PRACTICAL MANUAL

OF

FUNDAMENTALS OF PLANT BREEDING

(GPB 3.3)
THIRD SEMESTER B.Sc. (Hons.) Agriculture
(As per ICAR Fifth Dean Committee Recommendations)

FUNDAMENTALS OF PLANT BREEDING

(GPB 3.3)
THIRD SEMESTER B.Sc. (Hons.) Agriculture
(As per ICAR Fifth Dean Committee Recommendations)

PREPARED BY
Dr. R. S. Parmar, Assistant Professor
AND
Dr. M. A. Vaddoria, Principal and Dean
Department of Genetics and Plant Breeding
College of Agriculture,
Junagadh Agricultural University
Mota Bhandariya (Amreli) -365610
 Certificate

Roll No.: Reg. No.:

Certificate
Batch No.: Uni. Seat No.:

This is to certify that the practical work has been satisfactorily

carried out by Mr. /Ms. In
the course No. GPB 3.3 with course title “Fundamentals of Plant Breeding”
(2+1) of Third Semester B.Sc. (Hons.) Agriculture in the laboratory of Genetics
and Plant Breeding, during the academic year .

He/ She has certified practical exercises out in the

subject of Fundamentals of Plant Breeding.

External Examiner Course teacher

Place:

Date:
 Practical manual of
GPB 3.3- Fundamentals of Plant Breeding (2+1)
INDEX
Page Signature of course
Ex. No. Title of Exercise Date
No. teacher
1 Plant Breeder’s kit
Study of germplasm of various
2
crops
Mode of pollination; To work out
the mode of pollination in a given
3
crop and extent of natural
outcrossing
Consequences of inbreeding on
4 genetic structure of resulting
population
Estimation of heterosis and
5
inbreeding depression
Emasculation and hybridization
6 techniques in self and cross
pollinated crops
Concepts of population genetics
7
and Hardy – Weignberg Law
Methods of calculating mean,
8
range, variance, standard deviation
Component of Genetic variation –
9
Heritability and Genetic advance
Designs used in plant breeding
10
experiments
Analysis of Randomized Block
11
Design
Prediction of performance of
12
double cross hybrids

2
EXERCISE 1: PLANT BREEDER’S KIT Date:

Plant breeding is an art, science and technology of improving genetic makeup of

plants for benefits of the human kind. For this, a plant breeder often requires artificial
self-pollination, hybridization, evaluation of genotypes in the field. Following
tools/equipments are required for emasculation, pollination and field experimentation for
selection and filed observations.
Sr.
Tools/Equipments Purpose/Use
No.
1 Fine pointed forceps For emasculation, holding flowers and
removing anthers

2 Scissor For cutting the vegetable parts and

small/large size floral parts or flowers
during hybridization

3 Pointer/Needle For incising the floral parts and

removing the anthers during
emasculation

4 Alcohol or A small vial is required to sterilize

methylated spirit forceps, scissors, needles, brushes
during hybridization programme

5 Eye lens / To observe small flowers,

Magnifying lens stigmatic surface, dehiscence of
anthers

6 Automizer It is used for spraying the gametocides

during emasculation of flowers. e.g.
57% ethyl alcohol to kill Lucerne
pollens

7 Hair brush For transferring the pollen grains

without injuring to stigmas or pollens

1
8 Parchment / Kite For protecting small size flowers
paper bag during selfing and crossing
(white/red)

9 Butter paper bag For protecting the individual flowers

or small twigs during selfing or
crossing.

10 Brown paper bag To cover the panicles or large size

influences during selfing and crossing

11 Muslin cloth bag To cover the whole plant (Chilli,

Brinjal, Fennel) or twig (Mango)
while selfing or crossing

12 U-pins(u- clips) For fastening the bags on earheads or

flowers to keep the bag in proper
position

13 Soda straw tubes For protecting the emasculated or

pollinated flower buds and selfing

14 Wire ring/ Smooth For tying closed buds for selfing.

threads They are inserted / tied in axis of
flowers to identify selfed/crossed
flower.

2
15 Small white tag For identifying the flower or a small
twig during hybridization and writing
detailed information about crossing
with pencil and then inserted on
pedicel or peduncle
16 Pencil For writing field labels or field bags as
it will not erase or spread during rains,
dew or under intense light

17 Aluminum label For tagging the flowers in fruit crops

with wire or tree species after crossing. It is also
used for identification of selected trees

18 Luggage labels For tagging the large sized plants

(white or yellow) while rouging or during selection

19 Waxy threads For fastening the luggage labels on

plants

20 Sample bag For storing the crossed seeds in small

(Yellow) quantity

21 Field note books To note down all breeding activities

and daily observation in the field
regarding germination, flowering,
morphological, description, date of
emasculation, pollination, number of
cross attempted and seeds setting
22 Measure scale To measure plant height and prepare
field layout.

Exercise:
A. List the different tools/equipments of breeder's kit and write down its uses.

3
EXERCISE 2: STUDY OF GERMPLASM OF VARIOUS CROPS Date:

Germplasm: The sum total of hereditary material, i.e., all the alleles of various genes,
present in a crop species and its wild relatives.
Genetic resources: The sum total of genes in a crop species.
Germplasm is the basic material with which a plant breeder has to initiate his breeding
programme.

Important features of plant genetic resources are

1. Gene pool represents the entire genetic variability or diversity available in a crop
species.
2. Germplasm consists of land races, modern cultivars, obsolete cultivars, breeding
stocks, wild forms and wild species of cultivated crops.
3. Germplasm includes cultivated species, wild species and relatives of crop plants.
4. Germplasm is collected from the centres of diversity, gene banks, gene sanctuaries,
farmer’s fields, markets and seed companies.
5. Germplasm is the basic material for launching a crop improvement programme.
6. Germplasm may be indigenous (collected within country) or exotic (collected from
foreign countries).
Germplasm consists of the following five types of materials:
1. Land Races
These are primitive varieties, which had evolved without a systematic and
sustained plant breeding effort.
They are sources of many valuable genes including those for adaptation. They are
storehouses of genetic variability.
Adapted to the local soil type, climatic conditions etc.
2. Obsolete Varieties
These varieties were developed by systematic breeding effort, were once
commercially cultivated but are no more grown.
They have some desirable features.
For example, wheat varieties K 65, K 68, Ph 591, many NP series varieties etc. are
obsolete varieties.
3. Varieties in Cultivation
The varieties in cultivation are the easiest to use in breeding programmes.
They form a major part of a working collection.
They are good sources of genes for yield, quality etc.
They can be introduced in a new area, and directly released for cultivation.

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4. Breeding Lines
These are lines/populations developed in breeding programmes.
They often contain valuable gene combinations.
This includes nearly homozygous lines, mutant lines, lines derived from
biotechnology programmes and, now, transgenic lines.
5. Wild Forms
Wild forms are the wild species from which crop species were directly derived.
They are easy to cross with the concerned crop species.
6. Wild Relatives
Wild relatives include all other species, which are related to the crop species by
descent during their evolution.
Wild relatives are much more difficult to hybridize with crops than are the wild
forms.
7. Mutants
Mutants can be identified in nature as well as can be induced through the use of
physical and chemical mutagens.
Mutation breeding is non-conventional method which is used when the variability
for desired character is not found in the genetic material of cultivated species and
its wild relatives.
Genetic resources can be broadly grouped into two types, depending on the state of
their domestication as (1) cultivated germplasm and (2) wild germplasm.
Alternatively, they may be termed as (1) indigenous (from the country in question)
and (2) exotic (from another country) based on their place of origin.

GENE POOL CONCEPT

Gene pool: All the genes and their alleles present in the individuals, which hybridize
or can hybridize with each other.
In some sense, gene pool describes a concept similar to germplasm.
The gene pool is classified into three groups:
(1) Primary gene pool (GP1),
(2) Secondary gene pool (GP2)
(3) Tertiary gene pool (GP3)
1. Primary Gene Pool (GP1)

It includes all such strains and species, which hybridize readily with each other and
give rise to fertile hybrids.
It consists of all the different strains or varieties of a crop species and some related
species.
The members of primary gene pool are the most commonly used in breeding
programmes.
5
2. Secondary Gene Pool (GP2)
Members of secondary gene pool are all those species that hybridize with the
members of primary gene pool with some to considerable difficulty and the
hybrids are partially fertile.
These species are difficult to hybridize with those of GP1 due to ploidy differences,
chromosome alterations or genetic barriers.
Gene transfers from GP2 to GP1 are possible but usually difficult.
Members of this group are often used in breeding programmes.
3. Tertiary Gene Pool (GP3)
The species belonging to this group cross with the members of primary gene pool
with considerable to great difficulty, and hybrids, if produced, are anomalous,
lethal or completely sterile.
Gene transfers from this group to the primary gene pool are very difficult and
require special techniques.
Hybrids are invariably sterile.
Gene transfers from GP3 to GP2 are relatively easier.
GP3 is used only occasionally in breeding programmes.

GENETIC EROSION

Genetic erosion: The gradual loss of variability from cultivated species, and their wild
forms and wild relatives.
• Genetic erosion is a creation of man since his success in plant breeding is the chief
cause of genetic erosion.

The main causes of genetic erosion are briefly summarized below.

 Replacement of genetically variable land races ('Desi' varieties) by the improved,

genetically uniform pureline or hybrid varieties.
 Improved crop management practices have virtually eliminated the weedy forms of
many crops.
 Increasing human needs have extended farming and grazing into forests, the
habitats of most wild species. This has led to the extinction of many wild relatives
of crops.
 Developmental activities like hydroelectric projects, roads, industrial areas,
railways, buildings etc. have also disturbed the wild habitat. Often wild relatives of
crops are destroyed due to these activities.
 Ideal, solution to the problem of genetic erosion is collection and conservation of
the germplasm of cultivated plant species.

6
Germplasm regeneration: Growing seeds of the various entries in field and harvesting
fresh seeds for further storage. Germplasm collections have to be re-generated after every
few years since seeds of most species lose viability during storage.

ACTIVITIES IN GERM PLASM CONSERVATION

• The various activities in germplasm conservation can be grouped into the following
categories:
(1) Collection of germplasm
(2) Conservation
(3) Evaluation
(4) Cataloguing, i.e., data storage and retrieval,
(5) Multiplication and distribution, and
(6) Utilization.
In addition, at world level International Plant Genetic Resources Institute (IPGRI), Rome
looks after the Training of personnel and Global coordination.

COLLECTION OF GERMPLASM

 The process of obtaining the various germplasm accessions for a germplasm

collection is known as collection of germplasm.
This can be done in two chief ways
1. Exploration and Collection
• Trips for collection of various forms of crop plants and their related species are
termed as explorations.
• Explorations are the primary source of all the germplasm present in various
germplasm collections.
• Therefore, cultivated forms like land races, open-pollinated varieties etc., wild
forms and wild relatives are all collected.
2. Procurement from other Agencies
• Germplasm can be obtained from other agencies concerned with germplasm
conservation, from research institutions, individuals or companies.

GERMPLASM CONSERVATION

Conservation refers to protection of genetic diversity of crop plants from genetic erosion.
The germplasm has to be maintained in such a state that there is minimum risk for its loss
is called Germplasm conservation.

