Male sterility-classification, mechanism, site of action, utilization in plant breeding,advantages and disadvantages

1. STERILITY
IN
CROP PLANTS
ARUN CHACKO
PhD SCHOLAR
PLANT BREEDING AND GENETICS

2. • Sterility: An inability of a living organism to
effect sexual reproduction ie., both anthers and
ovules are non-functional.
• Male sterility: It is the failure of plants to
produce functional anthers, pollen, or male
gametes.
• Characterised by non-functional pollen grains.

3. Male Sterility
• Absence of male individuals in dioecious strain,
• Absence of male organ in a normal bisexual plant,
• Failure to produce normal sporogenous tissue in
stamens,
• Inhibition at various stages of pollen
development,
• Failure to mature, dehisce or function on a
compatible stigma,
• Pollen Sterility: Pollen abortion and Pollen
failure.

4. • 1694: Detection of sex in higher plants by
Camerarius.
• 1763: Anther abortion within species and
species hybrids (Koelreuter).
• ‘Male contabescens’ and Hybrid contabescens.
• Bateson et al.(1908) : ‘male sterility is not simply
a random sporadic event but a genetically
conditioned system in Sweet pea, where sterility
is controlled by one recessive gene’.

5. • Correns (1908): maternal control of gynodioecy.
• Evidence for non-mendalian inheritance and
studies came with gene-cytoplasm interactions
and led to discovery of sex types, sex
reversals, male sterility in higher plants.

6. Occurrence of male sterility
• Mutation of either nuclear or cytoplasmic genes
or both.
• Male sterility > Female sterility
• Male sporophyte and gametophyte are more
vulnerable to intrinsic and extrinsic forces than
the protected ovule and embryo sac.
• Pollen : ease of availability and detection
• Pollen: Carmine, lactophenol or iodine test
• Female sterility: artificial crossing and
developmental studies of seed formation.

7. Mechanism of Male Sterility
• Abnormal microsporogenesis leads to non-
viable microspores.
• Factors:
Mitochondrial mutation
Barrier of Tapetal layer
Improper timing of Callase activity
Operon concept of Gene Action

8. • Callase enzyme: for normal pollen development.
• Early or delayed callase activity leads to male
sterility.
• Heslop-Harrison (1967) : CMS results from operon
type control rather than due to cytoplasmic factors.
• Demonstrated that a specific condition guided by the
nuclear gene is propagated by in the cytoplasm thus a
permanent repression upon genetic system that
determines the pollen producing capacity of the plant.
• But failed to explain fertility restoration in male
sterility system.

9. Male Sterility
• Gableman (1956)
• All classifications is either based on
phenotypic or on genotypic basis.
Pollen
Staminal
Functional

10. General classification of Male Sterility
Male Sterility
Genetic
(Spontaneous or
induced)
Phenotypic
Structural, Sporogeneous,
Functional
Genotypic
Genic, Cytoplasmic and
Cytoplasmic-Genic
Non-Genetic
(Induced)
Chemical
Male Gametocide and other
gametocides
Physiological
> Temperature
> Photoperiod
> Enzyme balance
> Other biochemicals
Ecological
> Climatic
> Biotic
> Edaphic
>Physiographic
(Kaul, 1988)

11. Sterility Type Phenotypic
Alteration
Effect on
male sex
organs
Action of the male
sterility gene
Env.
Influence
on MS
gene
action
Influence
of other
genes on
MS gene
action
1. Structural a) No anthers
b) Transform
ed anthers
c) Faulty
developm
ent
Form and
function
impaired
During anther
development and
differentiation
before the onset of
microsporogenesis
High High
2.Sporogeneous a) No
meiosis
b) Abnormal
microspor
ogenesis
Function
impaired
Just before or
during or after
microsporogenesis
High Medium
3.Functional a) Viable
pollen but
not
liberated
b) Mechanic
al barrier
Functional
ability
inhibited
After
microporogenesis,
before anther
dehiscence
Low Low

12. Genic Male Sterility (g-mst)
• Occurrence: Spontaneous or artificial mutation
• In Self-pollinated sp., loss of chromosome
carrying mft (male fertile) gene results in male
sterility and the plants become weak, slow
growing and inviable.
• Observed in O. lamarckiana, Pisum sativum.

13. Origin of ms alleles
• Spontaneous mutation
• Chemical and physical mutagens
• X-rays, EMS, Et-Br, Acetone, Colchicine

14. Gene Control: Mostly by single recessive gene (ms)
0
10
20
30
40
50
60
70
Spontaneous
a : recessive gene
b: dominant gene
c : Polygene
d: unknown

15. • Dominant gene governing: eg. Safflower
• Both recessive and polygene: Medicago sativa
• Recessive, dominant and Polygene: T.aestivum
• Several different ms genes act monogenically
to produce male sterility.

