This document discusses male sterility in plant breeding. It defines male sterility as the inability of plants to produce or release functional pollen grains. There are several types of male sterility, including genetic male sterility (GMS), cytoplasmic genetic male sterility (CGMS), transgenic male sterility, and chemical-induced male sterility (CHA). Male sterility is useful in plant breeding as it allows for cross-pollination and the production of hybrid seeds without the need for labor-intensive emasculation. However, maintaining male sterile lines requires additional considerations like synchronizing flowering times and complete fertility restoration.

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C-4 MALE STERILITY IN PLANT BREEDING
By
MUHAMMAD ANAS
Roll No.250 BSc (Hon) 7th
semester
Department of Plant Breeding and Genetics
Submitted to
Prof. Dr. FARHATULLAH SIR
Acting Dean in the Faculty of Crop Production Sciences
The University of Agriculture Peshawar Pakistan
INDEX

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Table of contents
1. Introduction
1.1 Male Sterility
1.2 Features of Male Sterility
1.3 Expression of Male Sterility
2. Types of Male Sterility
2.1 Genetic Male Sterility, Types, Environment Insensitive and Sensitive GMS, TGMS, PGMS.
2.2 Cytoplasmic Genetic Male Sterility
2.2(a) Limitation of Cytoplasmic Genetic Male Sterility
2.3 Transgenic Male Sterility
2.4 Chemical Induced Male Sterility
2.4(a) Properties of an ideal CHA
2.4(b) Some important CHA’s
3. Hybrid seed production based on CHA’s
3.1 Condition required
3.2 Advantages of CHA’s
3.3 Limitations of CHA’s
4. Significance of Male Sterility in plant breeding
4.1 Limitations in using Male sterile line
5. Requirements of 3 lines in CMS system
5.1 A line
5.2 B line
5.3 R line
6. Hybrid seed production in CGMS
6.1 Steps
6.2 Maintenance of R line
6.3 Identification of potential hybrid parents (A, B & R line)
7. Role of Maintainer and Restorer line in Cytoplasmic Genetic Male Sterility
8. Main steps of Hybrid Seed Production in plant breeding
8.1 Choice and development of seed male sterile (A-line)
8.1(a) Two ways
8.1(b) The desirable attributes which should be considered for a male sterile
Or A line development
 Plant height
 Duration of Spain flowering
 Tilling ability
 Productivity potential
 Stable Male sterility
 Free from diseases
 Combining ability
8.2 Choice in development of restore or male parent (R – line)
8.2(a) Procedures
8.2(b) The choice of parent for restore line depends on few attributes
8.3 Maintenance and multiplication of parental seed (B-line)
8.4 Production and improvement of F1 Hybrid
9. Applications of male sterility in plant breeding
10. Conclusion
11. References

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Introduction:
Male Sterility
Male sterility is characterized by nonfunctional pollen grains, if and where
produced,while female gametes function normally. OR
The incapability of plants to produce or release functional pollen grains. OR
Male sterility is the failure of plants to produce functional anthers, pollen, or
male gametes.
A male sterile plant cannot pollinate other plants or itself.However, the
female reproductionstructures of the flower are still fertile. A male sterile
plant can therefore set seed if they are pollinated by a different(male
fertile) plant. This genetic anomaly is used in hybrid seed productionsince
it guarantees cross-pollinationon male sterile plants.
Features of Male Sterility
 Prevent self-pollination,permits cross-pollination.
 Leads to heterozygosity
 Female gametes function normally
 Assayed through staining techniques
 In nature, occur due to spontaneous mutation
 Can be induced artificially
Phenotypic Expression of Male Sterility
 Absence of Male sex organs
 Lake of normal anther sac
 Inability of the pollen to mature.
 Inability to develop normal pollen.

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Types of Male Sterility:
Male sterility in plants can be controlled by nuclear genes or cytoplasm or
by both. Therefore,broadlythere are at least three differentmechanisms
for control of male sterility in plants.
 Cytoplasmic male sterility (CMS) – governed by cytoplasmic genes
Genetic Male Sterility (GMS):
 Genetic Male Sterility is also called as Nuclear Male Sterility.
 It is mostly covered by single recessive gene (ms) but dominant gene
governing male sterility (safflower).
 Origin: Spontaneous mutation or artificial mutations (Gamma rays,
EMS are common).
S. No Mutagens Crops
1 Colchicine Jowar
2 Ethidium Bromide Groundnut, maize,
wheat.
3 Acetone Barley
 ‘ms’ alleles may affectstaminal initiation, stamen, or another sac
development,PMC formation, meiosis,pollen formation, maturation,
and dehiscence.
Types of GMS:
 Environmentinsensitive GMS: ms gene expressionis much less
affected bythe environment.

