Fundamental of Plant breeding

Practical Notes

Dr SARVAN KUMAR
Assistant Professor
Agriculture Botany
SGTB Khalsa College, Sri Anandpur Sahib
 Content
Lab. No. Experiments Page No.

Laboratory Exercise for plant breeding

1 Study of the Flowers, flower parts and inflorescence of plant
2 To Study about the Plant Breeder’s kit and its uses
3 To Study of germplasm of various crops

4 To Study floral structure of self-pollinated and cross pollinated

crops
5 To Study of Emasculation and hybridization techniques in self
and cross pollinated crops
6 Consequences of inbreeding on genetic structure of resulting
populations
7 To Study of male sterility system in plants
8
To Study the Handling of segregation populations/ generations
9 Methods of calculating mean, range, variance, standard deviation,
heritability
10 Designs used in plant breeding experiments, analysis of
Randomized Block Design, Laying out of Field Experiments

11 To work out the mode of pollination in a given crop and extent of

natural out-crossing
12 Prediction of performance of double cross hybrids
 Laboratory Exercise for plant breeding
This laboratory exercise is designed to provide the student with practical experience in the
breeding method known as hybridization. The student will learn how to emasculate the
female plant and cross pollinate it with pollen from the male plant. The parent plants which
students will use will be provided by the instructor from a population which he has
developed. The plants used in this exercise. They were chosen because they grow and
flower quickly.
Materials/ equipments required
1. Tweezers
2. Scissors
3. alcohol
4. Cotton swabs (alcohol)
5. Paper “boats”
6. Tags
7. Pencil
8. Two plants, Field note book etc

Procedure:
Step 1: Obtain 2 plants from the instructor. Designate one plant as a female and the other
male.
Step 2: On the female plant choose a raceme composed of 8-10 flowers. Trim the raceme
by removing 4-5 flowers at their bases
Step 3: The outer petals of each flower should be removed using the scissors. The inner
petals should be gently trimmed so as to allow the sexual column containing the ovary and
staminal column to be exposed. WARNING—Failure to be gently in trimming the inner
petals could result in cutting off the staminal column.
Step 4: You are now ready to begin the process of emasculating the flowers which is done
be remove the pollen. Take the tweezers and gently squeeze the base of the flower to trip
or release the sexual column. You should be able to see the pollen as tiny yellow grains on
the top of the column.
Step 5: Gently use the vacuum to remove the pollen from the sexual column. DO NOT
allow the vacuum to directly tough the column, it may kill the flower. If a vacuum is
unavailable alcohol swabbed on the pollen will kill it. Emasculation is complete.
Step 6: Choose a raceme from the male plant and remove it at the base of the stem. Gently
remove or trim the outer and inner petals being careful not to trip or cut-off the sexual
column.
Step 7: Gently use the tweezers to trip the sexual column to expose the pollen using a paper
“boat” collect the pollen by using the pointed end of the “boat” to pick-out the pollen.
Step 8: Take the “boat” filled with pollen and carefully deposit the pollen grains onto the
flowers which were emasculated on the female plant
Step 9: Tag the newly pollinated flower with a tag upon which you have noted the female
parent, male parent, and the date of the cross. Attach to the stem of the raceme.
Step 10: Within a few days you should be able to see if the cross was successful. If it was
you will notice the beginning of the seed pod formation.

Equipment commonly used in emasculation and crossing procedure

 Experiment No. 1
Object: Study of the Flowers, flower parts and inflorescence of plant
Flowering plants make new seeds inside the flower using male and female parts. Some
plants, such as a holly tree, may have either male or female flowers. If a tree has male
flowers it will never produce any berries: they are only produced on trees with female
flowers.
Not all green plants produce flowers. Mosses, ferns and conifers do not produce flowers.
The plants that have flowers belong to a group called the flowering plants.
A flower is the part of a plant that is responsible for making new seeds. It is often made
of petals and may have an attractive scent.

Physiological Function
A flower, sometimes known as a bloom or blossom, is the reproductive
structure found in plants that are floral (plants of the division Magnoliophyta, also
called angiosperms). The biological function of a flower is to effect reproduction,
usually by providing a mechanism for the union of sperm with eggs.

Composing of Flower

The essential parts of a flower can be considered in two parts: the vegetative part,
consisting of petals and associated structures in the perianth, and the reproductive or
sexual parts. A stereotypical flower consists of four kinds of structures attached to the
tip of a short stalk. Eachof these kinds of parts is arranged in a whorl on the receptacle.
 Parts of Flower:
A. Vegetative Parts of a Flower

The vegetative part of a flower consists of the following:

 Petals: This is a bright-coloured part that attracts bees, insects, and birds.
Colour of petals varies from plant to plant; some are bright while some are pale
coloured. Thus, petals help us to differentiate one flower from another.

 Sepals: Sepal is the green-coloured part beneath the petals to protect rising
buds. Some flowers have fused petals-sepals while a few have separated petals-
sepals.

B. Reproductive Parts of a Flower

Flowers contain the plant’s reproductive structures

In different plants, the number of petals, sepals, stamens and pistils can vary. The presence
of these parts differentiates the flower into complete or incomplete. Apart from these parts,
a flower includes reproductive parts – stamen and pistil. A flower may have only female
parts, only male parts, or both.

The reproductive parts of a flower consist of the following:

 Stamen: This is the male reproductive organ and is also known as Androecium. It
consists of two parts namely: anther and filaments.

 The anther is a yellowish, sac-like structure, involved in producing and storing the
pollens.

 The filament is a slender, threadlike object, which functions by supporting the

anther.

 Pistil: This is the innermost part and the female reproductive organ of a flower
which comprises three parts -stigma, style and ovary. This is collectively known as
the pistil.

 Stigma: It is the topmost part or receptive tip of carpels in the gynoecium of a

flower.
  Style: It is the long tube-like slender stalk that connects stigma and the ovary.

 Ovary: It is the ductless reproductive gland that holds a lot of ovules. It is the part
of the plant where the seed formation takes place.