16. • Sterility maintenance:
Sterile Fertile
1: 1 Male fertile
Male fertile: Male sterile
r r R R
R r r r
•Fertility Restoration
r r R r
R r

17. Site of action of ms allele
• Time,
• site,
• stage and sex specific.

18. STAGE MODE OF ACTION
Staminal initiation Rudimentary stamen, undifferentiated, forming
minute cellular protuberence.
Staminal
development and
differentiation
Stamens malformed, anther sacs rudimentary and
misformed, lump of unorganised cells are formed.
Anther sac
development and
archesporial
differentiation
misdifferentiation, non-functional cells
Microsporangial
differentiation and
development
Anther sacs formed, microsporangia
misdifferentiated.

19. STAGE MODE OF ACTION
PMC formation Anther sacs developed & differentiated but PMC
fail to separate out from each other and doesn’t
acquire the required genetic autonomy to undergo
meiosis.
-Their chromatin & nucleoli contract, fragment
and degenerate with cytoplasmic content.
Premeiosis PMC separate out, no chromosome despiralization
occurs, DNA synthesis drastically reduced, unique
meiotic histones are not produced.
Meiotic entry Chromatin compacts and fragments, PMC
degenerate.
Tetrad formation Unable to develop, separate and produce callose or
to be released from PMCs and finally degenerate.

20. STAGE MODE OFACTION
Microspore
liberation
Enzyme callase either absent/insufficiently
produced, microspore remain enlarged with the
PMCs and finally degenerate.
Primexine and
Sporopollenin
deposition
Either absence/misfunction of primexine or
abnormal deposition.
Pollen development Failure of nucleus to undergo meiosis, followed
by cytoplasmic disintegration, low enzyme
activity, rRNA and ascorbic acid content.
Pollen maturation Generative cells follows vegetative cells
degeneration, pollen remains sticky and
degenerates
Pollen liberation Exine sporopollenin becomes hydrophilic &
sticky, pollen release prevented.
Anther sac dehisence Anther sac becomes hard and non-dehisce.

21. Molecular mechanism of ms action
• Changes in content & proportion of amino acids.
• High deficiency in DNA & RNA
• Reduced carbohydrate and protein
• Reduction in activities of callase, cytochrome
oxidase.
• Altered proportion of growth regulators.
• Delay tapetum degeneration, so that no nucleotide
is provided to PMC causing degeneration and
male sterility.

22. Allelic relationship between genes
causing GMS
• Identified by crossing the male steriles from
different sources with their fertile counter
parts (MsMs)and intercrossing F1s.
• Intercross progenies (sterile:fertile)
• 3:1  Allelic to each other.
• 4:0  Non-allelic, all fertile.

23. Advantages of GMS
1.Large number of parents can be used in
crosses, because all the genotypes have
dominant genes for male sterility.
2.Only female parents of a good hybrid has to be
converted.
3.GMS generally does not have undesirable
agronomic characters.
4.It is possible to breed the varieties from
segregating population of GMS.

24. Disadvantages
1.GMS is less stable. Sometimes, sterile plants become
fertile under low temperature conditions.
2.In GMS, the lines segregate into male sterile and fertile
plants in 1: 1 ratio.
3.Conversion of a genotype into GMS needs selfing after
each backcross to isolate recessive genes and hence
more number of generations are required.
4.It requires more area as 50% of the population is fertile.
5.The quantity of seed produced is less.
6.There is possibility of admixture if fertile plants are not
properly rogued out.

25. Types of GMS
• Environment insensitive GMS: ms gene expression is much
lessaffectedby theenvironment.
• Environment sensitive GMS: ms gene expression occurs
within a specified range of temperature and /or photoperiod
regimes (Rice,Tomato,Wheat etc.).
• Temperature-sensitive GMS
• Photoperiod-sensitive GMS
• Transgenic GMS

26. Temperature-sensitive Genetic Male Sterility
(TGMS)
• Controlled by recessive genes tms1 and tms2.
• Complete male sterility by the ms gene at higher
temperatures.
• Temp. below critical temperatureNormal fertility.
• 1st TGMS line of indica rice: Annong S-1
(Spontaneous mutant in China)
• Critical Sterility Point in rice: 27-280C
• Temp. 240C or below can cause male fertile lines.

27. TGMS lines in rice:
• IR 68945, Pei-Ai 645, Hennong-S, Norin PL
12
• UPRI 95-140 and UPRI 95-167 : Spontaneous
mutant.
• Pei-Ai 645 : 23.30C or above can cause male
sterile lines.
• PMC stage to meiosis : Temperature sensitive.

