This document discusses male sterility and its use in hybrid seed production. It defines male sterility as the inability to produce or release viable pollen. There are several types of male sterility including genetic male sterility (GMS), cytoplasmic male sterility (CMS), and chemically-induced male sterility. CMS is determined by the cytoplasm and is stable across environments, while GMS expression can be influenced by temperature or photoperiod. Male sterility systems allow for efficient hybrid seed production by avoiding manual emasculation and pollination. The document provides various examples of crops where different male sterility systems have been utilized.

1. Mr. Devkumar Arya
(Lecturer)
Department of Genetics and Plant Breeding
O.P. Agriculture College Budhwal, Behror, Alwar (Raj..),India
Affiliated to Sri Karan Narendra Agriculture University,
Jobner,(Rajsthan),India
Email:devkumararya59@gmail.com
Male Sterility & Self-incompatibility and
their use in hybrid seed production

2.  Male sterility
 Manifestation of Male sterility
 History of Male sterility
 Need of Male sterility
 Creation of Male sterility
 Classification of Male sterility
 Cytoplasmic Male sterility (CMS)
 Genetic Male sterility (GMS)
 Cytoplasmic genetic Male sterility (CGMS)
 Transgenic Male sterility
 Chemical hybridizing agents (CHAs)
 Use of Male sterility
 Reference
Contents

3. Male Sterility
 Male sterility is characterized by nonfunctional pollen grains,
while female gametes function normally.
 Inability to produce or to release viable or functional pollen as
a result of failure of formation or development of functional
stamens, microspores or gametes.
 Main reason is mutation.
Sterile SterileFertile Fertile

4. Manifestations of Male Sterility
 Absence or malformation of male organs.
 Failure to develop normal microsporogenous tissue- anther
 Abnormal microsporogenesis (deformed or inviable pollen)
 Abnormal pollen maturation
 Non dehiscent anthers but viable pollen, sporophytic control
 Barriers other than incompatibility preventing pollen from
reaching ovule

5. History of Male Sterility
 J.K. Koelreuter (1763) observed anther abortion within species
& species hybrids.
 Genic male sterility has been reported in cabbage (Rundfeldt
1960), cauliflower (Nieuwhof 1961)
 Male sterility systems have been also developed through genetic
engineering (Williams et al. 1997) and protoplast fusion
(Pelletier et al. 1995)
 Male sterility were artificially induced through mutagenesis
(Kaul 1988)

6.  Use of genetically engineered “pollen killer” genetic system
Several forms of pollination control
 Manual emasculation
 Use of male sterility
 Use of self-incompatibility alleles
 Use of male gametocides

7. Why Male Sterility ???
 Reduced the cost of hybrid seed production.
 Production of large scale of F1 seeds.
 Avoids enormous manual work of emasculation and
pollination.
 Speed up the hybridization programme.
 Commercial exploitation of hybrid vigour.

8. Creation of Male Sterility
 Spontaneous mutations
 Interspecific hybridization
 Mutation induction (EtBr)
 Genetic Engineering
 Chemically induced male sterility (CHAs)

9. Classification of Male Sterility
Kaul (1988) Classified Male Sterility in three major groups
1. Phenotypic Male Sterility (Morphological)
 Structural or Staminal Male Sterility
 Pollen Male Sterility
 Functional Male Sterility
2. Genotypic Male Sterility
 Genetic Male Sterility (GMS)
 Environmental Sensitive (EGMS)
a) Thermo sensitive genetic male sterility (TGMS)
b) Photoperiod sensitive genetic male sterility (PGMS)
 Environmental non-sensitive
 Cytoplasmic Male Sterility (CMS)
 Cytoplasmic Genetic Male Sterility (CGMS)
 Transgenic Male Sterility (TMS)
3. Chemically Induced Male Sterility (CHA)

10.  Pollen sterility: In which male sterile individuals differ from
normal only in the absence or extreme scarcity of functional
pollen grains (the most common and the only one that has played
a major role in plant breeding).
 Structural or staminal male sterility: In which male flowers or
stamen are malformed and non functional or completely absent.
 Functional male sterility: In which perfectly good and viable
pollen is trapped in indehiscent anther and thus prevented from
functioning
Phenotypic Male Sterility