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 EnvironmentsensitiveGMS: ms gene expressionoccurs within a
specified range of temperature and/or photoperiodregimes (Rice,
Tomato, Wheat, etc.)
 TGMS:sterility is at particular temperature e.g. In rice TGMS line
(Pei-Ai645)at 23.30c (China).
 TGMS at high temperature is due to the failure of pairing of two
chromosomes at metaphase was evident.
 This abnormality led to the abnormal meiosis, abnormal or sterile
pollen.
 Anthers were shriveled and non-dehiscence-male sterile.
 However, these lines produce normal fertile pollen at low
temperature.
 Sensitive period: PMC formation of Meiosis
 PGMS:Governed by two recessive genes.
 Sterility is obtained in long day conditions while in short days, normal
fertile plant.
 Rice: Sterile under long day conditions (13hr.45 min + Temp.23-
290C) but fertile under short day conditions.
 Sensitive Period: Differentiation of secondary rachis branches
to PMC formation.
or
Cytoplasmic Genetic Male Sterility (CGMS):
 CGMS is also known as NucleoPlasmic Male Sterility.
 Case of CMS, where a nuclear gene (R) for restoring fertility in male
sterility line is known.
TGMS
PGMS
Any Restorer Line
Hybrid Seed

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 R (Restorergene) is generally dominant can be transferred from
related strains or species.
 This system is known in cotton, maize, jowar, bajra, sunflower,
cotton, rice and wheat etc.
Limitations of Cytoplasmic-Genetic Male Sterility:
 Undesirable effects of Cytoplasm
 Unsatisfactory fertility restoration
 Unsatisfactory pollination
 Spontaneous reversion
 Modifying Genes
 Contribution of cytoplasm by male gamete
 Environmental effects
 Non availability of a suitable restorerline.
Transgenic Male Sterility:
 RecombinantDNA techniques for disturbing any or number of
developmental steps required for the production of functional pollen
within the microscope or for the developmentof any somatic tissues
supporting the microscopes.
 Transgenes for the male sterility are dominant to fertility.
 Also, to develop effective fertility restoration system for hybrid seed
production.
 Example: Barnase/Baster System.
 Barnase is extracellular RNase; barster is inhibitor of barnase
(Bacillus amyloliquefaciens).
 Plants with TA29 promoter-Barnase construct are male sterile.
 Those with TA29-Barsterare not affected by the transgene barnase.
 Barster is a dominant over barnase.
 Fuse the barster and barnase genes to a TA29 promoter,TA29
promoteris a plant gene that has tapetum specificexpression.
 Cross male sterility (barnase) with male sterile (barster) to get hybrid
seed,which now has both barnase and barster expressed in tapetum
and hence is fully fertile.

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Chemical Induced Male Sterility:
 CHA is a chemical that induces artificial, non-genetic male sterility in
plants so that they can be effectivelyused as female plant in hybrid
seed production.
 Also, known as Male gametocides,male sterilant, selective male
sterilant, pollen suppressants,pollenocide,androcide etc.
 The first report was given by Moore and Naylor (1950), they induced
male sterility in Maize using maleic hydrazide (MH).
Properties of an Ideal CHA:
 Must be highly male or female selective.
 Should be easily applicable and economic in use.
 Time of application should be flexible.
 Must not be mutagenic.
 Must not be carried over in F1 seeds.
 Must consistently produce >95% male sterility.
 Must cause minimum reduction in seed set.
 Should not affectour crossing.
 Should not be hazardous to the environment.
Some Important CHA’s:
S.no CHAs Critical Stage Crop species
1 Zinc Methyl Arsenate Sodium
Methyl Arsenate
5 days before
heading
Rice
2 Ethephon/Ethel Depends on crop Bajra, Oat, Bajra,
rice
3 Mendok Depends on crop Cotton, Sugar
beat
4 Gibberellic acid 1-3 days before
meiosis
Maize, Barley,
Wheat, Rice,
Sunflower
5 Maleic Hydrazide Early
Microsporogenesis
Maize, cotton,
Wheat, Onion

