This document discusses male sterility in plants. It begins with definitions of male sterility and classifications of sterility systems. Male sterility is characterized by nonfunctional pollen grains while female gametes function normally. It is classified into phenotypic, genotypic, and chemically induced types. Genotypic male sterility includes genetic male sterility (GMS), cytoplasmic male sterility (CMS), and cytoplasmic genetic male sterility (CGMS). CMS is determined by the cytoplasm and is useful for hybrid seed production. The document then discusses various manifestations, history, need for male sterility, and methods for detection, creation, and transfer of male sterility systems. It provides examples of CMS sources and restorer genes in crops

1. Dr. Naveen Kumar K.L
Assistant Professor
Dept. of GPB

2. Male sterility
Manifestation of Male sterility
History of Male sterility
Need of Male sterility
Detection of Male sterility
Creation of Male sterility
Classification of Male sterility
Applications of Male sterility in Hybrid seed
production
Contents

3. 1. Sterility systems: spores and gametes
are absent or abnormal.
2. Incompatibility systems: male and
female gametes are normal and functional.
Ex: self-incompatibility, cross-
incompatibility.
INFERTILITY VS. INCOMPATIBILITY
“Male sterility is defined as the failure of plants to produce functional
anthers, pollen, or male gametes”.

6. Linke, Mitochondrion (2005)

7. 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 Sterile
Fertile Fertile

8. 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

9. 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)
 Texas (T) cytoplasmic male sterility discovered in 1940s; used
extensively throughout the 1960s.

10. 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.

11. Classification of Male Sterility
Kaul (1988) Classified Male Sterility in three major groups
I. Phenotypic Male Sterility (Morphological)
1. Structural or Staminal Male Sterility
2. Pollen Male Sterility
3. Functional Male Sterility
II. Genotypic Male Sterility
1. Genetic Male Sterility (GMS)
a) Thermo sensitive genetic male sterility (TGMS)
b) Photoperiod sensitive genetic male sterility (PGMS)
c) Transgenic Male Sterility (TrMS)
2. Cytoplasmic Male Sterility (CMS)
3. Cytoplasmic Genetic Male Sterility (CGMS)
III. Chemically Induced Male Sterility (CHA)

12.  Structural or staminal male sterility: in which male
flowers or stamen are malformed and non functional or
completely absent.
 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).
 Functional male sterility: in which perfectly good and
viable pollen is trapped in indehiscent anther and thus
prevented from functioning
Phenotypic Male Sterility

13. 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

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

16. Types of GMS
 Environment insensitive GMS: ms gene expression is much less affected
by the environment.
 Environment sensitive GMS: ms gene expression occurs within a
specified range of temperature and /or photoperiod regimes (Rice, Tomato,
Wheat etc.).
1. 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 failure of pairing of two
chromosomes at metaphase was evident.
 This abnormality led to abnormal meiosis, abnormal or sterile pollens.
 Anthers were shriveled and non-dehiscence-Male sterile.
 However, these lines produced normal fertile pollen at low temp.
Sensitive period : PMC formation to Meiosis

17. 2. PGMS: Governed by 2 recessive genes.
 Sterility is obtained in long day conditions while in short days,
normal fertile plant.
 Rice:- Sterile under Long day conditions (13 hr. 45 min +
Temp. 23-290 C) but fertile under short day conditions.
 Sensitive period: Differentiation of secondary rachis branches
to PMC formation

18. Inheritance & Maintenance Of male sterile line

19. 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.

21.  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, Maize
cabbage etc.
Utilization of CMS in Plant Breeding

22. Use of CMS lines

23. Transfer of CMS to new strains (Diversification)

24. MAIZE
1. CMS-T (Texas) (Rogers and Edwardson, 1952)
 Highly stable under all environmental conditions
 Characterized by failure of anther exertion and pollen abortion
 Susceptible to race T of the southern corn leaf blight - (Cochliobolus
heterostrophus = Bipolaris maydis)
 Widespread use of T-cytoplasm for hybrid corn production led to
epidemic in 1970 with the widespread rise of Race T.
 Toxin produced by C. heterostrophus = T-toxin.
 Fertility restoration is sporophytic
 Rf1 (chr. 3) & Rf2 (chr.9) are responsible for fertility restoration