Type of Stamen
1. Monadelphous : Fused into a single, compound structure
2. Diadelphous : Joined partially into two androecial structures.
3. Didynamous : Occurring in two pairs, a long pair and a shorter pair
4. Tetradynamous: Occurring as a set of six stamens with four long and two
shorter ones
5. Polyadelphous: Having united filaments so that they are arranging in three
or more groups.
6. Syngenesious: Having the stamens united by the anthers; of or pertaining to
the Syngenes
Types of pistil
1. Simple pistil : The female ovule-bearing part of a flower composed of ovary
and style and stigma.
2. Syncarpous pistil : Syncarpous (fruit or pistil), composed of several carpels
consolidated into one.
3. Apocarpous pistil : Having carpels that are free from one another. Used of a
single flower with two or more separate pistils as in roses.
 Type of flower
1. Complete flower : A flower having all four floral parts: sepals, petals, stamens, and
carpels.
2. Incomplete flower : A flower without one or more of the normal parts, as carpels, sepals,
petals, pistils, or stamens.

Inflorescence

An inflorescence is a group or cluster of flowers arranged on a stem that is composed

of a main branch or a complicated arrangement of branches. Morphologically, it is
the part of the shoot of seed plants where flowers are formed and which is accordingly
modified. The modifications can involve the length and the nature of the internodes and
the phyllotaxis, as well as variations in the proportions, compressions, swellings,
adnations, connations and reduction of main and secondary axes.
Type of Inflorescence
1. Spike - an elongate, unbranched, indeterminate inflorescence with sessile flowers.
2. Spikelet - a small spike, characteristic of grasses and sedges.
3. Raceme - an elongate, unbranched, indeterminate inflorescence with pedicelled flowers.
4. Panicle - a branched raceme.
5. Corymb - a flat-topped raceme with elongate pedicels reaching the same level.
6. Compound Corymb - a branched corymb.
7. Umbel - a flat-topped or rounded inflorescence with the pedicels originating from a
commonpoint. Umbels can be determinate or indeterminate.
8. Compound Umbel - a branched umbel, with primary rays arising from a
common point,andsecondary umbels arising from the tip of the primary rays.
9. Capitulum (or head) - a dense vertically compressed inflorescence with sessile
flowers on a receptacle and subtended by an involucre of phyllaries, characteristic of
the Asteraceae. Headscan be determinate or indeterminate.
10. Thyrse - a many-flowered inflorescence with an indeterminate central axis and
many opposite, lateral dichasia; a mixed inflorescence, with determinate and
indeterminate shoots.
 Definite inflorescence
A type of flowering shoot) in which the first-formed flower develops from the growing
region at the top of the flower stalk. Thus no new flower buds can be produced at the tip
and other flowersare produced from lateral buds beneath.
Type of Definite Inflorescence
1. Solitary cyme
When the apical or the axillary bud forms a single flower it does not form a real
‘inflorescence’ but this type is better included within the ‘definite’ group as further
development is limited.

2. Cyathium cyme
The common garden Poinsettia (Euphorbia) pulcherrima shows this specialized cymose
inflorescence which is covered by a cup-shaped green involucre formed by the union of
bracts. The extremely reduced florets are placed on a convex receptacle.
 3. Dichasium
This is the more normal cymose type where- two lateral branches develop on the two
sides of the terminal apical (oldest) flower. The lateral branches may again branch
similarly after the mannerof biparous cymose branching
4. Polychasium
These branches as in the multiparous cyme, there being more than two lateral branches
from the base of the apical flower. An example may be found in Calotropis of
Asclepiadaceae of Caprifoliaceae.

Complete vs. Incomplete Flower

Although all flowers are different, they have several things in common that make up
their basic anatomy. The four main parts of a flower are the petals, sepals, stamen, and
carpel (sometimes known as a pistil). If a flower has all four of these key parts, it is
considered to be a complete flower. If any one of these elements is missing, it is an
incomplete flower.
Complete : Rose , Hibiscus ,Tulip
Incomplete : Holly,Corn,Squash,Grasses

Perfect vs. Imperfect

The reproductive parts of the flower that are necessary for seed production are the
stamen (the male organ) and carpel (the female organ). If a flower has both of these
parts, it is called a perfect flower, even if it lacks some of the other key parts. If a flower
has only one of the reproductive parts, either a stamen or carpel, it is considered to be an
imperfect flower.
Perfect: Lilly,Apple,Orchid,Cherry blossom,Orange,Carrot,Legumes
Imperfect : Begonia,Squash,Cucumber,Corn,Walnut,Chestnut

Monoecious vs. Dioecious

Furthermore, plants that have imperfect flowers can be broken up into two categories.
These are monoecious and dioecious. Monoecious plants have imperfect flowers, both
male and female, on the same plant. This means that some of the flowers on the same
plant will only have a functioning stamen but lack carpel, while other flowers on the
same plant will have the reverse situation: functioning carpel but not stamen.
These plants make life easier for themselves, and for the gardener, as they are self-
pollinating and are therefore able to bear fruit and produce seed on their own. Dioecious
plants have imperfect male and female flowers on separate plants, so one plant will
have all-female imperfect flowers with carpel and no stamen, while another plant of the
same type will have all-male imperfect flowers: functioning stamen but no carpel. For
dioecious plants, it’s imperative that they are planted near each other to make pollination
more likely.
Perfect : Birch, Oak, Pine, Spruce, Banana, Passionflower, Corn
Imperfect: Mulberry bush, Poplar, Juniper, Pistachio, Willow, Papaya, Hop, Asparagus,
Mistletoe , Date palm
 Experiment No. 2

Object: To Study about the Plant Breeder’s kit and its uses

Material required: Forceps, scissors, alcohol, tags, pencil and butter paper bags,
Cotton, petri plate, field note book, hair/ paint brush. etc

Introduction: The plant breeder frequently use different tools/ instruments and
material to carry out selfing, artificial crossing and for taking field observations.
Object of the experiment to study about the plant breeder’s kit and its uses.

