MALE-STERILEMALE-STERILE

TaryonoTaryono

Faculty of AgricultureFaculty of Agriculture

Gadjah Mada UniversityGadjah Mada University

Several forms of pollination controlSeveral 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-sterileMale-sterilePlant that do not produce viable, functional pollen Plant that do not produce viable, functional pollen

grainsgrainsAn inability to produce or to release functional An inability to produce or to release functional

pollen as a result of failure of formation or pollen as a result of failure of formation or development of functional stamens, microspores or development of functional stamens, microspores or gametesgametesThree 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 carrotFlower phenotypes in carrot

a) Normal (N-cytoplasm, restored CMS plants)b) Brown anther CMS (Sa)c) Petaloid CMS (Sp)

Cytoplasmic male-sterileCytoplasmic male-sterile

Stamen (anther and filament) and pollen grains Stamen (anther and filament) and pollen grains are affectedare 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 genomeb. 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-

operationbetween nuclear genome of one species and the

organellar genome another

CMS can be a result of interspecific protoplast fusion

Cytoplasmic male-sterileCytoplasmic 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 sterile2. Difficulties in restoration system3. Difficulties with seed production4. Undesirable pleitropic effect

Cytoplasmic male-sterileCytoplasmic male-sterileOrigins:Origins:

1. Intergeneric crosses2. Interspecific crosses3. Intraspecific crosses4. Mutagens (EMS, EtBr) 5. antibiotic (streptomycin and Mitomycin) 6. Spontaneus

CMS CharacterizationCMS Characterization It has been traditionally characterized by the restore genes It has been traditionally characterized by the restore genes

required to overcome the CMS and to provide male sterile required to overcome the CMS and to provide male sterile progeny in the male sterile systemprogeny in the male sterile system

CMS restoration is by nuclear genes, frequently dominant in CMS restoration is by nuclear genes, frequently dominant in action, in many cases, few in numberaction, in many cases, few in number

The CMS restore genes temporarily suppress the expression The CMS restore genes temporarily suppress the expression of the CMS permitting normal or near-normalof the CMS permitting normal or near-normal pollen pollen productionproduction

CMS mechanism of actionAbnormal behavior of the tapetum in the anther

Genetic determinant of CMS reside in mitochondria

Nuclear gene control the expression of CMS CMS LimitationCMS Limitation

Pleiotropic negative effect of the CMS on Pleiotropic negative effect of the CMS on agronomic quality performance of plants in the agronomic quality performance of plants in the CMS cytoplasmCMS cytoplasm

Enhanced disease susceptibilityEnhanced disease susceptibility Complex and environmentally unstable Complex and environmentally unstable

maintenance of male sterility and/or male fertility maintenance of male sterility and/or male fertility restoration restoration

Inability to produce commercial quantities of Inability to produce commercial quantities of hybrid seed economically because of poor floral hybrid seed economically because of poor floral characteristic of cross pollinationcharacteristic of cross pollination

CMS UtilizationCMS Utilization

It provides a possible mechanism of pollination It provides a possible mechanism of pollination control in plants to permit the easy production of control in plants to permit the easy production of commercial quantities of hybrid seedscommercial quantities of hybrid seeds

It consists of a male sterile line (the A-line), an It consists of a male sterile line (the A-line), an isogenic maintainer line (The B line), and if isogenic maintainer line (The B line), and if necessary also restore line (the R-line)necessary also restore line (the R-line)

A lines are developed by back-crossing selected A lines are developed by back-crossing selected B-lines to a CMS A-line for 4 – 6 times to generate B-lines to a CMS A-line for 4 – 6 times to generate a new A-linea new A-line

B and R-lines are developed by similar back cross B and R-lines are developed by similar back cross procedures using a CMS R-line as female in the procedures using a CMS R-line as female in the original cross and a new line as the recurrent original cross and a new line as the recurrent parent in 4 – 6 backcrossesparent in 4 – 6 backcrosses

