MALE-STERILE

Taryono
Faculty of Agriculture
Gadjah Mada University
Several forms of pollination control

1. Manual emasculation
2. Use of male sterility
3. Use of self-incompatibility alleles
4. Use of male gametocides
5. Use of genetically engineered “pollen killer” genetic
system
 Male-sterile
Plant that do not produce viable, functional pollen grains
An inability to produce or to release functional pollen as a
result of failure of formation or development of functional
stamens, microspores or gametes
Three types of sterility:
1. “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)
2. “Structural or staminal male sterility” in which male flowers or stamen
are malformed and non functional or completely absent
3. “Functional male sterility” in which perfectly good and viable pollen is
trapped in indehiscent anther and thus prevented from functioning
 Type of Male-sterile
 Based on its inheritance or origin
 Cytoplasmic male sterility (CMS) = sterile cytoplasm (S)
Male steril comes about as a result of the combined action of nuclear genes
and genic or structural changes in the cytoplasmic organellar genome
maternally inherited

 Nuclear male sterility (NMS) = Genic, genetic, mendelian

Male sterility is governed solely by one or more nuclear genes
Nuclear inherited

 Non genetic, chemically induced male sterility

Application of specific chemical (gametocides or chemical hybridizing
agents)
Flower phenotypes in carrot

a) Normal (N-cytoplasm, restored CMS plants)

b) Brown anther CMS (Sa)
c) Petaloid CMS (Sp)
 Cytoplasmic male-sterile
 Stamen (anther and filament) and pollen grains are
affected
 It is divided into:
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, interpecific 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
 Cytoplasmic male-sterile
 The nuclear genetic control of CMS is predominantly governed by
one or more recessive genes, but can be also dominant genes as
well as polygenes
 The different mtDNA restriction endonuclease digestion patterns are
reflections of aberrant intra- or inter molecular DNA recombination
events in the mitochondrial genome which have either modified
existing genes or related new genes some of which are more or less
related to the male sterile phenotypes

 Some drawback:
1. insufficient or unstable male sterile
2. Difficulties in restoration system
3. Difficulties with seed production
4. Undesirable pleitropic effect
 Cytoplasmic male-sterile
Origins:
1. Intergeneric crosses
2. Interspecific crosses
3. Intraspecific crosses
4. Mutagens (EMS, EtBr)
5. antibiotic (streptomycin and Mitomycin)
6. Spontaneus

CMS Characterization
 It has been traditionally characterized by the restore genes required
to overcome the CMS and to provide male sterile progeny in the male
sterile system
 CMS restoration is by nuclear genes, frequently dominant in action, in
many cases, few in number
 The CMS restore genes temporarily suppress the expression of the
CMS permitting normal or near-normal pollen production
 CMS mechanism of action
Abnormal behavior of the tapetum in the anther
Genetic determinant of CMS reside in mitochondria
Nuclear gene control the expression of CMS

CMS Limitation
 Pleiotropic negative effect of the CMS on agronomic
quality performance of plants in the CMS cytoplasm
 Enhanced disease susceptibility
 Complex and environmentally unstable maintenance of
male sterility and/or male fertility restoration
 Inability to produce commercial quantities of hybrid seed
economically because of poor floral characteristic of cross
pollination
 CMS Utilization
 It provides a possible mechanism of pollination control in
plants to permit the easy production of commercial
quantities of hybrid seeds
 It consists of a male sterile line (the A-line), an isogenic
maintainer line (The B line), and if necessary also restore
line (the R-line)
 A lines are developed by back-crossing selected B-lines
to a CMS A-line for 4 – 6 times to generate a new A-line
 B and R-lines are developed by similar back cross
procedures using a CMS R-line as female in the original
cross and a new line as the recurrent parent in 4 – 6
backcrosses
Fertility restoration in maize
 Simple hybrid with cms and
restoration
C1 C1
CMS line (A-line) N1 x N1 Maintainer line (B-line)
CMS, rfrf N, rfrf

Large amounts C1

x Male line (C-line)

