Principles of Plant breeding and biotechnology.pptx

1
Dr. Zekeria Yusuf (PhD)
Plant Breeding and Biotechnology
(BIOG 541 & 522)

Introduction
• Plant breeding is a science based on principles of
genetics and cytogenetics. It aims at improving the
genetic makeup of the crop plants.
• Improved varieties are developed through plant
breeding.
• Its objectives are to improve yield, quality, disease-
resistance, drought and frost-tolerance and important
characteristics of the crops.
• Plant breeding has been crucial in increasing
production of crops to meet the ever increasing
demand for food. Some well known achievements are
development of semi-dwarf wheat and rice varieties,
noblization of canes (sugarcanes), and production of
hybrid and composite varieties of maize…..
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Introduction….
• Crop improvement means combining desirable
characteristics in one plant and then multiplying it. The
job of a plant breeder is to select plants with desired
characters, cross them and then identify the offspring
that combine the attributes of both parents. Then
multiply the progeny to supply to farmers, growers or
planters.
• The modern age of plant breeding began in the early
part of the 20thC, after Mendel’s work was
rediscovered. Today
• plant breeding is a specialized technology based on
genetics. It is now clearly understood that within a
given environment, crop improvement has to be
achieved through superior heredity.
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Introduction….
• Plant breeding is the art, science and technology of changing the
heredity of plants for human welfare.
Nature of Plant Breeding:
1. Art
•In earlier days man depends on his skills and judgement in selecting
better plants. He knew nothing about the inheritance of
characters, role of environment in producing them and the basis
of variation in various plant characters. His method of selection
was designed without the understanding of principles of
inheritance.
•Therefore during primitive time plant breeding was largely an art
and very less science was involved in that.
Even today success of selection depends upon ability of the person
involved in the selection. 4

Introduction….
2. Science
• •Plant breeding is considered as the current phase of crop evolution.
As the knowledge of genetics and other related science progresses
plant breeding become less art and more science.
•Especially, discovery of Mendel̕s work in 1900 added a lot to the
knowledge of science.
•Selection of desirable plant even today is an art it depends on the skill
of a person. But alone skill is not enough, modern plant breeding is a
combined effort of art and understanding and use of genetic
principles.
3. Technology
• Product of all plant breeding activities, whether dependent on the
art or science, is improved variety, hybrids, synthetics and
composites. This product is utilized by farmers for commercial
cultivation.
• Therefore, plant breeding can be rightly viewed as a technology since
it generates a useful product.
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Role of Plant Breeding:
Human beings are dependent on the plants for:
1. Food :- Breeding of field crops provides us food either directly (food grains)
or indirectly (meat and milk).
2. Shelter :- In addition to food by produce of agriculture farms are used in
making shelter by farmers of rural areas.
3. Clothing :- Breeding for fibre crops like cotton provides clothes for the human
population.
4. Fuels :- Crops like Euphorbia and Jatropha are used for Biofuel production.
Breeding of such crops tackles the problems of energy production for rapidly
increasing human population. Now a days, Maize is also used as an important
source of Ethanol production.
5. Drugs :- Breeding of medicinal plants plays an important role in production of
many important drugs. These drugs are used for treatment of various human
and animal diseases.
6. Entertainment:- Flowers play an essential role in peoples celebrations and
everyday lives like weddings, Christmas etc. most of the medicinal plants are
seasonal in nature. Shifting the seasonal timing of reproduction is a major
goal of plant breeding efforts to produce novel varieties that are better
adapted to local environments and changing climatic conditions.
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 Plant breeding is the process by which humans change certain aspects of
plants over time in order to introduce desired characteristics
Plant Breeding Concept
Increase crop productivity

Plant Domestication
• Domestication: The process by which people try to
control the reproductive rates of animals and plants.
Without knowledge on the transmission of traits from
parents to their offspring.
• Plant Breeding: The application of genetic analysis to
development of plant lines better suited for human
purposes.
– Plant Breeding and Selection Methods to meet the food,
feed, fuel, and fiber needs of the world
– Genetic Engineering to increase the effectiveness and
efficiency of plant breeding.
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Domestication
 Plant Breeding activities began at least 10.000 years ago in the Fertile Crescent
with plant domestication
Challenges: transition from nomadic
to a sedentary lifestyle
Increase plant yield
Increase number of edible plants
(reduce toxicity)

Geography of crop domestication
• Vavilov’s eight centers of origin where crops were
first tamed.
• Turns out that centers of diversity do not coincide
with Vavilov’s centers of origin.
• Areas with lots of wild relatives and primitive
versions of modern crops can be invaluable
sources of genes for plant breeders and
geneticists.
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• Concept first devised by Vavilov in 1919
• Archaeological evidence suggests that hunter-gatherers independently
began cultivating food plants in 24 regions,….” (Purugannan and Fuller,
2009)
Centres of Plant Domestication
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What is a domestication syndrome?
A domestication syndrome describes the properties that
distinguish a certain crop from it’s wild progenitor.
Typically such characteristics are:
• larger fruits or grains
• more robust plants
• more determinate growth / increased apical
dominance
• loss of natural seed disperal
• fewer fruits or grains
• decrease in bitter substances in edible structures
• changes in photoperiod sensitivity
• synchronized flowering 18

Tomato - Fewer and Larger Fruits
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Sunflowers - reduced branching, larger seeds,
increased seed set per head
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Wheat - reduced seed shattering, increased seed size
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Squash – larger, fleshier fruits
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Corn – reduced fruitcase, softer glume, more kernels per cob, no
dispersal, reduced branching, apical dominance
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Lettuce – leaf size/shape, fewer secondary compounds
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Rice – no shattering,
larger grains
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Crop consequences of domestication:
• More ‘yield’ of desirable part.
• Non-shattering - seed are easier to harvest.
• Big seeds - domesticated bean seed are 5-8 times as large as
their wild relatives.
• Improved quality - remove or lower toxic substances.
• Increased protein, oil, sugar concentration, which means
improved flavor, storage ability.
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Methods for identifying domestication genes
1. Biparental QTL mapping
2. Association Mapping Using Unrelated Individuals
3. QTL Mapping Using Advanced Intercross
Populations
4. Genomic scans
5. Genome Resequencing and Screening for Selection
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Classical Examples…
Teosinte branched 1 (tb1) QTL of maize controls the difference in apical
dominance in maize and teosinte
tb1, it acts as transcriptional regulators, a class of genes involved in the
transcriptional regulation of cell cycle
Teosinte glume architecture1 (tga1) was identified as a QTL controlling the
formation of the casing that surrounds the kernels of the maize ancestor,
teosinte
tga1 is a member of the squamosa-promoter binding protein (SBP) family of
transcriptional regulators
Fruitweight2.2 (fw2.2) was identified as a large effect QTL controlling 30%
of the difference in fruit mass between wild and cultivated tomato
fw2.2 acts as a negative regulator of cell division in the fruit, perhaps via
some role in cell-to cell communication
Q is a major gene involved in wheat domestication that affects a suite of
traits, including
The tendency of the spike (ear) to shatter,
The tenacity of the chaff surrounding the grain, &
The spike is elongated as in wild wheat or compact like the cultivated forms
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• shattering4 (sh4) is a major QTL controlling whether the seed fall off the
plant (shatter) as in wild rice or adhere to the plant as in cultivated rice
• sh4 encodes a gene with homology to Myb3 transcription factors.
• A single amino acid change in the predicted DNA binding domain converts
plants from shattering to non-shattering
• Rc is a domestication-related gene required for red pericarp in rice
• Two independent genetic stocks of Rc revealed that the dominant red allele
differed from the recessive white allele by a 14-bp deletion within exon 6 -
originated in japonica cultivar and spread into indica cultivars.
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Super-domestication
• The processes that lead to a domesticate with
dramatically increased yield that could not be selected
in natural environments without new technologies.
• The array of genome manipulations enable barriers to
gene exchange to be overcome and have lead to super-
domesticates with
– dramatically increased yields,
– resistances to biotic and abiotic stresses, and with
– new characters for the market place.
• Hybrid rice can be considered a super-domesticate
• Conversion of a crop from C3 to C4 photosynthesis
would certainly be a super-domesticate.
Plantbreeders+GenomicScientists⇨ Superdomestication
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Domestication
‘Domestication is the process by which humans actively
interfere with and direct crop evolution.’
• It involves a genetic bottleneck:
• Often only few genes are actively selected and account
for large shifts in phenotype.
• Crops exhibit various levels of domestication.
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selective sweep
• A selective sweep is the reduction or elimination of
variation among the nucleotides in neighboring
DNA of a mutation as the result of recent and
strong positive natural selection
• A strong selective sweep results in a region of the
genome where the positively selected haplotype
(the mutated allele and its neighbours) is
essentially the only one that exists in the
population, resulting in a large reduction of the
total genetic variation in that chromosome region.
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selective sweep
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Domestication is a process
• The distinction ‘domesticated’ or ‘not domesticated’ is an over-
simplification
• Some crops have moved further along this process further than
others.
• We can recognize different levels of domestication
• How can we decide which level?
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• Different domestication traits were selected for progressively
•Distinction between selection under domestication vs. crop
diversification  more targeted, ‘conscious’ selection during
diversification
• ‘Slow’ rate of evolution of different domestication traits
despite faster rates suggested by models
• Artificial selection can be “similar across different taxa,
geographical origins and time periods”
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• Parallel evolution for “sticky glutinous varieties” in rice and
foxtail millets, all through selection at the waxy locus
• Most QTL studies suggest that many domestication traits are
controlled by a few genes of large effect – not though in
sunflower
• Population genomic studies in maize suggest 2 – 4% of genes
show evidence of artificial selection
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Domestication of Maize
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The evolution of non-shattering
in the archaeological record
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The genetic basis of the evolution of non-shattering
Non-shattering is often regarded as the hallmark of
domestication in most seed crops because it renders a plant
species primarily dependent on humans for survival and
propagation:
• rice gene sh4 (similar to the genes encoding MYB-like
transcription factors in maize)
• rice quantitative trait locus (QTL) qSH1, which encodes a
homeobox-containing protein
• the wheat gene Q, which is similar to genes of the AP2
family in other plants
• In sunflower likely controlled by multiple genes
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Domestication genes in plants
• Maize and rice domestication seem to suggest few loci of large effect
are important
• Sunflower domestication seems to suggest many loci of small to
intermediate effect are important
• 9 domestication genes in plants so far, as well as 26 other loci known
to underlie crop diversity
• Of the 9 domestication loci, 8 encode transcriptional activators.
• More than half of crop diversification genes encode enzymes.
 Domestication seems to be associated with changes in transcriptional
regulatory networks, whereas crop diversification involves a larger
proportion of enzyme-encoding loci (lots of them loss-of-function
alleles).
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The role of polyploidy in domestication
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Towards resolving the genetic basis of
domestication in the Compositae
Artificial selection
through domestication
but HOW ?
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Some fundamental questions in domestication genetics
 Which genes show strong signs of selection in different crops?
Can we see common patterns in taxa that have been domesticated
for similar purposes?
 Can we see dissimilar categories of genes under selection in
different crop types despite their close phylogenetic relationship (e.g.
sunflower and jerusalem artichoke)?
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Bioinformatics pipeline:
Methodological ‘bottom-up’ approach
1.) Input: EST libraries of crop, progenitor and
outgroup
2.) Genes that are orthologous in all taxa are
identified
3.) These genes are scanned for signs of
strong positive selection
4.) Such genes are compared to all known
proteins in Arabidopsis
5.) Functional characteristics of best fits in Arabidopsis genomic database
(TAIR) are annotated 52