Fertility restoration in maizeFertility restoration in maize

Simple hybrid with cms and restorationSimple hybrid with cms and restoration

Maintainer line (B-line)N, rfrf

N1

C1

Large amountsof CMS line

xCMS line (A-line)CMS, rfrf

N1

C1

N1

C1C2x N2

Male line (C-line)N and RfRf

C1

Fertile F1 hybridCMS, Rfrf

Breeding hybrid carrotsBreeding hybrid carrots

CMS UtilizationCMS Utilization

Selfing the last backcross generation two Selfing the last backcross generation two successive times and selection of pure breeding successive times and selection of pure breeding male fertility restore line is required to complete male fertility restore line is required to complete the development of the new R-lines developed in the development of the new R-lines developed in the CMS the CMS

Current commercial hybrid seed production relies Current commercial hybrid seed production relies entirely on the block method (alternating strips of entirely on the block method (alternating strips of female and male genotypes female and male genotypes

Originated through spontaneous mutation or Originated through spontaneous mutation or mutation by ionizing radiation and chemical mutation by ionizing radiation and chemical mutagens such as ethyl methane sulphonate mutagens such as ethyl methane sulphonate (EMS) and ethyl imine (EI) or by genetic (EMS) and ethyl imine (EI) or by genetic engineering, protoplast fusion, T-DNA transposon engineering, protoplast fusion, T-DNA transposon tagging and affecting the synthesis of flavonoidstagging and affecting the synthesis of flavonoids

can probably be found in all diploid speciescan probably be found in all diploid speciesUsually controlled by mutations in genes in the Usually controlled by mutations in genes in the

single recessive genes affect stamen and pollen single recessive genes affect stamen and pollen development, but it can be regulated also by development, but it can be regulated also by dominant genesdominant genes

Nuclear male sterility

Variable (complete absence of male Variable (complete absence of male reproductive organs to the formation of reproductive organs to the formation of normal stamen with viable pollen that fail to normal stamen with viable pollen that fail to dehisce)dehisce)

It is not distinguishable from parent fertile It is not distinguishable from parent fertile plants with the exception of flower structureplants with the exception of flower structure

Male sterile flowers are commonly smaller Male sterile flowers are commonly smaller in size in comparison to the fertilein size in comparison to the fertile

The size of stamens is generally reducedThe size of stamens is generally reduced

Morphology

TemperatureTemperatureChanging the optimal temperature can induce Changing the optimal temperature can induce

sterilitysterility PhotoperiodPhotoperiod

It has a strong influence (Photoperiod sensitive)It has a strong influence (Photoperiod sensitive)

Changing the growth habit can stimulate the Changing the growth habit can stimulate the sterilitysterility

Determining factor

Breakdown in microsporogenesis can Breakdown in microsporogenesis can occur at a number of pre-or postmeiotic occur at a number of pre-or postmeiotic

stagesstagesThe abnormalities can involve aberration The abnormalities can involve aberration

during the process of meiosis, in the during the process of meiosis, in the formation of tetrads, during the release of formation of tetrads, during the release of tetrad (the dissolution of callose), at the tetrad (the dissolution of callose), at the vacuolate microspore stage or at mature vacuolate microspore stage or at mature

or near-mature pollen stageor near-mature pollen stage

Cytological Changes

Male sterility has been shown to be accompanied Male sterility has been shown to be accompanied by qualitative and quantitative changes in amino by qualitative and quantitative changes in amino acids, protein, and enzymes in developing antheracids, protein, and enzymes in developing anther

Amino acidsAmino acids

The level of proline, leucine, isoleucine, The level of proline, leucine, isoleucine, phenylalanine and valine is reduced, but phenylalanine and valine is reduced, but asparagine, glycine, arginine, aspartic acids is asparagine, glycine, arginine, aspartic acids is increasedincreased

