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 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
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
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
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
 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
  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
                  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
Fertility restoration in maize
      Simple hybrid with cms and
                     C1              C1
CMS line (A-line)   N1    x         N1     Maintainer line (B-line)
CMS, rfrf                                  N, rfrf

  Large amounts      C1
                                x             C2
                                                    Male line (C-line)
  of CMS line       N1                      N2
                                                    N and RfRf

                              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
 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

 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
  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
     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
   Faculty of Agriculture
  Gadjah Mada University
Biochemical means of producing
male sterile plants
 Feminizing hormones
 Inhibitors of anther or pollen
 a. acting on sporophytic tissue
 b. acting on gametophytic tissue
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
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
 There are at least 4 classes of chemical agents:
  a. Plant growth regulators and substances that disrupt floral
  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,
  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
  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
 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
 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
 It is 5-(aminocarbonyl)-1-(3-methylphenyl)-1H-pyrazole-
 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
 It is potassium 3,4-dichloro-5-isothiocarboxylate
 When applied underdeveloped anthers, they will fail to
 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
 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-
  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
 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.
  1-(4’-trifluoromethylphenyl)-4-oxo-5-fluorocinnoline-3-carboxylic acid
  1-(4’-chlorophenyl)-4-oxo-5-propoxycinnoline-3-carboxylic acid
  1-(4’-chlorophenyl)-4-oxo-5(methoxyethoxy) cinnoline-3-carboxylic acid
           Inhibitors of microspore
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
 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
  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

    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
  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
   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
                                 Induced GMS


                                    transformation                  male-sterile

   Promoter which        Gene which disrupts
induces transcription   normal function of cell
in male reproductive
            Induced GMS System

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

How to induce                                              How to restore
  sterility?                                               fertility?
                              F1 (Ss, Rfrf) (50%)
                              F1 (ss, Rfrf) (50%)

                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

 Selection by herbicide application

 Inducible sterility

 Inducible fertility

 Two-component system
            Selection by Herbicide Application

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

 TA29       Banase   NOS-T                       Gene for inhibitor of
                                                    barnase from
  35S        PAT     NOS-T                        B. amyloliqefaciens

     Gene for glufosinate
      resistance from S.
         Selection by Herbicide Application

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

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

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

 selfing                   Sterile parent     X    Fertile parent

Plants transformed
   by TA29-argE                         Fertile F1 plant

                      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

                       Sterile parent   X      Restorer

                 addition of

                       Fertile parent       Fertile F1 plant
                                             Fertile parent


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

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

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

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

Male sterile   parent   can   be   propagated     without
 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

 CMS is induced by the expression of phaA gene in
 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

                                            CH3             S-CoA

                                 CoASH                               b-ketothiolase    (phaA gene)
                                         O              O

          Acetoacetyl-CoA                   C               C
                                   CH3            CH2           S-CoA
                                  NADP+                              Acetoacetyl-CoA
                                                                                       ( phaB gene )
                                         HO                O            reductase
(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
Chloroplast Transformation

                      vector construction

         Transformation by
        Particle bombardment
                       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



        carboxylase      Malonyl-CoA      Fatty acid

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.

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