7
Germplasm can be conserved either
A. In situ Germplasm Conservation
• Conservation of germplasm in its natural habitat or in the area where it grows
naturally.
• This is achieved by protecting this area from human interference. This area is
called natural park, biosphere reserve or gene sanctuary.
• A gene sanctuary is best located within the Centre of origin of crop species
concerned, preferably covering the micro-centre within the Centre of origin.
• NBPGR, New Delhi, is making attempts to establish gene sanctuaries in
Meghalaya for Citrus and in the North-Eastern region for Musa, Citrus, Oryza,
Saccharum and Mangifera.
B. Ex situ Germplasm Conservation
• Conservation of germplasm away from its natural habitat.
This method has following three advantages:
(1) It is possible to preserve entire genetic diversity of a crop species at one place.
(2) Handling of germplasm is also easy.
(3) This is cheap method of germplasm conservation.
It can be achieved in the following 5 ways:
(1) Seed banks
(2) Plant or field banks
(3) Shoot tip banks
(4) Cell and organ banks
(5) DNA banks
1. Seed Banks
• In seed banks germplasm is stored as seeds of various accessions. Virtually all
gene banks are essentially seed banks. Seed conservation is quite easy, relatively
safe and ordinarily needs minimum space. Under suitable conditions, seeds of
many species can be stored for upto 50-100 years.
• Seeds are classified, mainly on the basis of their storability, into two major groups:
a. Orthodox Seeds.
• Seeds of this type can be dried to moisture content of 5% or lower without
lowering their viability.
• Most crop seeds belong to this category.
• Such seeds can be easily stored for long periods.
b. Recalcitrant Seeds.
• The viability of seeds drops drastically if their moisture content is reduced below
12-30%.
• Seeds of many forest and fruit trees, and of several tropical crops like Citrus,
cocoa, coffee, rubber, oil palm, mango, jack fruit etc. belong to this group.

8
 • Such seeds present considerable difficulties in storage.
• Therefore, germplasm of such plants are conserved by alternative approaches
(Field gene bank).
2. Plant Banks
• A plant bank is an orchard or a field in which accessions of fruit trees or
vegetatively propagated crops are grown and maintained.
Limitations
• Requires large areas
• Expensive to establish and maintain
• Prone to damage from disease, insects, man-made or natural disasters abd
human handling errors.
3. Shoot Tip Banks
• In such gene banks, germplasm is conserved as slow growth cultures of shoot-tips
and node segments.
Benefits
• Conservation free from disease and pests
• Used for seeds which either do not produce viable seeds or produce recalcitrant
seeds
• Consists of sub-culturing the cultures, which may be done every 6 months to 3
years and every time it requires short period of time for sub-culturing
4. Cell and Organ Banks
• A germplasm collection based on Cryo-preserved (at -196°C in liquid nitrogen)
embryogenic cell cultures, shoot-tips and or somatic/zygotic embryos may be
called cell and organ bank.
5. DNA Banks
• In these banks, DNA segments from the genomes of germplasm accessions are
maintained as cosmid clones, phage lysates or pure DNA (For relatively short
periods).
• These DNA segments can be evaluated and the desired ones may be used to
produce transgenic plants.
• Applicable for conservation of genetic materials of extinct species since DNA
extracted from well preserved herbarium specimens can often be cloned.
• However, it is very expensive and highly sophisticated. A world-wide network of
DNA banks for threatened/endangered species has been established.

GERMPLASM EVALUATION

• Evaluation consists of assessment of the germplasm accessions for their various

features or traits of some known or potential use in breeding programmes.

9
 • Generally, germplasm accessions are evaluated for morphological, physiological,
biochemical, plant pathological (i.e., disease resistance), entomological (i.e., insect,
resistance) and other features.
• IPGRI, Rome has developed model lists of descriptors (= characters) for which
germplasm accessions of various crops should be evaluated.

GERMPLASM CATALOGUING, DATA STORAGE AND RETRIEVAL

• Each germplasm accession is given an accession number.
• This number is prefixed, in India, with IC (indigenous collection), EC (exotic
collection) or IW (indigenous wild).
• Information on the species and variety names, place of origin, adaptation and on its
various features or descriptors is also recorded.
• Therefore, catalogues of the germplasm collections for various crops are published
by the gene banks.

GERMPLASM MULTIPLICATION AND DISTRIBUTION

• The germplasm accessions requested by breeders/researchers are multiplied and
supplied to them, usually without cost.
• Active collections are used for this purpose.

GERMPLASM UTILISATION
• The germplasm can be used in a breeding programme in the following 3 ways:
(1) Direct release as a variety,
(2) It may be subjected to selection for developing a variety and
(3) It may be used as parents in hybridization programmes.

NBPGR:
 National Bureau of Plant Genetic Resources was established by ICAR in 1976. in
New Delhi
 The basic function of NBPGR is to conduct research and promote collection,
conservation, evaluation, documentation and utilization of crop genetic resources
in India.
Types of seed collections
Based on the use and duration of conservation, seed collections are of three types
1. Base collections
2. Active collections
3. Working collections

10
 • Base collections: It is also known as principal collection.
• These consist of all the accessions present in the germplasm of a crop. They are
stored at about -180C or -200C with 5 + 1% moisture content; they are disturbed
only for regeneration.
• When the germination of an accession falls below, usually, 95% of its germination
at the start of storage, the accession is regenerated.
For reasons of safety, duplicates of base collections should be conserved in other
germplasm banks as well. High quality orthodox seeds can maintain good viability upto
100 years

2. Active collections:
• The accessions in an active collection are stored at temperatures below 150C
(often near 00C), and the seed moisture is kept at 5%.
• The storage is for medium duration, i.e., 10-15 years. These collections are
actively utilized in breeding programme.
• These collections are used for evaluation, multiplication and distribution of the
accessions. They are usually maintained by multiplying the seeds of their own
accessions. But from time to time, base collection material should be used for
regeneration of these collections.
• Germination test is carried out after every 5-10 years to assess the reduction in
seed viability.

3. Working collections: The accessions being actively used in crop improvement

programmes constitute working collection.
Their seeds are stored for 3-5 years at less than 150C and they usually contain about 10%
moisture. These collections are maintained by the breeders using them.

 Core collection
The concept of core collection was proposed by Franked it refers to a subset of base
collection which represents the large collection. Or a limited set of accessions derived
from an existing germplasm collections.

Exercise:
1. Define: Germplasm, Exploration, Recalcitrant seeds, Genetic erosion, Exotic
collections
2. Enlist the various forms of germplasm
3. Write down about gene pool concept and its type
4. Explain activities in germplasm conservation
5. Difference the followings.
a. Orthodox Seeds and Recalcitrant Seeds
b. In situ and Ex situ Germplasm Conservation
c. GP1 and GP2

11
EXERCISE 3: STUDY OF MODE OF POLLINATION AND LIFE CYCLE OF AN
ANGIOSPERMIC PLANT Date:

Flower: A flower is highly metamorphosed shoot meant essentially for reproduction in the
plants.
Pollination: Pollination refers to the transfer of pollen grains from anthers to stigmas.
Pollen from an anther may fall on to the stigma of the same flower leading to self-
pollination or autogamy. When pollen from flowers of one plant is transmitted to the
stigmas of flowers of another plant, it is known as cross-pollination or allogamy.
A third situation, geitonogamy, results when pollen from a flower of one plant
falls on the stigmas of other flowers of the same plant, e.g., Maize. The genetic
consequences of geitonogamy are the same as those of autogamy.
Self-pollination
• Believed to have originated from cross-pollinated ancestors.
• Must have hermaphrodite flowers.
• 100% self -pollination is not occur & cross -pollination may occur up to 5%.
• The degree of cross-pollination in self-pollinated species is affected by several
factors, e.g., variety environmental conditions like temperature and humidity and
location.
Mechanisms of self-pollination
1. Bisexuality: Presence of male and female organs in the same flower.
2. Homogamy: Maturation of anther and stigma of a flower at the same time
3. Cleistogamy: In this case, flowers do not open at all. This ensures complete self-
pollination since foreign pollen cannot reach the stigma of a closed flower. Occurs in
some varieties of wheat, oats, and barley.
4. Chasmogamy: In some species, the flowers open, but only after pollination has taken
place. This occurs in many cereals, such as, wheat, barley, rice and oats. Since the
flower does open, some cross-pollination may occur.
5. Position of anther: In crops like tomato and brinjal, the stigmas are closely
surrounded by anthers. Pollination generally occurs after the flowers open. But the
position of anthers in relation to stigmas ensures self-pollination.
6. In some species, flowers open but the stamens and the sigma are hidden by other floral
organs. In several legumes, e.g., pea, mung, urdbean, soybean and gram. The stamens
and the stigma are enclosed by the two petals forming a keel.
7. In a few species, stigmas become receptive and elongate through staminal columns.
This ensures predominant self -pollination.
Genetic Consequences of autogamy
• Leads to a very rapid increase in homozygosity. Therefore, populations are highly
homozygous.

12
 • Do not show inbreeding depression, but may exhibit considerable heterosis.
Therefore, the aim of breeding methods generally is to develop homozygous
varieties.
FEATURES OF AUTOGAMY
• Homozygous and have advantage of homozygosity
• No recessive deleterious genes
• Homozygous balance and show no inbreeding depression
• New gene combination are not possible due to regular self-pollination
• Inbreeders have generally narrow adaptation and are less flexible
List of self-pollinated crops
Cereals Legumes Vegetables Oilseeds
Wheat Pea Tomato Sesamum
Rice Cowpea Chilli Linseed
Barley Chickpea Brinjal Groundnut
Oat Mungbean Potato Soybean
Cross Pollination
• In cross-pollinating species, the transfer of pollen from a flower to the stigmas of
the others plant.
• Many of the crop plants are naturally cross-pollinated. In many species, a small
amount (up to 5-10 %) of selfing may occur.
Mechanisms promoting allogamy
1. Dicliny or unisexuality: Flowers are either staminate (male) or pistillate (female).
i. Monoecy. Staminate and pistillate flowers occur in the same plant, either in the
same inflorescence, e.g., Castor, mango and coconut, or in separate inflorescences,
maize, chestnut, strawberries, rubber, grapes and cassava.
ii. Dioecy. The male and female flowers are present on different plants, i.e., the plants
in such species are either male or female, e.g., papaya, date palm, pointed gourd,
hemp, asparagus, and spinach. In general, the sex is governed by a single gene, e.g.,
asparagus and papaya.
In some cases, there are hermaphrodite plants in addition to males and females, and
a number of intermediate forms may also occur.
2. Dichogamy: Stamens and pistils of hermaphrodite flowers may mature at different
times facilitating cross pollination.
i. Protogyny: Pistils mature before stamens e.g. Pearl millet (Bajra).
ii. Protandry: Stamens mature before pistils e.g. Maize and sugar beets
3. Herkogamy (Prevention by physical barrier): In Lucerne or alfalfa, stigmas are
covered with a waxy film. The stigma does not become receptive until this waxy film

