide use to the
minimum required for economic production, and applying the required pesticides in a manner that is
least disruptive to biological control agents. The need for pesticides can be reduced by use of
resistant varieties, cultural methods that reduce pest abundance or damage, methods of manipulating
pest mating or host-finding behavior, and, in some cases, physical methods of control. Many IPM
programs, however, have not been able to move beyond the first stage of developing sampling
methods and economic thresholds for pesticide application.

I. Selecting and using pesticides to minimize the effect on natural enemies:

The effect of a pesticide on natural enemy populations depends on the physiological effect of the
chemical and on how the pesticide is used -- how and when it is applied, for example. While
insecticides and acaricides are most likely to be toxic to insect and mite natural enemies, herbicides
and fungicides are sometimes toxic as well. Among the insecticides, synthetic pyrethroids are among
the most toxic to beneficial insects, while Bacillus thuringiensis and insect growth regulators were
among the least toxic. In general, systemic insecticides, which require consuming plant material for
exposure, and insecticides that must be ingested for toxicity affect natural enemies much less than

11
pests. Pesticides may also have more subtle effects on the physiology of natural enemies than direct
toxicity. Several fungicides, such as benomyl, thiophanate-methyl, and carbendazim, inhibit
oviposition by predacious phytoseiid mites. Certain herbicides (diquat and paraquat) make the
treated soil in vineyards repellent to predacious mites

II. Providing habitat and resources for natural enemies

Natural enemies are generally not active during the winter so they are re-released each year, and
must have a suitable environment for overwintering. Some parasitoids and pathogens overwinter in
the bodies of their hosts (which may then have overwintering requirements of their own), but others
may pass the winter in crop residues, other vegetation, or in soil. A classic example is the
overwintering of predacious mites in fruit orchards. Ground cover in these orchards provides shelter
over the winter, refuge from pesticides used on the fruit trees, and a source of pollen and alternate
prey.The adults of many predators and parasitoids may require or benefit from pollen, nectar or
honeydew (produced by aphids) during the summer. Many crop plants flower uniformly for only a
short time, so flowering plants along the edges of the field or within the field may be needed as
supplemental sources of pollen and nectar. However, diversification of plants within the field can
also interfere with the efficiency of host-finding, particularly for specialist parasitoids. Populations of
generalist predators may be stabilized by the availability of pollen and alternative prey, but the
effectiveness of the predators still depends on whether they respond quickly enough, either by
aggregation or multiplication, to outbreaks of the target pest. Thus, diversification of plants or other
methods of supplementing the nutrition of natural enemies must be done with knowledge of the
behavior and biology of the natural enemy and pest.

2. Introducing new natural enemies and establishing a permanent population

This is a process which requires extensive research into the biology of the pest, potential natural
enemies and their biology, and the possibility of unintended consequences (e.g. negative effects on
native species which are not pests or on other natural enemies of the pest). After suitable natural
enemies are found, studied, and collected, they must undergo quarantine to eliminate any pathogens
or parasites on the natural enemy population. Then, the natural enemies are carefully released, with
attention to proper timing in the enemy and pest life cycles, in a site where the target pest is
abundant, and where disturbance of the newly released enemies is minimized. Although this process
is long and complex, when it is successful, the results can be impressive and permanent, as long as
care is taken in production practices to minimize negative effects on the natural enemy.

3. Mass culture and periodic release of natural enemies

A. Seasonal inoculative release:

12
In some cases, a natural enemy is not able to overwinter successfully here in the Northeast, due to the
weather or the lack of suitable hosts or prey. In other cases, such as in greenhouses, all possible
habitats for the natural enemy is removed at the end of the season or production cycle. Thus,
particularly in annual crops, or in other highly disturbed systems, the natural enemy may need to be
reintroduced regularly in order to maintain control of the pest.

B. Biological insecticides or inundative release

These two approaches are fundamentally different from all the other approaches to biological control
because they do not aim to establish a population of natural enemies that multiplies to a level where
it reaches a long-term balance with the population of its hosts or prey. Instead, the idea is to use
biological agents like a pesticide -- to release them in quantities that will knock down the pest
population. Most commercially available formulations of insect pathogens are used inundatively.

2.2 Advantages of using natural enemies in pest management:

 It can be effective for larger area.
 Initial cost to establish biological control station may be high but in long run it becomes
cheaper.
 Hidden pests are also controlled by the parasites e.g., Stem borer are inside the stem even
though they are parasitized.
 The application of biotic agents is easy and possible even in inaccessible areas like dense
forest, tall trees, ponds, rivers, lakes etc.
 It is safe to non-target insect (beneficial).
 Pest resistance to NE is not known.
 Useful to reduce the use of chemical insecticides.
 No residue problems.
 No harmful effects on humans, livestock and other organisms.
 Non-hazardous and eco-friendly methods.
 It is a long-lasting control i.e.; biological agents will survive as long as the pest is prevalent.
 It is self-propagating and self-perpetuating in nature: Once the parasites/ predators are
established in the particular area/locality they multiply themselves.
 Biological control is close to permanent.
 Waiting period is not required for harvesting a crop after treatment with biocides.
 The grower does not require any special treatment procedure / application equipment
(sprayer, duster etc.) except for microbial preparations.
 Biocontrol is an economically viable method. If successfully deployed and converted.

13
  Biological control method can be integrated well with other methods namely cultural,
physical, mechanical, chemical (except use of broad-spectrum insecticides) methods and host
plant resistance.

2.3 Limitations of biological control in pest management

 Biological controls slow process and the exotic parasite need at least three years for proper
establishment. Further farmers do not like to wait for the natural enemies they want quick
results
 The work of the natural enemies cannot be restricted to particular crop or area. They may
migrate in nearby field.
 If parasites have another alternative host in a locality, it may not be effective for particular
pest.
 If certain season is not favorable for the normal development of natural enemy.
Replacement/changing will become necessary every year (To fill again).
 Progress of natural enemy becomes slow if hyperparasites are present in locality.
 Use of chemical pesticides adversely affects the population level of natural enemies.
 It is specific in control or effective only for one pest; so far, the controls of other pest farmers
have to adopt other methods of insect control.
 Not always applicable.
 Research costs are high.
 May be difficult or expensive to produce.
 Level of control may not be sufficient.
 Small farm holding is limiting factors in adopting the biocontrol programme.
 Farmers do not easily believe that insects can help in controlling harmful insect pests.
 The farmers are inclined to use chemical pesticides because of convenience

2.4 Characteristics of effective natural enemies:

 Narrow host range: Generalized predators may be good natural enemies but they don't kill
enough pests when other types of prey are also available
 Climatic adaptability: Natural enemies must be able to survive the extremes of temperature
and humidity that they will encounter in the new habitat.
 Synchrony with host (prey) life cycle: The predator or parasite should be present when the
pest first emerges or appears.
 High reproductive potential: Good natural enemies produce large numbers of offspring.
Ideally, a parasite completes more than one generation during each generation of the pest.
 Efficient search ability: In order to survive, effective natural enemies must be able to locate
their host or prey even when it is scarce. In general, better search ability results in lower pest
population densities.

14
  Short handling time: Natural enemies that consume prey rapidly or quickly have more time to
locate and attack other members of the pest population. Small populations of efficient natural
enemies may be more effective than larger populations of less efficient species.
 Survival at low host (prey) density: If a natural enemy is too efficient, it may eliminate its
own food supply and then starve to death. The most effective agents reduce a pest population
below its economic threshold and then maintain it at this lower equilibrium level.

2.5 Examples of successful use of natural enemies:

 Cottony cushion scale (Icerya purchasi): This pest of citrus is kept in check by Rodolia
cardinalis , a ladybeetle lady beetle introduced from Australia.
 Woolly apple aphids (Eriosoma lanigerum): In apple orchards of the northeastern and
northwestern U. S., these aphids are controlled by Aphelinus mali, a chalcid parasite native to
Europe.
 Alfalfa weevils (Hypera postica): Antachinidae wasp (Bathyplectes curculionis) parasitizes
this beetle’s larvae and a braconid wasp (Microtonus aethiopoides) parasitizes the adults.
 Cassava mealybugs (Phenacoccus manihoti): This pest spread throughout much of tropical
tropical Africa in the 1980 s’, but it has been largely brought under control by an achinid
wasp (Apoanagyrus lopezi) discovered in South America.

2.6 Beneficial Organisms Commercially Available for Pest Management

Beneficial organism Target pest
 Parasitic wasps White flies
 Encarsia formosa
 Parasitic wasps, Scales
 Aphytis melinus
 Leaf miner parasites Serpentine leaf miners
 Dacnusca sibiriica
 Predatory mites, Spider mites
 Amblyseius californicus, Phytoseiulus longipes
 Phytoseiulus persimilis
 Predatory mites, Thrips
 Amblyseius cucumeris
 Amblyseius mckenziei
 Lady beetles, Soft bodied insect and eggs
 Hippodamia convergens
 Cryptolaemus montrouzeri
 Green lacewings, Chrysoperla carnea Soft bodied insect and eggs

15
3.0 Defense mechanism against pathogens
parasites and insects
3.1 Introduction:
Disease is any physiological abnormality or significant disruption in the normal health of a plant.
Disease can be caused by living (biotic) agents, including fungi and bacteria, or by environmental
(abiotic) factors such as nutrient deficiency, drought, and lack of oxygen, excessive temperature,
ultraviolet radiation, or pollution.

Plants have developed a complex defense system against diverse pests and pathogens. Once
pathogens overcome mechanical barriers to infection, plant receptors initiate signaling pathways
driving the expression of defense response genes. Plant immune systems rely on their ability to
recognize enemy molecules, carry out signal transduction, and respond defensively through
pathways involving many genes and their products. Pathogens actively attempt to evade and interfere
with response pathways, selecting for a decentralized, multi-component immune system. Recent
advances in molecular techniques have greatly expanded our understanding of plant immunity,
largely driven by potential application to agricultural systems. Plants represent a rich source of
nutrients for many organisms including bacteria, fungi, protists, insects, and vertebrates. Plants have
developed a stunning array of structural, chemical, and protein-based defenses designed to detect
invading organisms and stop them before they are able to cause extensive damage. In order to protect
themselves from damage, plants have developed a wide variety of constitutive and inducible
defenses.

Constitutive (continuous) defenses include many preformed barriers such as cell walls, waxy
epidermal cuticles, and bark. These substances not only protect the plant from invasion, they also
give the plant strength and rigidity. In addition to preformed barriers, virtually all living plant cells
have the ability to detect invading pathogens and respond with inducible defenses including the
production of toxic chemicals, pathogen-degrading enzymes, and deliberate cell suicide. Plants often
wait until pathogens are detected before producing toxic chemicals or defense-related proteins
because of the high energy costs and nutrient requirements associated with their production and
maintenance.

Many plant pathogens act like “silent thieves”. Many pathogens establish intimate connections with
their hosts in order to suppress plant defenses and promote the release of nutrients. Pathogens that
keep their host alive and feed on living plant tissue are called biotrophs.. Pathogens often produce
toxins or tissue-degrading enzymes that overwhelm plant defenses and promote the quick release of
nutrients. These pathogens are called necrotrophs. Some pathogens are biotrophic during the early

16
stages of infection but become necrotrophic during the latter stages of disease. These pathogens are
called hemibiotrophs and include the fungus Magnaporthe grisea, the causative agent of rice blast
disease. Most biotrophic and hemibiotrophic pathogens can only cause disease on a relatively small
group of host plants because of the slightly different set of specialized genes and molecular
mechanisms required for each host-pathogen interaction. A plant species that does not show disease
when infected with a pathogen is referred to as a non-host plant species for that pathogen. Organisms
that do not cause disease on any plant species, such as the saprophytic bacterial species Pseudomonas
putida, are referred to as non-pathogens.

3.2. Pathogen and detection of pathogen:

Pathogen is capable of causing disease on a particular host species, two outcomes are
possible: A compatible response is an interaction that results in disease, while an incompatible
response is an interaction that results in little or no disease at all. Although a particular plant species
may be a susceptible host for a particular pathogen, some individuals may harbor genes that help
recognize the presence of the pathogen and activate defenses. For example, some tomato cultivars
show disease when infected with the bacterial pathogen Pseudomonas syringae (a compatible
response), but others (cultivar Rio Grande, for example) are capable of recognizing the bacteria and
limiting disease via resistance (an incompatible response). Disease resistance exists as a continuum
of responses ranging from immunity (the complete lack of any disease symptoms) to highly resistant
(some disease symptoms) to highly susceptible (significant disease symptoms).

3.2.1 Detection of microbial pathogens

Plants have developed multiple layers of sophisticated surveillance mechanisms that
recognize potentially dangerous pathogens and rapidly respond before those organisms have a
chance to cause serious damage. These surveillance systems are linked to specific pre-programmed
defense responses.

Basal resistance, also called innate immunity, is the first line of pre-formed and inducible
defenses that protect plants against entire groups of pathogens. Basal resistance can be triggered
when plant cells recognize microbe-associated molecular patterns (MAMPs) including specific
proteins, lipo-polysaccharides, and cell wall components commonly found in microbes. The result is
that living plant cells become fortified against attack. Non-pathogens as well as pathogens are
capable of triggering basal resistance in plants due to the widespread presence of these molecular
components in their cells. Pathogens have developed countermeasures that are able to suppress basal
resistance in certain plant species. If a pathogen is capable of suppressing basal defense, plants may
respond with another line of defense: the hypersensitive response (HR). The HR is characterized by
deliberate plant cell suicide at the site of infection. The HR may limit pathogen access to water and
nutrients by sacrificing a few cells in order to save the rest of the plant. The HR is typically more

17
pathogen-specific than basal resistance and is often triggered when gene products in the plant cell
recognize the presence of specific disease-causing effect on molecules introduced into the host by the
pathogen. Bacteria, fungi, viruses, and microscopic worms called nematodes are capable of inducing
the HR in plants.

Once the hypersensitive response has been triggered, plant tissues may become highly resistant to a
broad range of pathogens for an extended period of time. This phenomenon is called systemic
acquired resistance (SAR) and represents a heightened state of readiness in which plant resources are
mobilized in case of further attack. These substances are gaining favor in the agricultural community
because they are much less toxic to humans and wildlife than fungicides or antibiotics, and their
protective effects can last much longer. In addition to the hypersensitive response, plants can defend
themselves against viruses by a variety of mechanisms including a sophisticated genetic defense
system called RNA silencing. Many viruses produce double-stranded RNA or DNA during
replication in a host cell. Plants can recognize these foreign molecules and respond by digesting the
genetic strands into useless fragments and halting the infection. Plants that are infected with viruses
will often exhibit chlorosis and mottling, but disease symptoms may eventually disappear if RNA
silencing is successful, a process called recovery. In addition, the plant may retain a template of the
digested genetic strand that can be used to quickly respond to future attack by similar viruses, a
process analogous to the memory of vertebrate immune systems.

3.1.2 Detection of insects

Mechanical damage caused by insects is not generally considered “true” plant disease
although plants have developed surveillance systems designed to recognize insect pests and respond
with specific defense mechanisms. Plants can distinguish between general wounding and insect
feeding by the presence of elicitors contained in the saliva of chewing insects. In response, plants
may release volatile organic compounds (VOCs), including monoterpenoids, sesquiterpenoids, and
monoterpenoids. These chemicals may repel harmful insects or attract beneficial predators that prey
on the destructive pests. For example, wheat seedlings infested with aphids may produce VOCs that
repel other aphids. Lima beans and apple trees emit chemicals that attract predatory mites when
damaged by spider mites, and cotton plants produce volatiles that attract predatory wasps when
damaged by moth larvae. Feeding on one part of the plant can induce systemic production of these
chemicals in undamaged plant tissues, and once released, these chemicals can act as signals to
neighboring plants to begin producing similar compounds. Production of these chemicals exacts a
high metabolic cost on the host plant, so many of these compounds are not produced in large
quantities until after insects have begun to feed.

18
3.3 Structural Defenses
3.3.1 The Plant Cell

All plant tissues contain pre-formed structural barriers that help limit pathogen attachment, invasion
and infection. The cell wall is a major line of defense against fungal and bacterial pathogens. It
provides an excellent structural barrier that also incorporates a wide variety of chemical defenses that
can be rapidly activated when the cell detects the presence of potential pathogens. All plant cells
have a primary cell wall, which provides structural support and is essential for turgor pressure, and
many also form a secondary cell wall that develops inside of the primary cell wall after the cell stops
growing. The primary cell wall consists mostly of cellulose, a complex polysaccharide consisting of
thousands of glucose monomers linked together to form long polymer chains. These chains are
bundled into fibers called microfibrils, which give strength and flexibility to the wall. Many cell
walls also contain lignin, a heterogeneous polymer composed of phenolic compounds that gives the
cell rigidity. Lignin is the primary component of wood, and cell walls that become “lignified” are
highly impermeable to pathogens and difficult for small insects to chew. Cutin, suberin, and waxes
are fatty substances that may be deposited in either primary or secondary cell walls (or both) and
outer protective tissues of the plant body, including bark.

Cell walls contain proteins and enzymes that actively work to reshape the wall during cell growth yet
thicken and strengthen the wall during induced defense. When a plant cell detects the presence of a
potential pathogen, enzymes catalyze an oxidative burst that produces highly reactive oxygen
molecules capable of damaging the cells of invading organisms. Reactive oxygen molecules also
help strengthen the cell wall by catalyzing cross-linkages between cell wall polymers, and they serve
as a signal to neighboring cells that an attack is underway. Plant cells also respond to microbial
attack by rapidly synthesizing and depositing callose between the cell wall and cell membrane
adjacent to the invading pathogen. Callose deposits, called papillae, are polysaccharide polymers that
impede cellular penetration at the site of infection, and these are often produced as part of the
induced basal defense response. Some plant cells are highly specialized for plant defense. Idioblasts
(“crazy cells”) help protect plants against herbivores because they contain toxic chemicals or sharp
crystals that tear the mouthparts of insects and mammals as they feed.

3.3.2 Plant Tissues and Specialized Appendages

The epidermis constitutes the outermost protective tissue system of leaves, floral parts, fruits,
seeds, stems, and roots of plants until they undergo considerable secondary growth. It is the first line
of defense against invading pathogens and consists of both specialized and unspecialized cells. The
epidermal cells of aerial plant parts are often covered in a waxy cuticle that not only prevents water
loss from the plant, but also prevents microbial pathogens from coming into direct contact with
epidermal cells and thereby limits infection. The cuticle can be relatively thin (aquatic plants) or
extremely thick (cacti). The hydrophobic nature of the cuticle also prevents water from collecting on

19
the leaf surface, an important defense against many fungal pathogens that require standing water on
the leaf surface for spore germination. However, some fungal pathogens including Fusarium solani
produce cutinases that degrade the cuticle and allow the fungi to penetrate the epidermis.
Interspersed among the many unspecialized cells of the epidermis are guard cells which regulate gas
exchange through small openings called stomata. These pores allow carbon dioxide to enter the leaf
for use in photosynthesis while restricting excessive water loss from the plant. Stomatal pore size is
highly regulated by plants, and guard cells can participate in defense by closing in response to the
presence of MAMPs.

Trichomes (“leaf hairs”) are specialized epidermal cells found on aerial plant parts that may provide
both physical and chemical protection against insect pests. The velvety appearance of dusty miller
(Senecio cineraria) is caused by thousands of tiny trichomes covering the plant’s surface. Trichomes
on the surface of soybeans (Glycine max) prevent insect eggs from reaching the epidermis and the
larvae starve after hatching. The hook-shape of snap bean (Phaseolis vulgaris) trichomes impale
caterpillars as they move across the leaf surface, and glandular trichomes in potato and tomato
secrete oils that repel aphids. In woody plants, the periderm replaces the epidermis on stems and
roots. Outer bark (phellem) is an excellent example of a preformed structural barrier that contains
high amounts of water-resistant suberin and prevents many pathogens and insects from reaching the
living cells underneath.