28. Photoperiod-Sensitive GMS
• ms gene expressed at prevailing photoperiod and
provided the temp. is within a critical range.
• Governed by two recessive genes.
• For Rice,
• Sensitive stage: 20 rachis differentiation to
PMC formation.
• 23-290C.
• Long day condition (> 13hr 45min.)

29. An ideal PGMS line
• A low critical temperature for fertility
induction.
• A high critical temperature for strerility
induction.
• Wide temp. range for photoperiod sensitivity.
• Strong interaction between photoperiod and
temperature.

31. Advantages of EGMS
• No need for a maintainer line
• Any genotype can be utilized as pollinator
parent.
• Seed production programme is simple and
more efficient.
• Negative influence of male sterility inducing
cytoplasm on the F1 plants can be avoided.

32. Photo-thermo sensitive GMS
• Adaptability of PGMS line: Critical day length
and intensity of interaction between
photoperiod and temperature.
• Adapted PGMS line: strong interaction
• Higher temp. complement with short
photoperiod in low altitudes.
• Low temp. complement with higher
photoperiod in high altitudes.

33. Transgenic Genetic Male Sterility
• 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 themicrospores.
• Transgenesfor male sterility are dominantto fertility.
• Alsoto develop effective fertility restoration systemfor hybrid seed
production.
• Example:Barnase/Barstarsystem

36. • The first transgene designed to confer GMS was reported and
were used to transform Tobacco and oilseed rape plants.
• Tapetal-specific transcriptional activity of the tobacco TA29
gene.
• Upstream regulatory elements of TA29 gene used to drive the
expression of transgenes (extracellular RNAses from bacteria).
• Two genes were used:
 barnase from Bacillus amyloliquefaciens
 RNAse-T1 from Aspergillus oryzae
• RNase genes selectively destroyed the tapetal cells during
anther development and prevented pollen formation
• Herbicide (bialophos) resistant gene (bar) used as selectable
marker

43. Utilization of GMS in Plant Breeding
• Male Sterile (A): ms ms
• Maintainer line (B): Ms ms
• Progeny of [ms ms] x [Ms ms] used as female.
• Rouging of male fertile progeny required .
• In India,
• Pigeonpea hybrid: ICPH-8,-4, CoH1,2, AKPH4104,
AKPH2022.

44. Cytoplasmic Male Sterility
• In many gynodioecious populations, the male-sterile
trait is transmitted matrilineally, and the genetic
determinants are therefore believed to reside in the
cytoplasm.
• These cytoplasmic determinants usually coexist in a
population with autosomal genes that repress their
action and cause the restoration of pollen fertility.
(Kheyr-Pour 1980, 1981; van Damme 1983)

45. • Cytoplasmic male sterility (CMS) was first
discovered by Correns (1906).
• Cytoplasmic male sterility (CMS) is the
maternal transmission of failed pollen
production in hermaphroditic plants leading to
a mixture of male-sterile and hermaphroditic
individuals in the population (gynodioecy).

46. • CMS is the result of mutation in mtDNA leading to
nuclear-mitochondrial interaction or incompatability.
• Nuclear genes are not involved.
• CMSis not influenced by environmental factors
(temperature) sois stable.
• Sterility Maintenance:
Sterile A line x Fertile B line A line sterile progenies
• Fertility restoration is not feasible., useful where seed
is not the desired end product.

47. • TEXAS, OR T, CYTOPLASM (cms-T) of maize was first
described in Texas in the Golden June line of maize.
• Carries two cytoplasmically inherited traits, male sterility and
disease susceptibility,
• The two traits are inseparable and are associated with an
unusual mitochondrial gene, T-urf13, which encodes a 13-
kilodalton polypeptide (URF13).
• An interaction between fungal toxins and URF13, which
results in permeabilization of the inner mitochondrial
membrane, accounts for the specific susceptibility to the
fungal pathogens.

48. • Male sterility is characterized by the failure of
anther exertion and pollen abortion.
• Female fertility is not affected by CMS, so
male-sterile plants can set seed if viable pollen
is provided.
• cms-T had replaced detasseling as the chief
method of pollen control in hybrid corn
production

49. • After it was determined that cms-T was specifically
susceptible to Bipolaris maydis race T, the organism
responsible for the blight, its use by the hybrid seed
corn industry was largely terminated.
• Phyllosricra maydis, another fungal pathogen, is also
specifically virulent on cms-T.
• Susceptibility of cms-T maize to the fungal pathogens is
due to mitochondrial sensitivity to the pathotoxins,
whereas disease-resistant maize types have
mitochondria that are insensitive to the pathotoxins.