Details of equipments/instruments and their uses in crossing programme

Sr. no. Name of equipments Uses
1 Fine pointed forceps It is used for incising the floral buds and for
removing the anthers from it. e.g. Tobacco,
Sesamum etc
2 Small/ curved scissor for cutting the small florets in cereals and small
flowers in the crops like lucerne, guar etc.
3 Long straight scissor it is used for clipping, cutting the vegetable parts
and large size floral parts in cereals like wheat,
sorghum, bajra, and tobacco.
4 Sharp pointer it is used for incising the floral parts and for
removing the anthers from the crops like bajra

5 Eye lens or magnifying For observing the reproductive parts to confirm

lens that there should not be any part of the anther left
on the stigma or stigma is free from any foreign
pollens
6 U-pins(u- clips) It is used for fasting the bags on ear heads or
flowers to keep the bag in proper position.

7 Paint brush It is used for transferring the pollen grains in crops

like Castor, Sorghum etc.
Advantage: Without injuring to stigmas or pollens,
pollination is accomplished very smoothly.
8 Pencil It is used for writing field labels or field bags.
Sometimes it is also used for emasculating the
sorghum flowers. Compared to ink or ball pen
 writing, pencil writing should be preferred as it
will not erase or spread during rains, dew or under
intense light.
9 Washing bottle It is used for filling sterilizing agent like alcohol or
spirit to sterilize the scissors, pointers, forceps and
brush during crossing work.
10 Wire ring and smooth It is used for selfing in crops like Cotton, Okra, and
thread Sesamum etc. Thread is used for fastening (tying)
the bud, while ring is inserted in axis of flowers to
identify it. Compared to bags, this method is more
convenient and cheaper
11 Small white tag It is used for identifying the internal flower or a
small twig during crossing programme. The
detailed information about crossing is written on it
with pencil and then it is inserted on pedicel or
peduncle e.g. Cotton, Bajra, Wheat, Sorghum,
Sesamum etc.
12 Soda straw tubes it is used for protecting the emasculated or
pollinated flower buds of cotton, tobacco etc.
Advantage: compared to paper bags it is very
convenient, easy and cheaper method of selfing
and crossing.
13 Waxy threads It is used for fastening(tying) the luggage labels on
the plants
14 Luggage labels : It is used for tagging the large sized plants like
(white or yellow) Tur or Castor while rouging or during selection.

15 Aluminium label with It is used for tagging the flowers in fruit crops or
wire tree species after crossing. It is also used for
identification of selected trees.
16 Muslin cloth bag (large To cover the whole plant while selfing or crossing
size) in the crops like Chillies, Brinjal etc. In large sized
plants like Tur it can be used for protecting
individual branch also.

17 Yellow sample bag For storing the crossed seeds in small quantity.
18 Butter Paper Bag It is used for protecting the individual flowers or
small twigs during selfing or crossing e.g. Mung
bean, Tur, Cotton, Okra, Brinjal And Earheads of
Sorghum, Bajra, Wheat and Maize. Being white
and semi-transparent it is more suitable than paper
bags even enthesis or blooming can be seen from
outside.
19 Kite paper bag It is used for protecting small size flowers of
(white/red) Pulses, Oilseeds and Other food crops during
selfing and crossing.
20 Automizer It is used for spraying the gametocides during
emasculation of flowers e.g. 57% ethyl alcohol can
be filled in it for killing the pollens of Lucerne
21 Adhesive (Rubber It is used for selfing the individual flowers which
solution) open (bloom) by one suture or pore. e.g.
Papilionaceous flowers, Tobacco etc. When other
such adhesives like quickfix can also be used for
the same purpose.
22 WAX For selfing Tur, Wal, Udid and Mug flower buds.
23 Mud and cotton This is indigenous method of selfing when threads
or bags are not available it is used for selfing in
cotton and okra

1. Fine pointed
forceps
2. Small/ curved
scissor

3. Long straight
scissor

4. Sharp pointer

5. Eye lens or
magnifying lens

6. U-pins(u- clips)
 7. Paint brush

8. Pencil

9. Washing bottle

10 Wire ring and smooth

thread
11. Small white tag

12. Soda straw tubes

13. Waxy threads

14. Luggage labels

(white or yellow)
15. Aluminium label with wire

16 Muslin cloth bag (large size)

17. Yellow sample bag

18 Butter Paper Bag

19. Kite paper bag
(white/red)

20. Automizer

21. Adhesive (Rubber solution)

22. WAX
23. Mud and cotton
 Experiment No. 3
Object: To Study of germplasm of various crops
Principle: Germplasm is living tissue from which new plants can be grown. It can
be a seed or another plant part – a leaf, a piece of stem, pollen or even just a few cells that
can be turned into a whole plant. Various institutes with different objectives are engaged in
plant and/or germplasm collecting activities. The collecting of plant genetic resources
primarily aims at tapping germplasm variability in different crop (Agriculture- Horticulture)
plants, their wild relatives and related species. The germplasm so collected reveals the
nature and extent of variability in different species, within species, cultigens, etc. as also
their agro-ecological/ phyto-geographical distribution. Knowledge of agro-ecology, crops
and their distribution and harvesting time in areas of survey, local contacts, equipment
required, transport arrangements and routes to be followed, distances involved, places of
halt/camping sites available, transport of material, besides team-composition etc. is to be
acquired before setting out on a collecting expedition. Of equal importance is to acquire
knowledge on diversity in crop plants vis-a-vis its distribution to tap target areas and/or
target species and the variability contained thereof.
Germplasm collecting strategies
A. For seed collections (cultivated and wild species)
 Collect from (30- 100) individuals per site (50 seeds of each as one sample or less, if
necessary, at random. One inflorescence per plant is generally suitable.
 Sample as many sites as possible according to availability of time.
 Choose sampling sites over as broad an environmental range as possible. This should
capture all alleles with frequency of 5 percent or more in the population.
 Change tactics, where necessary, for wild species, that is, where individuals are
scattered, you may need to consider that a population for sampling spreads over several
square kilometres.
 If considerable morphological variation is present in a population, make separate
samples of each type.
 Add biased sampling if some morphotypes are not included in random sampling.
 Take whole inflorescences, as well as seeds, where necessary, as vouchers.
 Make herbarium specimens, where possible.
 Take photographs.
 Write meticulous field notes.
B. i) For vegetatively propagated cultivated species
1. Sample each distinct morphotype in a village.
2. Repeat at intervals over an area.
3. Supplement with seed collections, where possible, and give same collection numbers if
seeds come from the same plants as the vegetative samples. If they do not or are bulked
samples, give separate collection numbers.
ii). For collecting wild vegetatively propagated species
Collect just a single propagule from each of 10-15 individuals as a bulk sample (less if
organs are very large, more if smaller, from area of about 100 x 100 m).
Germplasm cataloguing, Data storage and Retrieval
Each germplasm accession is given an accession number. This number is pre fixed 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
feature or descriptors is also recorded in the germplasm maintenance records. Catalogues
of the germplasm collection for various crops are published by the gene banks. The
amount of data recorded during evaluation is huge. Its compilation, storage and retrieval is
now done using special computer programmes.
National Bureau of Plant Genetic Resources (NBPGR)

