What is Male Sterility?

Definition : Inability of flowering plants to produce

functional pollen.

Male sterility is Agronomically important for the

hybrid seed production.

Flower of male-fertile pepper Flower of male-sterile pepper

TRANSGENIC

MALE-STERILITY

TRANSGENIC MALE-

STERILITY

• Transgenes may be used to produce GMS

which is dominant to fertility.

• In these cases it is essential to develop

effective fertility restoration systems for hybrid

seed production.

• An effective restoration system is available in

at least one case, Barnase/Barstar system

Recombinant DNA techniques have made it

possible to engineer new systems of male sterility

by disturbing any or number of developmental

steps specifically required for the production of

functional pollen within the microspore or for the

development of any somatic tissues supporting

the microspores

I. Dominant Male-Sterility GenesTargetting the expression of a gene encoding a

cytotoxin by placing it under the control of an atherspecific promoter (Promoter of TA29 gene)

Expression of gene encoding ribonuclease (chemical synthesized RNAse-T1 from Aspergillus oryzae and natural gene barnase from Bacillusamyloliquefaciens)

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 co-suppression of endogenous gene that are essential for pollen formation or function

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

Reproducing mitochondrial disfunction, a general phenotype observed in many CMS

Barstar Barnase system• Barnase (110 amino acids) is a secreted

ribonuclease from Bacillus amyloliquefaciens. Barstar (89 amino acids) is a cytoplasmic barnaseinhibitor with which the host protects itself. RNase is linked with bar gene (glufosinatetolerant), so glufosinate tolerant plant will be male sterile.

• GM canola containing barstar/ barnase system composes about 10% of commercially cultivated crops in Canada and is one of the few GMO cleared for agricultural use in Europe.

Dominant nuclear male sterility (Barnase/Barstarsystem (Goldberg et al.1993) )

• Barnase is extracellular Rnase

• Barstar is inhibitor of Barnase

• Fuse the Barnase and Barstar genes to TA29

promoter (TA29 is a plant gene that has

tapetum specific expression

• Plants containing the TA29-Barnase construct

are male sterile

• Those withTA29-Barstar are not affected by

the transgene barnase.

• Barstar is dominant over the Barnase

Induced GMS

Promoter which induces transcription in male reproductive

specifically

Gene which disrupts normal function of cell

Agrobacterium-mediated

transformation

regeneration

male-sterile plant

Cell Ablation Strategy Using Chimeric Barnase and Barstar Genes with Overlapping Cell Specificities.Blocks represent cross-sections through a hypothetical organ system that has four different cell types. The circular cells in the lower right quadrants are the targets of the ablation experiment.(A) Blue represents transcriptional activity of the promoter (lectin gene) fused with the anti-cytotoxic barstar gene.(B) Red represents transcriptional activity of the promoter (TA56 gene) fused with the cytotoxic barnase gene.(C) Combined transcriptional activities of the chimeric barnase and barstar genes. Both chimeric genes are active within the dark gray cells in the upper right quadrant. Only the chimeric barnase gene is active in the target cells present in the lower right quadrant.(D) Selective ablation of the target cells. Barnase/barstar complexes are formed within the dark gray cells in the upper right quadrant protecting them from the cytotoxic effects of barnase. The target cells in the lower right quadrant have been ablated selectively due to the cell-specific activity

A Nove1 Cell Ablation Strategy Blocks Tobacco Anther Dehiscence

Sterile and fertile anthers

Strategies to Propagate Male-Sterile Plant

Selection by herbicide application

Inducible sterility

Inducible fertility

Two-component system

Selection by Herbicide Application

TA29 Barnase 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/-)

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

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)

Features of commercial value

dominant genetic male sterility system

• Efficient fertility restorer system

• Easy maintenance of male sterile lines

• Easy elimination of a male fertile plants from

male sterile lines

• Lack of adverse affects on other traits

• Stable male sterile phenotype over different

environments

• Satisfactory performance of f1 hybrids

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)

fertileA1 (B5/B5)

fertile

fertile

fertile

sterile

X A2 (Bn3/Bn3)

fertileA1 (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

Harmone induciblemale sterility

based on BCP1

• Most attractive system of transgenic male sterility

based on antisense construct of B. campestris

gene BCP1 drawn by BCP1 promoter linked with

a harmone inducible enhances sequence.