Soluble proteinsSoluble proteins

Male sterile anthers contain lower protein content Male sterile anthers contain lower protein content and fewer polypeptide bandsand fewer polypeptide bands

Some polypeptides synthesized in normal Some polypeptides synthesized in normal stamens were absent in mutant stamensstamens were absent in mutant stamens

Biochemical Changes

EnzymesEnzymes

Callase is required for the breakdown of callose Callase is required for the breakdown of callose that surrounds PMCs and the tetrad. Mistiming of that surrounds PMCs and the tetrad. Mistiming of callase activity results in premature or delayed callase activity results in premature or delayed release of meiocytes and microsporerelease of meiocytes and microspore

Esterases have also been related to pollen Esterases have also been related to pollen development. The activity of esterase is decreaseddevelopment. The activity of esterase is decreased

The activity of amylases is decreased and it The activity of amylases is decreased and it corresponds with high starch content and reduced corresponds with high starch content and reduced levels of soluble sugarslevels of soluble sugars

Accumulation of adenine due to the decrease of Accumulation of adenine due to the decrease of adenine phosphoribosyltransferase (APRT) activity adenine phosphoribosyltransferase (APRT) activity may be toxic to the development of microsporesmay be toxic to the development of microspores

Biochemical Changes

Plant growth substances play an Plant growth substances play an important role in stamen and pollen important role in stamen and pollen development. Aberrant stamen and pollen development. Aberrant stamen and pollen development is known to be accompanied development is known to be accompanied by changes in endogenous PGSby changes in endogenous PGS

GMS line was related to a change in the GMS line was related to a change in the concentration of gibberellins (rice), IAA concentration of gibberellins (rice), IAA (Mercurialis annua), ABA (soybean), and (Mercurialis annua), ABA (soybean), and cytokinin (Mercurialis annua) cytokinin (Mercurialis annua)

Male serility is associated with changes in Male serility is associated with changes in not one PGS but several PGSnot one PGS but several PGS

Hormones and male Sterility

Male sterile plants of monoecious or Male sterile plants of monoecious or hermaprodite crops are potentially useful in hermaprodite crops are potentially useful in hybrid program because they eliminate the hybrid program because they eliminate the

labor intensive process of flower emasculationlabor intensive process of flower emasculation

Use of genic male sterility in hybrid programs

The maintenance of the male sterile line. The maintenance of the male sterile line. Normally, a GMS line (A-line) is maintained by Normally, a GMS line (A-line) is maintained by backcrossing with the heterozygote B-lines backcrossing with the heterozygote B-lines (Maintainer lines), but the progeny produced (Maintainer lines), but the progeny produced are 50% fertile and 50% male sterileare 50% fertile and 50% male sterile

Solution:Solution:

1.1. Identify marker genes that are closely linked to Identify marker genes that are closely linked to ms genes and affect some vegetative ms genes and affect some vegetative characterscharacters

2.2. Use of environmental and chemical methods Use of environmental and chemical methods that can lead to production of 100% male-sterile that can lead to production of 100% male-sterile seedseed

Constraint of the use of genic male sterility

CHEMICAL INDUCED MALE-CHEMICAL INDUCED MALE-STERILE STERILE

TaryonoTaryono

Faculty of AgricultureFaculty of Agriculture

Gadjah Mada UniversityGadjah Mada University

Biochemical means of Biochemical means of producing male sterile plantsproducing male sterile plants

Feminizing hormonesFeminizing hormones Inhibitors of anther or pollen Inhibitors of anther or pollen

developmentdevelopment

a. acting on sporophytic tissuea. acting on sporophytic tissue

b. acting on gametophytic tissue b. acting on gametophytic tissue

(gametocides)(gametocides)Inhibitors of pollen fertilityInhibitors of pollen fertility

Chemical hybridizing agent (CHA)Chemical hybridizing agent (CHA)