C2

of CMS line N1 N2
N and RfRf

C1
Fertile F1 hybrid
CMS, Rfrf
Breeding hybrid carrots
 CMS Utilization
 Selfing the last backcross generation two successive times
and selection of pure breeding male fertility restore line is
required to complete the development of the new R-lines
developed in the CMS
 Current commercial hybrid seed production relies entirely
on the block method (alternating strips of female and
male genotypes
 Nuclear male sterility
 Originated through spontaneous mutation or mutation
by ionizing radiation and chemical mutagens such as
ethyl methane sulphonate (EMS) and ethyl imine (EI) or
by genetic engineering, protoplast fusion, T-DNA
transposon tagging and affecting the synthesis of
flavonoids
 can probably be found in all diploid species
 Usually controlled by mutations in genes in the single
recessive genes affect stamen and pollen development,
but it can be regulated also by dominant genes
 Morphology

 Variable (complete absence of male

reproductive organs to the formation of normal
stamen with viable pollen that fail to dehisce)
 It is not distinguishable from parent fertile
plants with the exception of flower structure
 Male sterile flowers are commonly smaller in
size in comparison to the fertile
 The size of stamens is generally reduced
 Determining factor

 Temperature
Changing the optimal temperature can induce sterility
 Photoperiod
It has a strong influence (Photoperiod sensitive)
Changing the growth habit can stimulate the sterility
 Cytological Changes

 Breakdown in microsporogenesis can occur at a

number of pre-or postmeiotic stages
The abnormalities can involve aberration during
the process of meiosis, in the formation of
tetrads, during the release of tetrad (the
dissolution of callose), at the vacuolate
microspore stage or at mature or near-mature
pollen stage
 Biochemical Changes
 Male sterility has been shown to be accompanied by
qualitative and quantitative changes in amino acids,
protein, and enzymes in developing anther
 Amino acids
The level of proline, leucine, isoleucine, phenylalanine
and valine is reduced, but asparagine, glycine, arginine,
aspartic acids is increased
 Soluble proteins
Male sterile anthers contain lower protein content and
fewer polypeptide bands
Some polypeptides synthesized in normal stamens were
absent in mutant stamens
 Biochemical Changes
 Enzymes
Callase is required for the breakdown of callose that
surrounds PMCs and the tetrad. Mistiming of callase
activity results in premature or delayed release of
meiocytes and microspore
Esterases have also been related to pollen development.
The activity of esterase is decreased
The activity of amylases is decreased and it corresponds
with high starch content and reduced levels of soluble
sugars
Accumulation of adenine due to the decrease of adenine
phosphoribosyltransferase (APRT) activity may be toxic
to the development of microspores
 Hormones and male Sterility
 Plant growth substances play an important role
in stamen and pollen development. Aberrant
stamen and pollen development is known to be
accompanied by changes in endogenous PGS
 GMS line was related to a change in the
concentration of gibberellins (rice), IAA
(Mercurialis annua), ABA (soybean), and
cytokinin (Mercurialis annua)
 Male serility is associated with changes in not
one PGS but several PGS
Use of genic male sterility in
hybrid programs
 Male sterile plants of monoecious or
hermaprodite crops are potentially useful in
hybrid program because they eliminate the labor
intensive process of flower emasculation
 Constraint of the use of genic
male sterility
 The maintenance of the male sterile line. Normally, a
GMS line (A-line) is maintained by backcrossing with
the heterozygote B-lines (Maintainer lines), but the
progeny produced are 50% fertile and 50% male
sterile
 Solution:
1. Identify marker genes that are closely linked to ms
genes and affect some vegetative characters
2. Use of environmental and chemical methods that can
lead to production of 100% male-sterile seed
CHEMICAL INDUCED
MALE-STERILE
Taryono
Faculty of Agriculture
Gadjah Mada University
Biochemical means of producing
male sterile plants
 Feminizing hormones
 Inhibitors of anther or pollen
development
a. acting on sporophytic tissue
b. acting on gametophytic tissue
(gametocides)
Inhibitors of pollen fertility
Chemical hybridizing agent (CHA)
 Could be used in the large scale commercial
production of hybrid seed
Are applied to plant only at certain critical
stage of male gametophyte development
 Their action could result from a range of mechanism:
1. Inherently selective action as male gametocides or inhibitors
of anther development
2. Selective transport of generally toxic or growth-inhibitory
substances to the anthers during these periods
3. Metabolic detoxification of generally toxic or growth-
inhibitory substances after they have suppressed male
fertility
The logic of chemical hybridization
 High degree of efficacy and developmental selectivity
 Persistence during the development of flower or spikes
 Low cost
 Acceptable levels of toxicity to people and the environment
 Low general phytotoxicity
 Agronomic performance of hybrid seed produced is not
inferior to equivalent crosses produced by genetic methods
 CHAs and pollen development
 Chemical inhibitors of pollen development are not familiar topic to the
majority of academic scientists. The most likely explanation for the
unfamiliarity is that these substances have been identified and
developed almost entirely within the industry
 Pollen comes into being through a sequential and determinate program
within the central cavity or locule of anther. These programmes are
biochemically controlled and may be affected by one or more chemical
agents
 There are at least 4 classes of chemical agents:
a. Plant growth regulators and substances that disrupt floral
development
b. Metabolic inhibitors
c. inhibitors of microspore development
d. inhibitors of pollen fertility
These categories have considerable conceptual overlap and do not
address the molecular action of the chemical male sterilants
 Plant growth regulators and substances
that disrupt floral development
 Plant hormones/hormones antagonists
a. auxins and auxin antagonists (NAA, IBA, 2,4-D, TIBA,
MH)
b. Gibberellins and antagonist (GA3, GA4+7, CCC: 2-
chloroethyl-trimethyl ammonium chloride)
c. Abscisic acid