Thorns are modified branches that protect plants from grazing vertebrates, and include the honey
locust tree (Gleditsia triacanthos). Many cacti produce thorn-like structures that are actually modified
leaves or parts of leaves (e.g., stipules) called spines which serve similar purposes, such as in the
barrel cactus (Ferocactus spp.). Botanically speaking, the “thorns” on the stem of rose plants (Rosa
spp.) are neither true thorns nor spines: they are actually outgrowths of the epidermis called prickles.

3.3.3 Chemical Defenses

Plant chemicals can be divided into two major categories: primary metabolites and secondary
metabolites. Primary metabolites are substances produced by all plant cells that are directly involved
in growth, development, or reproduction. Examples include sugars, proteins, amino acids, and
nucleic acids. Secondary metabolites are not directly involved in growth or reproduction but they are
often involved with plant defense. These compounds usually belong to one of three large chemical
classes: terpenoids, phenolics, and alkaloids.

Terpenoids: They are found occur in all plants and represent the largest class of secondary
metabolites with over 22,000 compounds described. The simplest terpenoid is the hydrocarbon
isoprene (C5H8), a volatile gas emitted during photosynthesis in large quantities by leaves that may
protect cell membranes from damage caused by high temperature or light. Terpenoids are classified

20
by the number of isoprene units used to construct them. For example, monoterpenoids consist of two
isoprene units, sesquiterpenoids (three units), diterpenoids (four units), and triterpenoids (six units).

 Monoterpenoids and sesquiterpenoids are the primary components of essential oils, which are
highly volatile compounds that contribute to the fragrance (essence) of plants that produce
them. Essential oils often function as insect toxins and many protect against fungal or
bacterial attack.
 Diterpenoids include gossypol, a terpenoid produced by cotton (Gossypium hirsutum) that
has strong antifungal and antibacterial properties. Triterpenoids are similar in molecular
structure to plant and animal sterols and steroid hormones. Phytoectysones are mimics of
insect molting hormones. When produced by plants such as spinach (Spinacia oleracea), they
disrupt larval development and increase insect mortality. Triterpenoids such as cardiac
glycosides are highly toxic to vertebrate herbivores, including humans, and can cause heart
attacks if ingested in high quantities. Foxglove (Digitalis purpurea) is the principal source of
the cardiac glycosides digitoxin and digoxin, which are used medicinally in small quantities
to treat heart disease in people. Some herbivores have overcome the dangerous effects of
cardiac glycosides and actually use these toxins for their own benefit. Monarch butterfly
caterpillars feed almost exclusively upon milkweed (Asclepias spp.) which contains high
amounts of these toxins in the milky latex of their sap. The caterpillars store these toxins
safely within their bodies, and when the caterpillars become adult butterflies, they are highly
poisonous to most predatory birds that eat them.

Phenolics: Phenolics are class of secondary metabolites produced by plants to defend themselves
against pathogens. They are produced primarily via the shikimic acid and malonic acid pathways in
plants, and include a wide variety of defense-related compounds including flavonoids, anthocyanins,
phytoalexins, tannins, lignin, and furanocoumarins. Flavonoids are one of the largest classes of
phenolics. Anthocyanins are colorful water-soluble flavonoids pigments produced by plants to
protect foliage from the damaging effects of ultraviolet radiation.

 Tannins are water-soluble flavonoid polymers produced by plants and stored in vacuoles.
Tannins are toxic to insects because they bind to salivary proteins and digestive enzymes
including trypsin and chymotrypsin resulting in protein inactivation. Insect herbivores that
ingest high amounts of tannins fail to gain weight and may eventually die. The sharp taste of
red wine is caused by grape tannins binding to salivary proteins in the mouth which results in
protein coagulation.
 Lignin is a highly branched heterogeneous polymer found principally in the secondary cell
walls of plants, although primary walls can also become lignified. It consists of hundreds or
thousands of phenolic monomers and is a primary component of wood. Because it is

21
 insoluble, rigid, and virtually indigestible, lignin provides an excellent physical barrier
against pathogen attack.
 Furanocoumarins are phenolic compounds produced by a wide variety of plants in response
to pathogen or herbivore attack. They are activated by ultraviolet light and can be highly
toxic to certain vertebrate and invertebrate herbivores due to their integration into DNA,
which contributes to rapid cell death. In fact, grapefruit juice contains small quantities of
furanocoumarins, which greatly increase the absorption of certain drugs into the bloodstream
from the intestines. Some medicines carry warning labels cautioning people to avoid drinking
grapefruit juice while taking the drugs in order to avoid an accidental overdose.

Alkaloids: Alkaloids are a large class of bitter-tasting nitrogenous compounds that are found in
many vascular plants and include caffeine, cocaine, morphine, and nicotine. They are derived from
the amino acids aspartate, lysine, tyrosine, and tryptophan, and many of these substances have
powerful effects on animal physiology. Caffeine is an alkaloid found in plants such as coffee (Coffea
arabica), tea (Camellia sinensis), and cocoa (Theobroma cacao). It is toxic to both insects and fungi.
In fact, high levels of caffeine produced by coffee seedlings can even inhibit the germination of other
seeds in the vicinity of the growing plants, a phenomenon called allelopathy. Allelopathy allows one
plant species to “defend” itself against other plants that may compete for growing space and nutrient
resources.

 Members of the nightshade family (Solanaceae) produce many important alkaloid

compounds. Nicotine is an alkaloid that is produced in the roots of tobacco plants (Nicotiana
tabacum) and transported to leaves where it is stored in vacuoles. It is released when
herbivores graze on the leaves and break open the vacuoles. Atropine is a neurotoxin and
cardiac stimulant produced by the deadly nightshade plant (Atropa belladonna). Although it
is toxic in large quantities, it has been used medicinally by humans in small amounts as a
pupil dilator and antidote for some nerve gas poisonings. Capsaicin and related capsaicinoids
produced by members of the genus Capsicum are the active components of chili peppers and
produce their characteristic burning sensation in hot, spicy foods.
 Cyanogenic glycosides are a particularly toxic class of nitrogenous compounds that break
down to produce hydrogen cyanide (HCN), a lethal chemical that halts cellular respiration in
aerobic organisms. Plants that produce cyanogenic glycosides also produce enzymes that
convert these compounds into hydrogen cyanide, including glycosidases and hydroxynitrile
lyases, but they are stored in separate compartments or tissues within the plant; when
herbivores feed on these tissues, the enzymes and substrates mix and produce lethal hydrogen
cyanide. Glucosinolates, also known as mustard oil glycosides, are sulfur-containing
compounds synthesized by members of the mustard family (Brassicaceae) and produce
cyanide gas when broken down by enzymes called thioglucosidases.

22
3.3.4 Proteins and Enzymes
Many plants and seeds contain proteins that specifically inhibit pathogen and pest enzymes
by forming complexes that block active sites or alter enzyme conformations, ultimately reducing
enzyme function. These proteins are generally small and rich in the amino acid cysteine. They
include defensins, amylase inhibitors, lectins, and proteinase inhibitors. Unlike simple chemicals
such as terpenoids, phenolics, and alkaloids, proteins require a great deal of plant resources and
energy to produce; consequently, many defensive proteins are only made in significant quantities
after a pathogen or pest has attacked the plant. Once activated, however, defensive proteins and
enzymes effectively inhibit fungi, bacteria, nematodes, and insect herbivores. Defensins are small
cysteine-rich proteins that display broad anti-microbial activity and were first isolated from the
endosperm of barley (Hordeum vulgare) and wheat (Triticum aestivum). They are widely distributed
and may be present in most plants. Defensins are best characterized in seeds, but can be found in
virtually all types of plant tissues including leaves, pods, tubers, fruit, roots, bark, and floral tissues.
They exhibit a wide range of biological activities that serve to inhibit the growth of many fungi and
bacteria. Some defensins also inhibit digestive proteins in herbivores. The precise mechanisms
employed by plant defensins to inhibit fungi and bacteria are still being characterized, but they
appear to act upon molecular targets in the plasma membrane of pathogens. These defensins may
inhibit pre-existing ion channels or form new membrane pores that disrupt cellular ion balance.

Digestive enzyme inhibitors are proteins that block the normal digestion and absorption of nutrients
by vertebrate and invertebrate herbivores. Alpha-amylase inhibitors are proteins commonly found in
legumes that bind to amylase enzymes and inhibit starch digestion. Lectins are non-enzymatic
proteins and glycoproteins that bind to carbohydrates and exhibit a wide range of functions including
disruption of digestion in insects and agglutination of blood cells in vertebrates. Ricin is a powerful
toxin produced in castor beans (Ricinus communis). It combines a lectin molecule with an N-
glycoside hydrolase that enters animal cells and inhibits protein synthesis. Ricin is a highly potent
toxin, having an average lethal dose of only 0.2 milligrams in humans.Protease inhibitors are
typically produced in response to herbivore attack and inhibit digestive enzymes including trypsin
and chymotrypsin. They occur widely in nature but have been well studied in legumes, solanaceous
plants, and grasses. Herbivore feeding often triggers a series of molecular signaling events that
induce systemic production of these compounds in distal tissues that contribute to the protection of
undamaged plant parts from subsequent attacks by a wide range of herbivore pests.

Hydrolytic enzymes are produced by some plants in response to pathogens and often accumulate in
extracellular spaces where they degrade the cell walls of pathogenic fungi. Chitinases are enzymes
that catalyze the degradation of chitin, a polymer with a backbone similar to cellulose that is present
in the cell walls of true fungi. Glucanases are enzymes that catalyze the degradation of glycosidic
linkages in glucans, a class of polymers similar to cellulose that is present in the cell walls of many

23
oomycetes (water molds). In vitro analysis has verified the anti-fungal properties of these
compounds, and transgenic plants expressing high levels of these enzymes exhibit increased
resistance to a wide range of both foliar and root pathogens. Lysozymes are hydrolytic enzymes that
are capable of degrading bacterial cell walls.

24
 4.0 A great diversity in mechanism for diversity

Introduction:

Plants possess an elaborate suite of defense components to protect themselves against pathogen
infection. Plant breeding for disease resistance is crucial to sustain global crop production. For
decades, plant breeders and researchers have extensively used host plant resistance genes (R-genes)
to develop disease resistant cultivars. However, the general instability of R-genes in crop cultivars
when challenged with diverse pathogen populations emphasizes the need for more stable means of
resistance.

4.1.1 Broad Resistance:

Broad resistance refers to resistance against more than one pathogen species or against most races or
strains of the same species

4.1.2 Non host resistance:

Non-host resistance is recognized as the most durable, broad-spectrum form of resistance against the
majority of potential pathogens in plants. Non-host resistance (NHR) can be defined as the immunity
of an entire plant species to all genetic variants of a non-adapted pathogen species. Since this type of
resistance is typically both broad-spectrum and durable, NHR has considerable value for crop
improvement and its practical application has been of recent interest. The majority of NHR studies
have been performed in model plant species such as Arabidopsis, rice and Nicotiana benthamiana.
Despite the important role of NHR in plant defense, the mechanisms underlying NHR are still not
clear in many cases. The potential application of NHR has been shown in multiple crops using
genetically engineering (GM) methods. In crop species, examples of genetically engineered NHR
applications include introduction of genes providing protection against fungal and bacterial
pathogens, such as lettuce downy mildew, barley rust, strawberry gray mold and anthracnose, peach
mildew, and tomato bacterial spot.

Basic Mechanisms of Non-Host Resistance and Their Overlap with Host Resistance

Plants defend themselves from pathogen attack through two major defense systems: the recognition
of pathogen associated molecular patterns (PAMPs) by pattern recognition receptors (PRR) and
effector-triggered immunity (ETI). The PRR type of resistance is characteristic of NHR and usually
triggers multi-layered basal resistance mechanisms, which commonly include peroxisome-based
biosynthesis, restriction of pathogen growth by nutrient limitation, papilla formation, and callose and
lignin deposition. ETI is the main mechanism for host resistance in which host R-proteins directly or
indirectly recognize complementary pathogen effector proteins. As a result of the specific

25
recognition in the host plant, a hypersensitive response (HR) involving cell death (or programmed
cell death) often occurs, limiting or halting disease development.

To utilize NHR mechanisms for crop improvement, identification of the underlying genes is the first
step. A number of studies have identified NHR genes effective against bacterial and fungal
pathogens. Many of the identified genes involved in NHR have multi-functional roles including plant
development and stomatal regulation, and are not only important for the non-host defense signaling
pathway itself, but also in basic plant metabolism.

Non-host resistance can be divided into two categories based on the presence and absence of visual
cell death: Type I and Type II NHR. Type I NHR does not show any visible cell death symptoms
while type II NHR shows localized hypersensitive response (HR) cell death in response to non-host
pathogens. This cell death is mediated through the generation of reactive oxygen species (ROS) and
shares similarities with effector-triggered immunity, the main mechanism for host resistance. The
plant defense-related genes responsible for initial defense response (pathogenesis-related protein
genes, oxidative burst-associated genes, and cell defense genes) are similarly expressed in both Type
II NHR and host resistance in tobacco, suggesting overlapping defense signaling pathways between
non-host and host resistance. Non-host resistance against pathogens of distantly related hosts usually
involves complex, multi-layered, quantitatively inherited mechanisms.

Applications of Non-Host Resistance for Crop Disease Resistance: Genetic Engineering

Approaches

It is evident that the components required for NHR can be genetically engineered to control host-
adapted pathogens in crops. Strawberry is an excellent example of an economically important fruit
crop worldwide that has also been recognized as a suitable model plant species for the Rosaceae
family. The Arabidopsis NPR1 (Non-expresser of PR genes 1) gene regulates the onset of systemic
acquired resistance through the salicylic acid defense pathway and via defense genes such
as PR (pathogenesis-related) genes for plant immunity.The ectopic expression of Arabidopsis
NPR1 in diploid strawberry (Fragaria vesca) increases resistance to anthracnose
(Colletotrichum spp.), powdery mildew (Podosphaera aphanis), and bacterial angular leaf spot
(Xanthomonas fragariae). In another example, allelic variation for powdery mildew resistance
genes, MILDEW-RESISTANCE LOCUS (MLO), is highly diverse among monocotyledonous and
dicotyledonous plant species. Barley, in particular, has highly effective immunity against powdery
mildew fungi, and this immunity is achieved by loss-of-function mutant alleles of the barley MLO
gene It is suggested that the resistance present among different plants is due to similar mlo-based
immunity, natural loss-of-function polymorphisms in MLO gene. For example, the PpMlo1 (Prunus
perisica Mlo) of peach has been identified for powdery mildew resistance and shares a similar
functional mechanism for resistance in the Rosaceae The antisense expression of the

26
peach MLO gene (PpMlo1) in octoploid strawberry conferred NHR to strawberry powdery mildew
(Podosphaera aphanis).

4.1.3 Host range:

Pathogens are often characterized as specialists or generalists based on the number of different host
species they infect, as well as the phylogenetic relatedness among hosts. Host range can be
associated with several factors, including the geographic ranges of pathogens and hosts, host and
pathogen phylogeny, and life history traits.

Host range is defined as the number of host species used by a pathogen, is a simple metric that is
central to understanding pathogen epidemiology and pathogenicity. Host range conditions the
transmission dynamics and survival of pathogens and is predicted to be a major factor in their
evolution. The host range of a pathogen affects how diversification and speciation occur, the
incorporation of new species into the number of hosts a pathogen can infect, shifting among hosts in
response to biotic and abiotic environmental variation, or the ability to persist in the environment
between epidemics. Host range describes the breadth of organisms a parasite is capable of infecting,
with limits on host range stemming from parasite, host, or environmental characteristics. Parasites
can adapt to overcome host or environmental limitations, while hosts can adapt to control the
negative impact of parasites.

4.2.1 Hypersensitivity Resistance

The hypersensitive defense response is found in all higher plants and is characterized by a rapid cell
death at the point of pathogen ingress. It is usually associated with pathogen resistance, though, in
specific situations, it may have other consequences such as pathogen susceptibility, growth
retardation and, over evolutionary timescales, speciation. Due to the potentially severe costs of
inappropriate activation, plants employ multiple mechanisms to suppress inappropriate activation of
HR and to constrain it after activation. The plant hypersensitive response (HR) is a rapid localized
cell death that occurs at the point of pathogen penetration and is associated with disease resistance. A
rapid cell death localized at the area of pathogen infection those results in some suppression of
disease progress. HR is a widespread phenomenon, found in most if not all higher plants and induced
by a number of classes of pathogen. HRs induced by fungi, oomycetes, bacteria and viruses are the
most commonly observed, but HR can be induced by other organisms, such as insects and
nematodes that form sustained intimate interactions with the host plant. Cell death has also been
observed in resistant interactions between parasitic plants and their hosts though whether this
represents an HR is unclear. HR is generally associated with race‐specific resistance to biotrophic
pathogens (pathogens which derive nutrition from living tissues). It is generally less effective
against, and may actually be beneficial to, necrotrophs which require dead host tissue to complete
their life cycle

27
When a plant detects the presence of a pathogen, it initiates a series of signaling events that lead to
the activation of defense mechanisms. In some cases, this response can be excessive, leading to the
hypersensitive response. The hypersensitive response is often associated with the release of reactive
oxygen species, defense hormones, and the production of antimicrobial compounds. While
hypersensitive responses can effectively restrict the growth and spread of pathogens, they can also
cause damage to the plant tissues involved in the response. This can lead to visible symptoms such as
wilting, necrosis, or tissue discoloration.

4.2.2 Partial Resistance

The resistance that results in a reduced epidemic built-up of the natural enemy, despite a susceptible, non
hypersensitivity infection type and is applied with biotroph, hemi biotroph plant-patho systems.

Many crops to PM and rust, in potato to phytophthora infestans and in rice to Xanthomonas oryzae and
Pyricularia oryzae.

Components of partial resistance

 Latency Period: it is the time between the onset of the infection process and the reproduction of
the pathogen.
 Infection frequency: is the Percentage of spores that result in a reproducing infection but also for
the number of infections observed per plant, per leaf or per cm2 tissue.
 Spore production per plant: As the production of spores varies significantly

For eg, partial resistance in rice against P. oryzae is not due to a prolonged latency period, but mainly due to
a reduction of the infection frequency and a lower rate of lesion growth. The level of partial resistance can
only be determined directly in polycyclic field experiments.

Partial resistance of host depends on the, degree of correlation of the component with the Partial
resistance, the errors present during the experiments known as experimental error. Furthermore it also
depends on ease and convenience of measurement of the components of resistance and finally towards
the degree of correlation between the individual components involved.

Mechanisms of partial resistance: Certain crop architecture may result in lower air humidity in the crop,
or in a lower deposition of spores in comparison to others and such effects may lead as with partial
resistance. Similarly in some cases the cell wall penetration of plants, growth and reproduction of the
pathogen are less successful than on more susceptible plants. It is also associated with the formation of
papillae. In many cases pathogens infect young plants and young plant tissue more effectively than
older plants and plant tissues. The latency period tends to be shorter on seedlings than on older plants,
and on young leaves shorter than on the older leaves.

Genetics of partial resistance: Partial resistance is governed by quantative traits, which means there is
presence of several genes with small but cumulative effects. Similarly when progeny of a cross obtained

28
 with parents of different genotype are crossed having different level of partial resistance then a
quantitative continuous segregation is found when they were tested.

4.3 Ecological Resistance or Pseudo resistance:

Apparent resistance resulting from transitory characters in potentially susceptible host plants due to
environmental conditions. Pseudo resistance may be classified into 3 categories

o Host evasion Host may pass through the most susceptible stage quickly or at a time
when insects are less or evade injury by early maturing. This pertains to the whole
population of host plant.
o Induced Resistance Increase in resistance temporarily as a result of some changed
conditions of plants or environment such as change in the amount of water or nutrient
status of soil
o Escape Absence of infestation or injury to host plant due to transitory process like
incomplete infestation. This pertains to few individuals of host.