13
 is broken. The waxy membrane is broken by the visit of honey bees which also effect
cross-pollination.
4. A combination of two or more of the above mechanisms may occur in some specie.
This improves the efficiency of the system in promoting cross-pollination. e.g. Maize:
Monoecy and Protandry.
5. Self-Incompatibility: It refers to the failure of pollen to fertilize the stigma of same
flower or other flowers on the same plant.
• Sporophytic self-incompatibility: Controlled by the genotype of the pollen
producing plant.
• Gametophytic self-incompatibility: Controlled by the genotype of the pollen
itself.
6. Male Sterility: Male sterility refers to the absence of functional pollen grains in
hermaphrodite flowers.
• Genetic male sterility : Controlled by nuclear genes
• Cytoplasmic male sterility : Controlled by cytoplasmic genes
• Cytoplasmic- Genetic male sterility : Controlled by both nuclear and
cytoplasmic genes
7. Heterostyly: Style and filaments in a flower are of a different length e.g. Linseed
Genetic consequences of allogamy
• Preserves and promotes heterozygosity in a population.
• Show mild to severe inbreeding depression and a considerable amount of heterosis.
• The breeding methods aim at improving the crop species without reducing
heterozygosity to an appreciable degree.
• Hybrid or Synthetic varieties are the aim of breeder wherever the seed production
of such varieties is economically feasible.
FEATURES OF ALLOGAMY
• Random matting
• Heterozygous & advantage of heterozygosity
• Contains some deleterious recessive genes
• Show considerable inbreeding depression
• Permits new gene combinations from different sources.
• Variability is distributed over entire population.
• Wide adaptability and more flexibility to environmental changes.
List of cross pollinated crops
Cereals Vegetables Oilseeds Forages Fruits
Maize Bitter gourd Castor Lucerne/Alfalfa Banana
Bajra Onion Sunflower Napier grass Mango
Rye Cabbage Oil palm Sudan grass Aonla

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Often cross pollinated species
• Cross-pollination often exceeds 5 per cent and may reach 30 -50 per cent. e.g.,
Sorghum, Cotton, Okra, Pigeon pea
• The genetic architecture of such crops is intermediate between self-pollinated and
cross-pollinated species.
• Consequently, in such species breeding methods suitable for both of them may be
profitably applied.
• Hybrids are superior to others.
List of often cross pollinated crops
Cereals Pulses Fibres Others
Sorghum Pigeon pea Cotton Tobacco

Pollination syndrome
Flower characteristics or traits which attracts particular type of pollinators.
• Combination of colour
• Odour
• Quantity of nectar
• Location and type of pollen and flower structure
Rewards for pollinators
• Nectar
• Pollen
• Shelter
• Heat
Types
1) Abiotic pollination syndromes
• Wind pollination (Anemophily)
• Water pollination (Hydrophily)
2) Biotic pollination syndromes
• Bee pollination (Melittophily)
• Wasp pollination
• Butterfly pollination (Psychophily)
• Moth pollination (Phalaenophily)
• Fly pollination (Myophily and Sapromyophily)
• Bird pollination (Ornithophily)
• Bat pollination (Chiropterophily)
• Beetle pollination (Cantharophily)

15
DETERMINATION OF MODE OF POLLINATION

• The first step in determining the mode of pollination of a species is to critically

examine its flowers.
• Mechanisms like dioecy, monoecy, protogyny, protandry and cleistogamy are
easily detected. They clearly indicated the mode of pollination.
• The second step consists of isolating single plants and recording seed set under
isolation. Space isolation, i.e. individual plants grown at sufficient distance to
prevent cross-pollination, is preferable to isolation by bags or cages since the latter
may create an environment unfavourable for pollination and seed set.
• Failure to set seed in isolation proves the species to be cross-pollinated. However,
setting of seeds is only indicative of self-pollination.
• Finally, the effects of selling (inbreeding) on the vigour of plants should be studied.
Loss in vigour due to inbreeding is common in cross-pollinators, but self-
pollinators show no inbreeding depression.

DETERMINATION OF AMOUNT OF CROSS-POLLINATION

• The amount of cross-pollination is determined by planting two strains of the
concerned species in a mixed stand.
• One strain is homozygous for a dominant character, preferably an easily
recognizable seed or seedling character, while the other strain has the recessive
form of the character.
• The two strains arc planted in such a manner that each plant of the recessive strain
is surrounded by plants of the dominant strain to provide abundant pollen.
• Seeds from the plants of only recessive strain are harvested. The percentage of
these seeds carrying the dominant allele represents the percentage of cross-
pollination in the species.
• The frequency of cross-pollination varies greatly with the variety, weather
conditions and location.
• For example, in a study on safflower the estimates of out crossing in different
varieties grown in the same year at the same location ranged from 0-8.7 per cent.
• Similarly, the amount of cross-pollination in a single variety grown at several
locations varied from 1.3 to 9.8 per cent.
• Therefore, such a study should include several varieties of the crop and the study
should be conducted at several locations for two or more years.

16
The life cycle of an angiospermic plant describe by following phases:
1. Production of spores and gametes by the action of sporogenesis and
gametogenesis.
2. Pollination
3. Fertilization
4. Formation of zygote followed by seed & fruit from ovule & embryo sac (ovary),
respectively.
5. Germination of plant from seed.
Fertilization: It is the fusion of male and female gametes.
Gametes: Sexual reproductive unit which is produced by sporogenesis and
gametogenesis.
Process of fertilization:
• After pollination the pollen grain germinates on the stigma and pollen tube
elongates through the style and enters the ovary to ovule through the microphyl.
• At this time, function of vegetative nucleus is over and it degenerates.
• Pollen after reaching at ovule enters one of the synergids.
• Tips of pollen tube dissolved enzymatically and one of the male gamete fuse with
egg cell to from the zygote (2n), which form a protective wall around itself to form
the oospore.
• The second male gamete fuses with the two polar nuclei which give rise to triploid
endosperm (3n) nucleus (This is called double fertilization).
• Two synergids degenerates immediately after fertilization and three antipodals may
degenerate even before fertilization.
• The oospore (2n) give rise to the embryo and the triploid endosperm nucleus give
rise to the endosperm (3n) and this is known as triple fusion.
• The ovule and ovary gives rise to seed and fruit respectively (Figure 3.1)

Exercise:
1. Define: Homogamy, Heterostyly, Double fertilization, Triple fusion
2. Explain mechanisms for self and cross pollination in plants
3. Enlist the types of pollination syndrome
4. Explain determination of mode of pollination
5. Describe in brief about life cycle of an angiospermic plant
6. Write down about determination of the amount of cross-pollination

17
18
Figure 3.1: Life cycle of an angiospermic
plant
EXERCISE 4: CONSEQUENCES OF INBREEDING ON GENETIC
STRUCTURE OF RESULTING POPULATION
Date:
Cross-pollinated species and asexually reproducing species are naturally highly
heterozygous. They generally show reduction in vigour and fertility due to inbreeding,
while, hybridization involving unrelated strains often leads to increased vigour and
fertility. These phenomena are of great significance in breeding of these crops as a
breeding scheme must keep inbreeding to the minimum.
 INBREEDING DEPRESSION
 Mating between individuals related by descent is called inbreeding.
 Degree of inbreeding may be higher for more closely related the individuals in the
population on selfing.
 The degree of inbreeding of an individual is expressed as inbreeding coefficient
(F). The value of F for an individual is the probability of the two alleles of a gene
present in an individual to have been derived from a single allele of a common
ancestor, i.e., an ancestor that occurs in the pedigree of both maternal and paternal
parents of this individual.
 In a random mating population, the value of F for any individual is 0, while that
for an individual produced by selfing of a plant from a random mating population
is 1/2. The value of F is cumulative over generations.
 The chief effect of inbreeding is an increase in homozygosity proportionate to the
degree of inbreeding. Cross-pollinated and vegetatively reproducing species show
reduced vigour and fertility upon selfing. This is termed as inbreeding
depression.
 In 1876, Darwin concluded that progeny obtained from self-fertilization were
weaker than those derived from outcrossing. Detailed information on inbreeding
depression in Maize was given by East (1908) and Shull (1909).
 EFFECTS OF INBREEDING
• Appearance of lethal and sublethal traits (=alleles), e.g., chlorophyll deficiency,
etc. Plants showing such traits usually can’t survive in nature.
• A general reduction in vigour and plant size.
• A rapid reduction in rate of reproduction and reproductive ability of an individual.
• The population rapidly separates into phenotypically distinct lines due to
increased homozygosity (7 selfing generations leads to >99% homozygosity) and
random fixation of alleles.
• A drastic decline in yield is a common feature. An inbred is a homozygous line in
cross-pollinated crop produced and maintained by close inbreeding. In maize,
performance of inbreds isolated from open-pollinated populations yielded 50% of
its parental varieties.
19
 DEGREES OF INBREEDING DEPRESSION

The degree of inbreeding depression depends on the plant species concerned. But within a
species, the extent of depression is related to the value of F and the relative fitness of the
trait in question. Inbreeding depression is common in the case of traits that form an
important component of fitness. Those traits that contribute little to fitness, usually show
little or no inbreeding depression. The extent of inbreeding depression observed in various
plant species may be grouped into the following four categories:
1. High Inbreeding Depression: A large proportion of plants produced by selfing does
not survive. e.g. Alfalfa, Carrot. Loss in vigour and fertility is so high that only few
lines can be maintained after 3-4 generations of inbreeding. The yields of surviving
inbred lines are usually <25% of that of the parent open-pollinated varieties.
2. Moderate Inbreeding Depression: In species like Maize, jowar (Sorghum), Bajra
etc., a large proportion of plants can be maintained under self-pollination. Many
lines may be lost due to reduced fertility. Inbred lines may yield as much as 50% of
the parent open-pollinated varieties.
3. Low Inbreeding Depression: In species like onion, many cucurbits, sunflower,
hemp, etc. only a small proportion of plants show lethal or sublethal traits. Some
rare lines can’t be maintained due to poor fertility. There is little or no reduction in
yield and some inbred lines may yield as much as the parent open-pollinated
varieties.
4. Zero - No Inbreeding Depression: Populations of self-pollinated crops do not
show any inbreeding depression. However, when the F1's from various crosses are
selfed, a variable degree of inbreeding depression is observed in the F2 generation;
this is estimated as follows.
̅F̅̅1̅−F ̅̅̅2̅
Inbreeding depression (%) = ̅̅̅1̅
X 100
F

Where,
̅̅̅̅
𝐹1 = Mean performance of F1 generation
̅̅̅̅
𝐹2 = Mean performance of F2 generation

Formula for calculating homozygosity and heterozygosity

Homozygosity (%) (AA or aa) = [2n-1/2n]m x 100
Heterozygosity (%) (Aa) = [1/2n]m x 100
Where, n = Number of selfing generations.
m = Number of gene pairs segregating.
This also gives the degree of homozygosity with respect to any number of genes.

20
Table 4.1: Effect of selfing on the frequency of homozygosity and
heterozygosity with respect of a single locus Aa.

No. of Frequency (%) Frequency (%)

generation of
AA Aa aa Homozygosity Heterozygosity
selfing
S0 - 100 - - 100
S1 25 50 25 50 50
S2 37.5 25 37.5 75 25
S3 43.75 12.5 43.75 87.5 12.5
S4 46.875 6.25 46.875 93.75 6.25
S5 48.437 3.125 48.437 96.874 3.125
S6 49.218 1.562 49.218 98.436 1.562
S7 49.608 0.781 49.508 99.216 0.781

Exercise:
1. Define: Inbreeding, Inbreeding depression
2. Explain the various categories of extent of Inbreeding depression
3. Calculate homozygosity (%) and heterozygosity (%) in population having eight
generations of selfing for two independently segregating gene pairs.