NBPGR establishment in 1976 is the nodal organization in India for planning, conducting,
promoting, coordinating and lending all activities concerning plant.
 Experiment No. 4
Object: To Study floral structure of self-pollinated and cross pollinated
crops
Material required:- Scissor, forceps, plant inflorescence , dissecting Microscope etc
A flower is a structure adapted to reproduce the species. Floral morphology of a
crop plant is studied with the following objectives.
(i) To get acquainted with different parts of the flower and their functions
(ii) To study the time of opening and closing of the florets
(iii) To know the time of anther dehiscence and stigma receptivity; and
(iv) To study the mode of pollination in a particular crop.
All this information is a pre-requisite for undertaking hybridization work in any crop
plant.
Study the inflorescence of the plant
(i) Study the various floral parts like Calyx Corolla, Androecium and Gynoeciun
(ii) On the basis of this information. Draw the floral diagram and note the floral formula.
(iiii) For knowing the anthesis and time of anther dehiscence, observations are to
be recorded after emergence of inflorescence in the plant grown in laboratory /field.
(iv) For procedure to determine the mode of pollination of a crop species.

1. Floral structure of Rice (Oryza sativa): A self pollinated crop

Panicle
 The inflorescence of rice plant, borne on terminal shoot and thus called as panicle.

 It is determinate type and at maturity, it is droopy in nature.

Spikelet
 A spikelet is the floral unit and consists of two sterile lemmas, a lemma, a palea and the
flower.

 Its parts are:

 Lemma: It is a 5- nerved hardened bract with a filiform extension (of the

middle nerve) known as awn.
 Palea: It is a 3- nerved bract slightly narrower than lemma.

 Flower: It consists of 6 stamens with two-celled anthers and a pistil with one
ovary and two stigmas. The pistil contains one ovule.
2. Floral Structure of wheat (Triticum aestivum): A self pollinated crop
Floral Biology

The inflorescence of wheat is a spike bearing two opposite rows of lateral spikelets
and a single terminal spikelet on the primary axis. The unit of spike is called
spikelet. Two to five florets are born in each spikelet, subtended by a pair of glumes.
Each floret contains three anthers and a pistil bearing two styles each with feathery
stigma and two ovate lodicules which are modified perianth structure. Florets at
anthesis are forced open by swelling of the lodicules. Flowering starts several days
after the wheat spike emerges from the boot. Florets on the main culm flower first
and those on the tillers flowering later. Flowering begins in the early morning and
continues throughout the day. Two to three days are required for a spike to finish
blooming. A wheat grain is caryopsis, a small dry, indehiscent, one seeded fruit with
a thin pericarp consisting of a germ or embryo and an endosperm.
3. Floral structure of Maize (Zea mays) A cross pollinated crop

Floral biology

It is monoecious plant bearing male flower are the growing tip as tassel and female
flower at the axial of the leaf on the shoot. The tassel is terminal with staminate
flowers in several roots. Each pairs of flower consist of
sessile and pedicillate spiklet. Each spiklet contains two male
infloresc
similar glumes. The flower contains membranous palea ence

with three stamens and two lodicules. The pollens remain

viable for 18 to 24 hours.
The female inflorescence is a spadex known as cob or
ear. It is modified lateral branch developed from lateral female
bud. The shoot is composed of compressed internodes infloresc
ence
from which husk rise and terminates in an ear on which
the sessile are borne. Spiklets are in pair. Each spiklet
having two flowers, the lower one is reduced to lemma
and palea is non-functional, while upper one contained
knob shaped ovary surrounded by broad lemma and thin
palea. One carpel is provided with long silky hair, which
behaves as style and style stigma throughout the length.