These defense mechanisms are principally based on avoidance, resistance or tolerance. Avoidance
operates before parasitic contact between host and parasite is established and decreases the frequency
of incidence. After parasitic contact has been established the host may resist the parasite by
decreasing its growth, or tolerate its presence by suffering relatively little damage. Avoidance is
mainly active against animal parasites and includes such diverse mechanisms as volatile repellents,
mimicry and morphological features like hairs, thorns and resin ducts. Resistance is usually of a
chemical nature. Little is known of tolerance; it is very difficult to measure and is usually
confounded with quantitative forms of resistance. Parasites classified as fungi, bacteria, viruses or
viroids are considered disease inciting parasites or pathogens. Resistance mechanisms are by far the
most important defense mechanisms employed by host plants, including our crops, against
pathogens. Avoidance and tolerance play a minor role here. In the never-ending arms race between
plant and pathogen, the latter have developed widely different host ranges.

29
5.0 Source and test of Resistance
Introduction:

Availability of suitable sources of resistance is a basic prerequisite for successful resistance breeding.
In the beginnings of resistance breeding sources of resistance were selected among cultivated crops.
Later on, attention was paid also to inter-specific, as well intergeneric crosses including wild species
related to crops. At present alien species represent an important part of genetic sources of disease
resistance.

Sources:
Land race: They are the primitive cultivar which were selected and cultivated by the farmers for
many generations but were not deliberately bred like modem cultivars. They evolved under
subsistence agriculture and have high level of genetic diversity which provides them high degree of
resistance to biotic and a biotic stresses. Biotic stress refers to hazards of diseases and insects,
whereas a biotic stress means, drought, salinity, cold, frost, etc. they also have broad genetic base
which again provides them wider adaptability and protection from epidemic of diseases and insects.

Cultivated Varieties: In some crops, resistance to disease may be found in cultivated varieties. For
example, cotton variety MCU 5 VT tolerant to Verticillium wilt was isolated from the commercial
variety MCU 5 of Gossypium hirsutum. Some commercial varieties of Asiatic cotton are good
sources of resistances to Fusarium wilt. Resistant plants to curly top in Sugarbeet and to mildew and
leaf spot in alfalfa have been isolated from the commercial variety of respective crop. Cultivated
varieties are the best sources of disease resistance, because they posses good agronomic characters
besides disease resistance.

Germplasm Collections: Germplasm collections are the potential source of disease and insect
resistance in all the cultivated crops. In cotton, several germplasm lines resistant to bacterial blight
and Fusarium wilt have been identified based on screening of large number of germplasm in India.
Generally, germplasm lines have poor agronomic characters. Hence their use in breeding
programmes poses some problems.

Wild Species: Related wild species are also potential sources of disease resistance. However,
utilization of wild sources poses many problems such as cross incompatibility, hybrid inviability,
hybrid sterility and linkage of several undesirable traits with desirable ones. Therefore, wild related
species are only used as source of resistance when the desired resistance is not found within the
cultivated species. Wild species of crops like wheat, barley, potato, tomato, Sugarbeet, cotton, etc.

30
are good of resistance to various diseases. Many disease resistant genes have been transferred from
wild species to cultivated species in these crops. These are naturally occurring plant species which
have common ancestry with crops and can cross with crop species and are important sources of
resistance to biotic (diseases and insects) and a biotic (drought, cold, frost, salinity, etc.) stresses.

Mutations: Both spontaneous and induced mutations are good source of disease resistance. Disease
resistance has been achieved in several crops through the use of induced mutations. Some examples
of disease resistances induced by mutagenic agents are: resistance to Victoria blight and crown rusts
in oats to strip rust in wheat, to mildew in barley, to flax rust flax, and to leaf spot and stem rust in
peanut.

Genetically modified organisms: GMOs that have had genes from other species inserted into their
genome (the full complement of an organism’s genes) are called transgenic. The production of
pathogen-resistant transgenic plants has been achieved by this method; certain genes are inserted into
the plant’s genome that confers resistance to such pathogens as viruses, fungi, and insects.
Transgenic plants that are tolerant to herbicides and that show improvements in other qualities also
have been developed

Test of resistance:
Depending on mode of spread of disease the screening technique may differ. The screening can be
done both at screen or glass house level and field level. The different screening techniques are as
follows.

 Soil borne diseases: Wilt, root rot are produced by soil borne fungi. In this case sick plot
technique is followed. Susceptible varieties can be grown and infected plants can be ploughed
insitu to maintain optimum condition for infection.
 Air borne diseases: E.g. Rust, Smut, mildews, blights. For ground nut rust, infector rows can
be sown 15 days earlier as border rows and the disease will infest the susceptible infector
rows. After 15 days the varieties tested to be are to be sown. Spraying the spore suspension
from affected leaves will also increase the load.
 Seed borne disease: Smut, bunt etc. Artificial inoculation can be done by soaking the seeds in
solution of pathogen under vacuum condition. Insect transmitted diseases.
 Insect transmitted diseases: E.g. virus diseases, Red gram sterility mosaic virus. Sap
transmitted. Here the stapling technique is used. Leaves from affected plants can be stapled to
the entries to be tested. The insect feeding in susceptible leaf will transmit virus to test
entries.

31
Test for the disease resistant varieties can be done in:

 Field test: The varieties to be evaluated or the segregating population to be screened for
resistance may be raised in fields under natural infestation, either in endemic areas or by
adopting techniques for increasing field infestation. Cultural practices such as closer spacing
to create the most desirable humid microclimate within the crop, application of additional
dose of nitrogen and irrigation to induce vegetative growth may be adopted. The test material
may be sown or planted early, before the adult emerge in an area already infested in the
previous season. In case of insects such as plant hoppers, flies etc. that have a tendency to
move at a rapid speed, fiber glass mesh cages may be kept on micro plots and artificially
reared insects released at a specified number per plant. The insect’s population should be able
to infest the plant population uniformly so that plants that escape infestation are not graded as
resistant. Highly susceptible plants can be interplanted as “spreader rows” along with rows of
the test material. Insect attractants, such as fish meal for sorghum shoot fly may also be used
to attract and increase the insect density in the field.

 Green house screening: Screening the varieties or segregants in green houses providing
conditions conductive for infestation is more rapid reliable than field screening. Special
methods have been developed to increase the insect population to provide sufficient insect
pressure for valid screening. Insects reared in the culture maintenance cages are released on
plants raised in seed boxes kept in green house. Fibre glass screen cages are used for each
seed box. Depending on the crop on one hand and the insect pest on the other, the stage of the
crop at which the insect is released, the stage of the insect, whether egg, larva or adult and the
number of the insects population vary, as also the symptoms on the host to differential and
grade the resistant plants from the susceptible.
 Laboratory Screening: Laboratory screening for resistance can also be done using plant tips
or leaf discs and allowing forced or free choice feeding by insects as in the case of lucerne
weevil.
 Bioassay technique: Bioassay techniques are also used to screen resistance to insect such
as Heliothis spp and pink boll worm in cotton. Lyophilized square powder is incorporated in
an artificial diet and dispensed into two-ounce plastic containers. Late first or early second
instar larvae of Heliothis are weighed and placed in the diet cup. By periodic observations,
larval survival, larval growth and percent pupation are recorded. Dates on fecundity and
longevity of emerging adults are obtained to screen resistant types.

32
Screening Techniques for Diseases Resistance
The first step in a resistance breeding programme is to rapidly screen all the available genetic stocks,
including the local land races, improved cuttings and exotic germplasm using empirical techniques in
glass houses, or by field tests. An escape from disease is a hazard of breeding for resistant varieties.
There are two major techniques to achieve the objectives.

 Artificial inoculation: The artificial inoculation of test genotypes is necessary to obtain a

more uniform disease – epidemic than world occur naturally such. Uniformity facilitates
comparison among disease genotypes. For a successful screening, an adequate amount of
inoculum is necessary, since selecting for resistance in the presence of an inadequate amount
of disease create an instant artifact. Among few methods developed, three main procedures
of artificial inoculation are generally employed.

 Spraying the spore suspension on test genotypes – using an auto-minyor the aqueous
suspension of the pathogens propagules is sprayed. High pressure spraying is most
productive.
 Injection of spore suspension into the plants surface or into the intercellular air spaces of a
leaf with the help of a hypodermic needle. A single or multi-needle pricking may be
employed.
 Immersion of seedlings of test genotypes in a spore suspension before transplanting them into
fields. Since the host tissue becomed water soaked, they predispose themselves to disease.

Though spore – suspension in sterile water (Inoculum) is often used, successful inoculum of many
pathogens can be achieved only if dry spores are employed. Spores are damaged quickly if they
became too wet. For good results, any one of these three methods of artificial inoculation can be
used. Depending upon the convenience and crop materials the procedure may vary from one disease
to another and from one crop to another. But similarities are not ruled out.

 Artificial epiphytotics: The manual operation of artificial inoculation can be dispensed with
once a sick plot or disease nursery is created. Artificial creation of such an epiphytotic is
necessary because the occurrence of natural disease is subject to chance. This can be attained
by seeding spores in the soil (in case of only soil borne disease caused by facultative
parasites) either through growing a highly susceptible genotype in a plot for successive years,
or through mixing infected debris in the soil of that plot, prior to sowing the test genotypes.
Maintenance of requisite humidity is essential for high effectiveness.

33
 The epiphytotic should be intense developed at both national and international levels. The
international wheat rust nursery at Mexico established since 1957 is an outstanding example
of such an international collaboration.
 Frequency of exposure: The test genotypes can be subjected to artificial inoculation or sick
plots once or more than area for desired results. Two tests are suggested.

 Poly-cyclic test: Exposure that initiates the natural occurrence of diseases with a recurrent
infection cycle attacking a crop on the farmer’s field. The artificially created sick-nursery is
the best form of such polycyclic tests, through repeated artificial inoculations can also
achieve the same results.
 Mono-cyclic tests: Exposure only once by artificial inoculation, preferably under closed/
controlled condition, such as green houses.

34
 6.0 Stage of development, Application of natural enemies,
Composition of inoculums and Evaluation aspects

Measuring Resistance
Selection for resistance implies measurements of plant resistance. Ideally one should measure the
amount of pathogen present at a given moment compared with the amount present on or in an
extremely susceptible cultivar. The larger the difference in amount the larger the difference in
susceptibility/ resistance. It is normally not possible to measure the amount of pathogen, because the
pathogen is either not visible or only partially so. However, one can evaluate the direct or indirect
effects of the pathogen on the host even if the pathogen itself is not visible (Parlevliet, 1993). The
quantitative or partial resistance of a host cultivar cannot be assessed in absolute terms; it is always a
relative measure compared with that of a well-known standard cultivar. This standard cultivar is
often the most susceptible cultivar available (Parlevliet, 1989). The amount of tissue affected is, in
general, a good estimator of the amount of pathogen present. The amount of pathogen present,
however, is not just dependent on the level of resistance of the host cultivar. Other factors may and
do interfere with it such as

Interplot interference:

Van der Plank (1963) stated: “Plots in the experiment are meant to represent farmers’ fields
receiving the same treatment as these fields receive”. But plots represent fields only when the plots
within an experimental area do not interfere with one another. The representational error - the error
of taking plots to represent fields when they do not can be large. The most frequent causes of errors
in interpreting results are experimental interplot interference (Bainbridge &Jenkyn, 1976; Jenkyn et
al.,1983; Parlevliet & Van Ommeren, 1984; Parlevliet & Van Ommeren, 1975) and/or interactions
between host or pathogen with the environment (Van der Plank, 1968; Colhoun, 1973; Jeger et al.,
1983; Fraser, 1985; Hunter et al.,1986; Falkhof et al. 1988). This applies especially to partial
resistance, which, together with the aggressivity of the pathogen in general, is a quantitative
characteristic and is influenced by changes in the environment. Parlevliet & Van Ommeren (1975)
observed a very strong interplot interference in barley evaluated for partial resistance to leaf rust. In
plots well isolated from one another the most susceptible cultivar had more than 2,000 times more
uredosori than the least susceptible one. In plots of less than 1m2 and adjacent to each other the
difference between the extremes was not more than 25 times. The interplot interference may not only
underestimate the partial resistance, but it may cause the ranking order of cultivars compared to the
one in large isolated plots (Norgaard Knudsen et al., 1986). The authors concluded that for a reliable
selection of partial resistance, plots of some 1,4 m2 were advisable. However, not all airborne
pathogens cause interplot interference.

35
Relation between disease symptoms and amount of the pathogen: True disease symptoms are
observed with several pathogens such as wilting caused by vascular pathogens, and leaf rolling,
mottling, stunting, etc., caused by viruses. These symptoms tend to be rather unreliable for assessing
resistance, since the relationship between the amount of pathogen present and the severity of
symptoms is often poor. In other cases, the pathogen itself is observed, making assessment much
easier and far more reliable. The ectoparasitic powdery mildews are good examples; their mycelia
remain on the surface of the host epidermis and are visible as white to grey spots. In rust diseases the
pathogen becomes visible when the sporulating infections rupture the epidermis, exposing powdery
masses of spores. A large number of pathogens fall between these two types. The pathogen itself is
not visible, but the symptoms of its presence are more or less easily discernible and restricted to the
parts of the host tissue invaded. Discoloration of the invaded tissue and immediately adjacent tissue
is the most general indication of the pathogen. The reliability of this measure of pathogen presence
or inversely, host resistance, varies with host and pathogen but tends to be fairly good in many cases.

4.3.2 Inoculum Density:

This factor may obscure real differences in quantitative resistance. In order to prevent escape of
genotypes from infection, there is a tendency to apply high inoculum densities. Complete resistance
in such cases is easily detectable, but small differences in susceptibility tend to disappear. The
optimal inoculum density is the density whereby escapes are largely prevented while only the most
susceptible cultivars are strongly affected (Parlevliet, 1989).

4.4.3 Earliness:
If the entries differ considerably in earliness the period of exposure to the pathogen varies greatly as
the assessment is usually done at the same moment for all entries. Resistance to head blight caused
by Fusarium in wheat is considerably overestimated in late cultivars due to this aspect (Parlevliet,
1993).

4.4.4 Plant Habitat:

In dense crops and short plants, the amount of tissue affected tends to increase. In loose crops and
tall plants, it tends to decrease. This is probably due to micro-climatic effects. Short wheat cultivars
are more affected than tall cultivars by Septoria leaf and glume blotch (Parlevliet, 1993).

36
7.0 Breeding for disease and insect resistance
7.1 Mechanism of Disease resistance:-
Disease escape:-The ability of susceptible host plants to avoid attack of disease due to environmental
conditions factors, early varieties, charge in the date of planting, change in the site of planting;
balanced application of NPK etc is called disease escape.

Disease endurance or tolerance:-The ability of the plants to tolerate the invasion of the pathogen
without showing much damage. This endurance is brought about by the influence of external
characters. Generally, tolerance is difficult to measure since it is confounded with partial resistance
and disease escape.

Immune reaction:-When the host does not show the symptoms of disease it is known as immune
reaction. Immunity may result from prevention of the pathogen to reach the appropriate parts of the
host e.g. exclusion of spores of ovary infecting fungi by closed flowering habit of wheat and barley.

Hypersensitivity:-Immediately after the infection several host cells surrounding the point of
infection are so sensitive that they will die. This leads to the death of the pathogen because the rust
mycelium cannot grow through the dead cells. This super sensitivity (hypersensitivity) behaves as a
resistant response for all practical purposes. Phytoalexins are specific polyphenolic or terpenoid
chemicals and are produced by the host in response to the infection by a pathogen.

7.2 Sources of Disease Resistance:

A known variety:-Disease reactions of most of the cultivated varieties are documented and a breeder
may find the resistance he needs in a cultivated variety.

Germplasm collection:-When resistance to a new disease or a new pathotype of a disease is not

known in a cultivated variety germplasm collection should be screened.

Related species:-Often the resistance to a disease may be found in related species and transferred
through interspecific hybridization. Eg. Resistance to stem, leaf & stripe rusts of wheat.

Mutation:-Resistance to diseases may be obtained through mutation arising spontaneously or

induced through mutagenic treatments. Eg, Resistance to Victoria blight in oats was induced by
irradiation with x-rays or thermal neutrons / also produced spontaneously, Resistance to stripe rust in
wheat, Resistance to brown rust in oats, Resistance to mildew in barley.

37
7.3 Methods of Breeding for Disease Resistance:-
Plant Introduction:-Process plants introduced fron their native plac to another place for crop
improvement. Eg; Early varieties of groundnut introduced from USA have been resistant to leaf spot
(Tikka). Kalyanasona and Sonalika wheat varieties originated from segregating material introduced
from CIMMYT, Mexico, were rust resistant. African bajra introductions have been used in
developing downy mildew resistant cms lines

Selection:-Kufri Red potato is selection from Darjeeling Red round , Pusa Sawani behind (yellow
mosaic) selection from a collection obtained from Bihar, MCU I was selection from CO4 for black
arm resistance in cotton.

Pedigree method;- In wheat Kalyana Sona, Sonalaka, Malvika 12, Malvika 37, Malavika 206,
Malavika 234, Laxmi in Cotton (Gadag 1 x CO2) for leaf blight resistance are agronomically highly
desirable variety. If the resistant parent is a wholly unadapted variety, backcross method is a logical
choice

Budding and Grafting:-The disease resistance in vegetatively propogated material is transferred by

adopting either by budding or grafting. By grafting or budding the resistant material, the resistance
can be transferred.

Mutation Breeding:-When adequate resistance is not available in the germplasm. Mutation breeding
is resorted to induce resistance. This is also us ed to break the linkages between desirable resistant
genes and other desirable genes.

7.4 Mechanism of Insect- Pest resistance:

Non-preference:- Various features of host plant which make the host undesirable or unattractive to
insects for food, shelter or reproduction, a mechanism of insect resistance; also called non-
acceptance or antixenosis.

Antibiosis:- Adverse effects of the host on feeding, development and reproduction of insect pest.

Tolerance:- Ability of a host to reproduce well despite the establishment of a pathogen in the host
tissues or the ability of a variety to produce more yield than susceptible variety at the same level of
insect attack.

Avoidance:- Escape of a variety from insect attack either due to earliness or its cultivation in the
season when insect population is very low.

38
8.0 Breeding for drought resistance
8.1 Introduction
Drought can be defined as an extended period of deficient rainfall relative to the statistical mean for
a region. The inadequacy of water availability including precipitation and soil moisture storage
capacity, in quantity and distribution during the life cycle of a crop to restrict the expression of its
full potential yield is known as drought stress. It seems difficult to define and quantify the drought
as under drought condition, water stress develops in the plants as the demand exceeds the supply of
water which may be due to atmospheric or soil conditions and is reflected in gradient of water
potential developed between soil, root surface and transpiring organs. Simply drought stress means
inability of plant to meet the evaporation demand. Drought may be of three types:

 Meteorological drought is qualified by any significant deficit of precipitation.

 Hydrological drought is manifest in noticeably reduced river and stream flow and critically
low groundwater tables.
 Agricultural drought: indicates an extended dry period that results in crop stress and crop
yield. The impact of drought on agriculture is due to a deficit of moisture in the soil, when the
moisture in the soil is no longer sufficient to meet the needs of growing crops. This results
from a lack of input of moisture from rainfall or irrigation for an extended period. It is
impossible to specify a period of time without rain as an agricultural drought, as the soil
moisture deficit depends on rate of loss as well as rate of input. Furthermore, the severity of
stress imposed on crops also depends on the susceptibility of different crops during different
stages of their development. When soil moisture is lacking, crop establishment may be
reduced, growth limited, normal development patterns disrupted and eventually, final yields
lowered.