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Hybrid Seed Production based on CHAs:
Conditions Required:
 Properenvironmental conditions (Rain, Sunshine, temp, RH etc.)
 Synchronization of flowering of male and female parents.
 Effective chemical emasculation and cross pollination.
 CHA at precise stage and with recommended dose.
 GA3 spray to promote stigma exertion.
 Supplementary pollination to maximize seed set.
 Avoid CHA spray on pollinator row.
Advantages of CHAs:
 Any line can be used as female parent.
 Choice of parents are flexible.
 Rapid method of developing the male sterile line.
 No need of maintaining the A, B&R lines.
 Hybrid seed productionline is based on only 2-line system.
 Maintenance of parental line is possible only by self-pollination.
 CHA based F2 hybrids are fully fertile as compared to few sterile
hybrids in case of CMS or GMS.
Limitations of CHAs:
 Expressionand duration of CHA is stage specific.
 Sensitive to environmental conditions.
 Incomplete male sterility produces selfed seeds.
 Many CHAs are toxic to plants and animals.
 Possess carry over residual effects in F1 seeds.
 Interfere with cell division.
 Affecthuman health.
 Genotype,dose application stage specific.

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Significance of male sterility in plant breeding:
 Male sterility a primary tool to avoid emasculation in hybridization.
 Hybrid production requires a female plant in which no viable pollens
are born. Inefficientemasculation may produce some self-fertile
progenies.
 GMS is being exploited (E.g. USA-Castor, India-Arhar).
 CMS/CGMS are routinely used in hybrid seed productionin corn,
sorghum, sunflower and sugar beat, ornamental plants.
 Saves lot of time, money and labor.
Limitations in using male sterile line:
 Existence and maintenance of A, B & R lines is laborious and difficult.
 If exotic lines are not suitable to our conditions,native/adaptive lines
have to be converted into MS lines.
 Adequate cross pollination should be there between A and R for good
seed set.
 Synchronization of flowering should be there between A and R line.
 Fertility restoration should be complete otherwise the F1 seed will be
sterile isolation is needed for maintenance of parental lines and for
producing hybrid seed.
Requirements for 3 lines in CMS system:
A-line:
 Stable sterility
 Well developed floral traits for outcrossing
 Easily, wide spectrum & strongly to be restored
B-line:
 Well developed floral traits with large pollen load
 Good combining ability
R-line:
 Strong restore ability
 Good combining ability

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 Taller than A-line
 Large pollen load, normal flowering traits and timing.
Hybrid seed production using CGMS:
Depends on cytoplasm that produce male sterility and gene restores the
fertility.
Steps:
1. Multiplication of CMS (A) line.
2. Multiplication of Maintainer (B) line and Restorer(R) line
3. Productionof hybrid seed (A*R).
Maintenanceof A and B lines:
 Grow A line and its corresponding B line together in an isolated plot.
 Isolation distance for A*B productionfields is at least 1000m.
 A ratio of 1A:1B row is maintained.
 Pollens produced by B line fertilize the male sterile plant (A) and
seeds producedthus.
 Give rise to A line again.
Maintenance of R line:
 Pearl Millet R line could be either an inbred line or an Open pollinated
variety which can be multiplied as variety.
 Seeds of R lines produced by multiplying seeds inisolated plot
having distance 1000m.
 Any plant in the R line plot appearing differentfrom the true R type
should be uprooted or rouged out before anthesis.
 Purity of the parental seed is very important because it effects the
quality of the hybrid seeds that is generated.
Identification of potential hybrid parents (A, B & R lines):
 Potential male and female parents for hybrid seed productionare
identified by crossing male fertile parent (Inbred, variety, germplasm,
breeding stocks in advanced generations) to a male sterile line (A line)
and observing their corresponding hybrids in small plots of an
observation nursery.

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 A few plants of each cross are subjected to the bagging test i.e.
covering the few panicles with the paper bags before anthesis and
observing the seed set under the bag after few weeks.
In cytoplasm genetic male sterility what are the role of
Maintainer & Restorer line?
Hybrid seed productionusing Cytoplasmic Male Sterility (CMS) is
based on three-line system i.e. A, B and R line.
Male Sterile (A-line): CMS line is called as A line. This is sterile due
to the genes in cytoplasm (mitochondrial DNA).
Maintainer (B-line):This is isogenic (genotypically identical except
one gene) to CMS line exceptfertility. It maintains A line. It means if
you want seed of A line, you have to cross it with B line otherwise A
line will be no more as it has no active male parts.
Restorer (R line): This line has restorergenes in nucleus to restore
fertility of A line. R line is entirely differentto that A and B line. R line
has very high SCA (Specific Combining Ability) effects.It is used to
produce hybrid seeds.
 A x A = No seed
 B x B = B line seed
 R x R = R line seed
 A x B = A line seed
 A x R = Hybrid seed
Cytoplasmic male sterility is used in hybrid seed production.These
CMS lines must be maintained by repeated crossing to a sister line
(known as the maintainer line) that is genetically identical except that
it possesses normal cytoplasm and is therefore male-fertile.
Main Steps of Hybrid Seed Production IN Plant Breeding
Step # 1. Choice and Developmentof Seed ParentMale Sterile (A-
Line):
 The seed parent or the female parent of a commercialhybrid should
be a male sterile line. But in case of non-availability of suitable male

