Dr. NAVEENKUMAR K.L Assistant Professor Dept. Genetics and Plant Breeding

1. Dr. NAVEENKUMAR K.L
Assistant Professor
Dept. Genetics and Plant Breeding
HYBRIDIZATION: Techniques and Consequences

2. Definition
 The mating or crossing of two plants or lines of
dissimilar or contrasting trait genotypes is known as
hybridization.
 Crossing of individuals of unlike genetic constitution
 A method of breeding new varieties which utilizes
crossing, to obtain genetic recombination
Parent 1  Parent 2
F1
Recombination
Hybridization

3. HISTORICAL PERSPECTIVES OF HYBRIDIZATION
 Hybridization is of more recent origin as compared to the process of selection
 700 BC – Babylonians and Assyrians hand pollinated date palm
 1694-Camararius discovered sex in plants
 1717 – Thomas Fairchild produced the first artificial hybrid – ‘Fairchild’s mule’
(sweet william x cornation)
 1760-66 – Joseph Koelreuter made many crosses in tobacco and emphasized
hybrid vigour in F1
 1756-1835 – T A Knight developed several varieties of apple, peach, pears, grapes
by artificial hybridization.
 Goss, Sargaret, Gaertner & Naudin noted uniformity & dominance in F1,
segregation and appearance of parental types in F2
 1865 – G. J Mendel – Law of inheritance of qualitative traits
 By the end of the 19th century hybridization was widely used for crop
improvement

4. 1. The main objective of hybridization is to create variation
2. The amount of variation created depends on the number of
genes for which the two parents used in hybridization differ in
in the parents F1.
OBJECTIVES OF HYBRIDIZATION
Parent 1  Parent 2
F1
Recombination
Hybridization
F2

5. AIM OF HYBRIDIZATION
1.Transfer of one or few qualitative traits - COMBINATION BREEDING
2.Improvement in quantitative traits - TRANSGRESSIVE BREEDING
3.Use of F1 as a hybrid variety

6.  Main aim is to transfer one/more characters into a single variety from another
variety/varieties
 The characters under consideration may be governed by oligogenes/ polygenes
 Increase in yield of the variety is achieved by correcting the weakness of the yield
contributing traits e.g., tiller number, grains/spike, test weight of the concerned
parent variety.
 The most common application of combination breeding is breeding for
disease/pest resistance – where a variety is good in all aspects except for
resistance.
 Breeding methods commonly used to fulfill this objective are – backcross
breeding/pedigree method
1. COMBINATION BREEDING

7.  Aims at improving yield or its contributing characters through transgressive
segregation
 Transgressive segregation? – refers to the process of appearance of plants in F2
generation that are superior to the both the parents for one/more characters.
 Usually such a phenomena occurs by accumulation of the plus/favourable genes by
recombination from both the parents.
 Breeding methods suitable are – pedigree method and its modifications
(population approach) AAbb x aaBB
(4) (4)
F1 AaBb (4)
F2 AABB: A_bb: aaB_: aabb
2. TRANSGRESSIVE BREEDING

8.  In most SPC, F1 is generally more vigorous and high yielding than
the parents.
 If commercially feasible the F1 may be directly used as a variety and
such a variety is called as hybrid variety
 Important – both the parents should combine well to produce as
outstanding F1
 Commercially hybrid varieties are being cultivated in rice, and other
self pollinated crops
OBJECTIVES OF HYBRIDIZATION
3. HYBRID VARIETIES
Parent 1 x Parent 2
F1

9. Based on taxonomic relationships of the parents involved,
hybridization may be classified as
1. Intervarietal hybridization
2. Distant hybridization
TYPES OF HYBRIDIZATION

10. 1. INTERVARIETAL HYBRIDIZATION/ INTRASPECIFIC HYBRIDIZATION
 The parents involved in intervarietal hybridization belong to the
same species; they may be two strains, varieties or races of the
same species
 The intervarietal crosses may be simple or complex depending on
the number of parents utilized .
 A. SIMPLE CROSS: in a simple cross, two parents are crossed to
produce the F1.
e.g., A x B ------ F1. In case of a hybrid variety, such a cross is called a
single cross
 The F1 may be selfed to produce F2 or it may be used in the
backcross breeding programme

11. B. COMPLEX CROSS:
 In a complex cross more than two parents are crossed to produce the hybrid,
which is then used either to produce the F2 or is used in the backcross
Also called as a ‘convergent cross – the crossing programme aims at converging
/bringing together genes from several parents into single hybrid’
Can be Three way cross, Double cross etc depending upon the characters to be
merged.
TYPES OF HYBRIDIZATION - 1. INTERVARIETAL HYBRIDIZATION
Parent 1 x Parent 2
F1 x Parent 3
Three way Cross
Parent 1 x Parent 2 Parent 3 x Parent 4
F1
F1
x
Double Cross