Effects of drought stress on crops

 Reduced seed germination and seedling development

 Poor vegetative growth
 Reproductive growth is severely affected
 Plant height and leaf area reduced
 Significantly reduction in leaf weight
 Reduced photosynthesis.
 Reduced stomatal conductance
 Significantly reduction in the total dry matter

39
8.2 Development of Resistance varieties:
Development of resistant varieties is the cheapest, easiest, highly dependable as well as most
ecofriendly approach to reduce the loss caused by stress. During the process high yielding varieties
could also be developed which could be drown in those areas where they were no grown previously.
Varieties could be drawn either by direct or indirect depending upon the conscious effort.

Indirect method for Stress Resistance:

In this system of breeding varieties were not developed directly for the stress environment but is only
evaluated for stress resistance after the variety has been developed. During the breeding period there
is no conscious effort towards the stress resistance but is believed that if the line is developed for
high potential in optimum condition than it would also perform well in stress environment as well.

Direct method for stress resistance:

In this system of breeding varieties were deliberately developed for stress resistance and the parents
are carefully selected and tested at some stage under stress condition. The test could be done in field
conditions or may be created in lab. During the process the varieties may be tested and selection may
be done based on survival, yield and characters responsible for stress resistance.

 Selection for survival: The stress environment is so severe that all the susceptible plants are
expected to die and only resistant survive. Aluminum (toxicity problem) resistance varieties
were grown in such an area where wheat could not be cultivated earlier. High stress condition
may not be desirable always as they may not lead to higher yield in stress condition.

 Selection for yield: Resistance to abiotic stress means the ability of plant to produce higher
yield than other plants subjected to same stress condition. Yield may not be best criterion for
selection to develop the resistant cultivars at stage of selection of parents as this could leads
to rejection of potential valuable sources of resistance. The selection should be initially based
on biological yield under stress condition. The harvest index of the selected material could be
improved later on as the harvest index and stress resistance are independent to each other.

 Selection for traits: It is the best selection system if some traits are selected based on the
stress resistance. It should be clear that some traits contribute to resistance to a given stress
and is also associated with higher yield.

8.3 Source of Drought Resistance in Plant Breeding:

Cultivated varieties:-Transfer of drought resistance is easy from cultivated variety and germplasm of
cultivated species, because such material can be easily used in the breeding programmes. Moreover,
there is no problem of cross incompatibility.

40
Land races: are developed in and adapted to drought environment are ordinarily valuable sources of
drought resistance attributes

Transgenes: various genes which may be source of resistance of abiotic stress have been identified,
isolated and cloned which can be used to develop resistant varieties.

Wild relatives and wild species:-When the source of drought resistance is a wild species, the transfer
of resistance poses several problems such as cross incompatibility, hybrid in viability, hybrid sterility
and linkage of several undesirable genes with desirable ones. Wild sources of drought resistance
have been reported in wheat, sugarcane, tomato, and several other crops.

Types of drought environment

Breeding procedure as well as resistance mechanism that should be used will depend on the type of
drought environment in which crop is grown.

 Stored moisture environment: Crop completes its life cycle on the moisture stored in soil
during rainy season/ wet period. The moisture available will depend on the amount of water
stored in soil, duration of crop and rate of evapo-transpiration. Crops face moisture stress
during their terminal phase of growth and development. Chances for success of breeding for
drought resistance are rather high and various traits can be exploited for this purpose.

 Variable moisture: Alternate dry and wet conditions of varying length during crop season.
Plants/crop must be able to take advantage of periodic rainfall to survive with minimum
detrimental effects. Due to alternate dry and wet period there is low chances for success of
breeding resistance varieties.

 Optimal moisture environments: Crops is grown with optimum moisture conditions in most
of their life cycle but drought occurs occasionally at high unpredictable periods of growth and
development. Breeding is extremely difficult as the effect of drought are likely to be severe

Drought resistance: The ability of crop plants to grow, develop and reproduce normally under
moisture deficit conditions is referred to as drought resistance.

8.4 Mechanisms of Drought Resistance:

Drought escape: - Plant avoids the injury of stress by regulating its life cycle to avoid meeting with
stress. This is not the kind of resistance – some short-lived, desert ephemeral plants germinate, grow
and flower very quickly following seasonal rains. They thus complete their life cycle during a period
of adequate moisture and form dormant seeds before the onset of dry season.

Drought tolerance:-The ability of crop plants to withstand low tissue water content is referred to as
drought tolerance. Drought tolerance is more desirable because the crop can produce more yields at
lower water potential.
41
Drought resistance:-Drought resistance is the sum of drought avoidance and drought tolerance. It
refers to the ability of crop plants to give higher yield under moisture stress conditions or survival of
plants under water deficit conditions without injury.

Dehydration avoidance: Drought avoidance refers to ability of the plant to maintain a favorable
internal water balance under moisture stress. In other words, plants which avoid drought retain high
water contents in their tissues. Drought avoidance can permit a longer growth period in the crop
through reduced water use or increased water uptake. The ability of plant to retain a relatively higher
level of hydration under conditions of soil or atmospheric water stress and protects from various
physiological, biochemical and metabolic processes of plant involved in growth and yield from being
exposed to water stress.

Mechanisms:

 Reduced transpiration: water saving mechanism is seen common in xerophytes and they show
poor biomass production. They reduce water transpiration mostly by closing of stomata
before wilting in water deficit condition.

 Osmotic adjustment: it is associated with dehydration avoidance. Osmoregulation is

positively associated with yield under water stress conditions as it allows growth and results
in delay in leaf death by maintaining turgor pressure.

 Abscisic acid: is also known as stress hormone as its concentration increase in response of
stress. Water stress is sensed by roots and it synthesize ABA as there in onset of stress which
is further transported to leaves via xylem and causes closing of stomata.

 Cuticular wax: as transpiration occurs from cuticle and the rate of transpiration depends on
the amount of wax deposited within or over the cuticle. The effect is small on transpiration
and can reduce within a range as it affects net radiation and leaf temperature.

 Leaf characters: leaf pubescence increases leaf reflectance and reduces net radiation resulting
in lower leaf temperature. Furthermore a leaf with erect or low angle receives low radiation
then horizontal leaves.

 Increased water uptake : depends on the root structure, when soil moisture is unlimited there
is deep root system whereas large root length density with small hairs are found when no
additional moisture is available

Dehydration tolerance: A condition where lower level of changes are seen in one than others due to
genotype when they are induced in same level of dehydration. When cell lose turgor there is
reduction in chemical activity of water, increased concentration of solutes and macromolecules,
removal of water of hydration from macromolecules and alteration in cellular mechanism. The

42
measurement is done by: Maintenance of membrane integrity: determined by solute leakages from
cells and by Plant growth: it is measured using seedling growth and survival.

8.5 Selection Criteria:

Dehydration avoidance:

 Leaf rolling: scored from 0-10 in rice and generally done in morning or at mid day. Leaf
rolling help to predict leaf water potential in species having low osmotic adjustment.

 Combination of leaf rolling and leaf firing:

 Canopy temperature

 Leaf attributes: like pubescence, heavy glaucousness are visibly scored.

 Leaf water retention: can be used as component of integrated selection index, by sampling
turgid leaves normally in morning and then determining their weight after certain period.

 Root characteristics

Dehydration tolerance

 Seedling growth under PEG stress

 Growth under stress in field

 Plant phenology

 Grain filling by translocated stem reserve

 Cellular membrane stability under stress

Creation of drought environment

 Green house environment

 Field environment

 Line source gradient

43
8.6 Water use efficiency (WUE)
Water use efficiency is the measure of a cropping system’s capacity to convert water into plant
biomass or grain. It includes both the use of water stored in the soil and rainfall during the growing
season.

Water use efficiency (or transpiration efficiency) describes the intrinsic trade-off between carbon
fixation and water loss that occurs in dryland plants because water evaporates from the interstitial
tissues of leaves whenever stomata open for CO2 acquisition.
Water use efficiency (WUE) is defined as the amount of carbon assimilated as biomass or grain
produced per unit of water used by the crop.

Water use efficiency relies on:

 The soil’s ability to capture and store water

 The crop’s ability to access water stored in the soil and rainfall during the season
 The crop’s ability to convert water into biomass
 The crop’s ability to convert biomass into grain (harvest index).

Plant available water capacity:

Plant available water capacity (PAWC) is the total amount of water a soil can hold that a particular
crop can extract from a particular soil. PAWC is related to the soil type and the crop being grown on
that soil. PAWC is less than the total water held in a saturated soil (Figure 1) and varies between
crops grown on the same soil type. It is defined by a soil’s drained upper limit (DUL) (the water
content of a soil when it is fully wet but drainage has ceased) and its crop lower limit (CLL) (the
water content of a soil when a crop has extracted as much water as it can). Not all soils are capable of
storing the same amount of water, or of releasing stored water to plants.

Water Use Efficiency:

Having conveyed water to the point of use and having applied it, the next efficiency concept of
concern is the efficiency of water use. It is expressed in kg/ha cm. The proportion of water delivered
and beneficially used on the project can be calculated using the following formula
𝑊𝑢
𝐸𝑢 = 𝑊𝑑 ∗ 100

where, Eu = water use efficiency, per cent Wu = water beneficially used Wd = water delivered

Water use efficiency is also defined as (i) crop water use efficiency and (ii) field water efficiency.

(a) Crop Water Use Efficiency: It is the ratio of yield of crop (Y) to the amount of water depleted by
crop in evapo-transpiration (ET).

44
 𝑌
𝐶𝑊𝑈𝐸 =
𝐸𝑇
where, CWUE = Crop water use efficiency Y = Crop yield and ET is evapo-transpiration

CWUE is otherwise called consumptive water use efficiency. It is the ratio of crop yield (Y) to the
sum of the amount of water taken up and used for crop growth (G), evaporated directly from the soil
surface (E) and transpired through foliage (T) or consumptive use (Cu)

𝑌
𝐶𝑊𝑈𝐸 =
𝐺+𝐸+𝑇

(b) Field Water Use Efficiency: It is the ratio of yield of crop (Y) to the total amount of water used
in the field.
𝐹𝑊𝑈𝐸 = 𝑌/𝑊𝑅
where, FWUE = field water use efficiency WR = water requirement This is the ratio of crop
yield to the amount of water used in the field (WR) including growth (G), direct evaporation
from the soil surface (E), transpiration (T) and deep percolation loss (D).

𝑌
𝐹𝑊𝑈𝐸 =
𝐺+𝐸+𝑇+𝐷

Where G + E + T + D = WR

It is expressed in kg/ha/mm (or) kg/ha/cm Deep percolation is important for rice crop. For other
crops seepage is important. Of the two indices defined, the crop water use efficiency is more of
research value whereas the field water use efficiency has grater practical importance for planners and
farmers.

Factors affecting water use efficiency:

 Nature of plant
 Varieties cultivated
 Agronomic practices applied
 Irrigation
 Fertilization applied
 Climatic conditions
 Soil conditions

Measures to increase water use efficiency

 Selection of crop/ varieties: Selection of plants should be done on the basis of total water
available and distribution in the area. C4 and CAM plants have more photosynthetic rate

45
 along with higher water use efficiency then C3 plants so they must be in priorities. Similarly
varietal characteristics like short duration, early vigor, deep rooting behavior, moderate
tillering should be of first priority.
 Time of plantation: Shifting of planting time from higher to lower evaporative demand may
be very useful.
 Method of planting: it helps to increase yield as well as reduce the total irrigation requirement
of crop.
 Row spacing and seed rate: Close plantation along with narrow row spacing can help in
higher water use efficiency as soil water evaporation is reduced with higher planting density.
 Tillage practice: it helps to increase water storage capacity by increasing infiltration rate,
similarly sub soiling and deep tillage facilitates root exposure and soil moisture abstraction.
 Nutrient management: Use of chemical fertilizers along with organic nutrient sources add
supports for higher water use efficiency. Similarly use of nitrogen and phosphorus
management and inclusion of legumes will be fruitful.
 Weed control: Weeds often have higher water requirement than the crop species so measure
of weed control and management plays critical role towards water use efficiency of field
crops.
 Water management: use of alternate wetting and drying system along with use of more
efficient irrigation system like drip irrigation will be more beneficial.

46
 9.0 Breeding for heat resistance

9.1 Introduction:
Heat stress due to increased temperature is an agricultural problem in many areas in the
world. Transitory or constantly high temperatures cause an array of morpho-anatomical,
physiological and biochemical changes in plants, which affect plant growth and development and
may lead to a drastic reduction in economic yield. The adverse effects of heat stress can be mitigated
by developing crop plants with improved thermo tolerance using various genetic approaches. Heat
stress affects plant growth throughout its ontogeny, though heat-threshold level varies considerably
at different developmental stages. For instance, during seed germination, high temperature may slow
down or totally inhibit germination, depending on plant species and the intensity of the stress. At
later stages, high temperature may adversely affect photosynthesis, respiration, water relations and
membrane stability, and also modulate levels of hormones and primary and secondary metabolites.
Furthermore, throughout plant ontogeny, enhanced expression of a variety of heat shock proteins,
other stress-related proteins, and production of reactive oxygen species (ROS) constitute major plant
responses to heat stress. In order to cope with heat stress, plants implement various mechanisms,
including maintenance of membrane stability, scavenging of ROS, production of antioxidants,
accumulation and adjustment of compatible solutes, induction of mitogen-activated protein kinase
(MAPK) and calcium-dependent protein kinase (CDPK) cascades, and, most importantly, chaperone
signaling and transcriptional activation. All these mechanisms, which are regulated at the molecular
level, enable plants to thrive under heat stress.

The adverse affect of temperature higher than the required for optimal growth and development is
considered as heat stress. It will affect the growth as well as growth and development along with
various physiological activities in plants. The nature and extent of effect of heat stress depends on
the temperature as well as the plant types.

Heat stress is often defined as the rise in temperature beyond a threshold level for a period of time
sufficient to cause irreversible damage to plant growth and development. In general, a transient
elevation in temperature, usually 10–15 ◦C above ambient, is considered heat shock or heat stress.
However, heat stress is a complex function of intensity (temperature in degrees), duration, and rate of
increase in temperature. The extent to which it occurs in specific climatic zones depends on the
probability and period of high temperatures occurring during the day and/or the night. Heat tolerance
is generally defined as the ability of the plant to grow and produce economic yield under high
temperatures.

47
9.2 Heat-stress threshold:
A threshold temperature refers to a value of daily mean temperature at which a detectable reduction
in growth begins. Upper and lower developmental threshold temperatures have been determined for
many plant species through controlled laboratory and field experiments. A lower developmental
threshold or a base temperature is one below which plant growth and development stop. Similarly, an
upper developmental threshold is the temperature above which growth and development cease. Base
threshold temperatures vary with plant species, but for cool season crops 0◦C is often the best-
predicted base temperature (Cool season and temperate crops often have lower threshold temperature
values compared to tropical crops. Upper threshold temperatures also differ for different plant
species and genotypes within species. However, determining a consistent upper threshold
temperature is difficult because the plant behavior may differ depending on other environmental
conditions.

9.3 Plant responses to heat stress

Plant under stress shows various types of unusual behavior in order to cope with the changing
environment.

9.3.1 Morphological symptoms:

In tropical climates, excess of radiation and high temperatures are often the most limiting factors
affecting plant growth and final crop yield. High temperatures can cause considerable pre- and post-
harvest damages, including scorching of leaves and twigs, sunburns on leaves, branches and stems,
leaf senescence and abscission, shoot and root growth inhibition, fruit discoloration and damage, and
reduced yield. Similarly, in temperate regions, heat stress has been reported as one of the most
important causes of reduction in yield and dry matter production in many crops, including maize.
High-temperature-induced modifications in plants may be direct as on existing physiological
processes or indirect in altering the pattern of development. These responses may differ from one
phenological stage to another. For example, long-term effects of heat stress on developing seeds may
include delayed germination or loss of vigor, ultimately leading to reduced emergence and seedling
establishment. Under diurnally varying temperatures, coleoptiles growth in maize reduced at 90◦C
and ceased at 95◦C.

9.3.2 Anatomical changes

Anatomical changes under high ambient temperatures are generally similar to those under drought
stress. At the whole plant level, there is a general tendency of reduced cell size, closure of stomata
and curtailed water loss, increased stomatal and trichomatous densities, and greater xylem vessels of
both root and shoot. In grapes heat stress severely damaged the mesophyll cells and increased
permeability of plasma membrane with the onset of high temperature regime, Zygophyllum
qatarense produced polymorphic leaves and tended to reduce transpirational water loss by showing

48
bimodal stomatal behavior. At the sub-cellular level, major modifications occur in chloroplasts,
leading to significant changes in photosynthesis. For example, high temperatures reduced
photosynthesis by changing the structural organization of thylakoids.

9.3.3 Phenological changes

Plant phenology in response to heat stress can reveal a better understanding of interactions between
stress atmosphere and the plant. Different phenological stages differ in their sensitivity to high
temperature; however, this depends on species and genotype as there are great inter and intra-specific
variations. Heat stress is a major factor affecting the rate of plant development, which may be
increasing to a certain limit and decreasing afterwards the developmental stage at which the plant is
exposed to the stress may determine the severity of possible damages experienced by the crop. It is,
however, unknown whether damaging effects of heat episodes occurring at different developmental
stages are cumulative Vulnerability of species and cultivars to high temperatures may vary with the
stage of plant development, but all vegetative and reproductive stages are affected by heat stress to
some extent. During vegetative stage, for example, high day temperature can damage leaf gas
exchange properties. During reproduction, a short period of heat stress can cause significant
increases in floral buds and opened flowers abortion; however, there are great variations in
sensitivity within and among plant species.

9.4 Physiological responses

9.4.1 Waters relations:

Plant water status is the most important variable under changing ambient temperatures. In general,
plants tend to maintain stable tissue water status regardless of temperature when moisture is ample;
however, high temperatures severely impair this tendency when water is limiting. Under field
conditions, high temperature stress is frequently associated with reduced water availability. In Lotus
creticus, for example, elevated night temperatures caused a greater reduction in leaf water potential
of water-stressed as compared to non-stressed plants. In sugarcane, leaf water potential and its
components were changed upon exposure to heat stress even though the soil water supply and
relative humidity conditions were optimal, implying an effect of heat stress on root hydraulic
conductance .Similarly, in tomato heat stress perturbed the leaf water relations and root hydraulic
conductivity .

9.4.2 Accumulation of compatible osmolytes:

A key adaptive mechanism in many plants grown under abiotic stresses, including salinity, water
deficit and extreme temperatures, is accumulation of certain organic compounds of low molecular
mass, generally referred to as compatible osmolytes. Under stress, different plant species may
accumulate a variety of osmolytes such as sugars and sugar alcohols (polyols), proline tertiary and
quaternary ammonium compounds, and tertiary sulphonium compounds. Glycinebetaine (GB), an

49
amphoteric quaternary amine, plays an important role as a compatible solute in plants under various
stresses, such as salinity or high temperature. Capacity to synthesize GB under stress conditions
differs from species to species. For example, high level of GB accumulation was reported in maize
and sugarcane due to desiccating conditions of water deficit or high temperature. In contrast, plant
species such as rice (Oryza sativa), mustard (Brassica spp.), Arabidopsis (Arabidopsis thaliana) and
tobacco (Nicotiana tabacum) naturally do not produce GB under stress conditions.

9.4.3 Photosynthesis:
Alterations in various photosynthetic attributes under heat stress are good indicators of thermo-
tolerance of the plant as they show correlations with growth. Any constraint in photosynthesis can
limit plant growth at high temperatures. Photochemical reactions in thylakoid lamellae and carbon
metabolism in the stroma of chloroplast have been suggested as the primary sites of injury at high
temperatures. Chlorophyll fluorescence, the ratio of variable fluorescence to maximum fluorescence
(Fv/Fm), and the base fluorescence (F0) are physiological parameters that have been shown to
correlate with heat tolerance. Increasing leaf temperatures and photosynthetic photon flux density
influence thermotolerance adjustments of PSII, indicating their potential to optimise photosynthesis
under varying environmental conditions as long as the upper thermal limits do not exceed.