Could be used in the large scale Could be used in the large scale commercial production of hybrid seedcommercial production of hybrid seed

Are applied to plant only at certain Are applied to plant only at certain critical stage of male gametophyte critical stage of male gametophyte developmentdevelopment

Their action could result from a range of mechanism:Their action could result from a range of mechanism:1.1.Inherently selective action as male gametocides or Inherently selective action as male gametocides or

inhibitors of anther developmentinhibitors of anther development2.2.Selective transport of generally toxic or growth-Selective transport of generally toxic or growth-

inhibitory substances to the anthers during these inhibitory substances to the anthers during these periodsperiods

3.3.Metabolic detoxification of generally toxic or growth-Metabolic detoxification of generally toxic or growth-inhibitory substances after they have suppressed male inhibitory substances after they have suppressed male fertilityfertility

The logic of chemical hybridizationThe logic of chemical hybridization

High degree of efficacy and developmental selectivityHigh degree of efficacy and developmental selectivity Persistence during the development of flower or spikesPersistence during the development of flower or spikes Low costLow cost Acceptable levels of toxicity to people and the environmentAcceptable levels of toxicity to people and the environment Low general phytotoxicityLow general phytotoxicity Agronomic performance of hybrid seed produced is not Agronomic performance of hybrid seed produced is not

inferior to equivalent crosses produced by genetic methodsinferior to equivalent crosses produced by genetic methods

CHAs and pollen developmentCHAs and pollen development

Chemical inhibitors of pollen development are not familiar topic Chemical inhibitors of pollen development are not familiar topic to the majority of academic scientists. The most likely to the majority of academic scientists. The most likely explanation for the unfamiliarity is that these substances have explanation for the unfamiliarity is that these substances have been identified and developed almost entirely within the industrybeen identified and developed almost entirely within the industry

Pollen comes into being through a sequential and determinate Pollen comes into being through a sequential and determinate program within the central cavity or locule of anther. These program within the central cavity or locule of anther. These programmes are biochemically controlled and may be affected by programmes are biochemically controlled and may be affected by one or more chemical agentsone or more chemical agents

There are at least 4 classes of chemical agents:There are at least 4 classes of chemical agents:a. Plant growth regulators and substances that disrupt floral a. Plant growth regulators and substances that disrupt floral developmentdevelopmentb. Metabolic inhibitorsb. Metabolic inhibitorsc. inhibitors of microspore developmentc. inhibitors of microspore developmentd. inhibitors of pollen fertilityd. inhibitors of pollen fertilityThese categories have considerable conceptual overlap and do These categories have considerable conceptual overlap and do not address the molecular action of the chemical male sterilantsnot address the molecular action of the chemical male sterilants

Plant growth regulators and substances that Plant growth regulators and substances that disrupt floral developmentdisrupt floral development

Plant hormones/hormones antagonistsPlant hormones/hormones antagonistsa. auxins and auxin antagonists (NAA, IBA, 2,4-D, a. auxins and auxin antagonists (NAA, IBA, 2,4-D, TIBA, MH)TIBA, MH)

b. Gibberellins and antagonist (GA3, GA4+7, CCC: 2-b. Gibberellins and antagonist (GA3, GA4+7, CCC: 2-chloroethyl-trimethyl ammonium chloride)chloroethyl-trimethyl ammonium chloride)

c. Abscisic acidc. Abscisic acid

Other substancesOther substancesa. LY195259a. LY195259

b. TD1123b. TD1123

Auxins and antagonistsAuxins and antagonists

It may differently affect some far-reaching It may differently affect some far-reaching process, such as blockade of nutrient process, such as blockade of nutrient transport to the development antherstransport to the development anthers

Male sterility induced was expressed in Male sterility induced was expressed in several ways several ways in situ pollen germination, in situ pollen germination,

in situ exudation of pollen cytoplasm,in situ exudation of pollen cytoplasm,modification of certain stamens into staminodesmodification of certain stamens into staminodesTapetum fails to enlarge (MH and IBA) or tapetal cells Tapetum fails to enlarge (MH and IBA) or tapetal cells enlarges atypically and was persistentenlarges atypically and was persistent