Figure 1: Maize
plant showing
position of male
and female
inflorescences

Fig.3 Male flowers

 anther

bracts enclosing a pair of flowers

and forming a spikelet

Figure 2: Part of male inflorescence

 Stigma

Ovary

Figure 4: Female
inflorescence

Figure 5: Longitudinal section through

female inflorescence
 Experiment No. 5
Object: To Study of Emasculation and hybridization techniques in self
and cross pollinated crops
Methods of Emasculation
1. Hand Emasculation
In species with large flowers, removal of anthers is possible with the help of
forceps. It is done before anther dehiscence. It is generally done between 4 and 6
PM one day before anthers dehisce. It is always desirable to remove other young
flowers located close to the emasculated flower to avoid confusion. The corolla
of the selected flower is opened with the help of forceps and the anthers are
carefully removed with the help of forceps. Sometimes corolla may be totally
removed along with epipetalous stamens e.g. gingelly.
In cereals, one third of the empty glumes will be clipped off with scissors to
expose anthers. In wheat and oats, the florets are retained after removing the
anthers without damaging the spikelets. In all cases, gynoecium should not be
injured. An efficient emasculation technique should prevent self pollination and
produce high percentage of seed set on cross pollination.
2. Suction Method
It is useful in species with small flowers. Emasculation is done in the morning
immediately after the flowers open. A thin rubber or a glass tube attached to a
suction hose is used to suck the anthers from the flowers. The amount of suction
used is very important which should be sufficient to suck the pollen and anthers
but not gynoecium. In this method considerable self-pollination, up to 10% is
like to occur. Washing the stigma with a jet of water may help in reducing self-
pollination; however self pollination cannot be eliminated in this method.
3. Hot Water Treatment
Pollen grains are more sensitive than female reproductive organs to both genetic
and environmental factors. In case of hot water emasculation, the temperature of
water and duration of treatment vary from crop to crop. It is determined for
every species. For sorghum 42-48OC for 10 minutes is found to be suitable. In
the case of rice, 10 minutes treatments with 40-44OC is adequate. Treatment is
given before the anthers dehiscence and prior to the opening of the flower. Hot
water is generally carried in thermos flask and whole inflorescence is immersed
in hot water.
4. Alcohol Treatment
It is not commonly used. The method consists of immersing the inflorescence in
alcohol of suitable concentration for a brief period followed by rinsing with
water. In Lucerne the inflorescence immersed in 57% alcohol for10 second was
highly effective. It is better method of emasculation than suction method.

5. Cold Treatment
Cold treatment like hot water treatment kills the pollen grains without damaging
gynoecium. In the case of rice, treatment with cold water 0.6OC kills the pollen
grains without affecting the gynoecium. This is less effective than hot water
treatment.
6. Genetic Emasculation
Genetic/ cytoplasmic male sterility may be used to eliminate the process of
emasculation. This is useful in the commercial production of hybrids in maize,
sorghum pearl millet, onion, cotton, and rice, etc.,
In many species of self-incompatible cases, also emasculation is not necessary,
because self-fertilization will not take place. Protogyny will also facilitate
crossing without emasculation (e.g.) Cumbu.

7. Use of Gametocytes
It is Also known as chemical hybridizing agents (CHA) chemicals which
selectively kill the male gamete without affecting the female gamete. eg. Ethrel,
Sodium methyl arsenate, Zinc methyl arsenate in rice, Maleic hydrazide for
cotton and wheat
Bagging
Immediately after emasculation the flower or inflorescence enclosed with
suitable bags of appropriate size to prevent random cross-pollination.
Crossing
The pollen grains collected from a desired male parent should be transferred to
the emasculated flower. This is normally done in the morning hours during
anthesis. The flowers are bagged immediately after artificial crossing.
Tagging
The flowers are tagged just after bagging. They are attached to the inflorescence
or to the flower with the help of a thread. The following may be recorded on the
tag with pencil.
1. Date of emasculation:
 2. Date of pollination:
3. Parentage :
4. No. of flower/ spike emasculated

Emasculation and hybridization techniques in Paddy crop ( Self pollinated

crop)

1. Emasculation:

It is done in the afternoon on preveious day or early in the morning on the day of
pollination. The ear just emerged is selected and all spiklets already opened are
clipped the spiklets which are likely to be opened are selected and six anthers from
each spiklet is removed with needle and fine pointed forceps. The emasculated ear
after examination with lens covered with perforated butter paper bag and labelled.
In mass emasculation method hot water having temperature 42 to 45 0C is carried
in thermos flask in the field. The panicle of the proper stage is selected and inserted
in the water for 2 to 3 minutes. The flask is unopened spiklets are clipped off.
Pollination:
It is done on next day morning. Matured anthers are collected from protected male
parent in petri dish and dusted on the stigma of emasculated flower with brush and
forceps and covered with butter paper bag to protect natural cross pollination.

Emasculation and hybridization techniques in Maize crop (cross

pollinated crop)
Emasculation:

The tassels of the female plants are removed immediately as soon as appeared. The
process is called as detasseling. It is always done in the morning. Ear shoot which
emerging from the leaf sheath is bagged 1 to 2 days below the tip of the preveious
day of pollination.
The tassels of selected male parents is also covered with bag on following day in
the morning between 9.00 to 10.00 a.m. pollens from tassel bag is dusted over the
silk of the female cob/eat. The bag covered ear shoot is torn and bag from the male
parent may be placed over the cob. Care should be taken to avoid contamination of
silk with foreign pollens.
Crossing technique

Female parent

a. Detassel
b. Cut the tip of the cob before the silks emerge and cover with a butter paper cover.

Male parent

a. Cover the tassel before anthesis begins or as soon as the tassel emerges.
When the silks emerges in the female parent in the form of a brush, pollination
is done by transferring the freshly shed pollen cover form the male parent and
inserting it over the cob of the female parent after removing the cover from the
cob.
The details like date of pollination, parentage and breeding programme to be
carried out are clearly written by water proof pencil. The date or pollination
will be one day later than the date of tasselling. Pollination should be
completed within one week of silk emergence. Isolation distance for maize
= 400M.
 Experiment no. 6
Object: Consequences of inbreeding on genetic structure of resulting
populations
Inbreeding is a form of mating system in sexual organism. It implies mating together of
individual that are close to each other by ancestral or pedigree relationship.

When the individuals are closely related E. g Full sib mating, half sib mating. The
highest degree of inbreeding is achieved by selfing. The chief effect of inbreeding is to
increase homozygosity in the progeny, which is proportionate to the degree of
inbreeding. Cross – pollinated and asexually reproducing species are highly
heterozygous in nature. These species show a severe reduction in fertility and vigour due
to inbreeding (inbreeding depression). It contrast to this hybridization between unrelated
strains leads to an increased vigour and fertility (hybrid vigour or heterosis). These two
aspects are of great significance in breeding of these species. In fact heterosis and
inbreeding depression may be considered as the two opposite sides of the same coin.