Some preliminary results
Preliminary results from candidate domestication gene search in
Compositae crops:
• Several stress response genes are under selection in leaf and oil
seed crops
• Other interesting candidate domestication genes:
 safflower: fatty acid metabolism
 sunflower: nitrate assimilation
 Jerusalem artichoke: lateral root formation
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What to do with candidate genes?
Confirm their role underlying traits
- functional analysis (introgression/transgenes)
- expression
- population genetic work
confirm associations with fitness
association mapping with traits of interest
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What to do with candidate genes?
Applications:
breeding / improvement
conservation of genetic diversity
identification of taxon boundaries
understanding adaptation/domestication
comparative analysis – other taxa
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Crop improvement
• Phenotype – based selection
– Slow, ineficient but can be effective
• Using genetics to inform breeding
– Marker-assisted selection
– Marker-assisted introgression
• Transformation
– Efficient (if you have the gene) but controversial
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Transgenics controversy
• Advantages:
– Targeted to specific gene
– Any gene can be changed / introduced from any species
– Fast and efficient
• Disadvantages:
– Safety issues
– Regulations / legal issues
– Requires expertise and technology
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Transgenics controversy
• Advantages:
– Huge improvements in phenotype of interest possible
– Yield improvements
– Health / nutrition benefits
– Reduce herbicide / pesticide / fertilizer use
– New products – pharmaceuticals, chemicals, etc.
• Disadvantages:
– Little regulation for health/environmental safety
– Loss of genetic diversity
– Reliance on big seed companies
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Types of crops:
• Grain crops - wheat, rice, corn, sorghum, barley, oats.
• Oil crops - olive, linseed, sesame, sunflower, soybean,
coconut, palm, corn, peanut, canola.
• Fiber crops - cotton, flax, hemp, jute, kenaf, sisal.
• Forage crops - alfalfa, clovers, other legumes, many
grasses, including tall fescue.
• Spice / drug crops - tobacco, black pepper, cinnamon.
• Fruit crops, vegetable crops, ornamentals, forest
trees, etc.
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9000 BC First evidence of plant domestication in the hills above the
Tigris river
1694 Camerarius first to demonstrate sex in (monoecious) plants and suggested
crossing as a method to obtain new plant types
1714 Mather observed natural crossing in maize
1761-1766 Kohlreuter demonstrated that hybrid offspring
received traits from both parents and were intermediate in
most traits, first scientific hybrid in tobacco
1866 Mendel: Experiments in plant hybridization
1900 Mendel’s laws of heredity rediscovered
1944 Avery, MacLeod, McCarty discovered DNA is hereditary
material
1953 Watson, Crick, Wilkins proposed a model for DNA
structure
1970 Borlaug received Nobel Prize for the Green Revolution
Berg, Cohen, and Boyer introduced the recombinant DNA
technology
1994 ‘FlavrSavr’ tomato developed as first GMO
1995 Bt-corn developed
Selected milestones in plant breeding
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Landmarks in Plant Breeding
1694 1866 1953
Camerarius
crossing as a method
to obtain new plant
types
Mendel
Empirical evidence
on heredity
Watson, Crick,
Wilkins &
Rosalind Franklin
model for DNA
structure
1923
Wallace
First commercial
hybrid corn

“The Green Revolution” (1960)
Norman Borlaug
Challenge: improve wheat and
maize to meet the production
needs of developing countries
High yielding semi-dwarf, lodging
resistant wheat varieties

Future Challenges
Challenge: Increase of human
population by 60-80%, requiring to
nearly double the global food
production
Multidisciplinary Field
Biometry/
Statistics
Pathology

Challenges before Plant Breeder :
1. Increasing population: at present, the world population
stand at 6.3 billion and will reach at 10-12 billion during
the next 50-70 years.
The main problem from breeding respect is that the
population is growing faster than increases in food
productivity, to reduce the use of harmful agrochemicals
and to produce nutritious and healthful food is greater
today.
2. Squeezing arable land : Day-by-day the total arable land
for agriculture is decreasing due to urbanization and
industrial development.
Breeders have to tackle this problem by releasing
improved varieties of major crops which gives better
production per unit area.
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Challenges before Plant Breeder…
3. Erratic rainfall : esp in tropics rainfall is erratic,
unpredictable and unevenly distributed. Over 80% of the
annual rainfall is received in the four rainy months of June to
September. Therefore, varieties which can tolerate dry spells
and perform better at low water availability are needed to be
develop by Indian Breeders.
4. Mechanization:- The variety developed by plant breeders
should give response to application of fertilizers, manures,
irrigation and should be suitable for mechanical cultivation.
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Research Institutes, Universities, Governmental
Services, Private Companies, Non-Governmetal
Organizations, Breeders, Farmers…
….are working hard to breed plants for a
better agriculture with less environmental
impacts
Take-Home Message