Gibberellins and antagonistsGibberellins and antagonists

GA affects on sexual determination and floral GA affects on sexual determination and floral developmentdevelopment

The response varies by speciesThe response varies by speciesGA interferes with the development of male floral GA interferes with the development of male floral

organs or promotes feminizationorgans or promotes feminizationGibberellin-synthesis inhibitors (CCC) at certain Gibberellin-synthesis inhibitors (CCC) at certain

concentration, selectively inhibits the concentration, selectively inhibits the development of stamen or otherwise suppresses development of stamen or otherwise suppresses pollen development . These effects are not pollen development . These effects are not sufficiently selectivesufficiently selective

Abscisic acidAbscisic acid

ABA caused effect on developing floral buds ABA caused effect on developing floral buds similar to CCCsimilar to CCC

ABA caused male sterility if applied to plant just ABA caused male sterility if applied to plant just prior to or during meiosis of pollen mother cells prior to or during meiosis of pollen mother cells (wheat). ABA may cause male sterility through (wheat). ABA may cause male sterility through more than one mechanismmore than one mechanism

LY195259LY195259

It is 5-(aminocarbonyl)-1-(3-methylphenyl)-1H-It is 5-(aminocarbonyl)-1-(3-methylphenyl)-1H-pyrazole-4-carboxylic-acidpyrazole-4-carboxylic-acid

It is an effective chemical hybridizing agentIt is an effective chemical hybridizing agent It is applied when the flower was quite short with It is applied when the flower was quite short with

high application rates, whereas lower dosages high application rates, whereas lower dosages resulted in progressively reduced inhibitionresulted in progressively reduced inhibition

Sterility at lower dosages was associated with Sterility at lower dosages was associated with smaller, abnormally twisted and intensively smaller, abnormally twisted and intensively pigmented loculespigmented locules

The hybrid seed appeared normal, and no other The hybrid seed appeared normal, and no other phytotoxic effects were visually evident from ratesphytotoxic effects were visually evident from rates

Uptake from soil was particularly effectiveUptake from soil was particularly effective

TD1123TD1123

It is potassium 3,4-dichloro-5-isothiocarboxylateIt is potassium 3,4-dichloro-5-isothiocarboxylateWhen applied underdeveloped anthers, they will When applied underdeveloped anthers, they will

fail to dehiscefail to dehisceA variety of morphological effects were observed A variety of morphological effects were observed

at higher treatment levelsat higher treatment levels

Metabolic InhibitorsMetabolic Inhibitors

There are halogenated aliphatic acids (alpha, beta-There are halogenated aliphatic acids (alpha, beta-dichloroisobutyrate and 2,2-dichloropropionate salts) dichloroisobutyrate and 2,2-dichloropropionate salts) and arsenicals (methanearsonate salts)and arsenicals (methanearsonate salts)

They affect mitochondrial protein by reducing the They affect mitochondrial protein by reducing the efficiency of normal metabolic processesefficiency of normal metabolic processes

Inhibitors of microspore Inhibitors of microspore developmentdevelopment

Copper chelatorsCopper chelatorsCopper deficiency causes the irregular or absent of pollen Copper deficiency causes the irregular or absent of pollen developmentdevelopmentCopper deficiency exerts the effects by inhibiting copper-Copper deficiency exerts the effects by inhibiting copper-requiring oxidases that function in auxin metabolismrequiring oxidases that function in auxin metabolism

EthyleneEthyleneIt is a natural regulator of the development and maturation of It is a natural regulator of the development and maturation of several floral organs. Filament and corolla growth (unfolding and several floral organs. Filament and corolla growth (unfolding and senescence) are inhibited by ethelene productionsenescence) are inhibited by ethelene production