9.4.4 Assimilate partitioning:

Under low to moderate heat stress, a reduction in source and sink activities may occur leading to
severe reductions in growth, economic yield and harvest index. Assimilate partitioning, taking place
via apoplastic and symplastic pathways under high temperatures has significant effects on transport
and transfer processes in plants. However, considerable genotypic variation exists in crop plants for
assimilate partitioning. It was determined that photosynthesis had a broad temperature optimum from
20 to 30 ◦C, however it declined rapidly at temperatures above 30 ◦C. The rate of 19C assimilate
movement out of the flag leaf (phloem loading), was optimum around 30 ◦C, however, the rate of
movement through the stem was independent of temperature from 1 to 50 ◦C. It was concluded that,
at least in wheat, temperature effects on translocation result indirectly from temperature effects on
source and sink activities. From such results, increased mobilization efficiency of reserves from
leaves, stem or other plant parts has been suggested as a potential strategy to improve grain filling
and yield in wheat under heat stress.

9.4.5 Cell membrane thermo-stability:

Sustained function of cellular membranes under stress is pivotal for processes such as
photosynthesis and respiration. Heat stress accelerates the kinetic energy and movement of
molecules across membranes thereby loosening chemical bonds within molecules of biological
membranes. This makes the lipid bilayer of biological membranes more fluid by either denaturation
of proteins or an increase in unsaturated fatty acids. The integrity and functions of biological
membranes are sensitive to high temperature, as heat stress alters the tertiary and quaternary

50
structures of membrane proteins. Such alterations enhance the permeability of membranes, as evident
from increased loss of electrolytes. The increased solute leakage, as an indication of decreased cell
membrane thermo-stability (CMT), has long been used as an indirect measure of heat-stress
tolerance in diverse plant species, including soybean , potato and tomato and barley. Electrolyte
leakage is influenced by plant/tissue age, sampling organ, developmental stage, growing season,
degree of hardening and plant species. In maize, injuries to plasma lemma due to heat stress were
much less severe in developing than in mature leaves. It was determined that an increase in saturated
fatty acids in mature leaves elevated melting temperature of plasma membranes and thus reducing
heat tolerance of the plant. In Arabidopsis plants grown under high temperature, total lipid content in
membranes decreased to about one-half and the ratio of unsaturated to saturated fatty acids decreased
to one-third of the levels at normal temperatures.

9.4.6 Hormonal changes:

Plants have the ability to monitor and adapt to adverse environmental conditions, though the degree
of adaptability or tolerance to specific stresses varies among species and genotypes. Hormones play
an important role in this regard. Cross-talk in hormone signaling reflects an organism’s ability to
integrate different inputs and respond appropriately. Hormonal homeostasis, stability, content,
biosynthesis and compartmentalization are altered under heat stress (Maestri et al., 2002). Abscisic
acid (ABA) and ethylene (C2H9), as stress hormones, are involved in the regulation of many
physiological properties by acting as signal molecules. Different environmental stresses, including
high temperature, result in increased levels of ABA. For example, recently it was determined that in
creeping bent grass (Agrostis palustris), ABA level did not rise during heat stress, but it accumulated
upon recovery from stress suggesting a role during the latter period. However, the action of ABA in
response to stress involves modification of gene expression. Under field conditions, where heat and
drought stresses usually coincide, ABA induction is an important component of thermo-tolerance,
suggesting its involvement in biochemical pathways essential for survival under heat-induced
desiccation stress.

9.4.7 Secondary metabolites:

Most of the secondary metabolites are synthesized from the intermediates of primary carbon
metabolism via phenylpropanoid, shikimate, mevalonate or methyl erythritol phosphate (MEP)
pathways. High-temperature stress induces production of phenolic compounds such as flavonoids
and phenylpropanoids. Phenylalanine ammonia-lyase (PAL) is considered to be the principal enzyme
of the phenylpropanoid pathway. Increased activity of PAL in response to thermal stress is
considered as the main acclimatory response of cells to heat stress. Thermal stress induces the
biosynthesis of phenolics and suppresses their oxidation, which is considered to trigger the
acclimation to heat stress for example as in watermelon, Citrulus vulgaris. Carotenoids are widely
known to protect cellular structures in various plant species irrespective of the stress type.

51
9.5 Molecular responses

9.5.1 Oxidative stress and antioxidants:

In addition to tissue dehydration, heat stress may induce oxidative stress. For example, generation
and reactions of activated oxygen species (AOS) including singlet oxygen (1O2), superoxide radical
(O2−), hydrogen peroxide (H2O2) and hydroxyl radical (OH−) are symptoms of cellular injury due to
high temperature. AOS cause the autocatalytic peroxidation of membrane lipids and pigments thus
leading to the loss of membrane semi-permeability and modifying its functions.

9.5.2 Stress proteins:

Expression of stress proteins is an important adaptation to cope with environmental stresses. Most of
the stress proteins are soluble in water and therefore contribute to stress tolerance presumably via
hydration of cellular structure. Although heat shock proteins (HSPs) are exclusively implicated in
heat-stress response, certain other proteins are also involved.

9.5.2.1 Heat shock proteins:

Synthesis and accumulation of specific proteins are ascertained during a rapid heat stress, and these
proteins are designated as HSPs. Increased production of HSPs occurs when plants experience either
abrupt or gradual increase in temperature. Induction of HSPs seems to be a universal response to
temperature stress, being observed in all organisms ranging from bacteria to human . Plants of arid
and semiarid regions may synthesize and accumulate substantial amounts of HSPs.

9.5.2.2 Other heat induced proteins:

Besides HSPs, there are a number of other plant proteins, including ubiquitin, cytosolic Cu/Zn-SOD
and Mn-POD, whose expressions are stimulated upon heat stress. For example, in Prosopis chilensis
and soybean under heat stress, ubiquitin and conjugated-ubiquitin synthesis during the first 30 min of
exposure emerged as an important mechanism of heat tolerance

9.6 Mechanism of heat tolerance

Plants manifest different mechanisms for surviving under elevated temperatures, including long-term
evolutionary phenological and morphological adaptations and short-term avoidance or acclimation
mechanisms such as changing leaf orientation, transpirational cooling, or alteration of membrane
lipid compositions. In many crop plants, early maturation is closely correlated with smaller yield
losses under high temperatures, which may be attributed to the engagement of an escape mechanism.
Plant’s immobility limits the range of their behavioral responses to environmental cues and places a
strong emphasis on cellular and physiological mechanisms of adaptation and protection. Also, plants
may experience different types of stress at different developmental stages and their mechanisms of
response to stress may vary in different tissues .The initial stress signals (e.g., osmotic or ionic
effects, or changes in temperature or membrane fluidity) would trigger downstream signaling

52
processes and transcription controls, which activate stress-responsive mechanisms to reestablish
homeostasis and protect and repair damaged proteins and membranes. Inadequate responses at one or
more steps in the signaling and gene activation processes might ultimately result in irreversible
damages in cellular homeostasis and destruction of functional and structural proteins and
membranes, leading to cell death.

9.7 Acquired thermo-tolerance:

Thermotolerance refers to the ability of an organism to cope with excessively high temperatures. It
has long been known that plants, like other organisms, have the ability to acquire thermo tolerance
rather rapidly, may be within hours, so to survive under otherwise lethal high temperatures. The
acquisition of thermo tolerance is an autonomous cellular phenomenon and normally results from
prior exposure to a conditioning pretreatment, which can be a short but sub-lethal high temperature.
The acquisition of high level of thermo tolerance protects cells and organisms from a subsequent
lethal heat stress. Thermotolerance can also be induced by a gradual increase in temperature to lethal
highs, as would be experienced under natural conditions, and induction in this way involves a
number of processes. Using Arabidopsis mutants, it was shown that, apart from heat shock proteins
(HSP32 and HSP101), ABA, ROS and SA pathways are involved in the development and
maintenance of acquired thermo-tolerance.

9.8 Temperature sensing and signaling:

Perception of stress and relay of the signal for turning on adaptive response mechanisms are key
steps towards plant stress tolerance. There are multiple stress perceptions and signaling pathways,
some of which are specific while others may be involved in cross-talk at various steps. General
responses to stress involve signaling of the stress via the redox system. Chemical signals such as
ROS, Ca2+ and plant hormones activate genomic re-programming via signal cascades.

9.9 Genetic improvement for heat-stress tolerance

Plants experience oxidative stresses during the initial period of adjustment to any stress. Response of
plant to stress is from general to specific. Specific responses require sustained expression of genes
involved in processes specific to individual stresses. These responses accommodate short-term
reaction or tolerance to specific stresses. However, genome plasticity in plants, including genetic
(e.g., directed mutation) and epigenetic (e.g., methylation, chromatin remodeling, histone
acetylation) changes, allows long-term adaptation to environmental changes/conditions. Such
adaptations may be necessary for long-term survival or establishment of plant genotypes/species in
particular environmental niches. Under agricultural systems, plants adaptation or their tolerance to
environmental stresses can be manipulated by various approaches. In general, the negative impacts
of abiotic stresses on agricultural productivity can be reduced by a combination of genetic
improvement and cultural practices. Genetic improvement entails development of cultivars which

53
can tolerate environmental stresses and produce economic yield. Adjustment/modifications in
cultural practices, such as planting time, plant density, and soil and irrigation managements,
however, can minimize stress effects, for example by synchronizing the stress sensitive stage of the
plant with the most favorable time period of the season. In practice, to be successful in improving
agricultural productivity in stress environments, both genetic improvement and adjustment in cultural
practices must be employed simultaneously.

9.9.1 Conventional breeding strategies

Physiological and genetic investigations indicate that most abiotic stress tolerance traits are complex,
controlled by more than one gene, and highly influenced by environmental variation. The
quantification of tolerance often poses serious difficulties. Direct selection under field conditions is
generally difficult because uncontrollable environmental factors adversely affect the precision and
repeatability of such trials. Often, no consistent high-temperature conditions can be guaranteed in
field nurseries, as heat stress may or may not occur in the field. Furthermore, stress tolerance is a
developmentally regulated, stage-specific phenomenon; tolerance at one stage of plant development
may not be correlated with tolerance at other developmental stages. Individual stages throughout the
ontogeny of the plant should be evaluated separately for the assessment of tolerance and for the
identification, characterization and genetic manipulation of tolerance components. Moreover, species
may show different sensitivity to heat stress at different developmental stages. For example, in
tomato, though plants are sensitive to high temperatures throughout the plant ontogeny, flowering
and fruit set are the most sensitive stages; fruit set is somewhat affected at day/night temperatures
above 26/20 ◦C and is severely affected above 35/26 ◦C . Thus, partitioning of the tolerance into its
developmental and physiological/genetic components may provide a better understanding of the
plant’s response to heat stress and facilitate development of plants with stress tolerance throughout
the plant’s life cycle.

Several other issues of concern when employing traditional breeding protocols to develop heat-
tolerant crop plants are as follows:

 Identification of genetic resources with heat tolerance attributes. In many plant species, for
example soybeans and tomatoes, limited genetic variations exist within the cultivated species
necessitating identification and use of wild accessions. However, often there are great
difficulties in both the identification and successful use of wild accessions for stress tolerance
breeding .
 When screening different genotypes (in particular wild accessions) for growth under high
temperatures, distinction must be made between heat tolerance and growth potential. Often
plants with higher growth potential perform better regardless of the growing conditions.
 When breeding for stress tolerance, often it is necessary that the derived lines/cultivars be
able to perform well under both stress and non-stress conditions. Development of such

54
 genotypes is not without inherent difficulties. In some plant species, heat tolerance is often
associated with some undesirable horticultural or agronomical characteristics. In tomato, for
example, two undesirable characteristics commonly observed in heat-tolerant genotypes are
small fruit and restricted foliar canopy. The production of small fruit is most likely due to
adverse effects of high temperature on the production of auxins in the fruit, and the poor
canopy is due to the highly reproductive nature of the heat-tolerant genotypes.

Breeding for heat tolerance is still in its infancy stage and warrants more attention than it has been
given in the past. It is unfortunate that the literature contains relatively little information on breeding
for heat tolerance in different crop species. However, despite all the complexity of heat tolerance and
difficulties encountered during transfer of tolerance, some heat-tolerant inbred lines and hybrid
cultivars with commercial acceptability have been developed and released, at least in a few crop
species such as tomato Nevertheless, to accelerate such progresses, major areas of emphasis in the
future should be:

 Designing/development of accurate screening procedures;

 Identification and characterization of genetic resources with heat tolerance;
 Discerning the genetic basis of heat tolerance at each stage of plant development;
 Development and screening of large breeding populations to facilitate transfer of genes for
heat tolerance to commercial cultivars.

The use of advanced molecular biology techniques may facilitate development of plants with
improved heat tolerance,

9.10 Selection criteria for heat resistance:

For the selection of heat resistance crops or plants various traits are checked and under specific
environmental conditions some of which are:

 Growth under stress: growth is one of the factors which are highly affected by heat stress and
growth would reflect the major consequences of heat stress. If the plant under stress has good
growth then we can assume that they are resistance to specific heat stress.
 Yield under heat stress: heat stress affects the reproductive growth which includes flower
development, pod/fruit set along with pollen fertility and grain filling, if any crop can cope
with these and produce normal yield then we can say such one is resistance to heat stress
environment.
 Flower and fruit sets along with pollen fertility: they are highly affected by heat stress so can
be effectively used for selection criteria in heat stress resistance.
 Seed germination under stress: germination on seed under seed is adversely affected and
should be taken in consideration for effective selection of stress resistance. Higher the level
of germination under stress condition shows greater the resistance for specific environment.

55
 Recovery after heat stress: it may be one of useful criterion for selection as the plant which is
able to perform well after the stress will have greater resistance then those which cannot cope
under the stress conditions.
 Sensitivity of the photosynthetic process: level of photosystem II and chloroplast can be
analyzed for the study of heat stress as chlorophyll fluorescence at 685nm and selection can
be done with this basis. Plants having nearly same chlorophyll can be determined as heat
stress resistance.
 Membrane stability under heat shock: is measured by electrical conductivity and lower the
membrane stability means low resistance in plants.

56
 10. Breeding for mineral stress

 Abiotic stress generated by mineral salts and ranks second after the moisture stress

 Increasing salinization caused by agricultural practice leads to loss of 30% or arable land by
2025 and 50% by 2050

 May occur in form of specific mineral deficiency or toxicity or accumulation of excess

amount of salt in root zone

10.1 Salt affected soils:

In salt affected soil there will be excess accumulation of salts in root zone which effects the growth
and development of plant. Soils may be of two types based on salt accumulation: Saline soils: soils
contain enough neutral salts which adversely affect growth and performance of plants. This type of
soil contains chloride and sulphates of sodium, calcium, magnesium etc which may be previously
present or carried out by water. Alkali soils: which contain more than 15% of exchangeable sodium
and affect plant growth and development. Most common one is sodium carbonate and PH is
generally higher than 8.2.

Management of salt affected soils

 Reclamation: removal of soluble salts or excess exchangeable sodium from root zone. Can be
done by leaching of soluble salts in case of saline soils whereas by application of calcium
amendments to remove excess exchangeable sodium in alkaline soils

 Application of Management practices: choice of crops, methods of planting and irrigation

with other inter cultural operations can give better environment for crop plants

 Crops tolerant to saline or alkali soils: selection of varieties or crops based on their tolerance
capacity

 Salt tolerant varieties

Effects of salinity stress:

 Crops or plants which are grown in saline condition faces three different stress situation
which affects its growth and development.

 Water stress by osmoticum: Water potential of wet saline soil may be around -24 bar or
higher and due to this plants develop large soil to leaf gradient of potentials and are unable to
meet transpirational demands and plants may wilt and desiccate. Water stress is also known
as component of salt injury. Plants in salinity show osmotic adjustment by accumulation of
ions and metabolites.

57
  Salt toxicity (stress by mineral toxicity and disturbances in mineral nutrition of plants): NaCl
affects the activity of enzymes of glycolytic pathway whereas salinity affects the oxidative
phosphorylation and ATP/ADP ratio in root tissue and reduces the supply of energy. It
interferes with protein synthesis and enhances the free amino acid pool. Also affects plasma
membrane integrity and cell walls may be involved in salt injury due to interaction of Na with
cell wall bound ions of Ca. Due to salinity stress less cytokinin is produced whereas ABA in
root increases (production of low cytokinin is indication of stress)

Salinity resistance:

 Resistance to salinity induced water stress: Osmoregulation is the common response to

salinity stress which helps to maintain the turgor and avoid leaf desiccation. Proline is
important solute in osmoregulation of halophytes whereas in some cases the proline content
may be up to 10-20% of total dry weight.

 Resistance to salinity induced ion toxicity: Enzymes and cellular processes of halophytes
(plant adapted to growing in saline conditions, as in a salt marsh) are more sensitive than that
of glycophytes (Any plant that cannot tolerate relatively high concentrations of salt).
Avoidance of ion toxicity involves a mechanism which can aid to maintain low non toxic
level of salts in cytoplasm which can be done by two ways: ion exclusion and cellular
compartmentation or salt excretion.

 Ion exclusion: some genotypes uptake smaller quantities of injurious ions like Na, Cl so that
their concentration is much lower in tissues than those of other genotypes is known as ion or
salt exclusion. Ion exclusion at root is an effective means for avoiding toxicity and can be
seen in halophytes and in salt tolerant lines of pearl millet, rice, soyabean.

 Cellular compartmentation: tolerance means the differential effect on life processes at same
salt concentration in different genotypes. In case of halophytes the tolerance is developed by
the dispose of excess salt by excretion through special salt glands or by cellular
compartmentation like vacuole

10.2 Genetics of salinity resistance

Resistance depends on the genetic variation present among the plant species along with the variation
within species. Although much information has not be found but Blum in 1988 said that for the
development of resistance, evaluation of yield or reduction of yield among the genotypes is reliable
one.

 Interspecific variation: variation for salinity resistance can be seen among the genotypes for
SY 100 and SY 50.(SY 100: maximum salinity level at which no reduction in yield and SY

58
 50 salinity level where 50 % yield is reduced). Allopolyploids are generally more tolerant to
saline/alkaline soil then diploids

 Intraspecific variation: genetic variation exits within species, highest level of salt tolerance is
found in indigenous varieties of salt affected region. In case of rice it was detected in tall,
photosensitive, coarse grained and late maturing varieties. Barley is more resistant than wheat

 Gene action and heritability: Information is limited on this topic may be due to complexity
nature of salinity resistance. It has been found that monogenic control of resistance is not
commonly observed but can been seen Cl exclusion from shoot of soyabean by dominant
gene (Ncl) whereas in grapes and citrus it is polygenic controlled

10.3 Measurement of Salinity resistance

 Saline environment

 Non-saline field with saline irrigation

 Micro plots

 Green house experiments

 Germination

 Filter papers

 Antibiotic agar plate

 Pots filled with saline soil

 Pots filled with sand/ gravel

 Saline hydroponic culture

 Seedling survival and growth

Estimation of salinity resistance and selection criteria

 Cell survival: It is measured by immersing tissue sections for 24 hrs in a solution of known
salinity after which tissue sections were transferred into hypertonic glucose sections, from
these the proportion of plasmolysed cells gives the ratios of surviving cells. This is often
regarded as better index of salt resistance.

 Germination: Seed germination in saline medium is used as sole selection criteria for salt
resistance which improves germination under salinity stress.

59
  Dry matter accumulation: Seedling or plant dry weight is a good index of salinity resistance
as it integrates the various effects of salinity.