Scientific disciplines and technologies
of plant breeding
• Genetics
• Botany
• Plant physiology
• Agronomy
• Pathology and entomology
• Statistics
• Biochemistry
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Importance of Plant breeding
• Plant breeding allowed civilization to form and
its continual success is critical to maintaining
our way of life
• Problem: Feeding 9 billion (+) people with the
same (or fewer) inputs
Same or less acreage
Same or less fertilizer, pesticides, water
Adapting to climate and environmental
change
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Goals of Plant breeding
•Plant breeding aims to improve the characteristics of plants so that
they become more desirable agronomically and economically. The
specific objectives may vary greatly depending on the crop under
consideration.
Increase the frequency of favorable alleles within a line ( favoring
additive effects)
• Increase the frequency of favorable genotypes within a line (with
dominance and interaction effects)
• Better adapt crops to specific environments
– Region-specific cultivars (high location G x E)
– Stability across years within a region (low year to-year G x E)
Food (yield and nutritional value), feed, fibre,
pharmaceuticals(antibodies),landscape, industrial need (eg.
Crops are being produced in regions to which they are not
native).
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Objectives of Plant Breeding
• Development of pure (i.e. highly inbred) lines with high
per se performance,
• Development of pure lines with high hybrid performance
(either with each other or with a testcross),
• Less emphasis on developing outbred (random-mating)
populations with improved performance
• Development of lines with high regional G x E, low year G
x E.
Note: Details among plant species vary because of
origin, mode of reproduction, ploidy levels, and
traits of greater importance and adjustments
were made to adapt to specific situations.
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Objectives of Plant Breeding...
• The prime objective of plant breeding is to develop superior plants
over the existing ones in relation to their economic use. The
objectives of plant breeding differ from crop to crop. A brief account
of some important objectives are:
1. Higher productivity/yield- Increased yield has been the ultimate
aim of most plant breeders. This can be achieved by developing
more efficient genotypes having greater physiological efficiency.
2. Improved quality- Improved quality of agricultural products has
contributed a lot to the human well-being. Quality characters vary
from one crop to another crop. For example, Grain size, colour ,
milling, and baking qualities in wheat (Triticum aestivum).
3. Disease and Insect Resistance- Resistance varieties offer the
cheapest and most convenient method of disease and insect
management. In some cases, they offer only feasible means of
control. eg. Rust in Wheat.
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4. Varieties for new seasons- The varieties for new seasons
have been developed by adjusting the growth cycle of the
variety to suit better to the available growing season.
5. Modification of agronomic characteristics- modification of
agronomic characteristics such as plant height, tillering,
branching, erect or trailing habit etc. is often desirable. For
example, dwarfness in cereals is generally associated with
lodging resistance and fertilizer responsiveness.
6. Change in maturity duration- it permits new crop
rotations and often extends the crop area. Development of
wheat varieties suitable for late planting has permitted rice-
wheat rotation. This objective is more desirable especially in
those areas where multiple cropping system has been
followed.
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7. Photo and thermo insensitivity- Development of photo
and thermo insensitive wheat and photo insensitive rice
varieties has permitted their cultivation in new areas.
8. Synchronous maturity- Synchronous maturity is highly
desirable in crops where several pickings are necessary. Eg.
Mungbean, pigeon pea, cotton etc.
9. Non-shattering characteristics- It would be of great value
in crops like mung, castor, soybean etc. where shattering is
a major problem in case of many commercial varieties.
10. Determinate growth- Development of varieties with
determinate growth is desirable in crops like mung, pigeon
pea, cotton, etc.
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11. Dormancy- Dormancy plays both beneficial and harmful
role according to the need of grower.
For example, if we want next crop immediate after harvesting
of previous crop, in such case dormancy is not required. But
if we want to store the seed for its future purpose, a period
of dormancy is essential.
12. Elimination of toxic substances: some crops have toxic
substances which must be eliminated to make them safe for
consumption.
For example, • Khesari (Lathyrus odoratus) seeds have a
neurotoxin, β- N- oxalyl - α-β- diaminopropionic acid (BOAA)
that causes paralysis in humans.
•Similarly, elimination of Erusic acid from Brassica oil and
Gossypol from seed cotton is necessary to make them fit for
consumption.
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13. Moisture Stress and Salt Tolerance:
development of varieties for a rainfed area and
saline soils would help to increase crop
production.
14. Wider Adaptability: it helps in stabilizing the
crop production over region and seasons.
15. Useful for Mechanical Cultivation: the variety
developed should give response to application of
fertilizers, manures and irrigation, suitable for
mechanical cultivation etc.
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Animal and tree breeding
• Similar goals, but since mostly outcrossing, the
goal is to create high-performing populations,
not inbred lines
• Generally speaking, inbreeding is bad in
animals and many trees
• Focus on finding those parents with the best
transmitting abilities (highest breeding values)
• Less of a G x E focus with animals, less of a
focus on line and hybrid breeding
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Special features exploited by plant breeders
• Selfing allows for the capture of specific genotypes,
and hence the capture of interactions between
alleles and loci (dominance and epistasis)
– Homozygous for selfed lines
– Heterozygous for crossed lines
• Often high reproductive output (relative to animal
breeding)
• Seeds allow for multigeneration progeny testing,
wherein individuals are chosen on the
performance of their progeny, or of their sibs
– Allows for better control over G x E by testing over
multiple sites/years
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Historical plant breeding
• Early origins
– Creation of new lines through species crosses
(allopolyploids)
– Visual selection
– Early domestication (selection for specific traits for
ease of harvesting)
• Biometrical school
– Using crosses to predict average performance
under inbreeding or crossing or response to
selection
– Better management of G x E
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Modern Breeding Tools
Increase of breeding effectiveness and efficiency
In vitro culture Genomic tools Genomic engineering

Classic/ traditional tools
• Emasculation
• Hybidization
• Wide crossing
• Selection
• Chromosome counting
• Chromosome doubling
• Male sterility
• Triploidy
• Linkage analysis
• Statistical tools
Advanced tools
• Mutagenesis
• Tissue culture
• Haploidy
• In situ hybridization
• Molecular markers
• Marker-assisted selection
• DNA sequencing
• Plant genomic analysis
• Bioinformatics
• Microarray analysis
• Primer design
• Plant transformation
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Plant Breeding Methods
Conventional breeding
• Mutation or crossing to introduce variability
• Selection based on morphological characteres
• Growth of selected seeds
Challenge: reduce the time needed to complete a breeding program

Plant Breeding Methods
Conventional Methods:
1.Plant introduction
2. Pureline selection
3. Mass selection
4. Pedigree method
5. Bulk method
6. Single Seed descent
method
7. Back cross method
8. Hetrosis breeding
Modern Methods
1. Mutation breeding
2. Polyploidy breeding
3. Transgenic breeding
4. Molecular breeding
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Symbols for basic crosses
• F: The symbol F (for filial) denotes the
progeny of a cross between two parents.
• Ⓧ: The symbol is the notation for selfing.
• S: The S notation is also used with numeric
• subscripts. In one usage S0 = F1; another
system indicates S0 = F2.
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Modern tools Used in Plant Breeding
• Molecular markers:
– Initially low density for QTL mapping, introgression of major
genes into elite germplasm
– With high-density markers, association mapping and
MAS/genomic selection
• New statistical tools:
– Mixed model methods
– Bayesian approaches to handle high-dimensional data sets
– New methods to deal with G x E
• Other technologies:
– Better standardization of field sites (laser-tilled fields, GPS,
better micro- and macro-environmental measurements)
– High throughput phenotypic scoring
– DH (double haploid) lines
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Integrated Approaches
• How do we best combine the rich history of
quantitative genetics and classical plant breeding
with the new tools from genomics and other
advances?
• Key: Quantitative genetics has all of the
machinery needed to fully incorporate these new
sources of information
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Basic steps in plant breeding
• Objective
• Germplasm
• Selection
• Evaluation
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Activities in plant breeding:
• 1. Creation of variation: variation means differences
among individuals of a population or species for a specific
character.
• Genetic variation is the source of raw material for
selection. These are heritable and are transmitted from
one generation to other. Such variation is useful in
selection.
• Success of a breeding program usually depends on the
desired genetic variation. It can be done in following ways
i.e. domestication, germplasm collection, plant
introduction, hybridization, polyploidy, mutation,
somaclonal variation and genetic engineering.
89

Activities in plant breeding….
2. Selection:
• During selection, the individual plant or group of plants
having the desired characters are picked up from a
population eliminating the undesirable ones.
• Those plants are selected which are looking promising for
the character on thee basis of phenotype.
• The selected plants are then allowed to grow for setting
their seeds.
• Seeds are selected and again a new crop is developed.
• This process is repeated again and again till the desired
result is achieved.
• Selection acts on the genetic variation present in a
population and produces a new population with improved
characters.
90

Activities in plant breeding….
3. Evaluation:
• The newly selected lines/strains/populations are tested
for yield and other traits and their performance is
compared with existing best varieties called Checks.
If the new lines/strain/population shows superior
performance to the checks, it is released and notified as
a new variety.
4. Multiplication:
• This step concerns with large scale certified seed
production of the released and notified variety.
5. Distribution:
• Certified seed is ultimately sold to the farmers who use it
for commercial crop cultivation.
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Scope of plant breeding
• From times immemorial, the plant breeding has been helping the mankind.
With knowledge of classical genetics, number of varieties have been evolved in
different crop plants.
• Since the population is increasing at an alarming rate, there is need to
strengthened the food production which is serious challenge to those scientists
concerned with agriculture.
• Advances in molecular biology have sharpened the tools of the breeders, and
brighten the prospects of confidence to serve the humanity.
• The application of biotechnology to field crop has already led to the field
testing of genetically modified crop plants. Genetically engineered rice, maize,
soybean, cotton, oilseeds rape, sugar beet and alfalfa cultivars are expected to
be commercialized before the close of 20th century.
• Genes from varied organisms may be expected to boost the performance of
crops especially with regard to their resistance to biotic and abiotic stresses.
• In addition, crop plants are likely to be cultivated for recovery of valuable
compounds like pharmaceuticals produced by genes introduced into them
through genetic engineering. It may be pointed out that in Europe hirudin, an
anti-thrombin protein is already being produced from transgenic Brassica
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What is Germplasm?
• Germplasm broadly refer to the hereditary
material (total content of gene)transmitted to the
offspring through germ cell.
• It can also be described as a collection of genetic
resources for an organism.
• For plants, the germplasm may be stored as a
seed ,stem, Callus, Whole plant in nurseries.
• In case of animals- Genes, Body parts stored in
gene bank/cryobank.
• Germplasm provide the raw material (genes)
which the breeder uses to develop commercial
crop varieties.
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Sources of germplasm for plant breeding
• Germplasm may be classified into five major types –
1. advanced (elite) germplasm,
2. improved germplasm (cultivars and varieties),
3. landraces,
4. wild or weedy relatives, and
5. Genetic stocks.
The major sources of variability for plant breeders may
also be categorized into three broad groups –
I. domesticated plants,
II. undomesticated plants, and
III. other species or genera.
95

1. Undomesticated plants
• When desired genes are not found in domesticated cultivars, plant
breeders may seek them from wild populations.
• When wild plants are used in crosses, they may introduce wild traits
that have an advantage for survival in the wild (e.g., hard seed coat,
shattering, indeterminacy)
• but are undesirable in modern cultivation. These undesirable traits
have been selected against through the process of domestication.
• Wild germplasms have been used as donors of several important
disease- and insect resistance genes and genes for adaptation to
stressful environments. The cultivated tomato has benefited from such
introgression by crossing with a variety of wild Licopersicon spp.
• Other species such as potato, sunflower, and rice have benefited from
wide crosses.
• In horticulture, various wild relatives of cultivated plants may be used
as rootstock in grafting (e.g., citrus, grape) to allow cultivation of the
plant in various adverse soil and climatic conditions.
96