11. Genetic Male Sterility (GMS)
 Also called as nuclear male sterility.
 Mostly governed by single recessive gene (ms) but dominant gene
governing male sterility (safflower).
 Origin: Spontaneous mutation or artificial mutations (Gamma rays,
EMS) are common.
 ‘ms’alleles may affect staminal initiation, stamen or anther sac
development, PMC formation, meiosis, pollen formation, maturation
and dehiscence.
S.No. Mutagens Crops
1 Colchicine Jowar
2 Ethidium Bromide Groundnut, Maize, wheat
3 Acetone Barley

12. GENETIC MALE STERILITY
ENVIROMENT SENSITIVE
MALE STERILITY
ENVIROMENT INSENSITIVE
MALE STERILITY
TEMPERATURE SENSITIVE GENETIC MALE STERILITY
PHOTOPERIOD SENSITIVE GENETIC MALE STERILITY

13. Temperature sensitive male sterility(TGMS)
In this Type of GMS complete male sterility is produced
by the ms gene at higher temperature. (e.g- male sterility
at 23.3º C or higher temperature for rice.)
Two lines (UPRI 95-140 TGMS and UPRI 95-167
TGMS) have been isolated as spontaneous mutants and
registered as germplasm.

14. Photoperiod senstitive genetic male sterility
(PGMS)
Expression of ms gene is drastically affected by the
prevailing photoperiod, provided the temperature is within a
critical range (e.g., 23-290 C for rice PGMS) within this
temperature range complete sterility obtained in rice plant
grown under long day condition (Day lengh more than 13 hr
45 min).

16. Nuclear male sterility and hybrid seed production
msms
MsmsX
Msms
Male fertile
P1
Male sterile
P2
Male fertile
msms
Male sterile
MsMs
Male fertile
X
F1
Msms
Male fertile

17. Cytoplasmic Male Sterility (CMS)
 Determined by the cytoplasm (mitochondrial or chloroplast genes).
 Result of mutation in mitochondrial genome (mtDNA)- Mitochondrial
dysfunction.
 Progenies would always be male sterile since the cytoplasm comes
primarily from female gamete only.
 Nuclear genotype of male sterile line is almost identical to that of the
recurrent pollinator strain.
 Male fertile line (maintainer line or B line) is used to maintain the
male sterile line (A line).
 CMS is not influenced by environmental factors (temperature) so is
stable.

18.  It is divided into types :-
a. Autoplasmic-
CMS has arisen within a species as a result of spontaneous
mutational changes in the cytoplasm, most likely in the
mitochondrial genome.
b. Alloplasmic-
CMS has arisen from intergeneric, interspecific or
occasionally intraspecific crosses and; where the male sterility
can be interpreted as being due to incompatibility or poor co-
operation between nuclear genome of one species and the
organellar genome another.
CMS can be a result of interspecific protoplast fusion .

19. Various CMS systems
 Raphanus or ogu system
 Polima or pol system
 Shiga-Thompson or nap system
 Diplotaxis muralis or mur system
 Tournefortii (tour) system
 Moricandia arvensis or mori system
 Chinese juncea or jun system
17 systems are available, only difference is the use of male
sterile cytoplasmic sources differs for each system.
 Nap system– B.napuus cross b/w winter & spring var.
 mur system--Diplotaxis muralis x B.campestris cv Yukina
 tour system– B.juncea collections

20.  CMS can used in hybrid seed production of certain ornamental
species or in species where a vegetative part is of economic value.
 But not for crop plants where seed is the economic part because the
hybrid progeny would be male sterile.
 This type of male sterility found in onion, fodder jowar, cabbage etc.
Utilization of CMS in Plant Breeding

21. Use of CMS lines

22. Transfer of CMS to new strains (Diversification)

23. Cytoplasmic Genetic Male Sterility (CGMS)
 Case of CGMS, where a nuclear gene (R) for restoring fertility
in male sterile line is known.
 R (restorer gene) 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.