12.  Distant hybridization refers to crosses between different species of the same
genus or of different genera.
If two species belong to same genus – Interspecific hybridization;
when two species belong to different genera – Intergeneric
hybridization.
 Chief objective is to transfer one/two simply inherited characters like disease
resistance/ pest resistance to a crop species.
Eg: Interspecific hybrids in cotton and sugarcane
 Intergeneric hybridization may also be used to develop new crop species –
Triticale hexaploide from a cross between Triticum turgidum x Secale cereale
(rye)
 The wild species often provide genes which are not present in the cultivated
species.
 In rice O. barthii: BLB resistance, O.rufipogan: Source of male sterility in
rice.
TYPES OF HYBRIDIZATION – 2. DISTANT HYBRIDIZATION

13. If two species belong to same genus – Interspecific hybridization, It is also
termed as intrageneric hybridization
Main features:
It is used when desirable character is not found within the species of a crop
Effective method of transferring desirable genes into cultivated plants from
their related cultivated or wild species
It is more successful in vegetatively propagated species like sugarcane and
potato than in seed propagated species
It gives rise to three types of crosses. Viz. a) Fully fertile: between those
species that have complete chromosomal homology-cotton, wheat, oats and
soybean
b) Partially fertile: between those species which differ in chromosome
number- wheat, cotton , tobacco
c) Fully sterile: between those species which do not have chromosome
homology-Brassica, cotton, tobacco
TYPES OF HYBRIDIZATION – 2. DISTANT HYBRIDIZATION

14. when two species belong to different genera – Intergeneric hybridization such
crosses are rarely used in crop improvement because of various problems
associated with them
Main features:
It is used when the desirable genes are not found in different species of the
same genus
This method is rarely used in crop improvement programmes
It has been generally used in asexually propagated species
F1 between two genera are always sterile. The fertility has to be restored by
doubling of chromosomes through colchicine treatment
It was used by some workers to develop new crop species
Example : Wheat x Rye crosses= Triticale
Radish Cabbage crosses
TYPES OF HYBRIDIZATION – 2. DISTANT HYBRIDIZATION

15.  Once the objectives of the programme are decided, a breeder
is ready to begin hybridization.
 There are totally 7 steps that a breeder would give importance
for
1.Choice of parents
2.Evaluation of parents
3.Emasculation
4.Bagging
5.Tagging
6.Pollination
7.Harvesting and storage of F1 seeds
HYBRIDIZATION PROGRAMME – PROCEDURE OF HYBRIDIZATION

16. HYBRIDIZATION PROGRAMME – PROCEDURE OF HYBRIDIZATION
1. Choice of parents
 Determines the success of the breeding programme
 Depends on the objective of the breeding programme. Generally higher yield is the common
objective.
 The parents should combine well with each other.
 Combining ability of the parents serves as a useful guide in selection of parents in
hybridization programme.
2. Evaluation of the parents
 Should be done if the performance of the parents in the area where breeding is to be done is
not known.
 Disease resistance is important as the introduced parent may be susceptible to the pathogen
occurring in the area/new disease present in the area for which the reaction is not known
 Physical purity/mechanical mixtures should be checked for in new strains
 If the parent is suspected to be heterozygous self pollination may be essential for few
generations

17. HYBRIDIZATION PROGRAMME – PROCEDURE OF HYBRIDIZATION
3. Emasculation
 The removal of stamens or anthers/male parts or killing of pollen grains of a
flower without affecting the female reproductive organs is called as
emasculation.
 The purpose of emasculation is to avoid self-fertilization of a line/variety.
4. Bagging
 Immediately after emasculation the flowers/inflorescences are covered with suitable
bags (butter paper/cloth bags/brown paper bags)to prevent random cross pollination.
Bags are tied to base of inflorescence/flower with the help of thread/wire/pin
5. Tagging
 Emasculated flowers are tagged after the bagging.
 Information recorded on tags:
1. Date of emasculation,
2. Date of pollination and
3. names of female and male parents