2. Domesticated plants
• Domesticated plants are those plant materials that have been
subjected to some form of human selection and are grown for food or
other uses. There are various types of such material:
1. Commercial cultivars:
• There are two forms of this material – current cultivars and retired
or obsolete cultivars.
• These are products of formal plant breeding for specific objectives. It
is expected that such genotypes would have superior gene
combinations, be adapted to a growing area, and have a generally
good performance.
• The obsolete cultivars were taken out of commercial production
because they may have suffered a set back (e.g., susceptible to
disease) or higher performing cultivars were developed to replace
them.
• If desirable parents are found in commercial cultivars, the breeder has
a head start on breeding since most of the gene combinations would
already be desirable and adapted to the production environment.
97

2. Breeding materials
• Ongoing or more established breeding programs maintain
variability from previous projects.
• These intermediate breeding products are usually genetically
narrow-based because they originate from a small number of
genotypes or populations.
• For example, a breeder may release one genotype as a
commercial cultivar after yield tests.
• Many of the genotypes that made it to the final stage or have
unique traits will be retained as breeding materials to be
considered in future projects. Similarly, genotypes with unique
combinations may be retained.
98

3. Landraces
• Landraces are farmer-developed and maintained cultivars. They are
developed over very long periods of time and have coadapted gene
complexes.
• They are adapted to the growing region and are often highly heterogeneous.
• Landraces are robust, having developed resistance to the environmental
stresses in their areas of adaptation.
• They are adapted to unfavorable conditions and produce low but relatively
stable performance.
• Landraces, hence, characterize subsistence agriculture. They may be used as
starting material in mass selection or pure-line breeding projects.
99

4. Plant introductions
• The plant breeder may import new, unadapted
genotypes from outside the production region, usually
from another country (called plant introductions).
• These new materials may be evaluated and adapted
to new production regions as new cultivars, or used as
parents for crossing in breeding projects.
100

Plant introductions…
Plant introduction is the process of importing new plants or cultivars
of well-established plants from the area of their adaptation to
another area where their potential is evaluated for suitability for
agricultural or horticultural use.
First, the germplasm to be introduced is processed through a plant
quarantine station at the entry port, to ensure that no pest and
diseases are introduced along with the desired material.
Once this is accomplished, the material is released to the researcher
for evaluation in the field for adaptation.
The fundamental process of plant introductions as a plant breeding
approach is acclimatization.
The inherent genetic variation in the introduced germplasm serves as
the raw material for adaptation to the new environment, enabling the
breeder to select superior performers to form the new cultivar.
101

Plant introductions…
• When the plant introduction is commercially usable as
introduced without any modification, it is called a primary
introduction.
• However, more often than not, the breeder makes
selections from the variable population, or uses the plant
introduction as a parent in crosses. The products of such
efforts are called secondary introductions.
• Some plant introductions may not be useful as cultivars
in the new environment. However, they may be useful in
breeding programs for specific genes they carry.
• Many diseases, plant stature, compositional traits, and
genes for environmental stresses have been introduced
by plant breeders.
• As a plant breeding method, plant introductions have had
a significant impact on world food and agriculture.
102

5. Genetic stock
Genetic stock: consists of products of specialized genetic
manipulations by researchers (e.g., by using mutagenesis to
generate various chromosomal and genomic mutants).
6. Other species and genera:
• Gene transfer via crossing requires that the parents be cross-
compatible or cross-fertile.
• Crossing involving parents from within a species is usually
successful and unproblematic.
• However, as the parents become more genetically divergent,
crossing (wide crosses) is less successful, often requiring special
techniques (e.g., embryo rescue) for intervening in the process in
order to obtain a viable plant.
• Sometimes, related species may be crossed with little difficulty.
103

Germplasm enhancement
• There are occasions when breeders are compelled to look beyond the
advanced germplasm pool to find desirable genes. T
• The desired genes may reside in unadapted gene pools.
• Breeders are frequently reluctant to use such materials because the
desired genes are often associated with undesirable effects (unadapted,
unreproductive, yield reducing factors). Hence, these exotic materials
often cannot be used directly in cultivar development.
• Instead, the materials are gradually introduced into the cultivar
development program through crossing and selecting for intermediates
with new traits, while maintaining a great amount of the adapted traits.
• To use wild germplasm, the unadapted material is put through a
preliminary breeding program to transfer the desirable genes into
adapted genetic backgrounds.
• The process of the initial introgression of a trait from an
undomesticated source (wild) or agronomically inferior source, to a
domesticated or adapted genotype is called prebreeding or germplasm
enhancement.
104

The major uses of germplasm enhancement may be summarized as follows:
1. Preventions of genetic uniformity and the
consequences of genetic vulnerability.
2. Potential crop yield augmentation. History teaches us
that some of the dramatic yield increases in major
world food crops, such as rice, wheat, and sorghum,
were accomplished through introgression of
unadapted genes (e.g., dwarf genes).
3. Introduction of new quality traits (e.g., starch,
protein).
4. Introduction of disease- and insect-resistance genes.
5. Introduction of environment-resistance genes (e.g.,
drought resistance).
Prebreeding can be expensive to conduct and time
consuming as well.
105

Genetic vulnerability
• Genetic vulnerability is brought about largely by the manner in which breeders go
about developing new and improved cultivars for modern society.
• Genetic vulnerability is a term used to indicate the genetic homogeneity and
uniformity of a group of plants that predisposes it to susceptibility to a pest,
pathogen, or environmental hazard of large-scale proportions. A case in point is the
1970 epidemic of southern leaf blight (Helminthosporium maydis) in the USA that
devastated the corn industry. This genetic vulnerability in corn was attributed to
uniformity in the genetic background in corn stemming from the widespread use of
T-cytoplasm in corn hybrid seed production.
• Genetic uniformity per se is not necessarily the culprit in vulnerability of crops. In
fact, both producers and consumers sometimes desire and seek uniformity in some
agronomic traits. The key issue is commonality of genetic factors.
• Genetically dissimilar crops can share a trait that is simply inherited and that
predisposes them to susceptibility to an adverse biotic or abiotic factor.
• A case in point is the chestnut blight (Cryphonectria parasitica) epidemic that
occurred in the USA in which different species of the plant were affected.
106

Conservation of Plant Genetic Resources
Why conserve plant genetic resources?
• There are several reasons why plant genetic resources should
be conserved:
1. Plant germplasm is exploited for food, fiber, feed, fuel, and
medicines by agriculture, industry, and forestry.
2. As a natural resource, germplasm is a depletable resource.
3. Without genetic diversity, plant breeding cannot be
conducted.
4. Genetic diversity determines the boundaries of crop
productivity and survival.
5. As previously indicated, variability is the life blood of plant
breeding. As society evolves, its needs will keep changing.
Similarly, new environmental challenges might arise (e.g.,
new diseases, abiotic stresses) for which new variability
might be needed for plant improvement.
107

Conservation of Plant Genetic Resources…
• When a genotype is unable to respond fully to the cultural environment, as well as to
resist unfavorable conditions thereof, crop productivity diminishes.
• The natural pools of plant genetic resources are under attack from the activities of
modern society – urbanization, indiscriminate burning, and the clearing of virgin land
for farming, to name a few.
• These and other activities erode genetic diversity in wild populations. Consequently,
The actions of plant breeders also contribute to genetic erosion as previously indicated.
High-yielding, narrow genetic-based cultivars are penetrating crop production systems
all over the world, displacing the indigenous high-variability landrace cultivars.
Genetic erosion
• Genetic erosion can be defined as the decline in genetic variation in cultivated or
natural populations largely through the action of humans.
• Loss of genetic variation may be caused by natural factors, and by the actions of crop
producers, plant breeders, curators of germplasm repositories, and others in society at
large.
1. Natural factors:
• Genetic diversity can be lost through natural disasters such as large-scale floods, wild
fires, and severe and prolonged drought. These events are beyond the control of
humans.
108

2. Action of farmers:
• Right from the beginnings of agriculture, farmers have engaged in activities
that promote genetic erosion.
• These include clearing of virgin land in, especially, germplasm-rich tropical
forests, and the choice of planting material (narrow genetic-based cultivars).
• Farmers, especially in developed economies, primarily grow improved seed,
having replaced most or all landraces with these superior cultivars.
• Also, monoculture tends to narrow genetic diversity as large tracts of land are
planted to uniform cultivars.
• Extending grazing lands into wild habitats by livestock farmers, destroys wild
species and wild germplasm resources.
3. Action of breeders: Farmers plant what breeders develop. Some methods used
for breeding (e.g., pure lines, single cross, multilines) promote uniformity and a
narrower genetic base.
When breeders find superior germplasm, the tendency is to use it as much as
possible in cultivar development.
For example: in soybean, most of the modern cultivars in the USA can be traced
back to about half a dozen parents. This practice causes severe reduction in
genetic diversity.
109

Problems with germplasm conservation
In spite of good efforts by curators of germplasm repositories to
collect and conserve diversity, there are several ways in which diversity
in their custody may be lost.
The most obvious loss of diversity is attributed to human errors in the
maintenance process (e.g., improper storage of materials leading to
loss of variability).
Also, when germplasm is planted in the field, natural selection
pressure may eliminate some unadapted genotypes. Also, there could
be spontaneous mutations that can alter the variability in natural
populations.
Hybridization as well as genetic drift incidences in small populations
are also consequences of periodic multiplication of the germplasm
holdings by curators.
110