24. Hybrid seed production using CGMS system

25. rr
S
Rr
S
rr
rr
S
Rr
S
rr
S
Rr
S
rr
S
RR S
rr
S
Rr
S
♂♀
StrainA Strain B
RR F×
×
♀ × rr
F
rr
F×
6-7 Back crosses
× RR S
11 : 2 :
CMS Restorer
F ♂Male fertile
Non restorer (Strain-C)
Male fertile ♀
×
Male fertileMale sterile
Discarded
Male sterile
Discarded
Male sterile
Discarded
Male sterile
Discarded
×Male fertile Self pollinated
Male sterile
Self pollinated
♂ Male fertile
(Strain-C)
♂ Male fertile
(Strain-C)
♀
Male fertile
Restorer line R is crossed to Male sterileA
Male fertile F1 is crossed to StrainC
in which R gene is to be transferred
Male fertility progenyis
back crossed to strain C
× Male fertility progenyis
back crossed to strain C
Male fertile progeny is self pollinated
Male fertile progeny is self pollinated.
Individual plant progenies grown in next generation
and non segregating progenies are selected
Transfer of Restorer gene ‘R’ to non restorerstrain

26. Inbred A
(Cytoplasmic
Male Sterile)
Inbred B
(Non restorer
male fertile)
Inbred C
(Cytoplasmic
Male Sterile)
Inbred D
(Non restorer
male fertile)
♂
♂
♀
♀
rr S
rr f
rr S
RR S
Single Cross-II
C×D
(Male Fertile)
rr S
Rr S
♀
Single Cross –I
A×B
(Male Sterile)
♂
Double Cross
(A×B) × (C×D)
rr S
Rr S
50%
50%
Production of Double cross maize hybrids using CGMS
(1:1 Segregation for
Male Fertility & Sterility)

27. Limitations of Cytoplasmic-Genetic Male
Sterility
 Unsatisfactory fertility restoration
 Unsatisfactory pollination
 Spontaneous reversion
 Modifying genes
 Environmental effects
 Non availability of a suitable restorer line

28. Sources of CMS & Restorer genes in some Crops
Crop species Cytoplasm Restorer Genes
Rice
CMS-CW O. spontanea
CMS-bo O. Sativa boroII (single dominant)
CMS-WA O. Spontanea (WA, four genes)
CMS-W18 O. rufipogon
Wheat (T.aestivum) T. timopheevi Rf1 and rf2
A. caudata -
T. Durum Aegilops ovata -
Maize
CMS-C Rf4
CMS-S Rf3
CMS-T Rf1 and Rf2

29.  Recombinant DNA techniques for disturbing any or number of
developmental steps required for the production of functional
pollen within the microspore or for the development of any
somatic tissues supporting the microspores.
 Transgenes for male sterility are dominant to fertility.
 Also to develop effective fertility restoration system for hybrid
seed production.
 Example: Barnase/Barstar system
Transgenic Male Sterility

31.  Barnase is extracellular RNase; barstar is inhibitor of barnase
(Bacillus amyloliquefaciens)
 Plants with TA29 promoter-Barnase construct are male sterile
 Those with TA29-Barstar are not affected by the transgene barnase.
 Barstar is dominant over the Barnase
 Fuse the barnase and barstar genes to TA29 promoter–TA29 is a plant
gene that has tapetum specific expression.
 Cross male sterile (barnase) with male fertile (barstar) to get hybrid
seed, which now has both barnase and barstar expressed in tapetum
and, hence, is fully fertile
Barnase/Barstar system

33. Hybrid seed production using Barnase/Barstar system

34.  Also called as Male gametocides, male sterilants, selective male
sterilants, 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).
 CHA is a chemical that induces artificial, non-genetic male
sterility in plants so that they can be effectively used as female
parent in hybrid seed production.
Chemical Induced Male Sterility

35. 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 affect out crossing.
 Should not be hazardous to the environment.

36. S.No. CHAs Critical stage Crop species
1. Zink Methyl Arsenate
Sodium MethylArsenate
5 days before heading Rice
2. Ethephon/ Ethrel Depends on crop Barley , oat, bajra,
rice
3. Mendok Depends on crop Cotton, sugarbeet
4. Gibberellic acid 1-3 days before meiosis Maize, Barley,
Wheat, Rice,
Sunflower
5. Maleic Hydrazide Early microsporogenesis Maize, wheat,
cotton, onion
Some important CHAs

37. Hybrid Seed Production based on CHAs
Conditions required:-
 Proper environmental conditions (Rain, Sunshine, temp, RH etc.)
 Synchronisation of flowering of Male & 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 maximise seed set
 Avoid CHA spray on pollinator row.