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sterile line, the fertile line or a self-incompatibleline can be used
where manual labor will be needed.
 In case of food grain crops where the number of seeds produced in
each pollination is restricted,there the cytoplasmic-genetic male
sterile line should be used as seed parent.
 But in case of vegetable crops where seed is not the economic
productand also only a single pollination will produce many seeds,
there fertile pure line can be used as seed parent. In case of
monoclines plants like maize, the male flowers can be removed easily
as these are borne on apices of the plants.
 After identification of a male sterile line (A-line), it should be
maintained by an isogenic B-line. Strains can be identified to have the
B-line reaction by crossing them to the A-line. Then a potential B-line
is converted to A-line by a process ofrepeated back- crossing till the
A and B lines become similar (isogenic).
 Transfer of male sterility is required when disease susceptibilityor
unwanted agronomic traits are associated with male sterile character.
This can be done in two ways:
1. Single phase repeatedback-crossing:
This method follows the repeated back- crossing of existing male
sterile line (non-recurrent parent) possessing the desirable
characteristics. In case of transferring cytoplasmic male sterility, it is
convenient through six generations of back-crossing.But in case of
developing cytoplasmic-genetic male sterility, it should possess
required agronomic characters and maintenance of male sterility is
needed through B-line or maintainer line.
2. Two phase limited back-crossing:
In this method instead of using A-line as non-recurrent parent, the B-
line is back-crossedwith a parent of desirable donors and the new
line formed is termed as IBC. Then these developed lines are again
crossed with A-line to seekout the male sterile line. These lines are

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then back-crossed repeatedlywith IBC lines which will help to develop
the male sterile line with desirable attributes.
The desirable attributes which should be considered for a male
sterile line or A-line development are:
 Plant Height:
Male sterile line should not be too tall or too dwarf. Since plant height
of the hybrid is the function of both the parents, so plant height of A-
line should be lower than that of R-line (male fertile parent).
 Duration and Span of Flowering:
This is an important attribute for commercialhybrid seed production.
Successfulhybrid seed productiondependson the synchrony
between the flowering of seed parent (A-line) and male fertile parent
(R-Line).
 Tillering Ability:
Profuse and synchronous tillering on a male sterile line is
advantageous both for harvesting male sterile seeds and also hybrid
seeds.
 Productivity Potential:
As the main objective is more hybrid seed production,thus male
sterile lines should possess ahigh productivity potential.
 Stable Male Sterility:
An ideal male sterile line should maintain its sterility under all
environmental conditions.
 Free from Diseases:
The male sterile line and also the R-line should be resistant to
probable diseases particularly seed borne diseases.
 Combining Ability:

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The male sterile line under use must have a high general combining
ability, so that it can combine well with R-lines to produce a large
number of hybrids.
Step # 2. Choice and Development of Restorer or Male Parent (R-
Line):
The second componentwhich is required for hybrid productionis the male
parent or restorerlines which are essentially inbred lines, like A-lines. Their
developmentis done through pure line selectionmethod where forced
selfing is practiced.
The procedure is as follows:
 From a heterozygous gene pool, the S0 is selected on the basis of
desirable attributes such as vigor, productivity, disease resistance,
etc.
 After self-pollination,the S1 plants are obtained and growing them
row-wise, after repeated selfing S2 seeds are obtained.
 This process is continued up to S5 or S6 generation
 A number of inbred lines are developedfora successfulhybrid
programme as to improvise the genetic potential of the source
material which can impart hybrid vigour. The inbreds are evaluated
for their agronomic traits and on the basis of general combining
ability.
 The unwanted inbred lines are discarded in the beginning of
inbreeding process and also the prospective R lines should be
chosenfor the developmentof F1 hybrids on the basis of combining
ability in S5 or S6 generation.
The choice of parents for restorer line specifically depends on
the presence of few attributes like:
 Ability to produce abundant pollen grains (profuse pollen
production),
 Maximum genetic diversity from the A-line chosen,