Germplasm collection
Planned collections (germplasm explorations or expeditions) are conducted by
experts to regions of plant origin.
These trips are often multidisciplinary, comprising members with expertise in
botany, ecology, pathology, population genetics, and plant breeding.
Familiarity with the species of interest and the culture of the regions to be
explored are advantageous.
Most of the materials collected are seeds, even though whole plants and
vegetative parts (e.g., bulbs, tubers, cuttings, etc.) and even pollen may be
collected.
Because only a small amount of material is collected, sampling for
representativeness of the population’s natural variability is critical in the
collection process, in order to obtain the maximum possible amount of genetic
diversity.
For some species whose seed is prone to rapid deterioration, or are bulky to
transport, in vitro techniques may be available to extract small samples from
the parent source.
Collectors should bear in mind that the value of the germplasm may not be
immediately discernible.
Materials should not be avoided for lack of obvious agronomically desirable
properties. It takes time to discover the full potential of germplasm.
111

Germplasm collection…
• Seed materials vary in viability characteristics. These have to
be taken into account during germplasm collection,
transportation, and maintenance in repositories.
• Based on viability, seed may be classified into two main groups
– orthodox and recalcitrant seed:
1. Orthodox seeds: These are seeds that can prolong their
viability under reduced moisture content and low
temperature in storage. Examples include cereals, pulses, and
oil seed. Of these, some have superior (e.g., okra) while
others have poor (e.g., soybean) viability under reduced
moisture cold storage.
2. Recalcitrant seeds: Low temperature and decreased moisture
content are intolerable to these seeds (e.g.,coconut, coffee,
cocoa). In vitro techniques might be beneficial to these species
for long-term maintenance.
112

Germplasm collection…
• The conditions of storage differ depending on the mode
of reproduction of the species:
1. Seed propagated species: these seeds are first dried to
about 5% moisture content and then usually placed in
hermetically sealed moisture-proof containers before
storage.
2. Vegetatively propagated species: these materials may
be maintained as full plants for long periods of time in
field gene banks, nature reserves, or botanical gardens.
Alternatively, cuttings and other vegetative parts may
be conserved for a short period of time under
moderately low temperature and humidity.
• For long-term storage, in vitro technology is used.
113

Types of plant germplasm collections
• There are four types of plant genetic resources maintained by germplasm
repositories – base collections, backup collections, active collections, and
breeders’ or working collections. These categorizations are only approximate
since one group can fulfill multiple functions.
1. Base collections:
• These collections are not intended for distribution to researchers, but are
maintained in long-term storage systems. They are the most comprehensive
collections of the genetic variability of species. Entries are maintained in the
original form. Storage conditions are low humidity at subfreezing temperatures
(−10 to −18°C) or cryogenic (−150 to −196°C), depending on the species.
Materials may be stored for many decades under proper conditions.
2. Backup collections:
• The purpose of backup collections is to supplement the base selection. In case
of a disaster at a center responsible for a base collection, a duplicate collection
is available as insurance. In the USA, the National Seed Storage Laboratory at
Fort Collins, Colorado, is a backup collection center for portions of the
accessions of the Centro Internationale de Mejoramiento de Maiz y Trigo
(CIMMYT) and the International Rice Research Institute (IRRI).
114

3. Active collections:
• Base and backup collections of germplasm are designed for long-term
unperturbed storage. Active collections usually comprise the same
materials as in base collections, however, the materials in active
collections are available for distribution to plant breeders or other
patrons upon request. They are stored at 0°C and about 8% moisture
content, and remain viable for about 10–15years. To meet this
obligation, curators of active collections at germplasm banks must
increase the amount of germplasm available to fill requests
expeditiously. Because the accessions are more frequently increased
through field multiplication, the genetic integrity of the accession may
be jeopardized.
4. Working or breeders’ collections:
• Breeders’ collections are primarily composed of elite germplasm that is
adapted. They also include enhanced breeding stocks with unique
alleles for introgression into these adapted materials. In these times of
genetic engineering, breeders’ collections include products of rDNA
research that can be used as parents in breeding programs.
115

Managing plant genetic resources
• The key activities of curators of germplasm banks
include:
1. Regeneration of accessions,
2. Characterization,
3. Evaluation,
4. Monitoring seed viability and genetic integrity
during storage, &
5. Maintaining redundancy among collections.
• Germplasm banks receive new materials on a
regular basis. These materials must be properly
managed so as to encourage and facilitate their
use by plant breeders and other researchers.
116

1. Periodic Regeneration
• The regeneration of seed depends on the life cycle and breeding system of the species
as well as cost of the activity.
• To keep costs to a minimum and to reduce loss of genetic integrity, it is best to keep
regeneration and multiplication to a bare minimum.
• It is a good strategy to make the first multiplication extensive so that ample original
seed is available for depositing in the base and duplicate or active collections.
• A major threat to genetic integrity of accessions during regeneration is contamination
(from outcrossing or accidental migration),which can change the genetic structure.
Other factors include differential survival of alleles or genotypes within the accession,
and random drift. The isolation of accessions during regeneration is critical, especially in
cross-pollinated species, to maintaining genetic integrity.
• This is achieved through proper spacing, caging, covering with bags, hand pollination,
and other techniques.
• Regeneration of wild species is problematic because of high seed dormancy, seed
shattering, high variability in flowering time, and low seed production.
• Some species have special environmental requirements (e.g., photoperiod,
vernalization) and hence it is best to rejuvenate plants under conditions similar to those
in the places of their origin, to prevent selection effect, which can eliminate certain
alleles.
Dr. Zekeria Yusuf (PhD) 117

2. Characterization:
• Curators of germplasm banks characterize their accessions, an activity
that entails a systematic recording of selected traits of an accession.
• Traditionally, these data are limited to highly heritable morphological
and agronomic traits.
• However, with the availability of molecular techniques, some germplasm
banks have embarked upon molecular characterization of their holdings.
For example, CIMMYT has used the simple sequence repeat (SSR)
marker system for characterizing the maize germplasm in their holding.
• Passport data are included in germplasm characterization. These data
include an accession number, scientific name, collection site (country,
village), source (wild, market), geography of the location, and any
disease and insect pests.
• To facilitate data entry and retrieval, characterization includes the use of
descriptors. These are specific pieces of information on plant or
geographic factors that pertain to the plant collection.
• The International Plant Genetic Resources Institute (IPGRI) has
prescribed guidelines for the categories of these descriptors. Descriptors
have been standardized for some species such as rice.
118

3. Evaluation:
• Genetic diversity is not usable without proper evaluation.
• Preliminary evaluation consists of readily observable
traits. Full evaluations are more involved and may include
obtaining data on cytogenetics, evolution, physiology, and
agronomy.
• More detailed evaluation is often done outside of the
domain of the germplasm bank by various breeders and
researchers using the specific plant traits such as disease
resistance, productivity, and quality of product are
important pieces of information for plant breeders.
• Without some basic information of the value of the
accession, users will not be able to make proper requests
and receive the most useful materials for their work.
119

4. Monitoring seed viability and genetic integrity:
• During storage, vigor tests should be conducted at appropriate
intervals to ensure that seed viability remains high. During
these tests, abnormal seedlings may indicate the presence of
mutations.
5. Exchange:
• The ultimate goal of germplasm collection, rejuvenation,
characterization, and evaluation is to make available and
facilitate the use of germplasm. There are various computer-
based genetic-resource documentation systems worldwide,
some of which are crop-specific.
• These systems allow breeders to rapidly search and request
germplasm information. There are various laws regarding,
especially, international exchange of germplasm.
• Apart from quarantine laws, various inspections and testing
facilities are needed at the checkpoint of germplasm.
120

6. Germplasm storage technologies:
• Once collected, germplasm is maintained in the most appropriate form
by the gene bank with storage responsibilities for the materials. Plant
germplasm may be stored in the form of pollen, seed, or plant tissue.
• Woody ornamental species may be maintained as living plants. Indoor
maintenance is done under cold storage conditions, with temperatures
ranging from −18 to −196°C.
i. Seed storage:
• Seeds are dried to the appropriate moisture content before being
placing in seed envelopes. These envelopes are then arranged in trays
that are placed on shelves in the storage room.
• The storage room is maintained at −18°C, a temperature that will keep
most seeds viable for up to 20 years or more.
• The curator of the laboratory and the staff periodically sample seeds of
each accession to conduct a germination test.
• When germination falls below a certain predetermined level, the
accession is regrown to obtain fresh seed.
121

ii. Field growing:
• Accessions are regrown to obtain fresh seed or to increase existing
supplies (after filling orders by scientists and other clients). To keep
the genetic purity, the accessions are grown in isolation, each plant
covered with a cotton bag to keep foreign sources of pollen out and
also to ensure self-pollination.
iii. Cryopreservation:
• Cryopreservation or freeze-preservation is the storage of materials
at extremely low temperatures of between −150 to −196°C in liquid
nitrogen. Plant cells, tissue, or other vegetative material may be stored
this way for a long time without loosing regenerative capacity.
• Whereas seed may also be stored by this method, cryopreservation is
reserved especially for vegetatively propagated species that need to be
maintained as living plants.
• Shoot tip cultures are obtained from the material to be stored and
protected by dipping in a cryoprotectant (e.g., a mixture of sugar and
polyethylene glycol plus dimethylsulfoxide).
122

iv. In vitro storage:
• Germplasm of vegetatively propagated crops is normally stored and distributed to
users in vegetative forms such as tubers, corms, rhizomes, and cuttings. However, it
is laborious and expensive to maintain plants in these forms.
• In vitro germplasm storage usually involves tissue culture. There are several types of
tissue culture systems (suspension cells, callus, meristematic tissues).
• To use suspension cells and callus materials, there must be an established system of
regeneration of full plants from these systems, something that is not available for all
plant species yet. Consequently, meristem cultures are favored for in vitro storage
because they are more stable.
• The tissue culture material may be stored using the method of slow growth
(chemicals are applied to retard the culture temperature) or cryopreservation.
v. Molecular conservation:
• The advent of biotechnology has made it possible for researchers to sequence DNA
of organisms. These sequences can be searched for genes at the molecular level.
Specific genes can be isolated by cloning and used in developing transgenic products.
123