21
EXERCISE 5: ESTIMATION OF HETEROSIS AND INBREEDING DEPRESSION
Date:
 The superiority of F1 hybrids in one or more characters over its parents is termed as
heterosis. The term hybrid vigour is used as synonym for heterosis.
 It is also defined as increase in fitness and yield over its parental values.
 The three main causes of heterosis are over dominance, dominance and epistasis, of
these dominance theory is the widely accepted one.
 The term heterosis was given by Shull in 1914
ESTIMATION OF HETEROSIS: Heterosis is estimated in three different ways
1. Average heterosis/Relative heterosis/ Mid-parent heterosis :
• When the heterosis is estimated over the mid parental value (i.e. mean value or
average of the two parents involve in hybrid (F1).
̅̅̅̅−MP
F1 ̅̅̅̅̅
Relative heterosis (RH) (%) = ̅̅̅̅̅
× 100
MP
̅̅̅̅+P2
P1 ̅̅̅̅
̅̅̅̅
MP =
2
2. Heterobeltiosis/ Better parent heterosis (Fonseca and Patterson, 1968):
• When the heterosis is estimated over the superior or better parent.
̅̅̅̅−BP
F1 ̅̅̅̅
Heterobeltiosis (HB) (%) = ̅̅̅̅
× 100
BP
Where, ̅̅̅̅
BP = mean value (over replications) of the better parent of the particular cross.
3. Standard /Economic /Useful heterosis (Meredith and Bridge, 1972):
• The term useful heterosis was used by Meredith and Bridge (1972).
• It refers to the superiority of F1 over the standard commercial check variety or
hybrid.
• This type of heterosis provides direct practical value in plant breeding.
̅̅̅̅−SC
F1 ̅̅̅̅
Standard heterosis (SH) (%) = ̅̅̅̅
× 100
SC
̅̅̅ = mean value (over replications) of the standard check.
Where, SC
Test of Significance for Heterosis
Standard errors (S.E) and critical differences (C.D.) for heterosis, heterobeltiosis
and standard heterosis were calculated by using following formulae.
𝟑𝑴𝒆
S.E. (M.P.) = √ 𝟐𝒓

C.D. (M.P.) = S.E. (MP) × table t0.05 and t0.01 at error d.f.
𝟐𝑴𝒆
S.E. (B.P./S.C.) = √ 𝒓

C.D. (B.P./S.C.) = S.E. (BP/SC) × table t0.05 and t0.01 at error d.f.
Where,
r = Number of replications
Me = Error mean square

22
t = Table value of ‘t’ at error degree of freedom corresponding to 5 per cent or 1 per cent
level of significance.
Alternatively, significance of heterosis value was tested using ‘t’ test.
̅̅̅̅
𝐹1 - ̅̅̅̅̅ 𝐵𝑃 or ̅𝑆𝐶
𝑀𝑃 or ̅̅̅̅̅ ̅̅̅
Calculated t = ---------------------------------------------
S.E. of heterosis over ̅̅̅̅̅ 𝐵𝑃 or ̅𝑆𝐶
𝑀𝑃 or ̅̅̅̅ ̅̅̅
Calculated t values were compared with tabulated ‘t’ values at error degree of
freedom for test of significance.
Conclusions:
 If table t0.05, error df is higher than calculated ‘t’ value then result is non-
significant.
 If calculated ‘t’ is higher than table t0.05, error df value then result is significant
(symbolized as ‘*’), but if calculated ‘t’ is higher than table t0.01, error df value then
result is highly significant (symbolized as ‘**’).

INBREEDING DEPRESSION:

• Inbreeding depression: reduction or loss in vigour and fertility as a result of

inbreeding.
• Since the maximum decline is reflected in F2 generation, the inbreeding depression
can be computed by relative data on F1 and F2 for any character as under :
̅F̅̅1̅−F ̅̅̅2̅
Inbreeding Depression (%) = ̅̅̅1̅
X 100
F
Where,
̅̅̅̅
𝐹1 = Mean performance of F1 generation
̅̅̅̅
𝐹2 = Mean performance of F2 generation

Exercise:
A total of 45 maize hybrids and their 10 inbred parents were evaluated in a Randomized
Complete Block Design (RCBD) with three replications. The following is the data on
grain yield per plant (grams). Work out the different types of heterosis per cent basis and
its test of significance. Comment on the results. Assume HIM 129 as standard check.
Genotype RI R II R III Genotype RI R II R III
IL112 × IL113 122.7 114.2 127.1 IL101 × IL109 119.9 126.4 135.1
IL112 × IL101 122.6 119.0 140.9 IL103 × HKI-193-1 156.5 143.9 131.1
IL112 × IL103 120.4 130.5 119.5 IL103 × IL105 125.0 115.4 120.7
IL112 × HKI-193-1 98.8 137.3 138.3 IL103 × CM140 160.5 176.7 169.3
IL112 × IL105 121.6 117.5 122.1 IL103 × IL111 135.0 122.1 141.2
IL112 × CM140 172.3 153.8 151.7 IL103 × IL104 143.6 141.2 159.3

23
 IL112 × IL111 142.3 149.8 130.5 IL103 × IL109 154.3 144.8 158.9
IL112 × IL104 139.4 137.1 146.7 HKI-193-1 × IL105 143.0 133.4 132.4
IL112 × IL109 115.3 124.1 137.3 HKI-193-1 × CM140 130.0 178.4 155.4
IL113 × IL101 104.7 122.0 121.8 HKI-193-1 × IL111 116.9 113.3 121.8
IL113 × IL103 110.9 96.2 118.2 HKI-193-1 × IL104 138.5 147.6 146.6
IL113 × HKI-193-1 115.0 125.8 108.5 HKI-193-1 × IL109 94.2 138.6 113.4
IL113 × IL105 136.7 129.4 133.1 IL105 × CM140 123.6 109.7 111.1
IL113 × CM140 141.6 128.7 126.8 IL105 × IL111 120.8 119.9 137.3
IL113 × IL111 140.4 146.6 150.0 IL105 × IL104 149.1 130.8 158.8
IL113 × IL104 133.0 119.6 97.5 IL105 × IL109 106.4 99.5 117.9
IL113 × IL109 130.7 137.3 136.4 CM140 × IL111 152.0 155.7 159.7
IL101 × IL103 123.5 112.2 120.9 CM140 × IL104 140.5 161.9 176.6
IL101 × HKI-193-1 112.8 116.1 123.6 CM140 × IL109 143.7 151.4 153.8
IL101 × IL105 129.1 107.4 122.8 IL111 × IL104 148.8 155.3 141.8
IL101 × CM140 148.3 144.7 156.6 IL111 × IL109 141.7 136.8 141.2
IL101 × IL111 168.4 141.8 149.7 IL104 × IL109 158.2 154.6 157.9
IL101 × IL104 144.2 141.5 135.9 HIM 129 (Check) 122.5 131.9 117.3

Genotype RI R II R III Genotype RI R II R III

IL112 104.7 115.5 116.4 IL105 100.3 119.4 111.3
IL113 87.9 110.7 102.3 CM140 121.5 129.8 114.4
IL101 102.4 102.7 100.9 IL111 126.8 106.2 122.3
IL103 125.3 121.7 126.6 IL104 120.6 131.9 136.4
HKI-193-1 115.6 130.5 115.9 IL109 115.2 119.4 118.5

ANOVA Table
Sum of Mean sum
Degree of freedom Calculated
Source squares (S.S.) of squares
(df) F
(M.S.)
Replication 2 385.88 192.94 1.98
Treatment 54 44469.98 823.52 8.46
Error 108 10517.35 97.38 -
Total 164 55373.21 - -
S.Em. = 5.70 C.D. (0.05) = 15.97 C.V. % = 7.53 General Mean = 131.13
Table 't' Value @ 5% = 1.98 and @ 1% = 2.62 for error degree of freedom (108).

24
EXERCISE 6: EMASCULATION AND HYBRIDIZATION TECHNIQUES IN
SELF AND CROSS POLLINATED CROP Date:

 Natural variability present in self-pollinated populations is exhausted quickly when

they are subjected to selection.
 For further improvement, therefore, new genetic variability has to be created, which is
easily and most commonly achieved by crossing two different purelines.
 The mating or crossing of two plants or lines of dissimilar genotype are known as
hybridization.
 In plants, crossing is done by placing pollen grains from one genotype, called the male
parent, onto the stigma of flowers of the other genotype, referred to as the female
parent.
 It is essential to prevent self-pollination as well as chance cross-pollination in the
flowers of the female parent. At the same time, it must be ensured that the pollen from
desired male parent reaches the stigma of flowers of the female parent for successful
fertilization.
 The seeds as well as the, progeny resulting from hybridization are known as hybrid.
 The progeny of F1, obtained by selfing or intermating of F1 plants, and the subsequent
generations are termed as segregating: generations. The term cross is often used to
denote the products of hybridization, i.e., the F1 as well as the segregating generations.

OBJECTIVES OF HYBRIDIZATION
 To create genetic variation
 To combine desirable characters into single plant (Combination breeding)
 To study the pattern of inheritance of the character
 To exploit and utilize hybrid vigour
 To produce transgressive segregants (Transgressive breeding)
 To produce hybrids in various crops for commercial cultivation by farmers
 To assess general combining ability of the parents and specific combining ability
of crosses
 TYPES OF HYBRIDIZATION

Based on the taxonomic relationships of the parents involved, hybridization may be

classified into two broad groups:
1) Inter varietal Hybridization
 The parents involved in inter varietal hybridization belong to the same species;
they may be two strains, varieties or races of the same species. It is also known
as intraspecific hybridization.
 Most commonly used for crop improvement

25
  Examples
o Wheat : GW 496 × GW 322
o Rice : IR 64 × IR 8
2) Distant hybridization
 It involves individuals belongs to same genus or different genus.
 Hybridization involving individuals belongs to distinct species of the same genus,
it is called interspecific or intrageneric hybridization.
o Examples : Wheat – Triticum aestivum × Triticum durum
 Hybridization involving individuals belongs to different genus, it is called
intergeneric hybridization.
o Examples : Triticale – Triticum aestivum × Secale cereal
o First man made cereal prepared by Rimpau (1890).
 These both interspecific and intergeneric hybridization are known as distant or
wide hybridization.
 In general, distant hybridization is much more difficult than intervarietal
hybridization; the difficulty increases with the taxonomic distance between the
parents involved.
 Usually, the aim of such crosses is to transfer few simply inherited characters like
biotic and abiotic stress tolerance genes from wild to cultivated species.
3) Introgressive hybridization
 It is one kind of inter-specific hybridization. In this type of hybridization, hybrids
may have repeatedly backcrossed to one of the parental species, so the most of the
nuclear genes of the parental species would be recovered along with few genes
from the other parental species.
o Example: Modern cultivated maize is developed by crossing Primitive
maize with Tripsacum (wild weedy species).