50. Ogura-CMS turnip:
• reduction in the size of the fleshy root,
• distinct defects in microspore development and tapetum
degeneration during the transition from microspore
mothercells to tetrads.
• Defective microspore production and premature tapetum
degeneration during microgametogenesis resulted in
short filaments and withered white anthers

51. • The mechanism regulating Ogura-CMS in turnip was
investigated using inflorescence transcriptome analyses
of the Ogura-CMS and MF lines.
• 5,117 differentially expressed genes (DEGs) were
identified including 1,339 up- and 3,778 down-
regulated genes in the Ogura-CMS line compared to the
MF line.
• A number of functionally known members involved in
anther development and microspore formation were
addressed in the DEG pool, particularly genes
regulating tapetum programmed cell death (PCD), and
associated with pollen wall formation.
• Additionally, 185 novel genes were proposed to
function in male organ development based on Gene
Ontology analyses, of which 26 DEGs were genotype-
specifically expressed.

53. Genetics of fertility restoration &Allelic
relationship of Rf genes
• Fertility restoration can be monogenic and
digenic.
• In digenic restoration, non-allelic interaction of
different types has been reported.
• Eg: Rice
• The fertility restoration controlled by a single
dominant gene have more practical value
where Rf gene is to be transferred to a
different genetic background.

54. • The allelic relationship b/w restorer genes can
be tested by crossing two restorers and using
F1 as pollinator to pollinate CMS line.
• 3:1 Non-allelic
• All progenies are fertile, if two restorer lines
carried genes are allelic.

55. Allelic condition
Restorer 1 x Restorer 2
R1R1 R1R1
F1 (R1R1) x r1 r1 CMS line
R1r1 All fertile

56. Non-allelic condition
Restorer 1 x Restorer 2
R1R1 r2 r2 r1r1 R2R2
F1 (R1 r1 R2r2) x r1 r1 r1 r1 CMS line
R1 r1 R2r2 R1 r1 r2r2 r1 r1 R2r2 r1 r1 r2 r2
Fertile Fertile Fertile Sterile
Fertile: Sterile 3:1

57. Molecular tagging and transfer of Rf gene
• Generate an appropriate mapping
population.F2 population from crossing CMS
and a restorer line.
• 150-200 F2 plants are selected randomly, DNA
isolated.
• Plants are individually tagged and screened for
pollen fertility as well as bagged to score
spikelet fertility.

58. • Based on pollen and spikelet fertility, 10 fully
sterile and fully fertile F2 plants are selected.
• DNA from these plants in each group is used
to create fertile and sterile bulks. These bulks
are used for Bulk Segregant Analysis (BSA).
• This helps to find molecular markers which are
polymorphic b/w bulks as well as b/w the
parents.

59. • The polymorphic markers can be used on the
individual plants to identify markers closely
linked to the Rf gene, which may be used for
marker-aided transfer of the Rf gene to a
desirable background.
• Eg: Rice, Sorghum, Brassica

60. Cytoplasmic-Genetic Male Sterility
• The male sterility which is governed by both nuclear
and cytoplasmic genes.
• Also called as Nucleoplasmic Male Sterility.
• When nuclear restoration of fertility genes (“Rf”) is
available for a CMS system in any crop, it is
cytoplasmic-genetic male sterility.
• There are commonly two types of cytoplasm
N (normal) and S (sterile).
• The genes for these are found in mitochondrion.
There are also restores of fertility (Rf) genes.

61. • Rf genes do not have any expression of their own,
unless the sterile cytoplasm is present. Rf genes are
required to restore fertility in S cytoplasm which is
responsible for sterility.
• So the combination of N cytoplasm with rfrf and S
cytoplasm with RfRf produces plants with fertile
pollens, while S cytoplasm with rfrf produces only
male sterile plants.
• R(restorer gene) is generally dominant canbe transferred
from related strains orspecies.

63. Hybrid Seed Production using CGMS

64. Transfer of Restorer gene ‘R’ to non-restorer starin

65. Production Double cross maize hybrids
using CGMS

66. Sources of CMS & Restorers

68. Hybrids released using CGMS
Sl.
No.
Crop Hybrid
1. CHILLI Arka Megha, MSH-149
MSH-96
2. CARROT Pusa Nayanjyoti
3. ONION Arka Kirtiman, Arka Lalit,
Hybrid-63, Hybrid-35

69. Merits of CGMS
1.In CGMS system, CMS is highly stable and is not
affected by environmental factors.
2.In CGMS system, CMS 'A' line gives only male
sterile plants.
3.Conversion of a genotype in CGMS system 'A' line is
quicker and direct.
4.CMS requires less area for maintenance.
5.The quantity of seed produced is more.
6.There is no chance of admixture.

