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MALE-STERILE. Taryono Faculty of Agriculture Gadjah Mada University. Manual emasculation Use of male sterility Use of self-incompatibility alleles Use of male gametocides Use of genetically engineered “pollen killer” genetic system. Several forms of pollination control.

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male sterile

MALE-STERILE

Taryono

Faculty of Agriculture

Gadjah Mada University

slide2

Manual emasculation

  • Use of male sterility
  • Use of self-incompatibility alleles
  • Use of male gametocides
  • Use of genetically engineered “pollen killer” genetic system

Several forms of pollination control

slide3

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

Male-sterile

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

slide4

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
Flower phenotypes in carrot

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

b) Brown anther CMS (Sa)

c) Petaloid CMS (Sp)

slide6

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

slide7

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

Cytoplasmic male-sterile

  • Some drawback:
  • 1. insufficient or unstable male sterile
  • 2. Difficulties in restoration system
  • 3. Difficulties with seed production
  • 4. Undesirable pleitropic effect
slide8

Origins:

1. Intergeneric crosses

2. Interspecific crosses

3. Intraspecific crosses

4. Mutagens (EMS, EtBr)

5. antibiotic (streptomycin and Mitomycin)

6. Spontaneus

Cytoplasmic male-sterile

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
slide9

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
slide10

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

CMS Utilization

simple hybrid with cms and restoration

x

N2

N1

N1

N1

C1

C2

C1

C1

C1

Simple hybrid with cms and restoration

CMS line (A-line)

CMS, rfrf

Maintainer line (B-line)

N, rfrf

x

Large amounts

of CMS line

Male line (C-line)

N and RfRf

Fertile F1 hybrid

CMS, Rfrf

slide14

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

CMS Utilization

slide15

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
slide16

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
slide17

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

slide18

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
slide19

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

slide20

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

slide21

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
slide22

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
slide23

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:
  • Identify marker genes that are closely linked to ms genes and affect some vegetative characters
  • Use of environmental and chemical methods that can lead to production of 100% male-sterile seed
chemical induced male sterile

CHEMICAL INDUCED MALE-STERILE

Taryono

Faculty of Agriculture

Gadjah Mada University

biochemical means of producing male sterile plants
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
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:
  • Inherently selective action as male gametocides or inhibitors of anther development
  • Selective transport of generally toxic or growth-inhibitory substances to the anthers during these periods
  • Metabolic detoxification of generally toxic or growth-inhibitory substances after they have suppressed male fertility
the logic of chemical hybridization
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
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 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
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
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
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
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
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
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
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 development37
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 development38
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
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

MALE-STERILITY THROUGH RECOMBINANT DNA TECHNOLOGY

Taryono

Faculty of Agriculture

Gadjah Mada University

i dominant male sterility genes
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
Fertility restoration
  • Restorer gene (RF) must be devised that can suppress the action of the male sterility gene (Barstar)
  • a specific inhibitor of barnase
  • Also derived from B. amyloliquefaciens
  • 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
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)

slide44

Induced GMS

regeneration

Agrobacterium-mediated transformation

male-sterile plant

Promoter which induces transcription in male reproductive specifically

Gene which disrupts normal function of cell

slide45

How to induce sterility?

How to restore fertility?

How to propagate

male-sterile plants?

Induced GMS System

X

Sterlie (Ss, rfrf)

Fertile (ss, RfRf)

(50%)

F1 (Ss, Rfrf)

fertile

(50%)

F1 (ss, Rfrf)

fertile

Sterile (Ss, rfrf)

X

Fertile (ss, rfrf)

(50%)

Sterile (Ss, rfrf)

(50%)

Fertile (Ss, rfrf)

slide46

Strategies to Propagate Male-Sterile Plant

  • Selection by herbicide application
  • Inducible sterility
  • Inducible fertility
  • Two-component system
slide47

Selection by Herbicide Application

Tapetum-specitic promoter

Gene for a RNase from B. amyloliqefaciens

TA29

Barstar

NOS-T

Gene for inhibitor of barnase from B. amyloliqefaciens

TA29

Banase

NOS-T

35S

PAT

NOS-T

Gene for glufosinate resistance from S. hygroscopicus

fertile

slide48

Selection by Herbicide Application

X

A (SH/-)

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/-)

slide49

Inducible Sterility

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

NH4+

accumulation

in tapetal cell

Male sterility

Glutamate

Glutamine

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

Glufosinate (toxic)

N-acetyl-L-ornithine 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.
slide50

Plants transformed by TA29-argE

Plants transformed by TA29-argE

Inducible Sterility

fertile

N-acetyl-L-phosphinothricin

X

Fertile parent

Sterile parent

selfing

Fertile F1 plant

fertile

slide51

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 F1 plant

Fertile parent

selfing

Sterile parent

slide52

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.

slide53

Two-Component System

X

A1 (B5/B5)

A1 (Bn5/Bn5)

A2 (Bn3/Bn3)

fertile

fertile

X

A (Bn5/Bn3)

B (- -)

sterile

fertile

selfing

selfing

F1 (Bn5/-)

fertile

F1 (Bn3/-)

X

A1 (Bn5/Bn5)

A1 (B5/B5)

A2 (Bn3/Bn3)

fertile

fertile

fertile

A (Bn5/Bn3)

sterile

Bn3 : 3’ portion of barnase gene

Bn5 : 5’ portion of barnase gene

slide54

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.
slide55

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.
slide56

Acetoacetyl-CoA

reductase

fertile

C

CH3

S-CoA

CH3

CH3

CH

CH

Reactions for the synthesis of PHB

Glucose

O

Acetyl-CoA

b-ketothiolase

CoASH

(phaA gene)

O

O

Acetoacetyl-CoA

C

C

CH2

CH3

S-CoA

NADPH

NADP+

( phaB gene )

O

HO

(R)-3-Hydroxybutyryl-CoA

CH

C

CH2

S-CoA

CH3

(phaC gene)

PHB synthase

O

O

Polyhydroxybutyrate (PHB)

C

O -

C

CH3

C

O

O

CH2

n

O

slide57

Transformation by

Particle bombardment

fertile

Chloroplast Transformation

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

slide58

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.

slide59

Illumination for 8 ~ 10 days

Male fertility

Reversibility of Male Fertility

Acetoacetyl-CoA

b-ketothiolase

Acetyl-CoA

Acetyl-CoA carboxylase

Malonyl-CoA

Fatty acid

slide60

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.