PROCEDURE OF HYBRIDIZATION
1. Choice of Parents
 The parents chosen for a hybridization must together possess all the traits, and in
sufficient intensity, which the breeder wishes to improve in the new variety.
 At least one parent must be well adapted in the region for which the variety is to be
developed. The other parent(s) should possess the traits, which one wishes to
improve in this well-adapted variety.
 The parents should be genetically diverse if the breeder aims to obtain
transgressive segregants.
 The general combining ability of the parents should also be considered, if the
estimates for the same are available. Otherwise, the level of phenotypic expression

26
 should be used as a guide since usually per se performance for a trait is positively
associated with the general combining ability for the trait.
2. Evaluation of Parents

 It is desirable to evaluate the performance of chosen parents in the area for which
breeding is to be done.
 Evaluation provides the following information about the parents: (1) performance
in terms of yield and yield traits, (2) reaction to the prevalent diseases and insect
pests, (3) presence of mechanical mixtures and (4) existence of heterozygosity.
3. Emasculation
 Removal of stamens/anthers or inactivation of pollen grains, before the pollen
grains become mature without any harm to the gynoecium, is called emasculation.
 Emasculation is done in the flowers of the parent to be used as female in a
hybridization program. It is usually done one day before anthesis of the concerned
flowers. The sole aim of emasculation is to prevent self-pollination in the flowers
of the female parent.
 The efficiency of emasculation can be tested by bagging the emasculated flowers
without pollination. The amount of seed set in such flowers will indicate the extent
of self-pollination, which occurred during emasculation.
 In crop improvement programmes, however, a small amount of self-pollination
may be permissible, but it should be kept to the minimum.
 An efficient emasculation technique should prevent self-pollination and produce a
high percentage of seed set on cross-pollination. The different techniques of
emasculation are as follows:
a) Hand Emasculation:
 In species with relatively large flowers, stamens or anthers are removed with the
help of forceps. Emasculation is usually done one day before the anthesis of
flowers during evening time.
 Generally, younger buds and older flowers, even pods close to the flower bud
selected for emasculation are removed to avoid confusion.
 The selected bud is then opened with the help of forceps, and all the anthers are
carefully removed.
 Care must be taken to remove all the anthers intact (i.e., without rupturing them)
from flower. Further, no damage should be caused to the female reproductive
organ, i.e., stigma and ovary.
b) Suction Method:
 This method is useful in species with small flowers, where hand emasculation is
tedious. Emasculation is done in the morning, on the day of anthesis, just before or

27
 immediately after the flowers open. If needed, petals may be removed with the
help of forceps.
 A thin rubber or glass tube is attached to the suction hose of a suitable suction
pump /aspirator. The tube is passed over the flowers to suck the anthers and also
the pollen grains that may be present on the stigmas. A considerable amount (up to
15%) of self-pollination may occur with this method.
c) Hot Water Method:
 Pollen grains are more sensitive than the female reproductive organs to both
genetic and environmental factors. Therefore, treatment with hot water of a
suitable temperature inactivates pollen grains without reducing female fertility.
 In case of jowar (sorghum), treatment with water at 42-44°C is optimum. Hot
water is generally carried in a thermos in which the whole panicle/spike (ear/head)
is immersed. It is generally highly effective, but regulating the water temperature
may present problems.
d) Cold Treatment:
 Exposure of wheat plants to 0-2°C for 15-24 hr inactivates pollen grains without
damaging gynoecium.
 In case of rice, treatment with cold water (0-6°C) kills the pollen grains. Cold
treatment is generally less effective than hot water treatment; it also shows a higher
frequency of self-pollination.
e) Chemical Treatment / Gametocides:
 Certain chemicals, which are sprayed at the time of flowering induces male
sterility plant.
 Gametocide: the chemicals which are used to kill the pollen grains are known as
gametocides.
Gametocide / CHA Chemical formulation Crops
Arsenicals Zinc methyl arsenate, Sodium Rice
methyl arsenate
Dalapon Sodium 2,2- dichloro propionate Cotton, Pearl millet, Wheat,
Onion
Ethepon 2-chloroethyl phosphonic acid Wheat, Barley, Rice, Oats
FW 450 (Mendok) Sodiumα,β-dichloto Capsicum, Cotton, Pearl
isobityrate millet, Sesame, Sunflower
OMT L-O-Methyl threonine Cotton
MH 1,2-dichloropyridazine-3,6- Maize, Wheat, Cotton
dione
GA Gibberellic acid Maize, Barley, Wheat, Rice,

28
  In clovers, a 10 second treatment with 57% alcohol was quite effective; the
frequency of self-pollination was less than 1%.
 Care must be taken that the chemical must not affect the function of the female
reproductive organ. The duration of treatment should be strictly controlled, since
even slightly longer duration would damage gynoecium and reduce seed set.
f) Genetic Emasculation:
 Prevention of self-pollination by genetic means is called genetic emasculation.
 This is achieved by using genetic (including transgenic) or cytoplasmic-genetic
male sterility, self-incompatibility or pistillate condition. This technique is of
great importance in hybrid seed production.
4. Bagging:
 The emasculated flowers or the whole inflorescences are enclosed in suitable bags;
this is called bagging.
 Bagging is done to prevent chance cross-pollination. Bags may be made of paper,
butter paper, glassine or fine cloth. The bags are tied to the base of inflorescence or
to the stalk of flower with the help of thread, wire or pins (of a suitable design).
 The bags are usually removed few days after pollination to prevent fungus growth.
In cross-pollinated crops like maize, the male flowers are also bagged to ensure the
purity of pollen used for pollination.
5. Tagging:
 The emasculated flowers are tagged just after bagging. In most crops circular tags
of about 3 cm diameter or rectangular tags of 3 × 2 cm are used.
 In crops like Maize, Bajra etc., larger tags of 6 × 3 cm are used. The tags are
attached to the base of inflorescence or the stalk of flowers with the help of a
thread.
 The following information is recorded on the tags with a carbon pencil:
o Date of emasculation,
o Date of pollination, and
o Names of female and male parents [in that order, i.e., A (female parent) x B
(male parent)].
6. Pollination:
 Transfer of mature and active pollen from the flowers of male parent on to the
receptive stigmas of emasculated flowers is called pollination.
 It is essential for seed set in the emasculated flowers. Pollination is usually done in
the morning at the time of anthesis, next day following emasculation.
 In Bajra, etc., pollens are collected in a bag and dusted onto the stigmas of female
flowers. In wheat, barley, etc., one mature anther about to burst is inserted in each
floret. Alternatively, the spike of male parent may be shaken over the emasculated
spike; this is done when lemma and palea are clipped off during emasculation. In

29
 species like Maize, the male inflorescence may be detached and enclosed in the
bag covering the female inflorescence.
 Mature anthers are collected, pollen liberated and applied to the stigmas using
camel hairbrush, tooth pick, pieces of paper, etc.
7. Harvesting and Storing the F1 Seed:
 The crossed heads/spikes/pods are harvested, threshed, dried and stored properly.
 Care should be taken to protect them from storage pests and from moisture,
especially during the monsoon.
 Every precaution should be taken to prevent contamination from other seed. Seeds
from each cross should be handled separately, and the original tags should be kept
with them.
Types of crosses
 Single Cross:
 It is a cross between to genetically dissimilar homozygous plants
e.g. A × B = F1.
 Concept was proposed by G. H. Shull (1909) in Maize.
 Number of possible single crosses without reciprocals = [n(n-1)] / 2
 Number of possible single crosses with reciprocals = [n(n-1)]
Where, n = Number of parents
 Double cross:
 It is a cross between two different single crosses.
e.g. (A × B) × (C × D) = F1 (Double cross)
 Concept was proposed by D. F. Jones (1909) in Maize.
 Number of possible double crosses = [n(n-1)(n-2) (n-3)] / 8
Where, n = Number of parents
 Three-way cross:
 When F1 from a single cross is mated to a third parent, it is called three-way cross.
e.g. (A × B) × C = F1 (Three way cross)
 Multiple cross or Complex Cross:
 When more than four parents are crossed to produce the F1 hybrid.
e.g. (A × B) × (C × D) × (E × F) × (G × H) = F1
 It aims at bringing together genes from several parents into a single hybrid and also
create large amount of genetic variability.
 Back Cross:
 A cross between F1 and any one of its parents.
e.g. (A × B) = F1 F1 × A = BC1 or F1 × B = BC2
 It is used to transfer specific deficient trait from donor parent to recipient parent.
 To develop isogenic lines for multiline development.

30
 To develop Near Isogenic lines (NILs) for mapping population.
 Test Cross:
 A cross between F1 and its homozygous recessive parents.
e.g. (AA × aa) = F1 F1 × aa = Test cross progeny
 It is used for linkage study.
 Top Cross:
 A cross made between an inbred line and open pollinated variety.
 It was proposed by Davis (1929).
 It is used to study the general combining ability of inbred lines.
 Double top Cross:
 A cross made between single cross hybrid (F1) and open pollinated variety.
 Poly Cross:
 The open pollination group of genotypes in isolation from other compatible
genotypes in such a way to promote random mating. In this, all the genotypes have
equal chance of pollination.
 It was proposed by Tysdal, Kiessel bach and Wastover (1942).
Exercise:
1. Define : Hybridization, Single cross, Interspecific hybridization, Genetic emasculation,
Gametocides, Double cross, Top cross
2. Objectives of hybridization
3. Explain procedure of hybridization in crops.
4. Explain different techniques of emasculation
5. Calculate the number of single crosses, three way crosses and double crosses
generated using ten parents.

31
EXERCISE 7: CONCEPTS OF POPULATION GENETICS AND HARDY –
WEIGNBERG LAW Date:

 Cross-pollinated crops are highly heterozygous due to the free intermating among their
plants. They are often referred to as random mating populations because each
individual of the population has equal opportunity of mating with any other individual
of that population. Such a population is also known as Mendelian population or
panmictic population.
 A Mendelian population may be thought of having a gene pool consisting of all the
gametes produced by the population. Thus, gene pool may be defined as the sum total
of all the genes present in a population.
Hardy - Weinberg Law
This law was independently developed by Hardy (1908) in England and Weinberg
(1909) in Germany. This law states that “In the large random mating population, gene
and genotypic frequencies remain constant generation after generation in the
absence of selection, mutation, migration and random genetic drift”.

FACTORS AFFECTTING THE EQUILIBRIUM IN THE POPULATION :

1. Selection: Differential reproduction rates of various genotypes is known as selection.
2. Migration: The movement of individuals into a population from a differential
population, and participation in the reproduction of this population. It may introduce
new alleles into the population or may change frequencies of existing alleles.
3. Mutation: A sudden and heritable change in any character of an organism and is
generally due to a structural change in the concerned gene. It is the ultimate source of
all the variation present in biological materials. It may produce a new allele that was
not present in the population or may change existing allelic frequencies. However,
since mutation rate is generally very low, i.e., approximately 10-6, the effects of
mutation on gene frequency would be detectable only after a large number of
generations. Therefore, in breeding populations such effects may be ignored.
4. Random genetic drift: It is a random change in gene and genotypic frequency due to
sampling error. In a smaller population if natural selection operates at random it will
lead to sampling error. This sampling error is greater in smaller population than in a
large one. Because of sampling the frequency of one of the alleles becomes zero and
that of the other alleles become one. The allele having the value one is said to be fixed
because there is no further change in its frequency and thus it becomes homozygous.
Thus, if the population is small genetic drift will occur. To overcome this, one has to
use larger population, which may not be possible because of limitations in space,
labour and finance.

32
 Apart from in smaller populations, a certain amount of inbreeding is bound to
occur and this will lead to homozygosity. Mating between individuals sharing a common
parent in their ancestry is known as inbreeding. Inbreeding reduces the proportion of
heterozygotes or heterozygosity and increases the frequency of homozygotes or
homozygosity. Thus, in small populations, even with strict random mating or even with
strict cross-pollination the frequency of homozygotes increases, while that of
heterozygotes decreases due to inbreeding.
 In a species for a single gene with two alleles, A and a in a random mating population,
there would be three possible genotypes AA, Aa and aa.
 If the frequency of allele A is denoted as “p” and of allele a as “q” in the population.
 The frequencies of these three genotypes would be AA = p2, Aa = 2pq and aa = q2
If, the sum of gene frequencies, i.e. p and q is one i.e. p + q =1 then sum of its
genotypic frequencies would be p2 + 2pq + q2 = 1.
When the gene and genotypic frequencies remain constant from one generation to next,
such a population would be called in equilibrium. This equilibrium is known as Hardy-
Weinberg equilibrium.
Whether the population is at equilibrium or not, it can be tested by chi-square test.
 Suppose the population has N individuals of which total number of individuals similar
to AA denoted as D, Aa denoted as H and aa denoted as R.
 So, N = D + H + R. The total number of alleles at this locus in the population would be
2N since each individual has two alleles at a single locus.
 The total number of A alleles would be 2D+H because AA Individuals have two A
alleles each, while each Aa individual has only one A allele.
 Therefore, the gene frequency of allele A is p = (2D+H)/2N or (D+1/2H)/N
 Similarly, the gene frequency of allele a is q = (2R+H)/2N or (R+1/2H)/N
 Whereas, genotypic frequency for AA is p2 = D/N and genotypic frequency for aa
is q2 = R/N.
 Gene frequency is the proportion of an allele, A or a, in a random mating
population or the proportion of gametes carrying an allele, A or a.
 Genotype frequency or zygotic frequency is the relative proportion of a
genotype, AA, Aa or aa, in the population.
Characteristics of random mating population
1. Each genotype in the population is different in some gegree from others.
2. Each genotype is highly heterozygous.
3. Each genotype is largely out crossing or random breeding or cross pollinating.
4. Each genotype gives a variable progenies.
Hence, gene and genotypic frequencies are important in cross pollinated crops rather
than genetic inheritance in self-pollinated crops.