Inbreeding Depression:
It refers to decrease in fitness and vigour due to inbreeding or it may be defined as the
reduction or loss in vigour and fertility as a result of inbreeding.
The most revealing impact of inbreeding is the loss of vigour and the physiological
efficiency of an organism characterized by reduction in size and fecundity. For example
selfing reduces heterozygosity, by a factor ½ in each generation. In fact the degree of
inbreeding in any generation is equal to the degree of homozygosity in that generation.
Inbreeding depression results due to fixation of unfavourable recessive genes in F2,
while in heterosis the unfavourable recessive genes of one line (parent) are covered by
favourable dominant genes of other parent.
The primary genetic consequence of inbreeding is increased homozygosity. Two
hypotheses for the genetic basis of inbreeding depression are put forth, both of which
depend on the idea that homozygosity wll increase with inbreeding. Either the over
dominance or partial dominance hypotheses are invoked to model the negative
consequences of inbreeding. In the over dominance hypothesis, inbreeding depression is
attributable to higher fitness of heterozygote versus homozygotes for the loci in question
.For the partial dominance hypothesis, negative fitness consequences for inbred lines are
due to the fixation of recessive or partially recessive deleterious alleles. Current thought
favors the latter hypothesis, where inbreeding depression is attributable to many genes of
small effect However, distinguishing between the two genetic mechanisms is
complicated by linked sets of deleterious recessives that imitate over dominance effects .
 Experiment no.7
Object: To Study of male sterility system in plants
Plant male sterility refers to the failure in the production of fertile pollen. It occurs
spontaneously in natural populations and may be caused by genes encoded in the
nuclear (genic male sterility; GMS) or mitochondrial (cytoplasmic male sterility;
CMS)
1. Palynology

This is the science involving the study of pollens. The pollen has a very minute
structure. It is unicellular and usually round although it may be oval, pyramidal,
polyhedral etc. It is provided with two coats-an inner, delicate, cellulose layer
called intine and an outer tough, cutinised layer called exine or extine. The exine
is often sculptured or provided with spines, warts etc., occasionally, it is smooth.
The exine may have a waxy coating to render the pollen more or less waterproof.
Very often, there are some definitely thinner circular spots or slits in the exine
called germ pores or slits. These weak spots are utilized during the germination of
the pollen.
2. Preparation of Acetocarmine Stain (C22H2O13)

It is one of the most widely used stain for pollen study. A mixture of 4 ml glacial
acetic acid and 55 ml of distilled water is boiled. A quantity of 1 g of carmine
(according to the strength required) is added to 100 ml of the above mixture at
about boiling point and then boiled for few minutes. After boiling, the contents are
removed from the flame and allowed to cool and filtered in a clean bottle. The
filtrate is reddish in colour and known as 1% acetocarmine. Ferric chloride or ferric
acetate may be added if necessary for deep staining and preservation.
Fertility and sterility in A, B, R and TGMS lines
Male sterility is characterized by nonfunctional pollen grains, while female
gametes function normally. It occurs in nature sporadically.
Types of male sterility, maintenance and uses:
Male sterility may be conditioned due to cytoplasmic or genetic factors or due to
interaction of both. Environment also induces male sterility. Depending on these
factors male sterility can be classified in to
a) Cytoplasmic male sterility (CMS)
b) Genetic male sterility (GMS)
 c) Cytoplasmic-genetic male sterility (CGMS)

d) Environmental induced male sterility which is again sub divided in to

i) TGMS (Theromosensitive)
ii) PGMS (Photo sensitive)
A line or ms line: It represents a male sterile line belonging to any one of the above
categories. The A line is always used as a female parent in hybrid seed production.
B line or maintainer line: This line is used to maintain the sterility of A line. The B line is
isogenic line which is identical for all traits except for fertility status.

R line or restorer line: It is other wise known as Restorer line which restores fertility in the
A line. The crossing between A x R lines results in F1 fertile hybrid seeds which is of
commercial value.
Pollen fertility count
a. Different crop species
No. of pollen grains Percentage of
Name of Crop Total
Unstained Stained pollen fertility
species
Wheat
( aestivum)

Wheat
(Durum)

b) A, B & R Lines of the crop-------------------

Number of pollen grains Percentage of
Lines Total
Unstained Stained pollen fertility

Formula: Pollen fertility test = No. of stained pollen grain / Total No. of pollen x100

Pollen Sterility test = No. of Unstained pollen grain / Total No. of pollen x100
 Experiment no.8
Object: To Study the Handling of segregation populations/ generations
Maintenance of Records of population
1. Accession Register
2. germplasm Bank
3. Descriptive blank register
4. Cropping programme
5. Single plant selection register
6. Row test
7. Replicated row test
8. Preliminary/Initial evaluation trial
9. Comparative yield/ yield evaluation trial
10. Multilocation I& II trials.
11. Quality observations Note book
12. Record of crosses
13. F1 generation
14. F2 segregation generation note book.

Accession Register
This will include the details of the seeds/ planting material with regard to receipt date,
source, their number, number assigned at the receiving unit, short description of the
planting material, to whom sent for evaluation, date, feed back information about the
genotype, now how maintained etc., Accession number given by the serial number
followed by the year of entry i.e. serial 125 in 2020. Then accession number will be
125202 or 20125. It will be mentioned as EC = Exotic collection IC = Indigenous
collection.
Proforma for Accession Register

Accessi on Name of Date of Source of Source Pedigree Description How

Feedback Re- marks
No. variety Receipt seed No. record of the disposed to
information
material whom sent
9 10
1 2 3 4 5 6 7 8
 Standard form of a Field Note Book
Each field note book should contain the following information.
A. Yield Trial
First Page
a) Number and title of the project
b) Season of raising the crop

c) Unit under which the trial is being conducted

Second page
a) A full plant of the field showing the location of the trial with the approach path.
b) North East directions should be specified.
Third Page
a) Plan of the experiment
b) Experiment details
1. Name of the experiment
2. Season
3. Number of variants
4. Design of the experiment
5. Replication
6. Size of the plot (Block/Plot/Row., etc.,)
7. Spacing (Between rows and within the row in cm)
8. Date of sowing/planting
9. Date of harvest
10. Name of the Principal Investigator
Fourth page
Details of cultural practices followed for the plot/ field
a. Date of ploughing
b. Date of layout of the trial
c. Manurial schedule adopted
Basal :
Topdressing :
d. Irrigation schedules with date from life irrigation onwards
e. Plant protection schedules followed
f. Details of intercultural operations A (hoeing, weeding, and earthing up etc.,)
 g. Date of harvest
h. Duration of processing till storage
i. Rainfall received during the crop growth
j. General remarks on the seasonal condition prevailed and its effects on crop
growth including the occurrence of pests and disease.
Fifth page
One page for each variant per replications allotted.
The following information have to be recorded in each page.
1. Date of germination
2. Date of gap filling
3. Initial stand on
4. Date of first flowering
5. Date of general flowering
6. Date of harvest