FenridazonFenridazonIt is 1-(-4chlorophenyl-1,4-dihydro-6-methyl-4-It is 1-(-4chlorophenyl-1,4-dihydro-6-methyl-4-oxopyridazine-3-carboxylic-acid. oxopyridazine-3-carboxylic-acid. The treated microspores had wavy surfaces and The treated microspores had wavy surfaces and progress to plasmolysis and abortion with the onset of progress to plasmolysis and abortion with the onset of the microspore vacuolation stagethe microspore vacuolation stagePollen wall was 80% thinner in treated plants Pollen wall was 80% thinner in treated plants

Inhibitors of microspore Inhibitors of microspore developmentdevelopment

Phenylcinnoline carboxylates (SC-1058, SC-1271 and SC-Phenylcinnoline carboxylates (SC-1058, SC-1271 and SC-2053)2053)

All capable of producing complete male sterility with minimal All capable of producing complete male sterility with minimal phytotoxicity and loss of seed yield when applied just prior to meiosisphytotoxicity and loss of seed yield when applied just prior to meiosis

They cause a general retardation of anther developmentThey cause a general retardation of anther development

Pollen development was generally arrested in the late prevacuolate or Pollen development was generally arrested in the late prevacuolate or early vacuolate microspore stageearly vacuolate microspore stage

The microspore often becomes wavy or wrinkled and the cytoplasm The microspore often becomes wavy or wrinkled and the cytoplasm degenerates and the cells become collapsed.degenerates and the cells become collapsed.SC-1058: SC-1058:

1-(4’-trifluoromethylphenyl)-4-oxo-5-fluorocinnoline-3-carboxylic acid1-(4’-trifluoromethylphenyl)-4-oxo-5-fluorocinnoline-3-carboxylic acid

SC-1271:SC-1271:

1-(4’-chlorophenyl)-4-oxo-5-propoxycinnoline-3-carboxylic acid1-(4’-chlorophenyl)-4-oxo-5-propoxycinnoline-3-carboxylic acid

SC-2053:SC-2053:

1-(4’-chlorophenyl)-4-oxo-5(methoxyethoxy) cinnoline-3-carboxylic acid1-(4’-chlorophenyl)-4-oxo-5(methoxyethoxy) cinnoline-3-carboxylic acid

Inhibitors of microspore Inhibitors of microspore developmentdevelopment

Genesis ® (MON 21200)Genesis ® (MON 21200)It provides good CHA activity over a very diverse range It provides good CHA activity over a very diverse range of genotypes, geographic regions and growing of genotypes, geographic regions and growing conditioncondition

Seed production has provided a high and reliable level Seed production has provided a high and reliable level of outcrossingof outcrossing

Hybrids produced with the aid of genesis are equivalent Hybrids produced with the aid of genesis are equivalent to conventional hybrids based on CMS technologyto conventional hybrids based on CMS technology

Inhibitors of pollen fertilityInhibitors of pollen fertility

Azetidine-3-carboxylate (A3C, CHA™)Azetidine-3-carboxylate (A3C, CHA™)It effectively induces male sterility in small grains, It effectively induces male sterility in small grains, particularly wheatparticularly wheat

The major effect of mature pollen is a structural The major effect of mature pollen is a structural alteration of cell wall precursor vesiclesalteration of cell wall precursor vesicles

Only 10% of the pollen grains showed normal pollen Only 10% of the pollen grains showed normal pollen tube growth in the first hour after pollination and none tube growth in the first hour after pollination and none penetrated the secondary stigmatic branchpenetrated the secondary stigmatic branch