18. HYBRIDIZATION PROGRAMME – PROCEDURE OF HYBRIDIZATION
6. Pollination
7. Harvesting and storage of F1 seeds
 The crossed heads/pods should be harvested and threshed separately
 The seeds should be dried and stored properly to protect them from storage pests
 Seeds from each cross should be maintained independently along with the original
tags
 Two important operations of hybridization are emasculation and pollination.
 During pollination mature fertile and viable pollen from the male parent is placed
on the receptive stigma of emasculated flowers to initiate fertilization
 The pollination procedure consists of collecting of the fresh and mature pollen
grains and dusting this pollen on the mature and receptive stigmas of emasculated
flowers

19. HYBRIDIZATION PROGRAMME – RAISING F1 GENERATION
 The breeder should note the differences between parents for any morphological
differences, which can be used as phenotypic markers (Eg: pigmentation,
hairiness).
 The parent with dominant form of character should be used as male parent as it
will allow for identification of selfed seeds in F1 generation
 Allard (1960) feels that 12 F1 plants are enough for most breeding programmes,
while Elliot (1958) is of the opinion that F1 should be as large as possible. Larger
the F1 greater the possibility of recombination to occur.
 Generally larger F1 is not used as
1. Hand crossing is tedious and time consuming
2. With large F1 population F2 becomes unmanageable
3. Many breeders feel the size of F1 is not important
4. Rare recombinations may not be detected due to masking influence of the
environment

20. HYBRIDIZATION PROGRAMME – CONSEQUENCES OF HYBRIDIZATION
 In SP species it is easy to permit SP in F1 to produce F2 generation
 Segregation and recombination will produce new genotypes in addition to the parental types.
 The number of genotypes produced in F2 increases rapidly with the number of segregating genes
No. of
genes
segregating
Types of
gametes
produced in F1
No. of different
genotypes in
F2
Smallest
perfect F2
population
No. of phenotypes in F2 No. of
completely
homozygous
genotypes
Full
dominance
No
dominance
1 2 3 4 2 3 2
2 4 9 16 4 9 4
3 8 27 64 8 27 8
4 16 81 256 16 81 16
5 32 243 1024 32 243 32
10 1024 59049 1048576 1024 59049 1024
15 32768 14348907 1073741824 32768 14348907 32768
20 1048576 3486784401 1099511627776 1048576 3486784401 1048576
n 2n 3n 4n 2n 3n 2n
No. of different types of gametes produced by F1 and the composition of F2
generation from crosses segregating for different number of genes

21. Barriers for wide Hybridization
1. Failure of zygote formation
May be due to failure in fertilization, inability of pollen tube to reach
embryo sac (Maize- long style), bursting of pollen tube in style of the other
spp(Datura)..
2. Failure of zygote development
It may be due to
 Lethal genes: Cause the death of the zygote. Ex: Aegilops umbellulata (has
lethal genes) x Diploid wheat
 Genotypic disharmony between the two parental genomes: Genetic imbalance
in the hybrid Ex: Gossypium gossipioids crossed with other spp of cotton
 Chromosome elimination: Generally, chromosomes from one genome are
successively eliminated due to mitotic irregularities Ex: Hoedeum.bulbosum x
wheat, the chromosomes of H. bulbosum fet eliminated from developing F1.

22. Barriers for wide Hybridization
3. Failure of zygote development
 Incompatible cytoplasm: Embryo development is blocked due to incompatible
reaction between cytoplasm of female and genome of male. This leads to
sterility / prevention of embryo development.
 Endosperm abortion ( poor development of endosperm): It is common feature
in wide hybridization. Poor development of endosperm leading to embryo
abortion.
4. Failure of F1 seedling development:
Seedlings may die at various stages of development and it may be due to complementary
lethal genes
 Chlorosis – improper chlorophyll development. It is due to presence of genes.
Interspecific and inergeneric crosses of wheat chow chlorosis due to Chl1 and Chl2
genes
 Hybrid necrosis: The F1 hybrids produced from rye and wheat show necrosis.

23. Techniques for production of wide hybrids
1. Incase of pollen tube unable to reach the embryo sac, the species
with shorter style should be used as the female parent. or style
may be cut off to make it shorter.
2. If lethal genes are involved,
 Different strains of a species may be crossed
 In some cases autopolyploidy may be helpful
 Embryo rescue technique may be applied to recover the embryo.
 Crosses may be attempted in different environments if it is the
cause for the failure of interspecific hybrid.

24. Techniques for production of wide hybrids
3. If there is a difference in ploidy level,
 They may be crossed directly using species with higher ploidy level as female
and diploid as pollen parent Chromosome doubling of either the wild species
or interspecific F1 hybrid to overcome the sterility.
 Chromosome number of the polyploid species may be doubled to obtain a
semisterile interspecific F1 which can be crossed to cultivated species Ex: B
oleracea x B campestris the hybridization possible at 4x level and not diploid
level
 Chromosome number of higher ploidy level species may be reduced. This
approach is extensively used in potato, where certain combination of seed
parents produce 35-80% dihaploid.