 Other substances
a. LY195259
b. TD1123
 Auxins and antagonists
It may differently affect some far-reaching
process, such as blockade of nutrient transport
to the development anthers
Male sterility induced was expressed in several
ways
in situ pollen germination,
in situ exudation of pollen cytoplasm,
modification of certain stamens into staminodes
Tapetum fails to enlarge (MH and IBA) or tapetal cells enlarges
atypically and was persistent
 Gibberellins and antagonists
 GA affects on sexual determination and floral
development
 The response varies by species
 GA interferes with the development of male floral organs
or promotes feminization
 Gibberellin-synthesis inhibitors (CCC) at certain
concentration, selectively inhibits the development of
stamen or otherwise suppresses pollen development .
These effects are not sufficiently selective
 Abscisic acid
 ABA caused effect on developing floral buds similar to
CCC
 ABA caused male sterility if applied to plant just prior to
or during meiosis of pollen mother cells (wheat). ABA
may cause male sterility through more than one
mechanism
 LY195259
 It is 5-(aminocarbonyl)-1-(3-methylphenyl)-1H-pyrazole-
4-carboxylic-acid
 It is an effective chemical hybridizing agent
 It is applied when the flower was quite short with high
application rates, whereas lower dosages resulted in
progressively reduced inhibition
 Sterility at lower dosages was associated with smaller,
abnormally twisted and intensively pigmented locules
 The hybrid seed appeared normal, and no other
phytotoxic effects were visually evident from rates
 Uptake from soil was particularly effective
 TD1123
 It is potassium 3,4-dichloro-5-isothiocarboxylate
 When applied underdeveloped anthers, they will fail to
dehisce
 A variety of morphological effects were observed at
higher treatment levels
 Metabolic Inhibitors
 There are halogenated aliphatic acids (alpha, beta-
dichloroisobutyrate and 2,2-dichloropropionate salts)
and arsenicals (methanearsonate salts)
 They affect mitochondrial protein by reducing the
efficiency of normal metabolic processes
 Inhibitors of microspore
development
Copper chelators
Copper deficiency causes the irregular or absent of pollen development
Copper deficiency exerts the effects by inhibiting copper-requiring
oxidases that function in auxin metabolism
Ethylene
It is a natural regulator of the development and maturation of several
floral organs. Filament and corolla growth (unfolding and senescence)
are inhibited by ethelene production
Fenridazon
It is 1-(-4chlorophenyl-1,4-dihydro-6-methyl-4-
oxopyridazine-3-carboxylic-acid.
The treated microspores had wavy surfaces and progress to
plasmolysis and abortion with the onset of the microspore
vacuolation stage
Pollen wall was 80% thinner in treated plants
 Inhibitors of microspore
development
 Phenylcinnoline carboxylates (SC-1058, SC-1271 and SC-2053)
All capable of producing complete male sterility with minimal phytotoxicity
and loss of seed yield when applied just prior to meiosis
They cause a general retardation of anther development
Pollen development was generally arrested in the late prevacuolate or early
vacuolate microspore stage
The microspore often becomes wavy or wrinkled and the cytoplasm
degenerates and the cells become collapsed.
SC-1058:
1-(4’-trifluoromethylphenyl)-4-oxo-5-fluorocinnoline-3-carboxylic acid
SC-1271:
1-(4’-chlorophenyl)-4-oxo-5-propoxycinnoline-3-carboxylic acid
SC-2053:
1-(4’-chlorophenyl)-4-oxo-5(methoxyethoxy) cinnoline-3-carboxylic acid
 Inhibitors of microspore
development
Genesis ® (MON 21200)
It provides good CHA activity over a very diverse range of
genotypes, geographic regions and growing condition
Seed production has provided a high and reliable level of
outcrossing
Hybrids produced with the aid of genesis are equivalent to
conventional hybrids based on CMS technology
 Inhibitors of pollen fertility
Azetidine-3-carboxylate (A3C, CHA™)
It effectively induces male sterility in small grains, particularly
wheat
The major effect of mature pollen is a structural alteration of
cell wall precursor vesicles
Only 10% of the pollen grains showed normal pollen tube
growth in the first hour after pollination and none penetrated
the secondary stigmatic branch
 MALE-STERILITY
THROUGH RECOMBINANT
DNA TECHNOLOGY