MALE-STERILITY THROUGH MALE-STERILITY THROUGH RECOMBINANT DNA RECOMBINANT DNA

TECHNOLOGY TECHNOLOGY

TaryonoTaryono

Faculty of AgricultureFaculty of Agriculture

Gadjah Mada UniversityGadjah Mada University

I. Dominant Male-Sterility I. Dominant Male-Sterility GenesGenes Targetting the expression of a gene encoding a cytotoxin by Targetting the expression of a gene encoding a cytotoxin by

placing it under the control of an ather specific promoter placing it under the control of an ather specific promoter (Promoter of TA29 gene)(Promoter of TA29 gene)Expression of gene encoding ribonuclease (chemical Expression of gene encoding ribonuclease (chemical synthesized synthesized RNAse-T1RNAse-T1 from from Aspergillus oryzaeAspergillus oryzae and natural and natural gene gene barnasebarnase from from Bacillus amyloliquefaciensBacillus amyloliquefaciens))RNAse RNAse production leads to precocious degeneration of production leads to precocious degeneration of tapetum cells, the arrest of microspore development and tapetum cells, the arrest of microspore development and male sterility. It is a dominant nuclear encoded or genetic male sterility. It is a dominant nuclear encoded or genetic male sterile (GMS), although the majority of endogenous male sterile (GMS), although the majority of endogenous GMS is recessiveGMS is recessiveSuccess in oilseed rape, maize and several vegetative Success in oilseed rape, maize and several vegetative speciesspecies

Used antisense or cosuppression of endogenous gene that Used antisense or cosuppression of endogenous gene that are essential for pollen formation or functionare essential for pollen formation or function

Reproducing a specific phenotype-premature callose wall Reproducing a specific phenotype-premature callose wall dissolution around the microsporogenous cellsdissolution around the microsporogenous cells

Reproducing mitocondrial dysfunction, a general phenotype Reproducing mitocondrial dysfunction, a general phenotype observed in many CMSobserved in many CMS

Fertility restorationFertility restoration Restorer gene (RF) must be devised that can Restorer gene (RF) must be devised that can

suppress the action of the male sterility gene suppress the action of the male sterility gene (Barstar)(Barstar)

1.1. a specific inhibitor of barnasea specific inhibitor of barnase2.2. Also derived from B. amyloliquefaciensAlso derived from B. amyloliquefaciens3.3. Served to protect the bacterium from its own RNAse activity Served to protect the bacterium from its own RNAse activity

by forming a diffusion-dependent, extreemely one to one by forming a diffusion-dependent, extreemely one to one complex which is devoid of residual RNase activitycomplex which is devoid of residual RNase activity

The use of similar promoter to ensure that it would The use of similar promoter to ensure that it would be activated in tapetal cells at the same time and to be activated in tapetal cells at the same time and to maximize the chance that barstar molecule would maximize the chance that barstar molecule would accumulate in amounts at least equal to barnaseaccumulate in amounts at least equal to barnase

Inhibiting the male sterility gene by antisense. But Inhibiting the male sterility gene by antisense. But in the cases where the male sterility gene is itself in the cases where the male sterility gene is itself antisense, designing a restorer counterpart is more antisense, designing a restorer counterpart is more problematicproblematic

Production of 100% male sterile Production of 100% male sterile populationpopulation

When using a dominant GMS gene, a means to When using a dominant GMS gene, a means to produce 100% male sterile population is produce 100% male sterile population is required in order to produce a practical required in order to produce a practical pollination control systempollination control system

Linkage to a selectable markerLinkage to a selectable markerUse of a dominant selectable marker gene (bar) that Use of a dominant selectable marker gene (bar) that confers tolerance to glufosinate herbicideconfers tolerance to glufosinate herbicideTreatment at an early stage with glufosinate during Treatment at an early stage with glufosinate during female parent increase and hybrid seed production female parent increase and hybrid seed production phases eliminates 50% sensitive plantsphases eliminates 50% sensitive plants

Pollen lethalityPollen lethalityadd a second locus to female parent lines consisting of an add a second locus to female parent lines consisting of an RF gene linked to a pollen lethality gene (expressing with RF gene linked to a pollen lethality gene (expressing with a pollen specific promoter)a pollen specific promoter)