70. Demeritsof Cytoplasmic-GeneticMale Sterility
•Undesirable effects of thecytoplasm
•Unsatisfactory fertility restoration
•Unsatisfactorypollination
•Spontaneousreversion
•Modifying genes
•Contribution ofcytoplasm by male gamete
•Environmentaleffects
•Non availability of asuitable restorerline

71. CHEMICALLY INDUCED MALE STERILITY
• CHAis a chemical that induces artificial, non-genetic male sterility
in plants so that they can be effectively used asfemale parent in hybrid
seedproduction.
• Also called as Male gametocides, male sterilants, selective male
sterilants,pollensuppressants,pollenocide,androcideetc.
• The first report was given by Moore and Naylor (1950), they
inducedmalesterility in Maizeusingmaleichydrazide(MH).

72. Propertiesof anIdeal CHA
•Must be highly male orfemale selective.
•Should be easily applicable and economic inuse.
•Time of application should beflexible.
•Must not bemutagenic.
•Must not be carried over inF1seeds.
•Must consistently produce >95%malesterility.
•Must causeminimum reduction inseedset.
•Should not affect outcrossing.
•Shouldnot be hazardousto the environment

73. Advantages of CHAs
 Anyline canbe usedasfemale parent.
 Choiceof parents isflexible.
 Rapidmethod of developing male sterileline.
 Noneed of maintaining A,B&Rlines.
 Hybrid seedproduction isbasedon only 2 linesystem.
 Maintenance of parental line ispossible by self pollination.
 CHAbased F2 hybrids are fully fertile as compared to few
sterile hybrids in caseof CMSorGMS.

75. HybridSeedProductionbasedon CHAs
• Conditionsrequired:-
1. Proper environmental conditions (Rain, Sunshine,temp,
RHetc.)
2. Synchronisationof flowering of Male & Female parents.
3. Effective chemical emasculation and crosspollination.
4. CHA at precise stageand with recommendeddose.
5. GA3 spray to promote stigmaexertion.
6. Supplementary pollination tomaximise seedset.
7. Avoid CHA spray on pollinator row.

76. Limitationsof CHAs
•Expression and duration of CHAis stagespecific.
•Sensitive to environmentalconditions.
•Incomplete male sterility produce selfedseeds.
•Many CHAsare toxic to plants andanimals.
•Possesscarryover residual effects in F1seeds.
•Interfere with celldivision.
•Affect humanhealth.
•Genotype, dose application stagespecific.

77. PISTILLATE CONDITION
• In some mutants, only pistillate flowers are
produced in place of both male and female
flowers.
• eg: Castor, Cucurbits
• In castor : it is governed by a single recessive
gene.
Types:
• N Type
• S Type
• NES Type

78. N Type Pistillate Lines
• Governed by single recessive gene (n).
• Produce only pistillate flowers.
• Maintaining pistillate lines : crossing them
with heterozygous monoecious (Nn) lines that
produce both male and female flowers.
• Progeny: 50% pistillate, 50% monoecious.
• Similar to GMS.

79. S Type Pistillate Lines
• Sex reversal variants: pistillate to begin with and
changes to monoecy.
• In S type, continued selection for increased expression
of pistillate condition within sex reversal variants.
• 50-70% plants are pistillate.
• Pistillate plants revert back to monoecious in different
stages of development.
• Use of pistillate lines in hybrid seed production requires
removal of monoecious plants and early revertants.
• Eg. Castor VP1 (female parent of GAUCH1)
Geeta (female parent of GCH1)

80. NES Type
• Temperature sensitive N lines.
• 100% pistillate when temp. during flowering <
350C., produce male flowers @ temp. > 350C
• Plants are multiplied during hot season.
• Comparable to TGMS and PGMS.
• Most suited for hybrid seed production.
• Eg. JP65 (female parent of GCH6)

81. Significanceof 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 borne. Inefficient emasculation may produce some self
fertile progenies.
• GMSisbeing exploited (Eg.USA-Castor,India-Arhar).
• CMS/ CGMS are routinely used in Hybrid seed production in corn,
sorghum, sunflower and sugarbeet, ornamentalplants.
•Saveslot of time, money andlabour.

82. LimitationsinusingMale Sterile line
• 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 havetobe converted into MSlines.
• Adequate crosspollination should be there between AandR lines for
goodseedset.
• Synchronizationof flowering should be there betweenA& Rlines.
• 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.