 Leaf death and senescence: Salinity stress induces death of leaf as well as senescence which
is generally measured by using total dead leaf area and number of dead leaves.

 Leaf ion content: The concentration of ions in plant leaves give information about the
measure of salt resistance.

 Leaf necrosis: Accumulation of ions like Cl- , Na+ and K+ causes’ leaf necrosis and magnitude
of leaf necrosis helps to measure the salinity resistance if it’s the case of ion exclusion.

 Root growth: Although there is no conclusive evidence for usefulness of this criteria for
selection but often this gives relative resistance of plants to mineral toxicity.

 Osmoregulation: This is measured as accumulation of proline or carbohydrate in response to

salinity. Furthermore it is determined as maintenance of turgor under stress and estimated as
leaf wilting and desiccation along with premature senescence. This is one of the highly
desirable criteria of selection.

 Yield: It is one of the important selection criteria for any index of salinity resistance whereas
yield per plant is estimated from segregating lines but in case of selected lines and germplasm
lines yield per unit area is more desirable for selection.

Problems in breeding for salinity resistance

 Creation of reliable and dependable variety as creation of environment is not a easy task

 No reliable criteria for scoring and selection criteria

 Genetic control mechanism of salinity is complex and is generally done by polygenes

Breeding approaches:

 Use of salinity resistance root stocks:

 Selection:
 Hybridization:
 Interspecific hybridization:
 Cell selection:
 Genetic Engineering:

60
Mechanism of mineral deficiency resistance:

 Mineral Redistribution: in this case minerals present in older leaves are mobilized to younger
leaves. That is plant redistribute the nutrients like N, P and K.
 Efficient mineral uptake: uptake of mineral is increased due to acidification of rhizospore due
to the secretion of organic salts by plant roots.
 Increased mineral transport: in some cases one nutrient may hamper the uptake of other and
can create deficiency. For e.g.: Ca competes or inactivates Fe.
 Increased root/ shoot ratio: Increased root/shoot ratio under stress is associated with
deficiency resistance.
 Increased root hair density/ length: Root hair density is associated with P and K uptake.

Genetics of mineral deficiency:

Large amount of variation has been found within the species towards the mineral deficiency and
generally this is due to the genetic variation present within and among the species. In many cases P
efficiency in crops has been found due to polygenic nature but these have very low heritability,
which shows that there is involvement of non specific, indirect effect on mineral nutrition. Similarly
in several cases it has been observed that mineral efficiency or inefficiency has been governed by
single gene which may be due to presence of dominant gene or due to recessive ones.

Sources:
These are the genotypes which were used and will be used for the development of resistant varieties to cope
the stress.

 Cultivated varieties
 Land races
 Mutants
 Related species

Selection criteria:
 Visible deficiency symptoms: Different symptoms were observed in plants due to mineral
deficiency but these symptoms may be sometime unrelated to deficiency but may be due to
environment.
 Mineral contents of plant tissue: the content of certain elements could be determined in target
tissues if plants are grown under mineral deficiency. They may be affected by growth habit,
tissue age and environmental factors.
 Biochemical test: Activities of certain enzymes help to evaluate the deficiency of specific
elements. These tests are of great importance and help towards the selection of genotypes.

61
  Yield: it is highly affected due to mineral deficiency so plays an important role in selection
criteria.

Creation of environment:
Filed: This type of experiment is more desirable for selection as they are cheap and easy to prepare
and selection program for mineral resistance is done in filed but for that the field soil must be of
homogenous nature for the deficiency of concerned minerals.

Pots: Use of pots are desirable for the evaluation of seedling responses whereas may not be more
beneficial with increment in seed number. While filling the pots care must be done as soil filled must
be of homogenous nature for deficiency of minerals further if they are placed in green house
temperature control should be of prime importance.

Hydroponics: It is expensive than other two methods but is of prime importance to evaluate the
parents which are to be used for hybridization and for lines which are selected from crosses.

Mineral Toxicity Resistance:

The plant will suffer for mineral toxicity stress and this will hamper in growth and development, and
the level of toxicity increases with the presence of toxic concentrations of minerals in root zone.
Acidic soil may contain high amount of Al and Mn which were the major reason for toxicity as
around 40% of the total arable area are facing the problem. So breeding for toxicity resistance is of
prime importance.

Mechanism for Aluminum Resistance:

Aluminum toxicity may be observed in soil where the PH level is below 5.5 although the
concentration of aluminum will rarely be higher the 4 ppm. The initial stage of aluminum toxicity is
generally observed due to Al induced membrane instability. The various disorders are seen due to Al
toxicity which is due to binding of Al to proteins and resultant conformational change. It has been
observed the conformational change in Calmodulin which was the major reason for toxicity in
eukaryotic cells. Al toxicity causes reduction in root growth and lateral root formation along with the
discoloration of roots. Seedlings were more sensitive towards aluminum toxicity incomparision to
older ones. Furthermore aluminum also induces the deficiency of other minerals like P, Ca of Fe.

Aluminum resistance is in form of avoidance and can be achieved by:

 Increasing the level of soil PH as it reduce the Al solubility and uptake.

 Exclusion of Al from entering the root due to root cell membrane properties or due to
interaction of Ai with mucilage.
 Compartmentation of AI in root and are prevented from their movement in shoots.
 Accumulation of AI in older leaves and relatively low content in younger leaves

62
  In some cases Al resistance is involved with nutritional aspect

Selection Criteria:

 As AI retards root growth and reduces shoot growth so shoot dry matter content is effective
for selection
 Root length and weight is best for selection from hydroponics
 Root deformation and discoloration is also beneficial for selection in hydroponics
 Yield is also an integral measure of resistance

Mechanism of Manganese Resistance:

Manganese is the second highest problem which generally comes with Aluminum and occurs in soil
with PH less than 5.5. Toxicity of Mn decreases the activity of various enzymes whereas increases
the activity of oxidases like IAA and reduce respiration. Toxicity of Mn occurs mostly in shoots and
causes necrosis and leaf crinkling. Mn resistance is generally due to avoidance and can be achieved
by

 Reduction of Mn transport from root to shoot eg: in Maize

 Distribution of Mn throughout the various parts of shoot
 Exclusion of Mn from shoot in resistant genotypes

Selection criteria: As Mn and AI come together evaluation must be done in independent way
generally under pot or in nutrient culture.

 As shoot growth is inhibited before root growth , estimation of shoot growth is better for
selection
 Parent lines and their progenies should be evaluated for shoot under Mn toxicity stress and
non stress conditions.
 Selection should be done in mean performances

63
64
 11. Breeding for cold resistance

Stress due to temperature above freezing ie above 0 degree Is chilling while below freezing or zero is
known as freezing stress.

Cold resistance: ability to stand low temperatures

Chilling stress: sensitive plants are typically tropical plants. Effects may occur at cellular or
subcellular level

11.1 Chilling stress at plants level

 Can be measured in terms of

 Seed germination

 Growth

 Fruit sets

 Yield

 Pollen fertility

 Fruit quality

11.2 Chilling stress at sub-cellular level

 Affects:

 Membrane stability

 Chlorophyll synthesis

 Photosynthesis

 Respiration

 Production of toxicity due to H2O2

11.3 Chilling tolerance

 Membrane lipid unsaturation

 Reduced sensitivity of photosynthesis

 Increased chlorophyll accumulation

 Improved germination

65
  Improved fruits sets/ seed set

 Pollen fertility

11.4 Genetics of chilling resistance

 Inheritance of several traits has been studied in maize, tomato, rice to understand about the
genetics

 Electrolyte leakage following chilling injuries and germination, seedling vigor, floret sterility
and plant growth has been found to have polygenic control

 Whereas in case of rice dominant gene Cts1 specifies chilling tolerance in terms of leaf
discoloration

 In some cases additive genes effects were predominant but dominant effects were also
present

11.5 Source of chilling resistance

 present in large number in:

 Well adopted breeding populations

 Germplasm

 Cold tolerant mutants

 Somaclonal variants

 Related wild species

 Genetic engineering

11.6 Selection criteria

 Germination

 Growth

 Chlorophyll loss

 Membrane stability

 Photosynthesis

 Seedling mortality

 Seed/fruit set

 Pollen fertility

66
11.7 Freezing stress
When plants are grown in subzero temperature a complex array of stress and strains develop within
the plants

 Winter survival is determined by interaction between plants and plant temperature plus that
between plant, temperature and pathogens

 Is most studied stress in plants

 And most extensively focused area

 Dormant state is conducive to freezing resistance while resistance in actively growing tissues
are rare

 Freezing resistance involves the survival of freezing stress in such a manner that it became
able to subsequent regrowth when temperature rises

Initiation/ Causes

 Ice formation: requires nucleation: Ice formation: requires nucleation (factors): for the
starting of ice formation nucleation is required, it may be external factors or internal factors.
Extracellular ice formation is caused by the outside sources which includes dust, organic
particle, bacteria or gas bubbles whereas internal are intercellular space, xylem vessels etc.
some strains of bacteria can cause ice formation in higher temperature of -1 or -2. Whereas
larger intercellular spaces contribute for intracellular ice formation. It can disrupts the sub
cellular membranes by formation of ice crystals whereas extracellular ice formation increases
the concentration of extracellular solutes due to which water is withdrawn from the cells
during ice formation and causes water stress in frozen tissues.

 Membrane disruption: freezing can cause disruption or alteration in semi permeable

properties of plasma membrane due to which they will be high loss of solutes from cell which
can cause frost plasmolysis even after thawing.

 Super cooling: the cooling of water below zero degree but without formation of ice crystals,
where water may cool down to -1 to -15 (in herbaceous) and up to -40 to -45 (in hardy) , this
may occurs due to absence of internal ice nucleators. (it is one the importance mechanism for
freezing avoidance)

 Stress due to external factors: formation of ice sheets in field above or below the ground
surface it is lethal as it causes depletion of reserve. The cells which are dead due to freezing
injury are highly prone to pathogens and so reduce the chances of recovery, whereas
surviving tissues may face auto toxicity produced from the dead cells.

67
11.8 Freezing resistance
 Ability of a genotype to survive freezing stress and to recover and regrow after thawing

 Is complex trait and involves various physiological, chemical and physical processes at tissue
and cell level

 Freezing avoidance

 The ability of plant tissues or organs to avoid ice formation at subzero temperature

 Super-cooling is mechanism for freezing avoidance

 Is controlled by lack of ice nucleators and is favored by small cell size little or no
intercellular space, low moisture content, barriers against external nucleators and presence of
anti nucleators

 It is common on meristematic tissues of flower buds of hardy plats

 May be effective up to even -47 degree Celsius

 (smaller the cell size or lesser the intercellular spaces higher the chances of avoidance)

 Freezing tolerance: it’s the ability of plants to survive the stress generated by extracellular ice
formation and to recover and re-grow after thawing. In this case intra cellular ice formation is
lethal and is affected by freezing hardening i.e. increase in tolerance to freezing due to an
earlier exposure to low temperatures from critical duration.

 Osmotic adjustment: during hardening there is increase in cellular solutes which avoids
intracellular ice formation and cellular dehydration. Accumulation of solutes like proline and
trehalose protects the membrane( higher the amount of solutes higher the level of tolerance)

 Bound water: part of cellular water which does not take part in osmometric response of plants
is known as bound water. Higher the level of bound water higher will be the tolerance.

 Plasma membrane stability: during hardening the fluidity of plasma membranes increases and
its sensitivity to freezing stress reduces furthermore the increased stability of plasma
membrane reduces the extension of ice formation. Injury can be measured by the loss of
electrolyte.(lesser the loss of electrolyte higher the tolerance)

 Cell wall properties: limited or lower the intercellular spaces lower will be the chances for
intracellular ice formation.

 Cold responsive proteins: proteins produced in response of low temperature. Anti freeze
proteins are produced by polar artic fishes whereas in some Solanium species accumulation

68
 of ABA increases due to hardening which shows that accumulation of ABA may help in
tolerance. (higher the cold responsive protein higher the tolerance)

11.9 Genetic resources:

 Cultivated varieties: generally cereals developed during first half of twentieth century are still
most winter hardy., Minhardy,Albidum varieties of wheat.

 Germplasm lines: lines generally collected from extreme winter environment having low
temperature margins are useful sources for development of freezing tolerance.

 Induced mutations: in oats induced mutations showed 76% winter survival as compared to
26% in parent variety.

 Related wild species: tolerance can be found in some wild relatives, in case of potato variety
kufri sheetman is good source. Trails have been done in transfer of gene from rye to wheat
but the expression has been suppressed by wheat genes.

 Transgene: chemically induced antifreeze proteins like ala3 when inserted in tobacco showed
tolerant to freezing.

11.10 Selection environment:

 Field environment: an area where subzero temperature occurs regularly over the periods of
years. There is high fluctuation in temperature condition over the period of years so it reduces
the effectiveness of selection.

 Controlled environment: the parts, organs/tissue or whole plant may be subjected to freezing
stress under controlled environment; they are generally done for evaluation work but rarely
for selection activities.

11.11 Selection criteria:

 Field survival: survival and recovery after freezing is major criteria for selection but the
effectiveness can be reduced by variable environment condition. Best results can be obtained
if they are evaluated over locations and years, generally done for parental lines and lines
isolated from crosses.

 Freezing test: subjected to various evaluations in lab

 Viability of thawed tissue is determined by reduction of TTC to red color formation

(Triphenyl Tetrazolium chloride) generally used for frozen twigs of ornamental plants

 Degree of browning is common index for injury, applied to potato at 0 degree for 24 hours

 Electrolyte leakage

69
 Chlorophyll fluorescence after freeze thawing applied in potato

 Recovery and regrowth in cereals crowns(base )

 Freezing of cereal crown: in case of cereals crowns are subject to freezing and then their
recovery is measured. The crown is removed from soil mix and then they are washed clean
and shoots in excess are removed (2-3cm) along with roots of excess (1 cm) are removed.
Then is stored at 2 degree Celsius, then the crown is frozen at 1-1.5 degree per hour till 8
hours (may depend on species) and can go up to -9 to -19 after which the crown are thawed at
2 degrees and kept at growth chamber of 16 degrees and regrowth is measured at 7-10 days
time.

 Osmoregulation: can be determined by hardening and before freezing. This measures the
sugar content in tissues, can be applied to limited number of samples.

Problems:

 Complex traits and involve many components so cannot be readily measured under field
conditions

 Breeding work has to be carried out under field conditions which is inherently variable and is
subject to biotic stress during growth and recovery

 Field survival shows poor heritability due to G*E interactions

 Shows high G*E interaction and it limits progress under selection

70
 12. Selection procedures

12.1 Back cross:

In backcross method of breeding, the hybrid and the progenies in subsequent generations are
repeatedly backcrossed to one of the parents. As a result, the genotype of the backcross progeny
becomes increasingly similar to that of the recurrent parent. The objective of backcross method is to
improve one or two specific defects of a high yielding variety.

Requirements of a back cross programme:

 Existence of a good recurrent parent variety which requires improvement is some

qualitatively inherited character or a quantitative character with high heritability.
 A suitable donor parent must be available possessing the character or characters to be
transferred in a highly intense form.
 High expressivity of the character under transfer through several back crosses in the genetic
back ground of the recurrent parent.
 The character to be transferred must have high heritability-preferably determined by one or
few genes
 Simple testing technique for detecting the presence of the character under transfer.
 Recovery of the recurrent genotype in a reasonable number of back crosses generations.

Applications of Back Cross method:

 Back Cross method is applicable to both S.P. & C.P. crops

 Inter varietal transfer of simply inherited characters: characters governed by one or two major
genes – Eg. Disease resistance, used color.
 Linkage drag: Failure of transfer of simply inherited characters like disease resistance by
B.C. method due to a tight linkage between the gene being transferred and some other
undesirable gene.
 Inter varietal transfer of Quantitative characters: Quantitative characters with high heritability
can be transferred. Eg. Earliness, Pl. height seed size, seed shape.
 Inter specific transfer of simply inherited characters: Mostly disease resistance from related
species into a cultivated species. Eg. 1. Leaf and stem rust resistance from Triticum
timopheevii, T. monococcum, Aegilops speltoides and rye (S. cereale ) to T. aestivum. 2.
Black arm resistance from several Gossypium species to G. hirsutum
 Transfer of cytoplasm: Back Cross method used to transfer cytoplasm from one variety or
species to another. This is especial desirable in cases of Cytoplasmic or Cytoplasmic-genetic
male sterility. Transfer of T. timopheevii cytoplasm to T. aesticem

71
  Transgressive segregation: Back cross method may be modified to obtain transgressive
segregants. It may be modified is one of the following two ways.
 The F1 may be back crossed only 1 or at most 2 times to the recurrent parent leaving much
heterozygosity for transgressive segregants to appear.
 To or more recurrent parents may be used in the back cross programme to accumulate genes
from them in to the back cross progeny. Such a modification of the back cross would produce
a new variety that would not be exactly like any one of the recurrent parents.
 Production of Isogenic lines: Isogenic lines are identical in their genotype, except for one
gene. Such lines are useful in studying the effects of individual genes on yield and other
characteristics. Isogenic lines and easily produced using the back cross method.
 Germplasm conversion: Conversion of photosensitive germplasm lines (using as recurrent
parent) to photo insensitive line (using a photo insensitive line as a donor or non-recurrent
parent.

Transfer of a Dominant Gene:

Let us suppose that a high yielding and widely adapted variety A is susceptible to stem rust. Another
variety B is resistant to stem rust, and that resistance to stem rust is dominant to susceptibility. A
generalized scheme of the backcross programme for the transfer of rust resistance from variety B to
variety A is given below.

 Hybridization : Variety A is crossed to variety B. Generally, variety A should be used as the

female parent. This would facilitate the identification of selfed plants, if any F1 Generation:
heterozygous for rust resistance, selection for rust resistance is not necessary.
 First Backcross Generation (BC1): half of the plants would be resistant and the remaining
half would be susceptible to stem rust. Rust resistant plants are selected and backcrossed to
variety A. BC1 plants resistant to rust may be selected for their resemblance to variety A as
well.
 BC2-BC5 Generations: In each backcross generation, segregation would occur for rust
resistance. Rust resistant plants are selected and backcrossed to the recurrent parent A.
Selection for the plant type of variety A may be practiced, particularly in BC2 andBC3.
 BC6- Generation: On an average, the plants will have 98.4 per cent genes from variety A.
 Rust resistant plants are selected and selfed; their seeds are harvested separately.
 BC6 F2 Generation: Individual plant progenies are grown. Progenies homozygous for rust
resistance and similar to the plant type of variety A are harvested in bulk. Several similar
progenies are mixed to constitute the new variety.
 Yield Tests: The new variety is tested in a replicated yield trial along with the variety A as a
check. Plant type, date of flowering, date of maturity, quality etc. are critically evaluated.
Ordinarily, the new variety would be identical to the variety A in performance.

72
Transfer of a Recessive Gene:-

When rust resistance is due to a recessive gene, all the backcrosses cannot be made one after the
other. After the first backcross, and after every two backcrosses, F2 must be grown to identify rust
resistant plants. The F1 and the backcross progenies are not inoculated with rust because they would
be susceptible to rust. Only the F2 is tested for rust resistance. A generalized scheme for the transfer
of a recessive gene for rust resistance is given below.

 Hybridization: The recurrent parent is crossed with the rust resistant donor parent. The
recurrent parent is generally used as the female parent.
 F1 Generation: F1 plants are backcrossed to the recurrent parent.
 BC1 Generation: Since rust resistance is recessive, all the plants will be rust susceptible.
Therefore, there is no test for rust resistance. All the plants are self-pollinated.