25. 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

26. Crop species Cytoplasm Restorer Genes
Tobaco
N. Debneyi -
N. Megalosiphon -
N. bigelovii -
Cotton
G. Anomalum -
G. Arboreaum -
G. harknesii -
Sunflower PET-1 (H. petalaris) 2 polymorphic genes (Rf1, Rf2)
Jowar Milo or A1 Msc from kafir race
Bajra Tift-23A -

27. S.No. Crop Hybrid Variety Seed Production
1. Maize Ganga 101, Ganga 1,
Deccan, Ranjit,
Trishulatha, DHM-107,
DHM-109
CMS
2. Sorgum CSH1 CMS
3. Bajra HB1 CMS
4. Sunflower BSH1 CMS
5. Rapeseed PGSH51 CMS
6. Red Gram ICPH-8 GMS
7. Rice PRH1 CMS
Male Sterility based Hybrids in Important Crops

28. 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 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.

29. Requirements for 3 Lines in CMS System
A-line
Stable Sterility
Well developed floral traits for outcrossing
Easily, wide-spectum, & 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
Taller than A-line
Large pollen load, normal flowering traits and timing

30. Hybrid seed production using CGMS system

31. Advantage & Disadvantage of 3-line hybrid rice system
Advantages
 Stable male sterility.
Disadvantages
 Limit germplasm source (CMS, Restorer)
 Dominant CMS cytoplasm in large area (WA)
 One more step for parental seed production
 Time consuming of CMS breeding

32. Hybrid seed production using CGMS
 Depends on the cytoplasm that produce male sterility and gene that
restores the fertility.
 Steps:
 Multiplication of CMS (A) line
 Multiplication of Maintainer (B) line and Restorer (R) line
 Production of Hybrid seed (A×R)
 Maintenace of A & B lines:
 Grow A line and its corresponding B line together in an isolated
plots.
 Isolation distance for A×B production fields is at least 1000m.
 A ratio of 1A:1B row is maintained.
 Pollens produced by the B line fertilize the male sterile plant (A)
and seeds produced thus
 Give rise to A line again.

33. rr
S
RR
F
Rr
S
rr
F
rr
S
Rr
S
rr
S
Rr
S
rr
S
RR
S
rr
S
Rr
S
♂
♀
♂
♀
Strain A Strain B
×
×
♀ × rr
F ♂
rr
F ♂
×
6-7 Back crosses
× RR
S
1 2 1
: :
CMS Restorer
Male fertile
Non restorer (Strain-C)
Male fertile
×
Male fertile
Male sterile
Discarded
Discarded
Discarded
Male sterile
Male sterile
Discarded
Male sterile
× Self pollinated
Male fertile
Male sterile
Self pollinated
Male fertile
(Strain-C)
Male fertile
(Strain-C)
♀
Male fertile
Restorer line R is crossed to Male sterile A
Male fertile F1 is crossed to Strain C
in which R gene is to be transferred
Male fertility progeny is
back crossed to strain C
× Male fertility progeny is
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 restorer strain

34. 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 –I
A×B
(Male Sterile)
Single Cross-II
C×D
(Male Fertile)
rr S
Rr S
♀
♂
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)

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

36. Detection of Male Sterility system
Whether a particular sterile genotype belongs to which MS
system can be detected by its progeny performance on crossing
with a few normal genotypes.
 Trend-I- All progenies in all the rows may be sterile- CMS
 Trend-II- Some rows may consist all fertile
Some rows sterile and fertile in 1:1 ratio- GMS
 Trend-III- Some rows fertile. Some rows sterile and some
rows sterile and fertile in 1:1 ratio - CGMS

37.  Undesirable effects of the cytoplasm
 Unsatisfactory fertility restoration
 Unsatisfactory pollination
 Spontaneous reversion
 Modifying genes
 Contribution of cytoplasm by male gamete
 Environmental effects
 Non availability of a suitable restorer line
Limitations of Cytoplasmic-Genetic Male Sterility

38.  A gene introduced into the genome of on arganism by recombinant
DNA technology or genetic engineering is called transgene.
 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 (TrMS)

39.  Barnase/Barstar system by Mariani et al (1990) –rape seed (Brassica napus )
 Barnase gene- Bacillus amyloliquefaciens- encodes is extracellular RNase.
 Barstar gene- Bacillus amyloliquefaciens- encodes a protein, which is a higly
specific inhibitor of Barnase Rnase.
 Barstar is dominant over the Barnase.
 TA29 Promoter –TA29 gene of tobacco.
 TA29- expressed only in tapetum cells causing their degeneration.
 Fuse the barnase and barstar genes to TA29 promoter–TA29 is a plant gene that
has tapetum specific expression.
 Barnase gene is tagged with gene bar (Streptomyces)- which confer resitance to
herbicide phosphinpthricin.
 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