33
Calculation of gene and genotypic frequency
Example I:
In a sample population, total numbers of individuals are 100 which includes AA = 30, Aa
= 10 and aa = 60
So, D = 30, H = 10, R = 60 and N = 100
Gene frequency of A Individual allele
𝟏
p = [D + (𝟐H / N]
𝟏
= [30 + 𝟐 (10) / 100]
= 35 / 100 = 0.35
Similarly for ‘a’ individual allele
𝟏
q = [R + (𝟐H / N]
𝟏
q = [60 + 𝟐 (10) / 100]
= 0.65
∴ Here allele frequency, p + q = 1 ∴ 0.35 + 0.65 = 1
Exercise:
1. What is the frequency of heterozygotes. (Aa) in mendelian population. If the frequency
of recessive phenotype (aa) is 0.16?
2. Answer the following based on given data
a) No. of Individuals
Population
AA Aa aa
1 0.64 0.32 0.04
2 0.46 0.24 0.36
Which of the above population is in Hardy-Weinberg equilibrium?
b) What are the expected equilibrium frequencies for those above population are not
in equilibrium?
c) How long will it take for this equilibrium value to be reached at random
population?
3. Answer the following questions based on the date given to you.
No. of Individuals
Population
AA Aa aa
1 600 459 207
2 250 400 750

a) Calculate the genotype frequencies for above genotypes.

b) Calculate the gene frequencies for above genes/alleles.

34
 c) What is the expected number of each genotype if the above population is in Hardy
Weinberg Equilibrium?
4. The following numbers of the human M-N blood groups were recorded in a sample of
Africans White.
MM MN NN
1787 3039 1303
a) What are the genotype frequencies observed in this sample?
b) What are the gene frequencies observed in this sample?
c) With the gene frequencies observed, What are the genotype frequencies expected
from the Hardy - Weinberg law ?
d) How well do the observed frequencies agree with the expected ?
5. Explain the factors disturbing the H-W equilibrium in the population.
6. Explain Hardy - Weinberg law with example.

35
 EXERCISE 8: METHODS OF CALCULATING MEAN, RANGE, VARIANCE,
STANDARD DEVIATION Date:
When different plants/lines of a species show different magnitudes of a character, it
is called variation.
Variation is essential for any improvement in a species. Therefore, the first step in
any breeding programme is to create variation if it is not already present in the
population to be improved.
Variation can be created by hybridization, mutation, polyploidy, domestication. plant
introduction, somaclonal variation, genetic engineering, etc.
1. Mean of the distribution (First degree statistics)
i. Arithmetic mean: It is defined as sum of all observation divided by the total number
of individual added.
∑𝑋
𝑋̅ = 𝑁 𝑖
Where, 𝑋̅ = Mean , ∑ 𝑋𝑖 = sum of the individual observations
N= total number of observations
ii. Mode: Most frequent value in the population.
iii. Median: The middle value in an array of observation.
∑ 𝒇𝒙
Frequency distribution = 𝑵
Where, f = Class frequency
x = Class value
2. Measures of dispersion (Second degree statistics)
i. Range : The difference between the values of the highest and the lowest
observations of a sample is called range. It is generally depicted by listing the values
of the highest and the lowest observations.
ii. Mean deviation or Average deviation : Mean deviation is the average of deviation
of individual observation from the mean.
∑(𝑥−𝑥̅ )
Mean deviation = 𝑁
Where, x is the individual observation
𝑥̅ = is the mean
∑ 𝑓𝑑
Mean deviation by frequency = 𝑁
Where, f= Class frequency
d= deviation of class value from the mean
iii. Standard deviation : It is the square root of variance and is designated as SD in
case of sample, or as 𝜎 in case of population.
2
∑𝑥 2 − (∑ 𝑥)
Standard Deviation (S.D.) = √ 𝑁
𝑁−1

36
iv. Variance : average of square deviation of all the individual observation from the
mean.
(∑ 𝑥)2
∑ 𝑥2−
𝑁
Variance = 𝑁−1
N- 1 = degree of freedom
v. Standard error : It is the measure of the mean difference between sample estimate
of mean (𝑋̅) and the population parameter (𝜇) i.e. it is the measure of uncontrolled
deviation present in a sample.
𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑑𝑒𝑣𝑖𝑎𝑡𝑖𝑜𝑛
Standard error =
√𝑁
It is necessary to describe any sample in terms of its mean ± standard error.
vi. Coefficient of variation (C.V. %) : It is a percentage ratio of standard deviation to
the arithmetic mean of a given series. It is without unit or unit less.

𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑑𝑒𝑣𝑖𝑎𝑡𝑖𝑜𝑛
C.V. % = 𝑀𝑒𝑎𝑛
× 100
vii. Covariance: Any pair of related characters may yield a covariance

Example:
The plant height of 30 rice genotypes is given below. Workout the biometrical
quantities viz., mean, variance, standard deviation, standard error and coefficient of
variation.
Plant Plant Plant
Genotype height Genotype height Genotype height
(cm) (cm) (cm)
1 115.9 11 102.5 21 98.6
2 120.8 12 111.5 22 107.3
3 101.2 13 125.2 23 115.6
4 106.7 14 104.9 24 128.3
5 113.3 15 87.7 25 93.7
6 110.7 16 110.6 26 108.3
7 96.9 17 102.7 27 112.6
8 117.9 18 98.3 28 100.5
9 128.1 19 120.6 29 115.4
10 97.7 20 89.7 30 96.7

Answer:
∑ 𝒙
(a) Arithmetic mean = 𝑵
𝟏𝟏𝟓.𝟗+𝟏𝟐𝟎.𝟖+⋯….+𝟗𝟔.𝟕
= 𝟑𝟎
𝟑𝟐𝟑𝟗.𝟗𝟎
= 𝟑𝟎
= 108.00

37
Frequency Distribution
Range Class value Frequency (f) fx d fd fd2
(x)
85-90 87.5 2 175.0 -20.83 -41.66 867.78
90-95 92.5 1 92.5 -15.83 -15.83 250.59
95-100 97.5 5 487.5 -10.83 -54.15 586.44
100-105 102.5 5 512.5 -5.83 -29.15 169.94
105-110 107.5 3 322.5 -0.83 -2.49 2.07
110-115 112.5 5 562.5 4.17 20.85 86.94
115-120 117.5 4 470.0 9.17 36.68 336.36
120-125 122.5 2 245.0 14.17 28.34 401.58
125-130 127.5 3 382.5 19.17 57.51 1102.47
Total 30 3250.0 0.10 3804.17

∑ 𝒇𝒙
Mean = ∑𝒇

(2 𝑥 87.5)+(1+92.5)………..+(3+127.5)
= 30

3250.0
= 30

= 108.33

(b) Range = 87.7 to 128.3

∑𝑥 2
2 ( )
N
(c) Variance = ∑ 𝑥 – N−1
( 3239.9)2
115.92 + 120.82 ………+ 96.72 –
30
= 29
353345.05−349898.40
= = 118.85
29
∑ fd2 3804.17
Variance by frequency distribution = ∑f
= = 126.81
30

(d) Standard deviation = √Variance = √118.85 = 10.90

Standard deviation
(e) Standard error =
√N
10.90
= 5.48
= 1.99
Standard devaition
(f) Coefficient of variation (C.V.%) = x 100
Mean
10.90
= x 100 = 10.09 %
108

38
EXERCISE 9: COMPONENT OF GENETIC VARIATION – HERITABILITY
AND GENETIC ADVANCE Date:
Variability:
It means a difference among the individuals belonging to a single species or different
species within population.
The variability may be due to genotypes, environment pr due to the interaction of
genotype with environments. There are two basic types of variability
1. Genotypic variability: It is the component of variability, which is due to genetic
differences among the individuals within a population. It is the main concern of the
plant breeder.
2. Phenotypic variability: It is the observable variation present in a character of a
population. It includes both genotypic and environmental components of variability
and as a result, its magnitude differs under different environmental conditions.
Total variability can be partitioned into following components as under:
A. Genotypic variability:
a. Additive component
b. Dominance component
c. Epistatic component
B. Environmental variability
Importance of genetic variability in Plant Breeding
 The progress of any plant breeding programme for crop improvement depends on the
extent of the genotypic variability present in the base population.
 The assessment of the genetic variability present in the base population is the pre-
requisite before the adoption of the selection pressure in the variable population and
efficiency of the selection depends upon the identification of genetic variability from
the phenotypic expression of the character.
 The study of the genetic variability is necessary to determine the relative importance of
various plant characters with respect to yield in terms of genetic variability.
 The parameter like range of variability, co-efficient of variability, heritability and
expected genetic advance are computed for the assessment of the genetic variability
and gene action in expressing the genotypes which reflects into effective selection in
crop improvement programme.
 Genetic variability for important agronomic traits in almost all the crop is mainly due
to the additive genetic variance. The non-additive variance also exists for many
important traits in nearly all the crop but it is generally smaller in magnitude than
additive component.
 The variation required for improvement in any crop can be generated through plant
introduction, hybridization, mutation, polyploidy as well as somatic variation.