38. Advantages of CHAs
 Any line can be used as female parent.
 Choice of parents is flexible.
 Rapid method of developing male sterile line.
 No need of maintaining A,B&R lines.
 Hybrid seed production is based on only 2 line system.
 Maintenance of parental line is possible by self pollination.
 CHA based F2 hybrids are fully fertile as compared to few sterile
hybrids in case of CMS or GMS.

39. Limitations of CHAs
 Expression and duration of CHA is stage specific.
 Sensitive to environmental conditions.
 Incomplete male sterility produce selfed seeds.
 Many CHAs are toxic to plants and animals.
 Possess carryover residual effects in F1 seeds.
 Interfere with cell division.
 Affect human health.
 Genotype, dose application stage specific.

40.  Male sterility a primary tool to avoid emasculation in hybridization.
 Hybrid production requires a female plant in which no viable
pollens are borne. Inefficient emasculation may produce some self
fertile progenies.
 GMS is being exploited (Eg.USA-Castor, India-Arhar).
 CMS/ CGMS are routinely used in Hybrid seed production in corn,
sorghum, sunflower and sugarbeet, ornamental plants.
 Saves lot of time, money and labour.
Significance of male Sterility in Plant Breeding

41.  Existence and maintenance of A, B & R Lines is laborious and
difficult.
 If exotic lines are not suitable to our conditions, the native/adaptive
lines have to be converted into MS lines.
 Adequate cross pollination should be there between A and R lines for
good seed set.
 Synchronization of flowering should be there between A & R lines.
 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.
Limitations in using Male Sterile line

42. Content of self incompatibility in plant
 Introduction
 History of self incompatibility
 General features of self incompatibility
 Classification of self incompatibility
 Use of SI for hybrid seed production
 References

43. Self-incompatibility (SI)
It refers to the inability of a plant with
functional pollen to set seeds when self
pollinated. It is the failure of pollen from a
flower to fertilize the same flower or other
flowers of the same plant.
43

45. General features of Self-incompatibility
 Prevents selfing and promotes out-breeding so increases the probability of
new gene combinations.
 Causes may be morphological, physiological, genetical or biochemical.
 Normal seed set on cross pollination.
 May operate at any stage between pollination and fertilization.
 Reduces homozygosity.
 In plants, self-incompatibility is often inherited by a single gene (S) with
different alleles (e.g. S1, S2, S3 etc.) in the species population

46. •Proposed by bateman in 1952.
•S.I due to absence of stimulation by the pistil on
pollen growth in the like genotype(S1S2xS3S4)
•Means S.I is due to absence of substance in the pistil
or pollen which are essential for pollen tube
penetration.

47. •It states that interaction between like alleles(S1S2xS1S2)
leads to production of inhibitor which inhibit the growth of
pollen tube in the pistil.
•Interaction between like alleles, a substance is produced in
pollen and pistil which property to interfere with normal
metabolism of pollen grain and tube(inhibit enzyme or
auxin, block pollen tube membrane, inhibit enzyme
necessary for penetration.)

48. Criteria Types
Flower
morphology
Heteromorphic
self
incompatibility
Distyly
Tristyly
Homomorphic
self
incompatibility
Sporophytic self
incompatibility
Gametophytic self
incompatibility
Classification of Self-incompatibility
48

49. Criteria Types
Genes involved
(number)
Monoallelic (governed by single
gene)
Diallelic (governed by two genes)
Polyallelic (governed by many
genes)
Cytology of
pollen
Binucleate (pollens with two nuclei)
Trinucleate (pollens with three
nuclei)
Expression site
Ovarian (expression site is ovary)
Stylar (expression site is style)
Stigmatic (expression site is stigma)
49

50. The genes responsible for
self-incompatibility in
heterostylous flowers are
strongly linked to the genes
responsible for flower
polymorphism, so these
traits are inherited together.
The associated concepts are
distyly and tristyly.
Heteromorphic self-incompatibility
50

51. What is Distyly ?
Here, both stamens and styles are of two
types.
 Stamens may be low and high
 styles short and long.
 It is determined by a single gene, with two
alleles.
 The flower with short style and high stamen
is called as thrum type and flower with long
style and low stamen is called as pin type.
Both thrum and pin flowers differ for six
characters in addition to stamen and style
length.
51