40. Hybrid seed production using Barnase/Barstar system

41. Classification of Male Sterility
Kaul (1988) Classified Male Sterility in three major groups
I. Phenotypic Male Sterility (Morphological)
1. Structural or Staminal Male Sterility
2. Pollen Male Sterility
3. Functional Male Sterility
II. Genotypic Male Sterility
1. Genetic Male Sterility (GMS)
a) Thermo sensitive genetic male sterility (TGMS)
b) Photoperiod sensitive genetic male sterility (PGMS)
c) Transgenic Male Sterility (TrMS)
2. Cytoplasmic Male Sterility (CMS)
3. Cytoplasmic Genetic Male Sterility (CGMS)
III. Chemically Induced Male Sterility (CHA)

42.  Many chemicals affecting the function of male reproductive organs
in plants; these compounds have been called as male gameticides /
male sterilants / pollen supressants / pollinocides / androcides.
 1985 McRae suggested the use of single term Chemical Hybridising
Agents (CHA)
 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.
 The first report was given by Moore and Naylor (1950), they induced
male sterility in Maize using maleic hydrazide (MH).
Chemical Induced Male Sterility

43. Properties of an Ideal CHA
1. Must be highly male or female selective.
2. Should be easily applicable and economic in use.
3. Time of application should be flexible.
4. Must not be mutagenic.
5. Must not be carried over in F1 seeds.
6. Must consistently produce >95% male sterility.
7. Must cause minimum reduction in seed set.
8. Should not affect out crossing.
9. Should not be hazardous to the environment.

44. S.No. CHAs Critical stage Crop species
1. Zink Methyl Arsenate
Sodium Methyl Arsenate
5 days before heading Rice
2. Ethephon/ Ethrel Depends on crop Barley , oat, bajra,
rice
3. Mendok Depends on crop
Cotton 35 days
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

45. Hybrid Seed Production based on CHAs
 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.
Conditions required:-

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

47. Limitations of CHAs
1. Expression and duration of CHA is stage specific.
2. Sensitive to environmental conditions.
3. Incomplete male sterility produce selfed seeds-cause of seed
certifications.
4. Many CHAs are toxic to plants and animals.
5. Possess carryover residual effects in F1 seeds (ex. Arsenicals
and WL84811)
6. Interfere with cell division (EX. RH531 & RH53)
7. Stimulate neoplasmic growth and Affect human health.
8. Genotype, dose application stage specific.

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

49.  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

50. Male sterility system in Rice hybrid seed production
 Male sterility: a condition in which the pollen grain is unviable or
cannot germinate and fertilize normally to set seeds.
 Male Sterility Systems (genetic and non-genetic):
Cytoplasmic genetic male sterility (CMS)
Male sterility is controlled by the interaction of a genetic factor
(S) present in the cytoplasm and nuclear gene (s).
Environment-sensitive genic male sterility (EGMS)
Male sterility system is controlled by nuclear gene expression,
which is influenced by environmental factors such as temperature
(TGMS), daylength (PGMS), or both (TPGMS).
Chemically induced male sterility
Male sterility is induced by some chemicals (gametocides)

52. Two Commercial MS Systems for Hybrid Rice

53. TGMS and two-line hybrid
Based on the
discovery of
P(T)GMS mutant
Male sterility
controlled by 1 or 2
pairs of recessive
gene(s)
Fertile
S-line
Multiplication
Critical Fertility Point
Critical Sterility Point
Reproductive Upper Limit
Reproductive Lower Limit
Sterile
F1 Seed
Production
Partial Sterility
Model of Sterility / Fertility Expression for TGMS Rice
Temperature
low
high

54. Advantage & Disadvantage of 2-line hybrid rice system
Advantages
Simplified procedure of hybrid seed production
Multiple and diverse germplasm available as parents
Any line could be bred as female
97% (2-line) vs 5% (3-line) of germplasm as male
Increased chance of developing desirable & heterotic hybrids
Multiple cytoplasm courses as female parents
Disadvantages
Environmental effect on sterility could cause seed purity
problem