39
 Estimation of phenotypic and genotypic variances helps in the computation of
heritability and genetic advance.
Estimation of genetic variability, Heritability and genetic advance:
The genetic variability present in breeding populations can be assessed by three ways:
a) By using simple measure of variability
b) By estimating the various components of variance
c) By studying the genetic diversity within a population
1. By using simple measure of variability
When the experimental material is not replicated, the phenotypic (total) variability
present in breeding material can be assessed by using following simple measures of
variability. e.g. range, arithmetic mean, standard deviation, coefficient of variation,
standard error
2. By estimating the various components of variance
The magnitude of phenotypic variation present in a breeding population may be
partitioned into several components of variation using appropriate experimental design in
replicated trials. In randomized block design (RBD), the structure of analysis of variance
(ANOVA) is as under.
Source d.f. S.S. M.S. Cal. F Expected M.S.
Replication r-1 Rss Mr = Rss/ r-1 Mr / M e 𝜎𝑒2 + 𝑔𝜎𝑟2
Genotype g-1 Gss Mg = Gss/ g-1 Mg / M e 𝜎𝑒2 + 𝑟𝜎𝑔2
Error (r-1) (g-1) Ess Me = Ess/ (r-1) (g-1) -- 𝜎𝑒2
Total rg-1 Tss -- -- --

𝐸𝑟𝑟𝑜𝑟 𝑀.𝑆.
A. S.Em. = √ 𝑟

B. C.D. = S.Em. x √2 x t0.05 at error d.f

√𝐸𝑟𝑟𝑜𝑟 𝑀.𝑆.
C. Coefficient of variation (CV %) = × 100
𝑋̅
D. Error variance (Environment variance) (𝜎𝑒2 ): It is the non-heritable variation which
is due to the environment which varies depending upon the environments. σ2 e = Me
E. Genotypic variance (𝝈𝟐𝒈 ) : It is only measures the amount of genotypic variability
present in a character of a population.
(𝑀𝑔 −𝑀𝑒 )
𝜎𝑔2 = 𝑟
F. Phenotypic variance (Total variance) (𝜎𝑝2 ) : It is the total variation which is
observable and is the sum total of genotypic and environmental variances.
𝜎𝑝2 = 𝜎𝑔2 + 𝜎𝑒2

40
G. Genotypic coefficient of variation (GCV %) :It is measure the range of genetic
variability present in a character of a population and also provides a measure to
compare the genetic variability among various plant characters within and between
different populations. it is calculated as
√𝜎𝑔2
× 100
𝑋̅
H. Phenotypic coefficient of variation (PCV %) :
√𝜎𝑝2
× 100
𝑋̅
GCV % and PCV % are classified as suggested by Sivasubramaniam and Madhavamenon
in 1973 as
Low Less than 10 %
Moderate 10 – 20 %
High More than 20 %
If the difference between GCV and PCV is less, it indicates that environment has little
influence on the characters and if the difference is more it states that environment plays a
substantial role in the expression of that character.
I. Heritability is the proportion of the total variability that is due to genetic causes (or)
it is the ratio of genotypic variance to total variance.
 It is a good index of transmission of characters from parents to their offspring.
Depending upon the components of variance used as numerator in the calculation of
the heritability, it is of two types:
1) Broad sense heritability is the ratio of genotypic variance to the total phenotypic
variance and is calculated as
Vg σ2 g
Broad sense heritability (ℎ2𝑏 ) (%) = x 100 or × 100
Vp σ2 p

Where, Vg = σ2 g = Genotypic variance and Vp = σ2 p = Phenotypic variance

The features of broad sense heritability:
1. It can be estimated from both parental as well as segregating populations
2. It is estimated from total genetic variance
3. It is more useful in animal breeding than in plant breeding
4. It is useful in the selection of elite types from homozygous material
2) Narrow sense heritability is the ratio of additive (or) fixable genetic variance to the
total phenotypic variance and is calculated as
V σ2 a
Narrow sense heritability (ℎ2𝑛 ) (%) = Va x 100 or × 100
p σ2 p

Where, Va = σ2 a = Additive genetic variance and Vp = σ2 p = Phenotypic variance

41
The features of narrow sense heritability:
1. For estimation of narrow sense heritability, crosses have to be made in definite fashion
2. It is estimated from additive genetic variance.
3. It is useful in both, plant breeding as well as animal breeding.
4. It is useful in the selection of elite types from segregating populations.
Difference between broad sense and narrow sense heritability are given as:
Broad sense heritability Narrow sense heritability
1. Estimated from total genetic Estimated from additive genetic
variance variance
2. Can be estimated from both Requires crossing in definite fashion
parental and segregating material
3. More useful in animal breeding Useful in both plant and animal
breeding
4. Useful in selection of elite types from Useful in selection of elite types from
homozygous lines segregating material
Heritability plays an important role in selection process in the plant breeding
especially in the selection of elite genotypes from segregating populations. Johnson et
al., (1955) categorized heritability as
Low Less than 30 %
Moderate 30 – 60 %
High More than 60 %
J. Genetic advance (GA):
It is a measure of genetic gain under selection. It refers to the improvement in
the mean genotypic value of the selected lines (or) families over the base or over
population.
The expected genetic gain (or) advance under selection may be estimated as suggested by
Johnson as
GA= k × 𝜎𝑝 × ℎ2𝑏
Where, ℎ2𝑏𝑠 = Broad sense heritability
k = Selection differential at 5% intensity of selection, k = 2.06
Genetic advance is also calculated by another formula

Genetic advance (GA) = k x √σ2 p x ℎ2𝑏

Where, VP = phenotypic variance

𝑮𝑨
K. GA as a percentage of mean : × 100
𝑋̅
The success of genetic advance under selection depends
 The magnitude of genetic variability present in base population
 Heritability of a character under selection.
 Intensity of selection

42
 The range of genetic advance is classified by Johnson et al., 1955 as
Low Less than 10 %
Moderate 10 – 20 %
High More than 20 %

Heritability and genetic advance are important selection parameters and

heritability estimate along with genetic advance are interrupted 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.
3. By studying the genetic diversity within a population
There are two important biometrical techniques used for this purpose are as under
a) Metroglyph analysis and index score method
b) D2 statistics by Mahalanobis, 1936
Exercise:
1. Estimate Variability parameters viz., Error variance, Genotypic variance, Phenotypic
variance, GCV, PCV, Heritability (broad sense), expected genetic advance and GA
expressed as % of mean from the following data and write conclusion based on the
result.
ANOVA
Source of Variation df S. S. M. S. CAL. F
Replication 2 3.22 1.6
Genotype 24 2708.59 112.86 58.18
Error 48 93.28 1.94
Total 74 2805.09 General mean = 50.76
2. Define variability, heritability, PCV, Genetic advance.
3. Differentiate : Broad sense and Narrow sense heritability

43
EXERCISE 10: DESIGNS USED IN PLANT BREEDING EXPERIMENTS
Date:
Terminologies:
 Treatment: The objects of comparison which an experimenter has to try out in the
field for assessing their values are known as Treatment. e.g., varieties, manures,
cultivation practices, methods of seed treatments, insecticides, etc.,
 Experimental material: The material on which the experiment is performed is called
experimental material. e.g. a set of varieties, a set of various sources of nitrogenous
fertilizer, a set of animals, etc.
 Experimental Units: The experimental material is divided into a number of ultimate
smaller units to which the treatments are applied is known as experimental Units. The
experimental unit may be a plot of land, a patient in hospital, a cow, a group of pigs in
pen, a batch of seeds, etc.
 Uniformity trial: A trial consists of growing in a field a particular crop with a uniform
treatment, dividing the field into small units, harvesting and recording the observations
from each of these units separately, is called uniformity trial. It is used to prepare
“Fertility Contour Map” representing appreciable variation in fertility and it does not
follow any systematic pattern and distributed over the field in an erratic fashion.
 Experimental error: Variation arises due to uncontrolled factors.
Basic principles of field experimentation:
There are three basic principles of field experimentation.
1) Replication :
 The repetition of the treatments under investigation is known as
Replication. The advantages of replications are as under.
 In order to obtain a greater precision in the field experiments, the most effective
method is to increases the number of replications. This will reduce the error. Increase
in replications increases the degree of freedom of error, which decreases the value of
t due to decrease in confidence interval. The shortening of confidence interval is
good proof of increased precision.
 The error of experiment arises from the difference between the plots of the same
treatment. Thus without replications estimates of error is not possible and without
estimates of error comparison is not possible.
 Another way of reducing the experimental error is to increase the plot size, but the
plot size should not increases beyond the 0.1 acre because further increase in plot
will increase the heterogeneity within the plot and affect the advantage of increase
plot size.

44
2) Randomization :
 The allocation of different treatments to the different experimental plots by a random
process is known as randomization of the treatments.
 It gives equal chance to all the treatments for being allotted to a more fertile plot or
plot with similar fertility.
3) Local Control :
 The principle of making use of greater homogeneity in groups of experimental units
(e.g. blocks of a number of plots homogeneous within themselves) for reducing the
experimental error is known as ‘Local Control’.•
 As a lower experimental error helps in detecting the smaller real differences between
the treatments, it is desirable that it should be reduced as far as practically possible.
This is possible by making use of the principle that the adjacent areas are relatively
more homogeneous than those widely separated. In the field we find that the fertility
of the field may be classified in two, viz.,
a) A major fertility variation which is usually marked by a fertility gradient.
b) Sporadic or scattered fertility variations, which are not systematic but are
distributed in patches throughout the field.
 Now, if we divide the whole field into blocks, which are homogeneous within
them, we can calculate the variance in yield due to the effects of the first type of
fertility variation and eliminate this variance for arriving at an estimate of the
variance due to chance error only.
 Randomizing the treatments within the blocks minimizes the effects of scattered
fertility variations.
Experimental design: The choice of experimental design depends on the number and
nature of the treatments under study. It also depends on the object of the experiment.
The following designs are used under specific situations:
1. Completely Randomized Design (CRD): Used when the experimental material is
limited and homogenous e.g. pot experiments on soil, laboratory experiments, etc.
2. Randomized Block Design (RBD): It is used when the fertility gradient in the field is
in one direction. It may be adopted up to 20 treatments without any appreciable loss of
efficiency.
3. Latin Square Design (LSD): It is used when the fertility gradient is in two directions.
It may adopt for number of treatments ranging from 5 to 12 in most of the situations.
4. Factorial Experiment: When there are several factors with different levels to be
studied simultaneously with the same precision.
5. Split Plot Design (SPT): It is used when the factors are such that some of them
require larger plots (like irrigation, depth of ploughing, sowing dates etc.) and some
require smaller plots may be studied with different precision.

45
6. Incomplete Block Design (IBD): It is used when the number of treatments is
sufficiently large.
Size and Shape of plots:
 There is no particular size or shape of the plots which may be said to be the best for all
circumstances.
 The plot size increases, coefficient of variation (CV) of plots decreases. Therefore, it is
better to increase the plot size upto the extent of 1/ 10 acre.
 If the plot size is greater than this, the increase in soil heterogeneity within the plots
affects any advantage obtained by increasing the plot size.
 However, an increase in the number of replications with reduced plot size leads to a
more precise treatment comparison, when the land and other facilities are limited.
 So far as the shape of the plot is concerned it can be anything, square, rectangle or a
narrow long strip.
 The dimensions of the plots are so chosen as to utilize the whole of the experimental
site effective and give a correct field lay out.
Border Effect:
 The yield or any other character of the plants is affected in case of those plants which
are nearer to the borders of the plots with the result that the border plant differ from the
plants of the central portions of the plots with respect to the yield or other character
under study.
Exercise:
1. Define : Treatment, Experimental material, Experimental Unit, Replication,
Randomization, Border effect
2. Write in brief about the basic principles of experimentation.
3. Enlist the advantages of replication during experimentation.
4. Enlist the use of various experimental designs in different situations.

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EXERCISE 11: ANALYSIS OF RANDOMIZED BLOCK DESIGN Date:
Lay out of RBD:
 The experimental material (field) is first divided into blocks consisting of
homogeneous (uniform) experimental units.
 Each block is divided into number of plots equal to the total number of treatments.
 Randomization is done within each block and the treatments are applied in random
manner.
Collection and analysis of data
 After the collection of data from the individual experimental units (treatments),
tabulation and analysis is done. ANOVA (Analysis of Variance) table is formed.
 The significance of ANOVA table is that it indicates the sources of variation exhibited
by the treatments, the magnitude of variation derived from different sources and their
significance/non-significance basis.
Computation of Critical Difference (C.D.):
 Critical Difference is the difference between the treatment means, which places the
treatments statistically as well as significantly apart.
 Otherwise if the difference of two treatment means is less than the C.D., it can be
concluded that both the treatments are at par.

Example: Seven wheat varieties were evaluated with RBD with seven replications at
governmental experimental farm. Analyse the data and write conclusions.