Taryono
Faculty of Agriculture
Gadjah Mada University
I. Dominant Male-Sterility Genes
 Targetting the expression of a gene encoding a cytotoxin by placing
it under the control of an ather specific promoter (Promoter of TA29
gene)
Expression of gene encoding ribonuclease (chemical synthesized
RNAse-T1 from Aspergillus oryzae and natural gene barnase from
Bacillus amyloliquefaciens)
RNAse production leads to precocious degeneration of tapetum cells,
the arrest of microspore development and male sterility. It is a
dominant nuclear encoded or genetic male sterile (GMS), although
the majority of endogenous GMS is recessive
Success in oilseed rape, maize and several vegetative species
 Used antisense or cosuppression of endogenous gene that are
essential for pollen formation or function
 Reproducing a specific phenotype-premature callose wall dissolution
around the microsporogenous cells
 Reproducing mitocondrial dysfunction, a general phenotype
observed in many CMS
 Fertility restoration
 Restorer gene (RF) must be devised that can suppress
the action of the male sterility gene (Barstar)
1. a specific inhibitor of barnase
2. Also derived from B. amyloliquefaciens
3. Served to protect the bacterium from its own RNAse activity by
forming a diffusion-dependent, extreemely one to one complex
which is devoid of residual RNase activity
The use of similar promoter to ensure that it would be
activated in tapetal cells at the same time and to
maximize the chance that barstar molecule would
accumulate in amounts at least equal to barnase
 Inhibiting the male sterility gene by antisense. But in the
cases where the male sterility gene is itself antisense,
designing a restorer counterpart is more problematic
 Production of 100% male sterile
population
 When using a dominant GMS gene, a means to
produce 100% male sterile population is required in
order to produce a practical pollination control system
 Linkage to a selectable marker
Use of a dominant selectable marker gene (bar) that confers
tolerance to glufosinate herbicide
Treatment at an early stage with glufosinate during female parent
increase and hybrid seed production phases eliminates 50%
sensitive plants
 Pollen lethality
add a second locus to female parent lines consisting of an RF gene
linked to a pollen lethality gene (expressing with a pollen specific
promoter)
 Induced GMS

regeneration

Agrobacterium-
mediated
transformation male-sterile
plant

Promoter which Gene which disrupts

induces transcription normal function of cell
in male
reproductive
specifically
 Induced GMS System

Sterlie (Ss, rfrf) X Fertile (ss, RfRf)

How to induce How to restore

sterility? fertility?
F1 (Ss, Rfrf) (50%)
fertile
F1 (ss, Rfrf) (50%)
fertile

Sterile (Ss, rfrf) X Fertile (ss, rfrf)

How to propagate
male-sterile plants?

Sterile (Ss, rfrf) (50%)

Fertile (Ss, rfrf) (50%)

Strategies to Propagate Male-Sterile
Plant

 Selection by herbicide application

 Inducible sterility

 Inducible fertility

 Two-component system
 Selection by Herbicide Application

Tapetum-
specitic Gene for a RNase from
B. TA29 Barstar NOS-T
promoter
amyloliqefaciens

TA29 Banase NOS-T Gene for inhibitor of

barnase from
35S PAT NOS-T B. amyloliqefaciens

Gene for glufosinate

resistance from S.
hygroscopicus
fertile
 Selection by Herbicide Application

A (SH/-) X B (-/-)

SH/- -/- -/-

-/- SH/- SH/-
glufosinate -/- -/-
-/- SH/- SH/- SH/- -/-
X C (R/R)
SH/- -/-
-/- -/-
-/- SH/- -/-
-/-

SH/- -/-
-/- SH/- -/- SH/-

pTA29-barnase : S (sterility)
p35S-PAT : H (herbicide resistance)
pTA29-barstar : R (restorer) Fertile F1 (SH/-, R/-)

Fertile F1 (-/-, R/-)

 Inducible Sterility

 Male sterility is induced only when inducible chemical is applied.