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 High specific combining ability of the cross between A-line and
the R-line.
Step # 3. Maintenance and Multiplication of Parental Seeds(B-
line):
In case of cross-pollinated crops,the hybrid programme is based on
3 lines: A-line (ms), B-line (Maintainer) and R-line (Restorer) — all
homozygous inbreds are maintained by the breeder.Any kind of
change in the population should be discarded by the breederwhich
may occur due to random pollination and mutation.
For propermaintenance, the A-lines and B-lines are grown in field in
ideal agronomic condition and in rows in 4: 2 ratios. In the border
rows also, the B-lines are grown which supply sufficientpollen for
pollinating A-line. On maturity the seeds of B-line are harvested first
and kept separately. Then the seeds of A-line are harvested. Thus in
the same field both A-lines and B-lines are maintained and multiplied
in the same block.
For maintenance of R-line, the plot should be completelyisolated
from others. The natural open pollination characteristic of cross-
pollinated crop will be helpful for its maintenance. There is no need to
maintain the R-line separately exceptfor the first use, as R- line is
automatically maintained in the hybrid productionblock.
Step # 4. Production and Improvementof F1 Hybrids:
 For F1 hybrid seed production,A-line and R-line are grown together in
4: 2 ratio in hybrid productionblock (Fig. 7.1). The arrangement is
same exceptthe B-lines have been substituted by R-lines. At maturity
the seeds of A-rows are harvested separately and carefully as these
hybrids and also the R-line seeds are harvested for next use.

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 For improvementof F1 hybrids, the parental lines should be
improved.If the incidence of diseases (forwhich the inbred lines are
not resistant) occurs, breakdown of male sterility in A-line and genetic
weakness (like combining ability, nutritional quality) appear, then
these drawbacks can be removed by changing the parental lines.
 In many crops like pearl millet, sorghum and maize, hybrid seed
productionprogramme has been pursued and many of the hybrid
varieties have been released.
Applications of Male Sterility in Plant Breeding
There are two main fields of application
 the production of hybrid varieties
 inter‐ and intraspecific hybridization and back crossing programs for
the introduction of genetic variation into crop varieties.
Several schemeshave beenproposed for usingCMS in h y b r i d breedingindifferentcrops.

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CONCLUSIONS
 Hybrid production require a female plant in which no viable gametes
are born.
 Emasculation is done to make a plant devoid of pollen so that it is
made female.
 Genetic male sterility is used in hybrid seed productionbut has
limitations due to the need to maintain female parent lines as
heterozygotes and segregationof fertile and sterile plant each
generation.
 Male sterility plays important role in plant breeding,firstly in the
productionof hybrid seed and secondlyas a plant breeding tool
facilitating population improvement,back-crossing,inter-specific
hybridization and other intermediate breeding procedures.Hybrid
breeding methods have been successfullyemployed in many plants
(maize, sorghum, sugar beet, sunflower and tomatoes etc.)
Cytoplasm male sterility is the most important system used in hybrid
seed productionand so far, with few expectations,the only one by
which hybrid seed can be produced both effectivelyand
economically.
 It has real advantages in certain ornamental species,as the non-
fruitful plants tend to bloom longer than their seeded counterparts,
and flowers remain fresh. Cytoplasmic male sterility is also useful in
producing single or double cross hybrids in crop speciesin which
some vegetative parts of the plant is the commercialproduct. Nuclear
male sterility may be used to make recurrent selectionalso available
to breeders ofself-pollinated crops.1. 4. 2 Self-Incompatibilityin
Plant Breeding Self-incompatibility has also been adapted to hybrid
varietal production.

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References
 D., BosemarkN. O., RomagosaI.
 Campman & Hall, London.DiscollC. J. (1986)
 C. R. C Critcal Rev. Plant Sci., 3, 227-256.Lasa J. M & BosemarkN.
O. (1993),Male sterility. In Plant Breeding Principles and Prospects.
 ayward M. D., Bosemark N. O., RomagosaI. (eds)Campman & Hall,
London. Pandey K. K (1994),Elimination of heterozygosity and
efficiencyof genetic system. Theor. Appl. Genet. 44, 199-205.
Simmonds N. W. (1979), Principles of crop improvement,Longman,
England.
 ResearchGate
 Google
 Study notes
 Questions and Answers Forum
 GRAYBOSCH,R.,and R. G.PALMER,1984: Is the mSf male sterile
mutant partially fertile?SoybeanGenet. Newslett.11,102—104