Induced GMS

Promoter which induces

transcription in male reproductive specifically

Gene which disrupts normal function of

cell

Agrobacterium-mediated

transformation

regeneration

male-sterile plant

Induced GMS System

Sterlie (Ss, rfrf) X

F1 (Ss, Rfrf)

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

Sterile (Ss, rfrf) (50%)

Fertile (Ss, rfrf)(50%)

Fertile (ss, RfRf)

fertile

F1 (ss, Rfrf)fertile

(50%)

(50%)

How to propagate male-sterile plants?

How to restore fertility?

How to induce sterility?

Strategies to Propagate Male-Sterile Plant

Selection by herbicide application

Inducible sterility

Inducible fertility

Two-component system

Selection by Herbicide Application

TA29 Banase NOS-T

TA29 Barstar NOS-T Gene for a RNase from

B. amyloliqefaciens

Tapetum-specitic

promoter

35S PAT NOS-T

Gene for glufosinate

resistance from S. hygroscopicus

Gene for inhibitor of barnase from B. amyloliqefaciens

fertile

Selection by Herbicide Application

pTA29-barnase : S (sterility)p35S-PAT : H (herbicide resistance)pTA29-barstar : R (restorer)

SH/-

SH/-

-/- SH/-

SH/-

-/- SH/-

-/-

SH/-

-/-

-/- SH/-

-/- SH/-SH/-

-/- -/-

-/-SH/-SH/-

-/- -/-

-/- -/-

-/--/--/-

-/- -/-

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

glufosinate

X C (R/R)

Fertile F1 (SH/-, R/-)

Fertile F1 (-/-, R/-)

Inducible Sterility

Male sterility is induced only when inducible chemical is applied.

Glutamate Glutamine

NH4+

N-acetyl- L-phosphinothricin (non-toxic)

Glufosinate (toxic)N-acetyl-L-ornithine

deacetylase (coded by argE)

Male sterilityaccumulationin tapetal cell

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

Sterile parent X Fertile parent

fertile

selfing

Plants transformed by

TA29-argE

fertile

Fertile F1 plant

N-acetyl-L-phosphinothricin

Plants transformed by

TA29-argE

Inducible Fertility

Sterile parent X Restorer

selfing

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.

addition of restricted metabolite

Fertile parent

Sterile parent

Fertile parentFertile F1 plant

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.

Each grandparent has each part of barnase.

Two proteins which are parts of barnase

Two proteins can form stable barnase

Two-Component System

X

F1 (Bn3/-)

A (Bn5/Bn3)

A2 (Bn3/Bn3)fertile

A1 (B5/B5)

fertile

fertile

fertile

sterile

X A2 (Bn3/Bn3)fertile

A1 (B5/B5)fertile

B (- -)

A1 (Bn5/Bn5)

A1 (Bn5/Bn5)

X

F1 (Bn5/-)

fertile

A (Bn5/Bn3)sterile

selfing selfing

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

PHB synthase

fertileAcetoacetyl-CoA

reductase

Glucose

C

S-CoACH3

O

C

CH3

O

C

S-CoACH2

CH3

HO

CH

O

C

S-CoACH2

O

C

CH2

CH

CH3

O

O

C

CH3

CH

CH3

O C

O

O -

CoASH

NADPHNADP+

O

Acetyl-CoA

Acetoacetyl-CoA

(R)-3-Hydroxybutyryl-CoA

Polyhydroxybutyrate (PHB)

n

(phaA gene)

( phaB gene )

(phaC gene)

-ketothiolase

Chloroplast Transformation

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

fertileTransformation 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

Acetoacetyl-CoA

Acetyl-CoA

Malonyl-CoA Fatty acid

Acetyl-CoA carboxylase

Illumination for 8 ~ 10 days

Male fertility

-ketothiolase

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.