7. Final stand
8. Wet weight of grain
9. Wet weight of haulms/ straw etc.,
10. Dry weight of produce after cleaning
11. Yield per ha in kg.
The page will also have additional information on observations about the variant, recorded by the
breeder in relation to the object of the project.
The fifth page will also contain the following information and their
modification depending upon the crop.

Rice : Date of earhead emergence in the main shoot number of tillers,

: Date of earhead emergence in tillers and
: Number of tillers.

Cotton : Number of sympodial branches

: Number of monopodial branches

Cumbu : Date of emergence of female flowers

: Date of emergence of male flowers
: Number of tillers
Sunflower : Duration of flower opening etc.Generation study
This field note book will contain the following information.
a. Plan for the segregation generation
b. Details of the generation
1. Name of the generation study
2. Number of crosses
3. Details of the cross
Cross No. Female parent, Male parent, Number of families,
number of seed sown.
4. Length of row
5. Spacing (cm)
6. Date of sowing
7. Dates of harvest
8. Name of the Principal Investigator

c. Plan for the segregation generation

d. Details of the generation
1. Name of the generation study
2. Number of crosses
3. Details of the cross
i. Cross No.
ii. Female parent,
iii. Male parent,
iv. Number of families,
v. number of seed sown
4. Length of row
5. Spacing (cm)
6. Date of sowing
7. Dates of harvest
8. Name of the Principal Investigator
 Experiment No. 9
Object: Methods of calculating mean, range, variance, standard deviation

Mean( Average)
The sample mean is the average and is computed as the sum of all the observed
outcomes from the sample divided by the total number of events. We use x bar as the
symbol for the sample mean. In math terms,

where n is the sample size and the x correspond to the observed valued.

Example
Suppose you randomly sampled six acres in the Desolation Wilderness for a non-
indigenous weed and came up with the following counts of this weed in this region:
34, 43, 81, 106, 106 and 115
We compute the sample mean by adding and dividing by the number of samples, 6.
34 + 43 + 81 + 106 + 106 + 115 / 6
= 80.83
We can say that the sample mean of non-indigenous weed is 80.83.

3. Variance, Standard Deviation and Coefficient of Variation

The mean, mode, median, and trimmed mean do a nice job in telling where the center
of the data set is, but often we are interested in more. For example, a pharmaceutical
engineer develops a new drug that regulates iron in the blood. Suppose she finds out
that the average sugar content after taking the medication is the optimal level. This
does not mean that the drug is effective. There is a possibility that half of the patients
have dangerously low sugar content while the other half have dangerously high
content. Instead of the drug being an effective regulator, it is a deadly poison. What
the pharmacist needs is a measure of how far the data is spread apart. This is what the
variance and standard deviation do. First we show the formulas for these
measurements. Then we will go through the steps on how to use the formulas. We
 define the variance to be

and the standard deviation to be

Variance and Standard Deviation: Step by Step Calculate the mean, x.

Write a table that subtracts the mean from each observed value. Square each of the
differences.
Add this column.
Divide by n -1 where n is the number of items in the sample This is the variance. To get
the standard deviation we take the square root of the variance.

Example
The owner of the Ches Tahoe restaurant is interested in how much people spend at the
restaurant. He examines 10 randomly selected receipts for parties of four and writes down
the following data.
44, 50, 38, 96, 42, 47, 40, 39, 46, 50
He calculated the mean by adding and dividing
by 10 to get x = 49.2
Below is the table for getting the standard deviation:
x x - 49.2 (x - 49.2 )2
44 -5.2 27.04
50 0.8 0.64
38 11.2 125.44
96 46.8 2190.24
42 -7.2 51.84
47 -2.2 4.84
40 -9.2 84.64
39 -10.2 104.04
46 -3.2 10.24
50 0.8 0.64
Total 2600.4
Now 2600.4 / 10-1 = 288.7
Hence the variance is 289 and the standard deviation is the square root of 289 = 17.
Since the standard deviation can be thought of measuring how far the data values
lie from the mean, we take the mean and move one standard deviation in either
direction. The mean for this example was about 49.2 and the standard deviation
was 17. We have:

49.2 - 17 = 32.2 and 49.2 + 17 = 66.2

What this means is that most of the patrons probably spend between $32.20 and $66.20.
 Experiment No. 10
Object: Designs used in plant breeding experiments, analysis of
Randomized Block Design, Laying out of Field Experiments

The basic objective of plant breeding is the ultimate crop improvement. It results in development
of high yielding varieties hybrids etc., over the existing cultivars and so on. The performances of
the new varieties are confirmed from the results obtained from the field experiments. To be
explained scientifically the field experiments are laid out following certain rules and the data thus
collected are analyzed statistically. The steps involved in this process are explained here under.

Any designing of experiments involves three major steps.

1. Selection of experimental units

The objects on which the treatments are applied is known as experimental units.
Eg. Plots in the field, plant, etc.,

2. Fixing of treatments

The objects of comparison are known as

treatments Eg. Varieties, spacing etc.,
3. Arrangement of treatments in the experimental Units.
It comprises of three basic principles of design
a) Replication: repetition of treatments
b) Randomization: unbiased allocation of treatments to the experimental units.
c) Local control: minimizing the effect of heterogeneity of the experimental
units.
The objective of replication, randomization and local control is to minimize the
Experimental Error (EE). EE is nothing but differences in the responses from the
experimental unit to experimental unit under similar environments. Apart from these,
EE can be reduced further by proper selection of the experimental units and choosing
of most appropriate experimental design for a given number of treatment.

