Plant breeding. Male sterility. cytoplasmic male sterility. Genetic male sterility. Cytoplasmic genetic male sterility.

1. PRESENTED BY-
CHUDAMANI PANT
ROLL. NO. WS-01M-2019
DEPARTMENT OF AGRONOMY
AGRICULTURE AND FORESTRY
UNIVERSITY, RAMPUR, CHITWAN

3. Male Sterility
 Male sterility is characterized by non-functional 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 Fertile

4. 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)

5. Classification of Male Sterility
Kaul (1988) Classified Male Sterility in three major groups
1. Phenotypic Male Sterility (Morphological)
3. Chemically Induced Male Sterility (CHA)
 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)
2. Genotypic Male Sterility
 Structural or Staminal Male Sterility
 Pollen Male Sterility
 Functional Male Sterility

6.  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).
Phenotypic Male Sterility
 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

7. Cytoplasmic Male Sterility (CMS)
 May be divided into autoplasmic CMS(mutation within species in mitochondrial genome) and
alloplasmic CMS (intergeneric, interspecific or occasionally intraspecific crosses and MS is due
to incompatibility or poor co-operation between the nuclear genome of one species and the
organellar genome of another).
 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.

8. 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

9. 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.

10.  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

11.  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.
 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).
Chemical Induced Male Sterility

12. 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

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

15.  Firstly in the production of hybrid seed, and
Male sterility plays an important role in plant breeding
 Secondly as a plant breeding tool facilitating population
improvement, backcrossing, interspecific hybridization and other
intermediate breeding procedures

16. 1. Hybrid seed production
Maize is the only important agricultural crop that can be manually
emasculated on a sufficiently large scale to permit production of hybrid
seed for commercial use
Maize, sorghum, sugar beet, sunflower, tomato, carrot, onion and several
other vegetable and ornamental crops
As maize is monoecious species the male inflorescence can easily be
removed without damaging the female flowers
Plant breeders have introduced different kinds of male sterility into plant
populations and used them to circumvent the restrictions to large scale
controlled hybridization imposed by flower morphology and breeding
systems.

17. S.No. Crop Seed Production
1. Maize CMS
2. Sorgum CMS
3. Bajra CMS
4. Sunflower CMS
5. Rapeseed CMS
6. Rice CMS
7. Red Gram GMS
Male Sterility based Hybrids in Important Crops

18.  CMS can used in hybrid seed production of certain ornamental species or in
species where a vegetative part is of economic value.
 Only one method by which hybrid seed can be produced both effectively
and economically.
 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

19.  It is only the combination of 'sterile' cytoplasm and homozygosity for the recessive
gene rf, (S)rfrf, that results in male sterility
 A genotype that masks the expression of the CMS trait and which, when used as a
pollinator on a CMS female, restores the pollen fertility of the progeny is called a
restorer.
 As in the case of CMS in onion, the restorer gene may be the dominant allele of the
recessive maintainer gene, and the restorer genotype thus (N)- RfRf.
 A genotype of the constitution (N)rfrf is called a maintainer since a male sterile
plant will produce a uniformly male sterile offspring only when pollinated by plants
of this genotype.

20. 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

21. Crop species Cytoplasm Restorer Genes
Sunflower PET-1 (H. petalaris) 2 polymorphic genes (Rf1, Rf2)
Jowar Milo or A1 Msc from kafir race
Bajra Tift-23A -

22. 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

23. Recessive NMS
Nuclear male sterility (NMS)
NMS does not permit the production of a uniformly male sterile
population seriously limits its use in hybrid seed production.
To permit the use of recessive NMS in large-scale hybrid seed production
it is necessary to rogue out 50% male fertile plants from the backcross
msms X Msms.
Since safe classification of fertile versus male sterile plants cannot
usually be made until shortly before flowering, such roguing becomes
far too difficult and costly in most crops to permit economic production
of large quantities of hybrid seed.

24. Vegetative propagation of MS plants to be used as females in hybrid
seed production has so far been used mainly in ornamental plants
where the price of commercial seed is high.
 Several solutions to this problem have been proposed
comprising:
 (1) vegetative propagation;
(2) marker assisted selection;
(3) cytogenetic methods;
(4) temporary restoration of fertility;
(5) use of functional male sterility.

25. Since then several similar systems of marker-assisted selection have
been developed, involving seed characters such as white endosperm in
maize and shrunken endosperm in barley and seedling characters such
as short leaf in sorghum and the woolly character in tomato could be
used as a marker for a linked ms gene.
However, with the development of cheap and efficient methods of
micro propagation, hybrid seed production based on vegetatively
propagated heterozygous male sterile genotypes is likely to increase
in importance, especially among vegetable crops.

26. Standard linkage analysis of the progenies of independent transgenic
plants would then be used to identify sufficiently tight linkage between
the marker and the ms gene.
The method should be applicable to any hybrid breeding programme
provided a reliable ms gene, a suitable marker and an efficient
transformation-regeneration system are available.
Cytogenetic methods combine ms genes with chromosomal selective
mechanisms to produce pure male sterile progenies.
Partial male sterility, sensitive to photoperiod, for a few days during
the critical period of pollen development, resulting in breakdown of
male sterility in rice and similarly gibberellic acid (GA3) has been
reported to restore pollen fertility in the 'corn grass' mutant in maize,
stamenless mutants in tomato as well as in a nuclear male sterile in
barley

27. Advantage of chemical reversion of male sterility is that it is not
necessary to achieve complete fertility restoration.
Side effects of the chemicals on the plants are also not a serious problem
since the treated plants are not used to grow the hybrid seed.
Functional male sterility in hybrid seed production have also been
made.
A recessive gene in tomato which produces normal pollen in anthers
that do not dehisce; because of this, natural selfing is prevented.
However, this mutant can be selfed by manually inducing dehiscence
of the anthers.
Similar mutants have been found in maize, eggplant and beans.
The male sterile mutant can thus be easily maintained and used
for hybrid seed production without emasculation.

28. Described in several crop species including carrot, cotton, wheat and
rapeseed.
Although dominant NMS may be used to facilitate crossing in recurrent
selection programmes, its inheritance normally prevents its use in hybrid
seed production.
Recently, scientists have transformed tobacco and rapeseed plants with a
chimeric gene consisting of an anther tapetum specific gene (TA29) and a
ribonuclease gene (RNase).
 Female fertility is not affected and transformed plants are normal in all
other respects.
By linking the TA29-RNase gene to a dominant herbicide resistance
gene, one can use the corresponding herbicide to produce uniform
populations of male sterile plants.
Dominant NMS

29. 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)

30.  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

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

32. Hybrid seed production using Barnase/Barstar system

33. 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:-

34. 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.

35. 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, 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

36. 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.

37. 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.

38.  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

39.  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