73
  BC1 F2 Generation: Plants are inoculated with rust spores. Rust resistant plants are selected
and backcrossed with the recurrent parent. Selection is done for the plant type and other
characteristics of the variety A.
 BC2 Generation: There is no rust resistance test. Plants are selected for their resemblance to
the recurrent parent A, and backcrossed with the recurrent parent.
 BC3 Generation: There is no disease test. The plants are self-pollinated to raise F2. Selection
is usually done for the plant type of variety A.
 BC3F2 Generation: Plants are inoculated with stem rust. Rust resistant plants resembling
variety A are selected and BC to variety A. Selection for plant of A is generally effective.
 BC4 Generation: There is no rust resistance test. Plants are back-crossed to variety A.
 BC5 Generation: There is no rust test. Plants are self -pollinated to raise F2 generation.
 BC5F2 Generation: Plants are subjected to rust epidemic. A rigid selection is done for rust
resistance and for the characteristics of variety A. Selfed seeds from the selected plants are
harvested separately.
 BC5F3 Generation: individual plant progenies are grown and subjected to rust epiphytotic. A
rigid selection is done for resistance to stem rust and for the characteristics of variety A.
Seeds from several similar rust resistant homogeneous progenies are mixed to constitute the
new variety.
 Yield Tests: It is the same as in the case of transfer of a dominant gene.

Merits:-

74
  The genotype of new variety is nearly identical with that of the recurrent parent, except for
the genes transferred. Thus the outcome of a backcross programme is known beforehand and
it can be reproduced any time in the future.
 It is not necessary to test the variety developed by the backcross method in extensive yield
tests because the performance of the recurrent parent is already known. This may save upto 5
years’ time and a considerable expense.
 The backcross programme is not dependent upon the environment, except for that needed for
the selection of the character under transfer. Therefore, off -season nurseries and green-
houses can be used to grow 2-3 generations each year. This would drastically reduce the time
required for developing the new variety.
 Much smaller populations are needed in the backcross method than in the case of pedigree
method.
 Defects, such as susceptibility to disease, of a well-adapted variety can be removed without
affecting its performance and adaptability. Such a variety is often preferred by the farmers
and the industries to an entirely new variety because they know the recurrent variety well.
 This is the only method for inter-specific gene transfers, and for the transfer of cytoplasm.
 It may be modified so that transgressive segregation may occur for quantitative characters

Demerits:

 The new variety generally cannot be superior to the recurrent parent, except for the character
that is transferred.
 Undesirable genes closely linked with the gene being transferred may also be transmitted to
the new variety.
 Hybridization has to be done for each backcross which is often difficult, time taking and
costly.
 By the time the backcross programme improves it, the recurrent parent may have been
replaced by other varieties superior in yielding ability and other characteristics.

12.2 Recurrent selection:

Recurrent selection is defined as reselection generation after generation with inter- breeding of
selects to provide genetic recombination. It is a cyclic selection used to improve the frequency of
desirable alleles for a character in a breeding population. It is also an important method of population
improvement. The idea was given by Hayes and Garber (1919) and East and Jones (1920). Jenkins
1940 described the procedure of recurrent selection. However, the word recurrent selection was
coined by Hull 1945.

75
Main features:

It is a modified form of progeny selection and however it differs from progeny selection in two
ways.

1. The selected plants are selfed in recurrent selection whereas they are open pollinated in progeny
selection.

2. The selected plants are inter-mated in all possible combinations in recurrent selections but they
are open pollinated in progeny selection.

The characteristic features of recurrent selection are given below:

 Application: This method is more commonly used in cross pollinated species than in self
pollinated species.
 Base population: To start recurrent selection a heterogeneous population is required. In cross
pollinated species, a base population may be either of the five populations, viz.,
o an open pollinated variety,
o a synthetic variety,
o progeny of inter crosses among selected inbreds,
o a double cross, and
o a single cross.
 Important steps: A simple recurrent selection scheme consists of five main steps: (1) selection
of superior plants from base population, (2) selfing of selected plants (3) growing progeny of
selected plants in the next season from selfed seed, (4) intermating among progeny and (5)
bulking of crossed seed in equal quantity. This completes original cycle of recurrent
selection. The bulk seed is used for next cycle of selection which also involves above five
steps.
 Use of end product: The population developed by recurrent selection can be used in three
main ways: (a) In producing homozygous inbreds by selfing, (b) in the production of hybrids
varieties and (c) in the production of synthetic varieties.
 Basic assumption: Recurrent selection is based on three basic assumptions, viz., absence of
epistasis, absence of multiple alleles and absence of linkage disequilibrium. However, none
of these assumptions is considered valid.
 Impact: Recurrent selection is used to improve the frequency of desirable alleles for a
character in a population. In this method the heterozygosity that is lost due to selfing is
recovered by intermating of selected progeny.

76
TYPES OF RECURRENT SELECTION:

There are four types of recurrent selection, namely,

 Simple Recurrent Selection: A type of recurrent selection that does not include tester is
referred to as simple recurrent selection. It is also known as phenotypic recurrent selection.
This method is an extension of mass selection.

Main features of simple recurrent selection are given below:

 The tester is not used in this scheme.

 It does not measure the combining ability.
 The selection is based on phenotype or simple test.
 This method is useful only for those characters which have high heritability.
 This method requires only two seasons for the completion of one selection cycle
 Recurrent selection for general combining ability: A form of recurrent selection that is used
to improve the general combining ability of a population for a character and includes
heterozygous tester is referred to as recurrent selection for general combining ability
(RSGCA). It is also known as half sib recurrent selection with heterozygous tester. This is an
extension of the Jenkin’s (1940) scheme used for development of short term synthetics.

Main features of this scheme are given below:

 This method is used for genetic improvement of quantitative characters.

 The selection is made on the basis of test cross performance.
 A heterozygous tester with broad genetic base is used for testing general combining
ability. Generally, an open pollinated variety is used as a tester.
 This method is used for improving general combining ability of population for a
character.
 This method is more effective with incomplete dominance and less effective with over-
dominance.
 This method is used for the improvement of those characters which are governed by
additive gene action.
 This method requites three seasons or years for completion of each cycle of selection
 Recurrent selection for specific combining ability: A form of recurrent selection that is used
to improve the SCA of a population for a character by using homozygous tester is referred to
as recurrent selection for specific combining ability (RSSCA). It is also known as half sib
recurrent selection with homozygous tester. This method was originally proposed by Hull in
1945. The selection procedure of this method is same as for RSGCA except that the tester is
inbred lines in this case which has narrow genetic base. The differences in the performance of
test crosses are due to differences in their specific combining ability.

77
 Main features of this scheme are presented below:

 This method is used for the genetic improvement of polygenic character.

 Selection is made on the basis of test cross performance.
 A homozygous tester with narrow genetic base is used for testing specific combining
ability. In other words, an inbred is used as a tester.
 This method is used for improving specific combining ability of the population for a
character.
 This method is more effective with over dominance and less effective with incomplete
dominance.
 This scheme is used when a character is governed by non additive (dominance and
epistasis) gene action.
 This method required three seasons or years for completion of each cycle of selection.
 Reciprocal recurrent selection: A form of recurrent selection that is used to improve both
GCA and SCA of a population for a character using two heterozygous testers is known as
reciprocal recurrent selection (RRS). It is also termed as recurrent reciprocal half sib
selection. This scheme was proposed by Comstock, et al. in 1949.

Main features of this method are given below:

 This scheme is also used for the improvement of polygenic characters.

 Selection is made on the basis of test cross performance.
 Two heterozygous populati8ons each of which is the tester for other are used in this
method. These two populations may be designated as A and B.
 This method is used for improving a population both for GCA and SCA for a specific
character. 5. This method is equally effective with incomplete, complete and over
dominance.
 This method is used for the improvement of those characters which are governed by both
additive and non-additive gene action.
 This method also requires three seasons or years for completion of each cycle of
selection

Morphological marker:

A morphological marker is expressed as a specific and distinct morphological trait. Morphological

marker may be affected by environment. Generally it is incompletely linked with the gene of interest.
Its phenotypic expression may be dependent on growth stage. These markers are rare in a natural
population and show extremely low level of polymorphism.

78
12.3 Molecular markers:
Molecular markers are specific fragments of DNA that can be identified within the whole genome.
Molecular markers are found at specific locations of the genome. They are used to ‘flag’ the position
of a particular gene or the inheritance of a particular character. Molecular markers are phenotypically
neutral.

Characteristics of Ideal Markers:

 Should exhibit high degree of polymorphism

 Marker loci should be extensively distributed within genome. Loci should occur at short
intervals

 Should be co-dominant in nature as they provide more information then dominant markers

 Should be highly reproducible

 Detection should be easy, simple and rapid

 Cost of detection should be cheap

 Should be amenable to automation

Marker categories:

Depending on the technique used for detection and amplification of markers there can be different
classes of markers. Restriction fragment length polymorphism (RFLP) is based on restriction site
changes in the target DNA and subsequent hybridization with probe DNA. Random amplified
polymorphic DNA (RAPD), Sequence characterized amplified region (SCAR) and Sequence tagged
sites (STS) are based on mutation at primer annealing site in the target DNA. Cleaved amplified
polymorphic sequence (CAPS) and Amplified fragment length polymorphism (AFLP) are based on
both restriction site changes and mutation at primer annealing site in the target DNA. Additionally,
there are Simple sequence repeat (SSR), Inter simple sequence repeat (ISSR) and Single nucleotide
polymorphism (SNP) markers. Highly specialized techniques are required to detect SNP’s.

Dominant marker:

A marker is called dominant if only one form of the trait (which is targeted to be marked) is
associated with the marker, whereas the other form of the trait is not associated with any marker.
Such markers cannot discriminate between heterozygote and homozygote marker allele.

Co-dominant marker:

A marker is designated as co-dominant if both forms of the trait (which is targeted to be marked) are
associated with the marker. It can discriminate between heterozygote and homozygote marker allele.

79
The main uses of these molecular markers in crop genetic studies are as follows:

 Assessment of genetic variability and characterization of germplasm

 Identification and fingerprinting of genotypes
 Estimation of genetic distances between population, inbreeds, and breeding materials
 Detection of monogenic and quantitative trait loci (QTL)
 Marker-assisted selection
 Identification of sequences of useful candidate genes

Types of markers:

Random amplified polymorphic DNA (RAPD):

RAPD is a PCR based method, which employs single primers of arbitrary nucleotide sequence with
10 nucleotides to amplify anonymous PCR fragments from genomic template DNA. In RAPD
analysis, the target sequence(s) (to be amplified) are unknown. In RAPD, PCR is generally carried
out with arbitrary primers. The amplifications are visualized through agarose gel electrophoresis. For
amplification to occur it is essential that primers anneal in a particular orientation (such that they
point towards each other) and the primers must anneal within a reasonable distance to one another.

Advantages

 No prior knowledge of DNA sequences is required

 Random distribution throughout the genome
 The requirement for small amount of DNA (5-20 g)
 Easy and quick to assay
 The efficiency to generate a large number of markers
 Commercially available decamer primers are applicable to any species
 The potential automation of the technique
 RAPD bands can often be cloned and sequenced to make SCAR (sequence-characterized
amplified region) markers
 Cost effectiveness as compared to other markers.

Limitations:

 Dominant nature (heterozygous individuals cannot be separated from dominant homozygous)

 Sensitivity to changes in reaction conditions, which affects the reproducibility of banding
patterns
 Co-migrating bands can represent non-homologous loci
 The scoring of RAPD bands is open to interpretation
 The results are not easily reproducible between laboratories.

80
Applications of RAPD:

 Measurements of genetic diversity

 Genetic structure of populations
 Germplasm characterization
 Verification of genetic identity
 Genetic mapping
 Development of markers linked to a trait of interest
 Cultivar identification
 Identification of clones (in case of soma-clonal variation)
 Inter-Specific hybridization
 Verification of cultivar and hybrid purity
 Clarification of parentage

Restriction fragment length polymorphism (RFLP):

RFLP is a molecular marker based on the differential hybridization of cloned DNA to DNA
fragments in a sample of restriction enzyme digested DNAs (Fig. 4). RFLPs involve digestion of
genomic DNA with restriction enzymes (bacterial enzymes that cut DNA at specific sequences
known as restriction sites). The resulting DNA fragments are size fractionated on gel electrophoresis,
transfer of fractionated DNA fragments on Nylon membranes (a process known as Southern blotting)
and finally hybridization with labeled probe to visualize DNA polymorphisms. The first step in
RFLP analysis is to derive a set of clones that can be used to identify RFLPs. The two primary
sources of these clones for RFLP mapping of plants are cDNA clones and PstI-derived genomic
clones. RFLP markers are defined by a specific enzyme-probe combination. This technique is highly
reproducible, and the markers are co-dominant in their inheritance therefore, allows the
differentiation of heterozygotes from homozygotes. RFLP procedure is time consuming and
expensive but they have been used to generate saturated genetic map. RFLPs behave like any other
Mendelian trait. Each band seen in a Southern blot indicates the presence of one or more restriction
sites in a sequence. The sequence containing a restriction site is one allele, while the corresponding
sequence missing the restriction site is the other allele. The “phenotypes” of these alleles are the
differences in banding patterns, due to presence or absence of bands. RFLP loci are co-dominant
(twice as much informative in a genetic cross as compared to dominant markers like RAPDs).

Amplified fragment length polymorphism (AFLP):

Amplified fragment length polymorphisms (AFLPs) are polymerase chain reaction (PCR)- based
markers for rapid screening of genetic diversity. AFLPs are DNA fragments with different nucleotide
sequence of which large number of copies have been amplified via PCR. This technique is a
combination of the RFLP and PCR techniques. Like RFLP, the AFLPs are highly heritable and

81
polymorphic. The technique involves restriction digestion of DNA with two different enzymes and
ligation of two adopters, selective amplification of sets of restriction fragments and gel analysis of
amplified fragments. The amplified products are generally separated on a denaturing polyacrylamide
gel and visualized using autoradiography. The technique is more skill demanding than RAPD and
also requires more amount of DNA. The reproducibility of AFLP is ensured by using site specific
adopters. AFLP method rapidly generates hundreds of highly replicable markers from DNA of any
organism, and thus, they allow high resolution genotyping of fingerprinting quality. The time and
cost efficiency, replicability and resolution of AFLPs are superior or equal to those of other markers
[allozymes,random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism
(RFLP), microsatellites], except that AFLP methods primarily generate dominant rather than co
dominant markers. Because of their high replicability and ease of use, AFLP markers have emerged
as a major new type of genetic marker with broad application in systematic, pathotyping, population
genetics, DNA fingerprinting and quantitative trait loci (QTL) mapping.

Simple sequence repeats (SSR):

Marker Microsatellite or Simple sequence repeats (SSRs) provide fairly comprehensive genomic
coverage. They are amenable to automation, they have locus identity and they are multi-allelic.
Many agronomic and quality traits show quantitative inheritance and the genes determining these
traits have been quantified using Quantitative trait locus (QTL) tools. SSR markers have wide
applicability for genetic analysis in crop improvement strategies. They are widely used in plants
because of their abundance, hyper-variability, and suitability for high throughput analysis.

Factors influencing efficiency of a marker:

Efficiency of markers depends on their closeness to the linked trait; how the phenotype of marker is
affected by environment; consistency in phenotypic expression; how easy is to score the phenotype;
and level of polymorphism. Ideally, a marker should be polymorphic, tightly linked with the trait of
interest, highly heritable, co-dominant, easily score able and it should not affect the fitness of the
individual. DNA markers have many advantages over the morphological markers. DNA markers are
phenotypically neutral which is a significant advantage compared to traditional phenotypic markers,
highly polymorphic, abundant, usually randomly distributed throughout the genome, easily score
able and as such DNA markers are not affected by environment but the gene of interest may be
sensitive to GxE and hence its (DNA marker) association with phenotype of the gene may vary with
change of environment.

Mapping population:

Mapping population consists of individuals of one species, or in some cases they are derived from
crosses among related species. It is a group of individuals on which genetic analysis is carried out. It
can be either segregating for traits under study or a set of near homozygous lines representing a F2

82
variation. In both situations mapping population is generally derived from a single cross whose
parents were polymorphic for the trait of interest.

Different types of mapping populations:

Mapping population may comprise F2 , backcross, recombinant inbred lines (RIL), doubled haploid
lines (DHL), F2 derived F3 (F2 :F3 ) populations and near-isogenic lines (NILs). Specialized
populations are required in cases of cross pollinated species that can’t tolerate inbreeding, perennials
and the trees.

Doubled haploid population:

Doubled haploids (DH) are also the products of one meiotic cycle, and hence comparable to F2 in
terms of recombination information. DHs are permanent mapping population and hence can be
replicated and evaluated over locations and years and maintained without any genetic change. These
are useful for mapping both qualitative and quantitative characters. It provides opportunity to induce
homozygosity in single generation and instant production of homozygous lines. But in DH lines
recombination from the male side alone is accounted. Since it involves in-vitro techniques, relatively
more technical skills are required in comparison with the development of other mapping populations.
It is often suitable culturing methods / haploid production methods are not available for a number of
crops, and different crops differ significantly for their tissue culture response. In addition, tissue
culture induced variation should be taken care.

Near isogenic lines (NIL):

NILs can be generated through two different breeding procedures. It is developed through repeated
selfing and selecting heterozygous individuals until sufficient homozygosity is attained for all traits
except for the trait of interest. NILs can also be generated by backcrossing the F1 plants to the
recurrent parents and selecting the trait of interest in each generation. NILs developed through
backcrossing are similar to recurrent parent except for the gene of interest, whereas the NILs
generated though selfing are produced in pairs of near identical individuals (identical for all traits
except for the loci of interest). Like DHs and RILs, NILs are also immortal mapping population.
NILs are quite useful in functional genomics. NILs have disadvantages too. They require many
generations for development. These are directly useful only for molecular tagging of the concerned
gene but not for linkage mapping. Linkage drag is a potential problem in constructing NILs.

Use of Molecular markers in breeding program:

Marker aided selection is a tool for breeding, wherein genetic marker(s) tightly linked with the
desired trait/gene(s) are utilized for indirect selection for that trait in segregating/non-segregating
generations. In its simplest form it can be applied to replace evaluation of a trait that is difficult or
expensive to evaluate. When a marker is found that co-segregates with a major gene for an important

83
trait, it may be easier and cheaper to screen for the presence of the marker allele linked to the gene,
than to evaluate the trait. From time to time the linkage between the marker and the gene should then
be verified.

Marker assisted selection (MAS):

MAS is most useful for traits that are difficult to select e.g., disease resistance, salt tolerance, drought
tolerance, heat tolerance, quality traits (aroma of basmati rice, flavor of vegetables). The approach
involves selecting plants at early generation with a fixed, favorable genetic background at specific
loci, conducting a single large scale marker assisted selection while maintaining as much as possible
the allelic segregation in the population and the screening of large populations to achieve the
objectives of the scheme. No selection is applied outside the target genomic regions, to maintain as
much as possible the Mendelian allelic segregation among the selected genotypes. After selection
with DNA markers, the genetic diversity at un-selected loci may allow breeders to generate new
varieties and hybrids through conventional breeding in response to targets set in breeding
programme.

Material required for MAS:

Molecular markers are a set of authentic lines carrying trait of interest and a population to validate
the markers to be used e.g., F2 or BCF2 for each of the individual traits/genes.

Following are the basic pre-requisites for MAS:

 Search of molecular markers that are linked to the trait of interest

 Validate the available markers in parents and breeding population
 If markers are not available, it has to be designed and validated before use (if mapping
populations are not available in hand it may take 2-4 years to generate and validate markers)
 Design a selection scheme and breeding strategy
 Fix the minimum population to be assayed to capture all beneficial alleles
 Meticulous record keeping
 Progeny testing for fixation of traits.

Steps involved in MAS:

 Validation of molecular markers: Extract the DNA from test individuals and find out whether
there is one to one relationship with marker and the trait.
 Extract the DNA of breeding population at the seedling stage and apply MAS. Select the
individuals on the basis of presence of desired molecular markers for the concerned trait. For
other traits, selection is based on classical breeding methods. Minimum individuals to be
assayed should be as per the defined strategy and statistical considerations.