25. Techniques for production of wide hybrids
4. Bridge species.
 If two species can not be crossed directly, a third species is used as
bridge species. Which is compatible to both the species.
 Ex. To transfer genes from ‘A’ to ‘C’, ‘A’ is first crossed to ‘B’, the
resulting F1 is crossed to ‘C’. Here ‘B’ is bridge species compatible with
both ‘A & C is used to hybridize between A’ and ‘C’.
5. Use of growth regulators.
 2, 4-D & Liberalism acid is used get intergeneric crosses between
Hordeum vulgare & Avena, Triticum Lolium.
6. Embryo culture technique may be used, where endosperm abortion is
the cause for failure of the interspecific crosses.

26. Application of wide hybridization in crop improvement
1. Alien-addition line:
It carries one chromosome pair from a different species in addition to the
normal somatic chromosome complement of the parent species. Objective is to transfer the disease
resistance from related wild species. e.g.., i. Wheat, oats, tobacco. ii. tobacco, mosaic resistance is
transferred from N. glutinosa.
2. Alien-substitution line:
Has one chromosome pair from a different species in the place of chromosome pair of the recipient
species. e. g., Wheat, Cotton, tobacco, oats etc.,
 Alien substitution lines show more undesirable effect than Alien addition lines, hence are of no
direct use in agriculture
 Both are effectively viable in polyploid species, hence widely used in polyploid species.
3. Transfer of small chromosome segments.
 To transfer only the desirable gene from the related alien species.
 Ex. Transfer of Black arm resistance gene from Egyptian cotton to American upland cotton.
 Transfer of an entire desirable and undesirable linkage.

27. Techniques for production of wide hybrids
Transfer of gene for resistance is accomplished by three ways
1. Spontaneous crossing over
 Between the alien chromosome and chromosome of the recipient species.
2. Promotion of homologous pairing and C.O.
 Homologous Pairing may be promoted by certain genetic / cytogenetic manipulations.
 Ex. Deletion / Lack of Ph gene in T. aestivum promotes homeologous chromosome
pairing.
3. Induction of translocations using X-rays and gamma-rays.
 Many transfers have been made in wheat and tobacco.
 The techniques used for promoting homeologous pairing and translocations are known
as ‘chromosome manipulation techniques’.
 Examples of transfer of gene/ chromosomal segments from alien species include,
disease resistance, wider adaptability, Quality traits, yield etc.,

28. Techniques for production of wide hybrids
4. Transfer of Cytoplasm
It is achieved by repeated backcrossing of the F1 hybrid with the species to
which the cytoplasm is to be transferred.
5. Utilization as New Varieties
 If the distant hybrid is highly productive and easily produced on
commercial scale, it may be released for commercial cultivation.
 Ex. First interspecific cotton hybrid, Varalaxmi; a cross between G.
hirsutum & G. barbadense.
 Commercial varieties of Sugarcane, Napier-Bajra, Hybrid between B.
napus & B. campestris etc.,

29. Techniques for production of wide hybrids
5. Development of New Crop Species
 Allopolyploids may serve as new crop species
i. Ex. Triticale, Raphanobrassica.
 Synthetic allopolyploid may serve as bridging species between two
species with different levels of ploidy.
i. Ex. Hybrid between N. tabacum (2n=48) & N. gultinosa (2n=72)
is sterile. But the amphidiploid, N. digluta (2n=72) from the cross
N. tabacum x N. glutinosa is reasonably fertile and produces
partially fertile hybrids with N. tabacum and makes it possible to
transfer genes and chromosomes from N. gultinosa to N.
tabacum.

30. Limitations of Wide hybridization
1. Incompatible Crosses: common at the inter genetic level but at interspecific
level it is successful.
2. F1 sterility:Additional step of chromosome doubling is needed.
3. Problem in creating New Species
1. Lower economic yields,
2. Poor agronomic characteristics
4. Lack of homology between chromosomes of the parental species
5. Undesirable linkages; Linkage drag
6. Problems in the transfer of recessive oligogenes and quantitative traits
7. Lack of flowering in F1
1. Ex. Arachis, Glycine species.
8. Problems in using improved varieties in distant hybridization
Normally wild species cross easily with land races than with highly
improved varieties.
10. Dormancy
Some interspecific hybrids in Arachis remain dormant for 5-10 years regardless
of the treatments applied for inducing germination