What is germplasm conservation?
• Plant germplasm is the genetic source material used by
the plant breeders to develop new cultivars.
They may include :
• Seeds
Other plant propagules such as
• Leaf
• Stem
• Pollen
• Cultured cells
Which can be grown into mature plant?
• Germplasm provide the raw material (genes) which the
breeder used to develop commercial crop varieties.
124

Need for Conservation of plant Germplasm
•• Storage of Economically important, endangered, rare species and
make them available when needed.
• The conventional methods of storage failed to prevent losses caused
due to various reasons.
• Human dependence on plant species for food and many different
uses. E.g. Basic food crops, building materials, oils, lubricants,
rubber & other latexes, resins, waxes, perfumes, dyes fibres and
medicines.
• Species extinction and many others are threatened and endangered
– deforestation.
• Great diversity of plants is needed to keep the various natural
ecosystems functioning stably– interactions between species.
• Aesthetic value of natural ecosystems and the diversity of plant
species.
125

In-situ Preservation
Preservation of the germplasm in their natural
habitat
The conservation of domesticated and cultivated
species in the farm or in the surroundings.
However, there is a heavy loss or decline of
species, populations and ecosystem composition,
which can lead to a loss of biodiversity, due to
habitat destruction and the transformations of
these natural environments; therefore, in situ
methods alone are insufficient for saving
endangered species.
128

Ex-situ preservation
1. To maintain the biological material outside their
natural habitats.
2. Storage in seed banks, field gene collections, in
vitro collections and botanical gardens
3. Ex situ conservation is a viable way for saving
plants from extinction, and in some cases, it is the
only possible strategy to conserve certain species
4. In vitro conservation is especially important for
vegetatively propagated and for non-orthodox
seed plant species
130

Disadvantages of Ex-situ Conservation
• Some plants do not produce fertile seeds.
• Loss of seed viability
• Seed destruction by pests, etc.
• Poor germination rate.
• This is only useful for seed propagating plants.
• It’s a costly process.
133

In vitro method for germplasm conservation
• In vitro method employing shoots, meristems and
embryos are ideally suited for the conservation of
germplasm. The plant with recalcitrant seeds and
genetically engineered can also be preserved by this in
vitro approach.
There are several advantages associated with in vitro
germplasm conservation
Large quantities of material can be preserved in small
space
The germplasm preserved can be maintained in an
environment free from pathogens.
It can be protected against the nature’s hazards
From the germplasm stock large number of plants can be
obtained whenever needed.
134

CRYOPRESERVATION
• Cryopreservation (Greek, krayos-frost) literally mean in the frozen
state.
• The principal involved in cryopreservation to bring the plant cells and
tissue cultures to a zero metabolism or nondividing state by reducing
the temperature in the presence of caryoprotectants.
• Cryopreservation broadly means the storage of germplam at very low
temperature.
1. Over solid carbon dioxide (at 79oC)
2. Low temperature deep freezers (at -80oC)
3. In liquid nitrogen (at -196oC)
•Among these the most commonly used cryopreservation is by
employing liquid nitrogen. At the temperature of liquid nitrogen (-
196oC), the cell stay in a completely inactive state and thus can be
conserved for longer period.
In fact cryopreservation has been successfully applied for germplasm
conservation . Plant species e.g. rice, wheat, peanut, sugarcane
,coconut.
136

Principle of Cryopreservation
Cryopreservation is the only technique that ensures the safe
and cost-efficient long-term conservation of various
categories of plants, including non-orthodox seed species,
vegetatively propagated plants, rare and endangered species
and biotechnology products.
Storage of Biomaterial at ultra low temperature by means of
slow freezing.
In all cryopreservation processes, water removal plays a
central role in preventing freezing injury and in maintaining
post-thaw viability of cryopreserved material.
There are two types of cryopreservation protocols that
basically differ in their physical mechanisms:
1. Classical cryopreservation
2. Vitrification
137

Technique of cryopreservation
• The cryopreservation of plant cell culture followed
the regeneration of plants broadly involves the
following stages
1. Development of sterile tissue culture.
2. Addition of cryoprotectant and pretreatment
3. Freezing
4. Storage
5. Thawing
6. Reculture
7. Measurement of survival/viability
8. Plant regeneration
138

Classical cryopreservation
• In this procedure, cooling is performed in the presence of ice.
• It involves cryoprotection by using different cryoprotective solutions
combined or not with pregrowth of material and followed by slow cooling
(0.5–2.0°C/min) to a determined prefreezing temperature (usually around
−40°C),rapid immersion of samples in liquid nitrogen, storage, rapid thawing
and recovery.
• They are generally operationally complex, since they require the use of
sophisticated and expensive programmable freezers.
• Cryopreservation following classical protocols induces a freeze dehydration
process using a slow freezing regime. As temperature decrease slowly, ice is
initially formed in the extracellular solution and this external crystallization
promotes the efflux of water from the cytoplasm and vacuoles to the
outside of the cells where it finally freezes.
• Therefore, cell dehydration will depend on the cooling rate and the
prefreezing temperature set up before immersion of samples to liquid
nitrogen.
• Classical cryopreservation techniques have been successfully applied to
undifferentiated culture systems of different plant species, such as cell
suspensions and calluses.
141

Vitrification
• In this procedure, cooling normally takes place
without ice formation
• The process where formation of ice cannot take
place because of the Concentrated aqueous
solution which permit ice crystal nucleation.
Instead, water solidifies into an amorphous ‘glassy’
state.
• The vitrification-based procedures involve cell
dehydration prior to cooling by exposure of
samples to highly concentrated cryoprotective
media (usually called plant vitrification solutions,
PVS) and/or by air desiccation.
142

Vitrification…
• Cooling rate may be rapid or ultra-rapid, depending on
how samples are immersed into liquid nitrogen.
• Vitrification per se is a physical process, defined as the
transition of the liquid phase to an amorphous glassy solid
at the glass transition (Tg) temperature .
• This glass may contribute to preventing tissue collapse,
solute concentration and pH alterations during
dehydration.
• Therefore, the freeze-induced dehydration step
characteristic of classical procedures is eliminated and the
slow freezing regime is replaced by a rapid or ultra-rapid
cooling process, characteristic of the vitrification-based
protocols.
143

1. Development of sterile tissue culture
• The selection of plant species and the tissue with particular
reference to the morphological and physiological characters
largely influence the ability of the explants to survive in
cryopreservation .
• Any tissue from a plant can be used for cryopreservation
e.g. meristems, embryos, endosperm, ovules, seeds,
culture plants.
144

2. Addition of cryoprotectant
• Cryoprotectant are the compound that can be prevent the
damage caused to cells by freezing or thawing.
• There are several cryoprotectant which include (DMSO),
glycerol, ethylene, propylene, sucrose, mannose, glucose,
proline and acetamide. Among these DMSO, sucrose & glycerol
are most widely used.
145

3. Freezing
• The sensitivity of the cell to low temperature is variable
and largely depends on the plant species.
• Four different types of freezing method are used:
1. Slow freezing method: the tissue is slowly frozen at 0.5-
5°C/min from 0°C to -100°C, and then transferred to liquid
nitrogen.
2. Rapid freezing method: decrease in temperature up to -
300 to -1000°C.
3. Stepwise freezing method: intermediate temperature for
30 min. and rapidly cool.
4. Dry freezing method: reported that non-germinated dry
seeds can survive freezing at low temperature in contrast
to water imbibing seeds which are susceptible to
cryogenic injuries.
146

4 : Storage
• Maintenance of the frozen cultures at the specific
temperature is as important as freezing .
• In general the frozen cells/tissues are kept for storage at
temperatures in the range of -72 to -196°C. Storage is ideally
done in liquid nitrogen refrigerator – at 150°C in the vapour
phase, or at -196°C in the liquid phase.
• The ultimate objective of storage is to stop all the cellular
metabolic activities and maintain their viability. For long term
storage temperature at -196°C in liquid nitrogen is ideal.
147

5 : Thawing
• Thawing is usually carried out by plunging the frozen
samples in ampoules into a warm water (temp 37 – 45°C)
bath with vigorous swirling.
• By this approach, rapid thawing (at the rate of 500-750°C
min¯¹) occurs, and this protects the cells from the
damaging effects ice crystal formation.
• As the thawing occurs (ice completely melts) the ampoules
are quickly transferred to a water bath at temperature 20-
25°C. This transfer is necessary since the cells get
damaged if left for long in warm (37-45°C) water bath.
148