84
Limitations of MAS:

 Cost factor
 Requirement of technical skill  Automated techniques for maximum benefit
 Per se, DNA markers are not affected by environment but traits may be affected by the
environment and show G x E interactions. Therefore, while developing markers, phenotyping
should be carried out in multiple environments and implications of G x E should be
understood and markers should be used judiciously.
 DNA marker has to be validated for each of the breeding population. Any prior assumption
regarding the validity of markers may be disastrous

Marker assisted backcross breeding:

A backcross breeding programme is aimed at gene introgression from a “donor” line into the
genomic background of a “recipient” line. The potential utilization of molecular markers in such
programmes has received considerable attention in the recent past. Markers can be used to assess the
presence of the introgressed gene (“foreground selection”) when direct phenotypic evaluation is not
possible, or too expensive, or only possible late in the development. Markers can also be used to
accelerate the return to the recipient parent genotype at other loci (“background selection”). It is
assumed that the introgressed gene can be detected without ambiguity, and the theoretical study was
restricted to background selection only. The use of molecular markers for background selection in
backcross programmes has been tested experimentally and proved to be very efficient. Introgressing
the favourable allele of QTL by recurrent backcrossing can be a powerful mean to improve the
economic value of a line, provided the expression of the gene is not reduced in the recipient genomic
background. Yet, recent results show that for many traits of economic importance QTLs have rather
small effects. In this case, the economic improvement resulting from the introgression of the
favourable allele at a single QTL may not be competitive when compared with the improvement
resulting from conventional breeding methods over the same duration. Marker assisted introgression
of superior QTL alleles can then compete with classical phenotypic selection only if several QTLs
could be manipulated.

Application of DNA markers in crop improvement

QTL mapping: Some of the most difficult tasks of plant breeders relate to the improvement of traits
that show a continuous range of values. Genetic factors that are responsible for a part of the observed
phenotypic variation for a quantitative trait are called quantitative trait loci (QTLs). The term QTL
was coined by Gelderman. Conceptually it can be a single gene or may be a cluster of linked genes
of the trait. Although similar to a gene, a QTL merely indicates a region on the genome comprised of
one or more functional genes. Among such quantitative traits like yield, plant length and days to
flowering etc., are important ones. Selection for quantitative traits is difficult, because the relation

85
between observed trait values in the field (the phenotype) and the underlying genetic constitution
(the genotype) is not straight forward. Quantitative traits are typically controlled by many genes,
each contributes only a small part to the observed variation.

Tagging of disease resistance genes: DNA based markers have shown great promises in expediting
plant breeding methods. The identification of molecular markers closely linked with resistance genes
can facilitate expeditious pyramiding of major genes into elite background, making it more cost
effective. Once the resistance genes are tagged with molecular marker the selection of resistant plant
in the segregating generations becomes easy.

Tagging of male sterility genes: Cytoplasmic male sterile system is desirable for use in hybrid seed
production, as it eliminates the need of hand emasculation. CMS is a maternally inheritable trait
characterized by the inability to produce viable pollen but without affecting the female fertility, and
it is often associated with mitochondrial DNA rearrangements, mutations and editing. Several
restorer locus have been identified using RAPD and STS in different crop and DNA markers linked
to these locus enable the molecular study of the CMS system. These co-dominant markers are useful
in identifying the homozygous restorer genotypes after the backcrossing for production of restorer
lines. In this way, the restorer lines could be produced in a shorter period than by conventional
methods.

Diversity evaluation: Stability and identity of crop variety has assumed great importance for
predicting plant breeder’s right/farmers right. Traditionally, evaluation and conservation of bio-
diversity/genetic variability is based on comparative anatomy, morphology, embryology, physiology,
etc., which provide informative data but of low genetic resolution. Recent advances in molecular
biology have provided powerful genetic tools, which can provide rapid and detailed genetic
resolution. Molecular marker based genotyping involves the development of marker profile unique to
an individual. This unambiguous pattern of crop varieties obtained using DNA a marker is termed as
“DNA Fingerprinting”

Heterosis breeding: Another important application of DNA markers is their prediction of heterosis in
hybrids. Evaluation of hybrids for heterosis or combining ability in field is expensive. Molecular
markers have been used to correlate genetic diversity and heterosis in several cereal crops (like rice,
oat, and wheat). It has been reported that measures of similarity based on RFLP and pedigree
knowledge could be used to predict superior hybrid combinations. However, both low and high
correlations between heterosis and DNA based genetic distance have been observed.

86
 13. Durability of Resistance

Introduction:

Durable disease resistance is defined as resistance that has remained effective while a cultivar
possessing it has been widely cultivated in an environment favoring the disease. This characteristic
of resistance is recognized retrospectively, as are all other characteristics of interactions between
hosts and pathogens. Resistance that has been shown to be durable should be considered as a
valuable resource for further breeding and for investigation of the causes of durability.

Durable resistance is often achieved against diseases that show little or no specialization into races
pathogenic to particular cultivars. For such diseases, the control of resistance may be by single genes
or by several genes, each of small effect, and the resistance may be either complete or incomplete
(quantitative). For pathogens that possess Pathogenicity specific for particular cultivars, failures of
resistance can often be shown to be due to race-specificity of resistance genes and the evolution and
spread of pathogen races with matching Pathogenicity. These race-specific genes may have large
(major) or small (minor) effects, giving either complete or quantitative resistance, and may operate at
any stage of host development. Despite failures of resistance due to race-specificity in some
cultivars, other cultivars remain resistant for relatively long periods, though this does not prove that
their resistance will be permanent. For some diseases such durable resistance may be controlled by
one or few genes of large effect. More usually a number of genes, not necessarily a large number,
giving cumulative and often quantitative resistance, may appear to control durable resistance.

Resistance is considered durable when it remains effective for a considerable time, despite wide
exposure. In this sense, it is a quantitative concept. The Rpg1 gene was durable, but did not last
forever. And in the evolutionary sense, no resistance will last forever.

It is possible to discern three groups of resistances that are predominantly durable.

 Resistance to pathogens with a wide host range, generalists are usually of a quantitative
nature (Bruehl, 1983) and nearly always durable (Parlevliet, 1993). But there are exceptions,
such as the major resistance genes Mi in tomato and Rk in cow pea against the root knot
nematode, M. incognita (Roberts, 1995).
 QR against specialists and based on some to several genes with additive effects seems
durable. In the few cases where reported QR broke down, the resistance appeared to be
monogenic, like the field resistance against rice blast, M. grisea, in the rice cultivar St-1. The
resistance became ineffective within a few years and appeared to be based on a single
dominant gene Pi-f (Toriyama, 1975).
 Monogenic resistance against specialists of a non-hypersensitive nature. Such resistance is
often quite durable. The non-hypersensitive resistance genes Rpg2 (sr-2) and Rpr34 (Lr34) of

87
 wheat to stem rust and leaf rust respectively and the mlo-gene of barley to powdery mildew
have already lasted for a considerable time.

Usually, the presence of race-specific resistance effects is considered as evidence of non-durable

resistance. This idea, however, cannot be maintained. There are too many examples of race-specific
resistance that lasted a long time or that are still effective. The Rpg1 gene of barley discussed above
is such an example. The hypersensitive resistance genes Nx and Nb to virus X in potato is still
effective in Europe, despite the fact that the corresponding virulent strains have been present for
many years but at very low frequencies. Polygenic resistance is usually considered to be non-race-
specific and durable. The two models showed that the polygene-for polygene system was more
durable (less liable to adaptation in the pathogen population) than the system where the polygenes
were not operating in a gene-for-gene system, assuming all other variables, such as resistance
mechanisms, are the same. The durability of resistance also may depend on the circumstances. The
major gene resistance of flax-to-flax rust was not durable in the USA, but it has been durable in The
Netherlands (Parlevliet, 1993). In the latter situation, flax has been a small crop and all flax grown is
completely resistant. The pathogen population was, therefore, reduced so strongly that new races
have little chance to emerge.

Non Durable Resistance:

In nature there is a constant race of arms between the attacking parasite and the defending host, and
in the evolutionary sense, all resistance is transitory. But large differences exist in the ease by which
parasites can overcome a resistance. In agriculture, too the durability of a resistance varies greatly.
Resistance may already be neutralized in the last stages of the breeding program (at zero years) and
may, still be effective after more than 130 years and wide exposure, as the case of the Phylloxera
aphid resistance of grape (Vitis vinifera L.) rootstocks. Resistance is considered durable if it remains
effective when used for many years over a substantial area. Much of the resistance used by breeders
has not broken down; multiple major gene resistance and all QR based on some to several genes
appeared to be durable.

Application of Non-Durable resistance:

Gene Pyramiding:

Gene pyramiding or ideotype involves the assembly of multiple traits of interest derived from
various parental lines into a single variety. Gene pyramiding refers to the process of stacking
multiple genes into a single genotype to combine desirable traits through recombinant DNA
technology or conventional breeding. This approach has resulted in the so-called 'second generation'
of GE plants. In gene pyramiding scheme, strategy is to cumulate into a single genotype, genes that

88
 have been identified in multiple parents. The use of DNA markers, which permits complete gene
identification of the progeny at each generation, increases the speed of pyramiding process. In
general, the gene pyramiding aims at the derivation of an ideal genotype that is homozygous for the
favorable alleles at all n loci. The gene pyramiding scheme can be distinguished into two parts. The
first part is called a pedigree, which aims at cumulating of all target genes in a single genotype called
the root genotype. The second part is called the fixation step which aims at fixing the target genes
into a homozygous state i.e. to derive the ideal genotype from the one single genotype. Each node of
the tree is called an intermediate genotype and has two parents. Each of this intermediate genotype
variety can resist. Moreover, pyramiding can also improve becomes a parent in the next cross. The
intermediate genotypes are not just an arbitrary offspring of a given cross but it is a particular
genotype selected from among the offspring in which all parental target genes are present. Although
the pedigree step may be common, several different procedures can be used to undergo fixation.

Gene pyramiding is a crop-breeding technique that can be applied in conventional and advanced
molecular breeding programs to introduce novel lines. The conventional technique of crop breeding
develops new crop varieties by employing traditional techniques and routine natural processes, as
compared to modern and sophisticated tools of the current era . The technique involves sequential
gene pyramiding deployed in the same plant. The conventional pyramiding technique involves
backcross breeding: crossing a hybrid with one of the parental lines, followed by selection for the
desired characteristic. The inherited traits and resistant genes are transferred from donor parents into
recipient lines by backcrossing, pedigree breeding, or recurrent selection. With backcrossing, the
traits of interest are identified via the selection process. The backcrossing method is also used for
resistance gene pyramiding. Pedigree breeding is a method of genetic improvement of self-pollinated
89
species in which superior genotypes are selected from segregating generations and proper records of
the ancestry of selected plants are maintained at each stage of selection. Recurrent selection is an
efficient and modified form of progeny selection, where selection for some specific trait(s) is
conducted within consecutive segregating progeny generations on the basis of phenotypic
characteristics

Multilines:

Multiline variety is a mixture of several pure lines of similar phenotype(height, seed color flowering
time, maturity time and various other agronomic characteristics) but have different genes for the
character under consideration the disease resistance means these are isogenic lines. At the same time
they do not reduce the yielding ability of each other when grown in mixture. At the time of disease
outbreak, only one or few lines of the mixture get attacked, others remain resistant. So the loss to the
farmer is comparatively less. Multiline varieties are more adaptive to environmental changes than
individual pure line.

Types:

 Mixtures of Isolines: Isolines are genotypes having one gene difference only. Isolines are
developed by backcross method. The resistant genes for different races of a disease are
transferred into one popular variety by separate backcross programmes. Six to ten Isolines are
developed and their seeds are mixed in equal quantity to constitute a multiline. Such varieties
have been developed in oat, soybean and wheat in USA.
 Mixtures of Closely Related Lines: Sometimes, multiline cultivars are constituted by mixing
the seed of closely related lines. Closely related lines are developed from crosses having one
parent in common. This approach of multi-lines development is being followed now-a-days
at important breeding centers like CIMMYT.
 Mixtures of Unrelated Lines or Cultivars: Multi-lines are also constituted from seed mixtures
of distinctly different cultivars. Such multi-lines are developed when phenotypic uniformity
is not essential. First pure-lines are developed by pedigree, bulk or single seed descent
methods. The performance of each pure-line is evaluated and then superior pure-lines each
with different gene for resistance are mixed together to constitute multiline. This is almost
similar to varietal blends

Procedure of Developing Multiline Breeding:

 Selection of Recurrent Parent: The recurrent parent should be a high yielding popular variety.
The recurrent parent should be the best cultivar of a region.

90
  Selection of Donor Parents: Parents with resistance to various races of a disease should be
chosen as donor parents. The resistance should be thoroughly examined under artificial
epiphytotic conditions before use of the donor parents in the crossing programmes. The donor
parents should be adapted varieties as far as possible. Because in un-adapted parents disease
resistance is sometimes linked with several un-desirable characters and transfer of resistant
genes from such parents to the recurrent parent becomes difficult task. Several donor parents
are selected to incorporate different resistant genes against various races.
 Transfer of Resistance: The resistant genes are transferred from donor parents to the recurrent
parent through a series of several separate backcross programmes. Generally 4-5 backcrosses
are sufficient to retain the genotype of recurrent parent with added resistance in the backcross
derivatives. The backcross derivatives are evaluated for disease resistance during
backcrossing and also at the end of backcrossing. The desirable lines from each backcross are
mixed to form an isoline.
 Mixing of Isolines: The various isolines developed by various backcrosses are mixed together
to constitute a multiline cultivar. Generally 6-10 isolines are mixed to constitute a multiline
cultivar.

Cultivar mixture
Cultivar mixtures are "mixtures of cultivars that vary for many characters including disease
resistance, but have sufficient similarity to be grown together." Cultivar mixtures do not cause major
changes to the agricultural system, generally increase yield stability, and in some cases can reduce
pesticide use. They are also quicker and cheaper to formulate and modify than "multilines," which
are defined as mixtures of genetically uniform lines of a crop species (near-isogenic lines) that differ
only in a specific disease or pest resistance. Cultivars used in the mixture must possess good
agronomic characteristics and may be phenotypically similar for important traits including maturity,
height, quality and grain type, depending on the agronomic practices and intended use. Cultivar
mixtures in barley for the control of powdery mildew are an example of phenotypically similar
mixtures, whereas red- and white-grained sorghum mixtures used in Africa are an example of
phenotypically different mixtures.

Mechanisms by which cultivar mixtures suppress disease

Cultivar mixtures do not completely suppress or eliminate the disease. Rather, mixtures reduce the
rate of disease progress by eliminating large numbers of spores at each cycle of pathogen
multiplication. Spores are eliminated from the epidemic process by deposition on resistant plants and
by dilution because of the greater distance between plants of the same genotype. Moreover, the
infection process may be slowed by the induction of defense responses in susceptible plants by
strains of the pathogen that are avirulent on specific host genotypes. The result is a level of disease

91
suppression owing to multiple epidemiological and physiological mechanisms. The four mechanisms
by which cultivar mixtures suppress disease are summarized as follows:

 Dilution effect. Increasing the distance between susceptible plants reduces/slows the rate of
plant to plant spread. A decrease in the density of susceptible plants slows disease
development. If plants within the population possess different race-specific genes, the relative
ability of any given pathogen race to spread from plant to plant is reduced because distance
between plants of the same genotype is increased.
 Barrier effect. The presence of resistant plants in the canopy provides a physical barrier
against spore dispersal, interrupting spore movement. The number and size of the resistant
plants and the physics of spore dispersal influence the strength of the barrier effect. the
barrier effect is caused by the presence of a resistant plant that acts as a barrier to the spread
of pathogen propagules (e.g., spores). For both of these mechanisms, the size of the host plant
influences the effectiveness of the cultivar mixture. In general, mixture effectiveness
decreases with increasing size of the host individuals.
 Induced resistance. Induced resistance occurs when biochemical host defenses are triggered
by inoculation with an avirulent race. Triggering these defenses slows the infection processes
of virulent pathogen races to which the host is normally susceptible. Induced resistance
occurs when spores of an avirulent strain or race land on and trigger a biochemical defense
response on an incompatible host. This induction of defense responses reduces partially the
susceptibility of the host plant to infection by spores of a virulent strain or race. Either the
infection efficacy or the number of new spores produced as a result of infection can be
reduced. Induced resistance is a non-specific, general mechanism in many pathosystems and
its characteristics vary from disease to disease. Some mechanisms of induced resistance are
localized to tissues in the vicinity of an infection, but other mechanisms may affect a larger
part of the plant. It has been suggested, however, that even very localized induction of
resistance by an avirulent spore may result in significant disease reduction at the epidemic
level
 Modification of the microclimate. The presence in the component cultivar of plant attributes
(i.e. plant height, canopy traits, etc.) that modify the microclimate towards less favorable
conditions for the disease can help the suppression of the disease.

Effect of cultivar mixtures on epidemic development

The development of a plant disease epidemic is a function of the initial inoculum, the rate of disease
development and the duration of crop growth. Cultivar mixtures affect the epidemic factors as
follows

92
 a) The initial inoculum level can be reduced. Compared to a pure-line susceptible population,
the initial inoculum is lowered when a given race or strain is not completely virulent on all
genotypes in the mixture.
b) The rate of secondary infection can be reduced. As the proportion of susceptible tissue
available for a given race of the pathogen is lowered, the number of new, secondary
infections by this race is reduced, which results in a decrease in the observed 'apparent
infection rate.'

Effect of cultivar mixtures on the evolution of pathogen races

 The lower level of exposure to the pathogen population of the resistance gene in a mixture
compared with monoculture will reduce selection pressure on the pathogen population and
therefore increase the gene's durability.
 Mixtures support more diverse pathogen populations than do pure stands, and that diversity is
positively related to the degree of disease control provided by the mixture. The mixtures lead,
generally, to the evolution of complex pathogen races potentially capable of overcoming
many resistance genes and therefore reduce the efficacy of the crop mixture as a measure to
reduce disease. However, the appearance of complex races does not mean that those races
will be selected for within the mixtures. There are several factors that can prevent the
prevalence of complex races. The relative efficacy and durability of the resistance genes
deployed in crop mixtures compared with the other two options, depends on the rate of
progress of those races in the mixture:
 Diversity within pathotype. Increasing diversity reduces the rate of increase of complex
pathotype in crop mixtures
 Fitness cost associated with virulence. The ability of complex races to attack multiple host
genotypes is countered by a reduction in fitness associated with the lack of avirulence genes.
 Differential adaptation. Complex races can infect different host genotypes but have reduced
infection efficiency compared with the specific simple races corresponding to each
component of the mixture, reducing therefore their rate of progress.
 Density dependence. It is the decrease in pathogen multiplication rate associated with an
increase in lesion density. It could affect simple and complex races differentially during an
epidemic and reduce selection for complex races.

The progress towards complex pathogen races may be relatively slow. Single races or pathotype
capable of overcoming a single resistance gene should progress faster than complex races capable of
overcoming multiple resistance genes, precluding the former of becoming dominant within the
pathogen population.

93
Integrated Control:

Integrated control is the practice of using a range of measures to prevent and manage diseases in
crops. Hazard analysis is used to identify the potential for infection so that preventative or curative
measures can be put in place to minimize the risk of disease infection and spread. Incorporate as
many different control strategies as possible including the use of synthetic insecticides, biological
insecticides, beneficial insects (predators/parasites), cultural practices, transgenic plants (where
allowed), crop rotation, pest-resistant crop varieties and chemical attractants or deterrents. It deals
with:

 Exploitation without crop

 Crop rotation and phyto-sanitation
 Combination with resistance with biological control

94