52. Stigma
Anther
Cross Result
Ss (thrum) X Ss (thrum) Incompatible
ss (pin) X ss (pin) Incompatible
Ss(thrum) X ss (pin) 1:1
ss (pin) X Ss(thrum) 1:1
Distyly
Thrum Pin 52

53. What is Tristyly?
In tristyly, styles and stamens have three
different positions.
It is determined by two genes S and M, each
with two alleles.
 S gives rise to short style,
 S and M to medium style and
 s and m to long style.
 The number of possible genotypes is greater,
but a 1:1 ratio exists between individuals of
each SI type.
53

54. Short Style Medium Style Long Style
54

55. HOMOMORPHIC SELF INCOMPATITBILITY
The homomorphic system is found in the majority of
Self incompatibility. The incompatibility is not associated
With morphology differences among flowers. SI reaction is
Controlled by a single complex locus, the S locus, but in some
Cases. More than one gene is involved.
It is divided into two group
Gametophytic self incompatibility
Sporophytic self incompatibility

56. Gametophytic self incompatibility
Gametophytic incompatibility was first described by
East and Mangelsdorf in 1925 in Nicotiana sanderae.
In this system, the Incompatibility reaction of pollen
is determined by its own Genotype, and not by the
genotype of the plant on which it Is produced.
The incompatibility reaction may be controlled by one
or two Gene.
It is two type
 Monofactoreal gametophytic self incompatibility
 Bifactorial gametophytic self incompatibility

57. MONOFACTORIAL GAMETOPHYTIC SYSTEM
 SI reaction is controlled by single gene designated by S
Which usually has 50 or more gene
 This system is very efficient at preventing fertilization on
Self pollination but it is not very efficient at prevnting
fertilizationOn sib pollination
 This system is found in Trifolium, Nicotiana, Solanum etc.

58. BIFACTORIAL GAMETOPHYTIC SYSTEM
 SI reaction is controlled by two genes designated as S and Z
Which are unlinked and exhibit multiple alleles
 Homozygosity at one of the two loci is common
 Homozgyosity at both the loci are not found
 Heterozygotes at both the loci will show partial compatibility
When used as males in matings with females heterozygus at
Only one locus, but having the same alleles at the two loci as
Present in the males; however, when they are used as females,
There is complete incompatibility.
 Polyploidy has no effect on SI reaction

59. Sporophytic self incompatibility
 This type of SI was first reported by Hughes and Babcock
In 1950 in Crepis foetida, and by Gerstel in Parthenium
Argentatum (in the same year)
 In sporophytic system, self incompatibility is governed
By a single gene, S, with multiple alleles;
 The incompatibility reaction of pollen is governed by the
Genotype of plant on which the pollen is produced, and
Not by the genotype of pollen.

60. Gametophytic self-incompatibility (GSI)
Sporophytic self-incompatibility (SSI)
(Buchanan, et al., 2005)

61. Use of SI for hybrid seed production

62. Basic steps in the use of SI
• Identification of self-incompatible plants in diverse
population/genotypes
• Development of homozygous self-incompatible lines
•Identification of S-alleles in the homozygous self-incompatible lines
•Establishment of inter-allelic relationship among the S-alleles
•Identifying the best combining lines
•Maintenance of parental self-incompatible lines
•Commercial hybrid seed production

64. To fulfill the demand of food to the growing world
population.
To identify the stable SI and CMS lines.
There is need to acquire deeper knowledge about these
two important mechanisms for developing location/
environmental specific SI and MS lines.
64
Future Prospects

65. References
 Singh B.D.2009,Plant Breeding, Kalyani Publications, Ludhiana.
 Prasad B.K. 2006,Plant Breeding, Kalyani Publications, Ludhiana.
 Singhal N.C.2003,Hybrid seed production in field crops, Kalyani
Publications, Ludhiana.
 Mukharjee B.K.1995,Heterosis Phenomenon, Kalyani Publications,
Ludhiana.
 Singh R.K. 2006,Plant Breeding, Kalyani Publications, Ludhiana.
 Singh Phundan.2005,Plant Breeding,Kalyani Publications, Ludhiana.
 By Google.

66. Any query ?
Thank you for listening