6. Reculture:
• In general thawned germplasm is washed several times
to remove cryoprotectant. The material is then
recultured in a fresh media.
7. Plant regeneration:
• The ultimate purpose of cryopreservation of germplasm
is to regenerate the desired plant.
• For appropriate plant growth and regeneration, the
cryopreserved cell/tissues have to be carefully nursed,
grown.
• Addition of certain growth promoting substances,
besides maintenance of appropriate environmental
conditions is often necessary for successful plant
regeneration.
149

Applications of germplasm conservation
• Plant materials (cell/tissue) of several species can be cryopreserved and
maintained for several years, and used as and when needed.
• Cryopreservation is an ideal method for long term conservation of cell
culture which produce secondary metabolites e.g. medicines
• Disease (pathogen) free plant material can be frozen and propagated
whenever required.
• Recalcitrant seeds can be maintained for long.
• Conservation of somaclonal and gametoclonal variation in culture.
• Plant material from endangered species can be conserved.
• Cryopreservation is a good method for the selection of cold resistant
mutant cell lines which could develop into frost resistant plant .
• Disease free plants can be conserved and propagated.
• Recalcitrant seeds can be maintained for long time.
• Endangered species can be maintained.
• Pollens can be maintained to increase longitivity.
• Rare germplasm and other genetic manipulations can be stored.
150

Limitations of germplasm conservation
• The expensive equipment needed to provide
controlled and variable rates of cooling/warming
temperatures can however be a limitation in the
application of in vitro technology for large scale
germplasm conservation.
• Formation of ice crystal inside the cell should be
prevented as they cause injury to the cell.
• Sometimes certain solutes from the cell leak out
during freezing.
• Cryoprotectant also effect the viability of cells.
151

Concept of gene pools of cultivated crops
Harlan and de Wet proposed a categorization of gene pools of cultivated crops
according to the feasibility of gene transfer or gene flow from those species to
the crop species. Three categories were defined, primary, secondary, and tertiary
gene pools:
1. Primary gene pool (GP1). GP1 consists of biological species that can be
intercrossed easily (interfertile) without any problems with fertility of the
progeny. That is, there is no restriction to gene exchange between members of
the group. This group may contain both cultivated and wild progenitors of the
species.
2. Secondary gene pool (GP2). Members of this gene pool include both cultivated
and wild relatives of the crop species. They are more distantly related and have
crossability problems. Nonetheless, crossing produces hybrids and derivatives
that are sufficiently fertile to allow gene flow. GP2 species can cross with those
in GP1, with some fertility of the F1, but more difficulty with success.
3. Tertiary gene pool (GP3). GP3 involves the outer limits of potential genetic
resources. Gene transfer by hybridization between GP1 and GP3 is very
problematic, resulting in lethality, sterility, and other abnormalities. To exploit
germplasm from distant relatives, tools such as embryo rescue and bridge
crossing may be used to nurture an embryo from a wide cross to a full plant and
to obtain fertile plants.
152

Seed Technology
• Seed technology is the creation and application of the knowledge on seed
for its better usage in agriculture.
• Seed technology refers to methods or techniques used to maintain the
quality of seed from harvest till it is germinated.
Scope of Seed Technology
1. Seed technology encompasses all activities carried out to enhance
storability, germinability, vigour and health of the seed.
2. Activities include harvesting, transporting, handling, storage, testing,
grading, documentation, processing of seeds and germination of seeds.
Classes of seed
1. Nucleus or Basic Seed
2. Breeder seed
3. Foundation seed
4. Certified seed
155
Haramaya University

Nucleus or Basic Seed
• Nucleus seed (or basic seed) is the original or first seed (= propagating
material) of a variety available with the producing breeder or any other
recognized breeder of the crop.
• This seed has 100% genetic & physical purity along with high standards
of all other seed quality parameters.
• When a new variety is released there is very little seed. There may be
only a handful of seed selected by the breeder from individual plants.
This seed is the basis of a variety and is known as the Nucleus Stock.
• This nucleus stock must be managed with great care so that all seed
produced from it remains true to the new variety. This is a most
important step and is the responsibility of the plant breeder who
developed the variety.
• The nucleus stock seed is not available to farmers. The next step in the
chain from plant breeder to farmer is that the plant breeder develops
Breeder Seed.
157
Haramaya University

BREEDER SEED
• Breeder seed is the seed of the highest purity of the new variety.
• It is produced by the breeder and provided by the breeder’s institution
to agencies for further multiplication.
• If you are from a non-governmental organization (NGO) seed business
or a private company that is producing seed, you may need to purchase
breeder seed from a research institution. Breeder seed is the most
expensive seed to buy.
• Breeder seed : seed or vegetative propagating material directly
produced or controlled by the originating plant breeder or institution.
• Breeder seed provides the source for the increase of foundation seed. It
is usually limited in quantity.
• Breeder seed is the progeny of the nucleus seed and is the source for
foundation seed.
• Its production is directly controlled by the originating plant breeder
who developed the variety, or any other institution or qualified breeder
recognized by the authorities.
158
Haramaya University

FOUNDATION SEED
• Foundation seed is the seed produced from growing breeder
seed. It is produced by trained officers of an agricultural station to
national standards and handled to maintain the genetic purity of
the variety. It may be produced by a government seed production
farm or a private organization – this will depend on the
regulations of the country. Foundation seed is less expensive than
breeder seed.
• Also know as elite or basic seed. It is the direct increase form
breeder seed. The genetic identity and purity of the variety is
carefully maintained in foundation seed.
• Foundation seed is the source of certified seed.
159
Haramaya University

REGISTERED SEED
Registered seed is produced from growing foundation seed. It is
grown by selected farmers in a way that maintains genetic purity.
Production has undergone field and seed inspections by Seed
Inspectors to ensure conformity with standards.
160
Haramaya University

CERTIFIED SEED
• Certified seed is produced from growing foundation, registered
or certified seed. It is grown by selected farmers to maintain
sufficient varietal purity.
• Production is subject to field and seed inspections prior to
approval by the certifying agency. Harvest from this class is used
for producing again.
• Certified seed is the seed, which is certified by a Seed
Certification Agency
• Generally, it is known as the progeny of foundation seed and its
production is so handled as to maintain specified genetic
identity and purity standards as prescribed for the crop being
certified.
161
Haramaya University

Requirements of Certified seed
• Seed has to meet certain rigid requirements before it can be
certified for distribution.
• Seed must be of an improved variety released by either Central
or State Variety Release Committee for general cultivation and
notified by the Ministry of Agriculture.
• Genetic purity.
• Physical purity.
• Germination.
• Freedom from Weed seeds.
• Freedom from Diseases.
• Optimum Moisture Content.
162
Haramaya University

QUALITY DECLARED SEED
• Truthfully labeled seed or Quality Declared Seed is produced
from foundation, registered or certified seed.
• It is not subject to inspection by a certifying agency. As this
seed is not inspected, its quality is dependent on the good
reputation of the farmer who has grown the seed. His good
name in the village is important.
163
Haramaya University

MODE OF REPRODUCTION
• Reproduction refers to the process by which living organisms produces
offsprings of similar kind (species). In crop plants, the mode of reproduction is
of two types: viz. 1) asexual reproduction & 2) sexual reproduction.
• Asexual reproduction refers to the multiplication of plants without the
fusion of male and female gametes .It can occur either by vegetative plant
parts or by vegetative embryos which develop without sexual fusion
(apomixis). Thus asexual reproduction is of two types, viz., a) vegetative
reproduction and (b) apomixis.
• Vegetative reproduction refers to multiplication of plants by means of various
vegetative plant parts. Vegetative reproduction is again of two types, viz., a)
natural vegetative reproduction and (b) artificial vegetative reproduction.
• Natural vegetative reproduction is the multiplication of certain plants by
underground stems, sub aerial stems, roots and bulbils naturally. In some crop
species, underground stems (a modified group of stems) give rise to new
plants.
164

• Artificial vegetative reproduction occurs by cuttings of stem and roots, and by
layering, grafting etc.
• Stem cuttings: Sugarcane , grapes, roses etc.
• Root cuttings: Sweet potato, citrus, lemon, etc.
• Layering, grafting: Fruit and ornamental crops.
Example for underground stems
• Rhizome: Turmeric, Ginger
• Tuber: Potato
• Corm: Colocasia
• Bulb: Garlic, onion
Example for sub aerial stems
• Runner: Sweet potato ,Strawberry
• Sucker: Banana
• Stolon: taro, passion flower
• Bulbils: Garlic
165

• Apomixis refers to the development of seed without fertilization.The embryos
are developed without fertilization. Apomixis is found in many crop species and
is of two types based on nature.
• Reproduction in some species occurs only by apomixis. This apomixis is termed
as obligate apomixis. But in some species sexual reproduction also occurs in
addition to apomixis. Such apomixis is known as facultative apomixis.
• There are four types of apomixis based on origin: viz. 1) parthenogenesis, 2)
apogamy, 3) apospory and 4) adventive embryony.
• Parthenogenesis refers to development of embryo from the egg cell without
fertilization.
• Apogamy –when the embryo originates from either synergids or antipodal cells
of the embryo sac it is called as apogamy.
• Apospory- In apospory, some diploid cells of ovule lying outside the embryo
sac develops into another unreduced embryo sac through a series of mitotic
divisions and without meiosis. The embryo then develops directly from the
diploid egg cell of such an embryo sac without fertilization.
• Adventive embryony - The development of embryo directly from the diploid
cells of ovule lying outside the embryo sac belonging to either nucellus or
integuments is referred to as adventive embryony. It does not involve the
production of another embryo sac. 166