Types of basic experimental designs

1. Completely Randomized Design (CRD)
2. Randomized Block Design (RBD)
3. Latin Square Design (LSD)
Among these, RBD is the widely used design.
Laying Out of RBD
A. The experimental material (field) is divided first into blocks consisting of
homogenous (uniform) experimental units. Each block is divided into number of
treatments equal to the total number of treatments.
B. Randomization should be taken within each block and the treatments are
applied following the random number table.
C. Collection and analysis of data: After the collection of data from the
individual experimental unit (treatments) ANOVA (Analysis of Variance) table is
formed.
The significance of the ANOVA table is that it indicates the sources of variation exhibited
by the treatments, the magnitude of variation derived from different sources and their
worthiness (significant/ non significant).

D. Computation of Critical Difference (CD)

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 treatments mean is less than CD it can be concluded both the treatments are on
par.

RT: Row trial

Row trial is generally conducted in F3 and F4, when the seeds are not sufficient for
replication with individual plant progeny rows. Each row consists of about 20 or
more plants. Individual plants with desirable characteristics are selected from
superior progeny rows. Pest, Disease and lodging susceptible progenies with
undesirable characteristics are eliminated.
RRT – Replicated Row Trial
It is generally conducted from F3 generation onwards. Depending on availability of
seeds, 3-4 more rows are grown for each progeny to facilitate comparison among
progenies adopting suitable replications. Families, which have become reasonably
homozygous may be harvested in bulk. From those families showing segregation,
single plants are selected for characters under study. The breeder has to visually
assess the yielding potential of progenies and reject the inferior ones in the field
and the yield potential has to be assessed in the laboratory for confirmation.
PYT – Preliminary Yield Trial Or (IYET) Initial Yield Evaluation Trial

It is conducted from F5 generation onwards. Preliminary yield trial with three or

more replications are conduct to evaluated the comparative performance of the
culture and to identity the superior cultures among them. The cultures are
evaluated for plant height, lodging, pest and disease resistance, flowering time,
duration and yield, etc., Quality tests may also be carried out. Standard commercial
varieties must be included as checks for comparison. Ten to fifteen outstanding
cultures, if superior to checks, would be advanced to the Advanced yield trails.
AYT – Advanced Yield Trial
Advanced Yield Trial is conducted from F8 generation on wards. The superior
cultures identified from Preliminary Yield Trial are tested in Replicated Yield
Trial. In this trial, the cultures are evaluated for yield, pest, disease and lodging
resistance, duration, quality, etc.
Multi location trial is conducted from F13 onwards for 3 years by the Research
Station Scientists. Multilocation Trial are useful for suitability studies i.e. whether
a particular culture is able to perform well in all the locations or not. Stable
performance of a culture over all the locations will be promoted to ART.
ART – Adaptive Research Trial

It is conducted after MLT for 3 years by the Department of Agriculture. Nearly 3-4
cultures are tested and based on the performance of 3 Years in the farmers field,
the best culture over the check may be proposed to SVRC (State Variety Release
Committee) for releasing.
If the SVRC finds that the cultivar is suitable for any particular area or throughout
the state, then the variety is released and is notified by the State Department of
Agriculture.

Statistical Analysis of Randomized Block Design

This is one of the most commonly used designs in agricultural research, particularly in
plant breeding programmes. Its primary distinguishing feature is the presence of blocks
(replications) of equal size, each of which contains all the treatments.
The calculated F value is more than the table value at 4 and 12 degrees of freedom at P =
0.01. Therefore, difference between mean values of 5 varieties is highly significant.
 Experiment no.11
Object: To work out the mode of pollination in a given crop and extent of
natural out-crossing
There are more than a few approaches:
a) Morphological assessment of flowers:

Mechanism like dioecy , monoecy, protogyny, protandry and cleistogamy are easily
detected. They indicate the mode of pollination.
b) Space isolation:
Growing single plant of a crop in isolation and recording the seed sit, determines the extent
of pollination. Failure o set seeds in isolation proves the crop to be cross pollinated and seed
set is indicative of self-pollination.
c) Effects of selfing (inbreeding):
Vigour due to inbreeding is common in cross pollinated species while self-pollinated crops
show no inbreeding depression.
2) To work out the extent of out crossing:
The amount of cross-pollination is determined by planting two strains of the concerned
species in a mixed stand. One of these two strains is homozygous for a dominant character,
preferably an easily recognizable seeding or other phenotypic character, while other strain is
recessive for that character. The two strains are planted in such a manner that each plant of
the recessive strain is surrounded by plants of dominant strain to provide abundant pollen.
Seeds produced on the recessive strain are harvested and grown in the next generation. The
percentage of plant carrying the dominant allele of the character represents the percentage
of cross-pollination
 Experiment no. 12
Object: Prediction of performance of double cross hybrids
The performance of double cross hybrids can be predicted by comparative evaluation of
the predictions based on the performance of single cross.

The method was developed by Jenkins (1934). According to this method, the predicted
performance of any double cross is the average performance of the four non-parental
single crosses involving the four parental inbred.

For example:
If the 4 inbred are I1, I2, I3 and I4. The possible single cross among these inbred would
be 6, viz I1 × I2, I2 × I3, I3 × I4, I1 × I3, I1 × I4 & I2 × I4.
These single crosses can combine to produce 3 double crosses, Viz, (I1 × I2) × (I3 ×I4)
(I1 × I3) × (I2 ×I4) (I1 × I4) × (I2 × I3)
The performance of any of these double crosses can be predicted from the performance of
the four single crosses, not involved in producing that particular double cross.
For example:
The performance of double cross (I1 × I2) × (I3 ×I4) would be the average of the
performance of the four single crosses (I1 × I3), (I1 × I4), (bv+I2 × I3) and (I2 ×I4)