Replication
Genotype I II III IV V VI Total Avg.
GW 322 148 132 148 132 1322 124 816 136.00
GW 496 132 156 124 100 116 124 752 125.30
GW 451 132 156 116 76 132 116 728 121.30
GW 273 132 164 100 68 100 124 688 114.67
GW 278 124 124 148 44 76 100 616 102.67
GW 173 100 124 124 92 68 92 600 100.00
GW 452 116 124 160 44 100 100 636 106.00
Total 884 980 920 556 716 780 4836 (G.T.)

Answer:
4836
 General Mean (G. M.) = = 115.14
42
(GT)2 (4836)2
 Correction factor (C.F.) = = = 556830.86
N 42
 Total sum of square

47
 Total SS = ∑ x2 – C.F. = [(148)2 + (132)2 + (132)2 + …..+ (100)2] – 556830.86
= 34289.14
∑ T2 3378640
 Treatment sum of square = – C.F. = - 556830.86 = 6275.81
r 6
∑ R2 4018448
 Replication sum of square = – C.F. = - 556830.86 = 17233.14
t 7
 Error SS = Total S.S. –Treat S.S. – Rep. S.S.= 34289.14 - 6275.81 – 17233.14
= 10780.19
ANOVA Table
Source df S.S. M.S. Cal. F Table F
Replication (r-1)= 5 17233.14 3446.63 9.6 2.53
Treatment (t-1) =6 6275.81 1045.97 2.91 2.42
Error (t-1) (r-1) = 10780.19 359.34
30
Total (rt-1) =41 34289.14 836.32

Calculated F for the treatment 2.91> Table F 0.05,6,30 value i.e. 2.42
Observed difference is significant. We conclude that treatment means are significantly
differed from each other.
𝑀𝑆𝑒 359.34
S.Em. = √ = √ = 7.74
𝑟 6

C. D. = table t (0.05) (30) × √2 × S.Em.

= 2.042 × 1.41 × 7.74
= 22.348
1 2 3 4 7 5 6
136.00 125.30 121.30 114.67 106.00 102.67 100.00

Treatment 1 gives higher yield and treatment 6 gives lower yield.

Conclusions: Treatments 1, 2, 3, 4 were at par; treatments 2, 3, 4, 7 were at par;
treatments 3, 4, 7, 5, 6 were at par.
√𝑀𝑆𝑒 √359.34
C.V. % = × 100 = × 100 = 16.46 %
𝐺.𝑀. 115.14

48
EXERCISE 12: DEVELOPMENT OF HYBRIDS & PREDICTION OF
PERFORMANCE OF DOUBLE CROSS HYBRIDS

Date:
DEVELOPMENT OF HYBRID VARIETIES
Development of hybrid varieties differs from species to species. The production of hybrid
varieties in cross pollinated crops consists of three main steps.
1. Development of Inbreds :
 Development of inbreds is an important step in the production of hybrids. There are
two methods of developing inbred lines.
 One by selfing of heterozygous populations and another by doubling of haploids.
Various populations, viz., open pollinated varieties, synthetic varieties or any other
heterozygous population can be used for selfing. Superior plants on the basis of
vigour, disease resistance and yield are selected and selfed. Progeny of selected
plants are grown separately from the selfed seed in the next season.
 Again superior plants are selected in each progeny and selfed. In this way, selfing is
continued for 6-7 generations to get superior homozygous inbreds. The main purpose
of selfing is to fix desirable genes in homozygous condition and eliminate genotypes
with undesirable deleterious genes.
 Inbreds can also be developed from haploids by doubling the chromosome number
through colchicine treatment. This is the short cut method of developing inbreds.
 The vigour which is lost during inbreeding is regained when two unrelated or diverse
inbred lines are combined to develop F1 hybrid.
2. Evaluation of Inbred lines :
 The value of an inbred is assessed from its performance in hybrid combination with
other inbreds. The inbred lines are evaluated on the basis of their general combining
ability (gca) and specific combining ability (sca).
 Two methods, viz. (i) Top cross method, and (ii) Single crosses are commonly used
to measure the combining ability of inbreds.
(i) Top Cross Method :
 Top cross refers to a cross between an inbred line and an open pollinated variety.
Several inbred lines say 100 are crossed to a common tester (open pollinated variety)
to produce 100 single crosses.
 The yield performance of these crosses is evaluated in replicated trials on multiple
locations. Those lines which produce high yielding single cross with tester are
selected. This method was suggested by Davis in 1927.
 A large number of lines can be evaluated by this method at a time. The yield is
compared from the mean yield of all the crosses.
 Inbred lines which give high yield in top crosses generally produce high yielding

49
 single crosses. This method is used for measuring general combining ability.
 Here combining ability refers to yield performance and not to gca variances and
effects. The top cross seed is produced by planting alternate rows of inbred and open
pollinated variety and removing the tassel (male inflorescence) of inbred line.
(ii) Single Cross Method:
 This method is used to measure the specific combining ability of those inbreds which
are selected on the basis of top cross performance.
 The selected lines are crossed in all possible combinations viz, n(n - 1)/2, where n is
the number of inbred lines.
 With 10 inbred lines, there would be 10(10 - 1)/2 = 45 single crosses excluding
reciprocals.
 These single crosses are evaluated in replicated trials over several locations for yield
performance. The best performing single crosses are identified for release as a
variety or for use in the production of double cross hybrids.
 This method can evaluate only limited number of inbreds at a time, because
inclusion of more inbreds in crossing increases the number of single crosses in such
a way that their handling becomes difficult.
 Use of 50 inbreds, it will give rise to 50(50 - 1)/2 = 1225 single crosses.
Time of Testing:
 The testing of inbreds for general combining ability should be started from 3rd, 4th
and 5th generation of selfing.
 This will help in retaining of inbreds with good combining ability and elimination of
lines with poor combining ability.
 Some workers suggest that visual selection is effective in improving combining
ability in early inbred generations.
3. Production of Hybrid Seed :
 After identification of superior inbred lines, the hybrid seed is produced. Any type of
the hybrids can be developed.
 In case of single and three way cross hybrids, the rows of female and male parents
are planted in 2: 1 or 3: 1 ratio (Planting ratios are crop specific).
 In case of three way cross hybrid, the single cross hybrid is used as female parent
and inbred lines as male parent. In case of double cross hybrid, the rows of female
and male parents are planted in 3: 1 or 4: 1 ratio.
 The seed production is carried out in isolation to prevent crossing with other
compatible genotypes and maintain the high genetic purity.
Predicting Double Cross Performance:
 Single crosses are used to predict the performance of double cross hybrid. The yield of
a double cross can accurately be predicted from the mean yield of the four non parental
single crosses.
50
 The use of single cross performance for the prediction of double cross performance has
become a standard breeding procedure.
 The average performance of single crosses A x C, A x D, B x C and B x D is used to
predict the performance of the double cross (A x B) (C x D).
 Multilocational or multiseasonal testing is desirable to predict the performance of a
double cross hybrid.
 The predicted yield is worked out as follow:
(A x C) + (A x D) + (B x C) + (B x D)
Predicted yield = 4
Exercise:
1. Write down procedure for development of hybrids.
2. Explain predicting double cross performance.

51
EXERCISE 13: STUDY OF GENERAL COMBINING ABILITY & SPECIFIC
COMBINING ABILITY OF BREEDING LINES
Date:
Combining Ability:
 The concept of general and specific combining ability as a measure of gene action
was first proposed by Sprague and Tatum (1942).
 Combining ability is the relative ability of a strain to transmit desirable
performance to its progeny upon hybridization with other strains.
Types of combining ability
1) General combining ability (GCA)
 It is the average performance of a genetic strain in a series of cross combinations,
estimated from the performance of F1’s from crosses OR
 It is the average ability of a parent to combine with a set of other parents in
hybridization.
Characteristics of General combining ability
 GCA is related to parents only
 GCA effect of a parent (P) is calculated as (G-M)
Where, G = mean value of crosses in which P is a common parent and
M = General mean.
 GCA variance is attributed to additive and additive x additive interaction variance
which is considered as fixable.
 The knowledge of GCA helps to evaluate the parents of broad genetic base.
2) Specific combining ability (SCA)
 It is the deviation in the performance of a specific cross combination from the
performance predicted on the basis of general combining ability of the parents
involved in the cross.
 It is used to designate those cases in which certain combinations do relatively
better or worse than that would be expected on the basis of average performance of
the parents involved in the cross.
Characteristics of specific combining ability
 SCA is related to crosses (hybrid) only
 The knowledge of SCA helps to identify the hybrids.
 High SCA may be due to good x good or good x poor or poor x poor combiners.
 If high SCA is due to high x high GCA effects, one can exploit the hybrid vigour
in F1.
 If high SCA is due to high x low GCA effects, such parents are used for
development of sister lines which are used for the development of synthetic

52
 varieties. Based on this knowledge, further breeding programme is to be
formulated.
 If high SCA is due to low x low GCA effects, there are least chance of improving
such parents.
 SCA effects of a cross is calculated as (S – G1 – G2 – M)
Where, S = mean value of a specific crosses
G1 & G2 = GCA effects of two parents involved in a cross, and
M = General mean
 SCA variance is attributed to dominance genetic variance and additive ×
dominance and dominance × dominance interaction variance which are considered
as non-fixable.
 Kempthorne (1957) defined GCA and SCA variances in terms of covariance of
half and full sibs in a random mating population.
o Progeny produced by GCA is half-sibs (H.S.), because
1
𝜎2𝜎𝜎𝜎 = Cov. (H.S.) = 4 𝜎2𝜎
o Progeny produced by SCA is full-sibs (F.S.), because
1
𝜎2𝜎𝜎𝜎 = Cov. (F.S.) – 2 Cov. (H.S.) = 𝜎2𝜎
4
Methods to estimate combining ability:
1. Inbred –variety cross (Jenkins & Brunson , 1932)
2. Polycross (Tysdal et al., 1942)
3. Diallel cross analysis (Griffing, 1956)
4. Line × Cross analysis (Kempthorne, 1957)
Utility of combining ability in plant breeding
 It helps to identify the parents having good potential to transmit desirable
characteristics to their progenies.
 It helps to sort out the promising crosses for yield and its components.
 It also provides the information regarding the nature and magnitude of gene action
involved in the inheritance of traits.
 It is valuable for adoption of efficient breeding programme.
Selection of parents based on GCA is more desirable as compared to that based
on per se performance. Why?
 Parents are selected on the basis of per se performance such parents may be good
but transmission ability to their progenies may or may not be good. While parents
selected on the basis of GCA denotes good transmission ability to their progenies.
 In addition nicking ability of parents is also based on GCA. Therefore, parents
selected on the basis of GCA are more desirable as compared to selection of
parents on the basis of per se performance.

53
 How Combining ability is superior over simple hybridization between any two
parents at a time?
 The Knowledge of combining ability of parents helps to predict the performance of
resulting hybrids and accordingly one can formulate the breeding programme,
while in case of simple hybridization between any two parents; one can’t formulate
the breeding programme.
Relationship between combining ability and gene action:
1) If only GCA variance is significant: Additive gene action is important. In such case,
pedigree method of selection may be effective and that should be followed.
2) If only SCA variance is significant: Non- additive gene action is important. In such
cases, exploitation of heterosis in F1 should be followed.
3) If both GCA and SCA variances are significant: Both additive and non-additive
gene actions are important. In such cases, parental mating approach or reciprocal
recurrent selection by alternate selfing and inter mating should be followed which
helps in isolation of desirable transgressive segregants in segregating generations.

Exercise:
1. Differentiate general and specific combining ability.
2. Enlist the methods to estimate combining ability.
3. Enlist the utility of combining ability in plant breeding.

54