Sexual reproduction:
• Reproduction by which embryo is developed by the fusion of
male and female gamete is known as sexual reproduction. All
the seed propagating species belong to this group.
MODE OF POLLINATION
• The process by which pollen grains are transferred from anthers
to stigma is referred to as pollination.
• Pollination is of two types, viz., 1) Autogamy or self
pollination and 2) Allogamy or cross pollination.
A. Autogamy
• Transfer of pollen grains from the anther to the stigma of same
flower is known as autogamy or self pollination. Autogamy is
the closest form of inbreeding.
• Autogamy leads to homozygosity. Such species develop
homozygous balance & do not exhibit significant inbreeding
depression.
167

There are several mechanisms which promote autogamy.
1. Bisexuality is the presence of male and female organs in the same
flower. The presence of bisexual flowers is a must for self pollination.
All the self pollinated plants have hermaphrodite flowers. Eg. Rice
2. Homogamy Maturation of anthers and stigma of a flower at the same
time is called homogamy which is essential for self-pollination Eg.
Bhindi
3. Cleistogamy is when pollination and fertilization occur in an unopened
flower bud. It ensures self pollination and prevents cross pollination.
Eg. cowpea
4. Chasmogamy is the condition when flower opening occurs only after
the completion of pollination. This also promotes self pollination. Eg.
sesame.
5. Position of Anthers: When stigmas are surrounded by anthers self
pollination is ensured. Eg. tomato and brinjal. In some legumes, the
stamens and stigma are enclosed by the petals in such a way that self
pollination is ensured. Eg. greengram, blackgram, soybean, chickpea
and pea.
168

B. Allogamy
• The transfer of pollen grains from the anther of one plant to
the stigma of another plant (cross pollination).
• Allogamy is the common form of out-breeding and leads to
heterozygosity. Such species develop heterozygous balance
and exhibit significant inbreeding depression on selfing.
The conditions which promote allogamy are as follows:
1. Dicliny refers to unisexual flowers. This is of two types: viz.
i) monoecy and ii) dioecy.
• When male and female flowers are separate but present in the
same plant, it is known as monoecy.
• In some crops, the male & female flowers are present in the
same inflorescence such as in coconut, mango, castor &
banana. In some cases, they are on separate inflorescence as
in maize, cucurbits, cassava and rubber. When staminate and
pistillate flowers are present on different plants, it is called
dioecy as in papaya, nutmeg and date palm
169

2. Dichogamy refers to the maturation of anthers and
stigma of the same flowers at different times.
• Dichogamy promotes cross pollination even in the
hermaphrodite species.
• Dichogamy is of two types: viz. i) protogyny and ii)
protandry.
• When pistil matures before anthers, it is called
protogyny such as in black pepper and pearl millet.
• When anthers mature before pistil, it is known as
protandry as in coconut and several other species.
170

3. Heterostyly: When styles and filaments in a flower are of
different lengths, it is called heterostyly. It promotes cross
pollination, such as linseed.
4. Herkogamy:
Hinderance to self-pollination due to some physical barriers such
as presence of hyline membrane around the anther is known as
herkogamy. Such membrane does not allow the dehiscence of
pollen and prevents self-pollination such as in alfalfa.
5. Self incompatibility is the inability of fertile pollens to fertilize
the same flower.
It prevents self-pollination and promotes cross pollination. Self
incompatibility is found in several crop species like Brassica,
Radish, Nicotiana, and many grass species. It is of two types
sporophytic and gametophytic.
171

6. Male sterility: in some species, the pollen grains are non
functional. Such condition is known as male sterility. It
prevents self-pollination & promotes cross pollination. It
is of three types: viz. genetic, cytoplasmic and
cytoplasmic genetic. It is a useful tool in hybrid seed
production.
• Self incompatibility and male sterility are the two genetic
mechanisms favouring cross pollination.
• The mode of pollination plays an important role in plant
breeding. It has impact on five important aspects :
1) gene action
2) genetic constitution
3) adaptability
4) genetic purity
5) transfer of genes. 172

Estimation of Genetic variability parameters
173

VARIANCES AND COVARIANCES
• The variability present in a population is of polygenic
nature and this polygenic variation is of three types
• 1) Phenotypic
• 2) Genotypic
• 3) Environmental
• The statistical procedure which separates (or) splits the
total variation into different components is called
analysis of variance (or) ANOVA.
• ANOVA is useful in estimating the different
components of variance. It provides basis for the test of
significance and it is carried out only with replicated
data obtained from standard statistical experimental
results.
175

Dr. Zekeria Yusuf (PhD) 176
Genetic variability at a single location

F (calculated) is compared with F(Table) value by looking at the F table for
replication df(r-1) and error df values(r-1)(t-1). If the calculated F value is
greater than F(Table value) then it is significant.
Genotypic variance: It is the inherent variation which remains unaltered by the
environment. It is the variation due to genotypes. It is denoted by VG and is
calculated using the formula:
177

178
Genetic variability at multilocations

Heritability and genetic advance are important selection parameters and
heritability estimate along with genetic advance are interpreted as
follows
1) High heritability accompanied with high genetic advance indicates
heritability is due to additive (or) fixable variation and selection may
be effective.
2) High heritability accompanied with low genetic advance indicates non
additive gene action and selection for such characters may not be
rewarding.
3) Low heritability accompanied with high genetic advance reveals that
characters are governed by fixable gene effects and low heritability is
due to high environmental influence and selection may be effective.
4) Low heritability accompanied with low genetic advance indicates that
character is highly influenced by environment and selection is
ineffective.
183

ESTIMATION OF HETEROSIS AND INBREEDING DEPRESSION
• refers to the superiority of F1 in one or more characters over its
parents. It is also defined as increase in fitness and yield over its
parental values.
• Heterosis, or hybrid vigor, or outbreeding enhancement, is the
improved or increased function of any biological quality in a
hybrid offspring. It is the occurrence of a genetically superior
offspring from mixing the genes of its parents.
• It is also called as hybrid vigour. The three main causes of
heterosis are over dominance, dominance and epistasis, of this
dominance is the widely accepted one.
• In crop plants there are three main ways for fixation of heterosis
i.e. asexual reproduction, polyploidy and apomixis
185

Manifestations of Heterosis
1. increased heterozygosity
2. increased size and productivity in plants
3. Greater resistance to diseases, insects and environmental
factors
4. Early maturity when compared to either of the parents.
186

. Four different methods are used to estimate heterosis.
187

• Giving the mean yield of two inbred strains A=80kg , B= 50 and
F1 is 90kg, calculate i. Hmp; ii. Hbp
Solution:
1. mp =(80+50)/2= 65
Hmp = (F – mp)/mp= (90 – 65)/65 =0.3846
• This implies that the hybrid vigour is 38.46%
2. Hbp = (F – bp)/bp= (90 – 80)/80 =0.125
• Herobeltiosis is 12.5%
• The better parent heterosis is more significant as far as breeding is
concerned because individual progenies are more superior to the
better parent.
188

Gene action
• Alleles may interact with one another in a number of ways to produce
variability in their phenotypic expression. The following models may help us
understand various modes of gene action.
• Additive gene action: absence of DOMINANCE in case of single locus.
• Therefore, the breeding procedure chosen for a crop genotype will depend on
the prevalence of gene action e.g additive gene action will be effective in
accumulating favourable alleles in breeding materials especially in self-
pollinating crops.
• Heritability in this narrower sense is the ratio of the additive genetic variance
to the phenotypic variance:
189

Additive gene action
• When both alleles contribute for the phenotype.
• Thus, offspring phenotype resemble their parents
• It can be directly inherited from parents to their offsprings as
phenotype is effect of each alleles from both parents being added
together.
• It can be fixable
Dominance gene action:
 In which one allele contributes more or less to the final phenotype.
 Dominance variance is not directly inherited from parents to their
offsprings. Since it is due to interaction of genes from both parents
within individuals, and of course only one allele is passed from
each parent to offsprings.
191

• Dominance variance has two components : variance due to
homozygous alleles (w/c is additive) and variance due to
heterozygous genotypic values.
• Dominance effects are deviations from additivity that make
heterozygote resemble one parent more than the other.
• When dominance is complete heterozygote is equal to
homozygotes in effects (i.e. Aa=AA).
• Breeder can’t distinguish between heterozygous and homozygous
phenotypes as a result both Aa and AA will be selected.
• Thus fixing superiour genes will be less effective with dominance
gene action since Aa will segregate in next generation.
192

The variance component method of estimating heritability uses the statistical procedure of
analysis of variance (ANOVA). Variance estimates depend on the types of populations
in the experiment.
Estimating genetic components suffers from certain statistical weaknesses. Variances are
less accurately estimated than means. Also, variances are unrobost and sensitive to
departure from normality. An example of a heritability estimate using F2 and backcross
populations is as follows:
197

Overdominance gene action
• The hetrozygote is more valuable than either homozygotes.
• Exists when each allele at a locus produces a separate effect on
the phenotype and either combined effect exceeds independent
effect of the alleles.
• breeder can fix overdominance effects only in F1 generation
through apomixis or through chromosome doulbling of the
product of wide cross.
Epistasis gene action (nonalleleic interaction)
-interaction between alleles at different loci.
VG = VA + VD + VI
Vp= VG + VE
For inbreeding population, Vp =Ve, since Vg= 0
Vp = VA + VD + VI + VE + Vgxe
Narrow sense heritability =
199

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