NH4+ accumulation
Male sterility
in tapetal cell
Glutamate Glutamine

N-acetyl-
L- Glufosinate
N-acetyl-L-ornithine (toxic)
phosphinothricin
deacetylase
(non-toxic)
(coded by argE)

 Plants of male sterile line were transformed by a gene,

argE, which codes for N-acetyl-L-ornithine deacetylase,
fused to TA29 promoter.
 Induction of male sterility can occur only when non-toxic
compound N-acetyl-L-phosphinothricin is applied.
 Inducible Sterility

Plants transformed
by TA29-argE
fertile
N-acetyl-L-
phosphinothricin

selfing
Sterile parent X Fertile parent

Plants transformed
by TA29-argE Fertile F1 plant

fertile
 Inducible Fertility

If sterility was induced by inhibition of metabolite (amino acids, biotin,

flavonols, jasmonic acid) supply, fertility can be restored by application
of restricted metabolite and male sterile plant can be propagate by
selfing.

Sterile parent X Restorer

addition of
restricted
metabolite

Fertile parent Fertile

Fertile F1 plant
parent

selfing

Sterile parent
 Two-Component System

Male sterility is generated by the combined action of two genes

brought together into the same plant by crossing two different
grandparental lines each expressing one of the genes.

Two proteins which are

parts of barnase

Two proteins can form

stable barnase

Each grandparent has each part of barnase.

 Two-Component System

A1
A1(Bn5/Bn5)
(B5/B5) X A2 (Bn3/Bn3)
fertile fertile

A (Bn5/Bn3) X B (- -)
sterile fertile
selfing selfing

F1 (Bn5/-)
fertile
F1 (Bn3/-)
A1
A1 (Bn5/Bn5)
(B5/B5) X A2 (Bn3/Bn3)
fertile
fertile fertile

A (Bn5/Bn3)
sterile
Bn3 : 3’ portion of barnase gene
Bn5 : 5’ portion of barnase gene
 Advantages of CMS Engineering

Male sterile parent can be propagated without

segregation.
 Transgene is contained via maternal inheritance.
 Pleiotropic effects can be avoided due to subcellular
compartmentalization of transgene products.
 Non-transgenic line can be used as maintainer.
 Engineering CMS via the Chloroplast
Genome

 CMS is induced by the expression of phaA gene in

chloroplast.
 Fertility is restored by continuous illumination.
 Non-transgenic plants are used as the maintainer for
the propagation of male sterile plants.
 Reactions for the synthesis of PHB
Glucose

O
Acetyl-CoA
C
CH3 S-CoA

CoASH -ketothiolase (phaA gene)

O O

Acetoacetyl-CoA C C
CH3 CH2 S-CoA
NADPH
NADP+ Acetoacetyl-CoA
( phaB gene )
HO O reductase
fertile
(R)-3-Hydroxybutyryl-CoA CH C
CH3 CH2 S-CoA

PHB synthase (phaC gene)

O CH3 O CH3

Polyhydroxybutyrate C CH C CH O -

(PHB) CH3 O CH2 O C

n
O
Chloroplast Transformation

pLDR-5’UTR-phaA-3’UTP
vector construction

Transformation by
Particle bombardment
fertile
 Mechanism for CMS

Pollens of untransformed plant

Pollens of transgenic plant

Microspores and surrounding tapetal cells are particularly active in lipid

metabolism which is especially needed for the formation of the exine
pollen wall from sporopollenin.
High demand for fatty acid in tapetal cells cannot be satisfied because
of the depletion of acetyl-coA.
 Reversibility of Male Fertility

Acetoacetyl-CoA
-ketothiolase

Acetyl-CoA

Acetyl-CoA
carboxylase Malonyl-CoA Fatty acid

Illumination
for 8 ~ 10 days
Male fertility
 Prospects for CMS Engineering

 In present, chloroplast transformation is not efficient

for most of the crops except for tobacco.
 Although mitochondrial transformation has been
reported for single-celled Chlamydomonas and yeast,
there is no routine method to transform the higher-
plant mitochondrial genome.
 If the routine methods to transform organellar DNA of
crops are prepared, various systems for the CMS
engineering may be attempted.