• This system is ready for use in hybrid seed

production in Brassica oleracea

Sporophytic genes controlling male sterility. Such genes cause malesterile expression by disrupting functioning in Sporophytic tissues.

Expected site of transcription of Gametophytic genes is in haploidmicrospores following meiotic division. Ex: pollen specific geneLAT52 in tomato.

Anther specific gene Bcp1 shows different pattern of expression inboth diploid Tapetum and haploid microspores.

The Gametophytic and Sporophytic activity of this gene wasobserved.

Bcp1 m RNA in both diploid Tapetum and haploid microspores.

Bcp1 expression in Tapetal cells initiated shortly after microsporeformation and continued until Tapetum degeneration.

In unicellular microspores the activity was first detected in the lateVacuolate stage.

To confirm the role of Bcp1 in fertility control, LAT52 promoter from

pollen specific tomato gene was fused to 0.5 kb of a Bcp1-c DNA

insert in a reverse orientation and cloned into p B 1101.

The second antisense construct was devoloped by fusion of 0.77 kb of

a Bcp1 gene regulatory region with 0.5 kb of an antisense Bcp1

construct.

Resulting antisense constructs was subsequently introduced into A.

thaliana via 2 separate A. tumefaciens transformation events.

The anthers of transformants carrying LAT52 (pollen specific)

promoter fused to a reverse Bcp1 c DNA construct had 1: 1

segregation of viable or aborted pollen, indicating Gametophytic male

fertility control.

Complete male sterility and the absence of typical 1: 1 segregation of

fertile and sterile pollens in the anthers of transgenic plants carrying

Bcp1 promoter suggested that sterility was the result of down

regulating Bcp1 gene expression in the diploid tapetum.

Co segregation of male sterile phenotype and presence of antisense

insert.

In Bcp1 promoter antisense plants low level of male fertility

reduction appeared to correlate with dosage levels of antisense gene.

2 copies of antisense gene were needed to ensure complete male

sterility expression, while presence of a single copy resulted in leaky

male sterile phenotype.

o Male sterility system based on Bcp1 promoter antisense

technology and linked to a harmone inducible promoter is

now ready for use in hybrid seed production in B. oleraceae.

This trasgenic male sterile line is self-maintainable and

remains male fertile so long as it is not sprayed with the

concerned harmone. (after this spray, it shows complete

male sterility).

Attractive alternative to 3- line CMS or barnase- barstar

system , as male sterile lines are self maintainable and any

line would act as fertility restorer.

Pollen Self-Destructive Engineered Male Sterility

• Theoretically, it is feasible to genetically engineer plants having

altered endogenous auxins indole acetic acid (IAA) levels with pollen

exhibiting self-destructive mechanisms.

• To achieve this, Mc Cormick et al. (1989) and Wood (1990)

transformed plants with a chimeric gene consisting of pollen-specific

promoter (LAT59) and a gene(fins2) that converts indole acetamide

(IAM) into IAA.

• Although the detailed characterization of such a transgenic was not

reported, it was argued that, if this system works, plants carrying the

LAT59-fms2 gene when sprayed with IAM will selectively convert

IAM into IAA at very high concentrations to kill the pollen and render

the plants male sterile.

• Another possibility of inducing such male sterility

is the transformation of plants with chimeric gene

involving TA-29 promoter and coding region of β-

glucuronidase (GUS).

• Such transformants, when sprayed with protoxins

like sulfonyl urea or maleic hydrazide, cause

malesterility through their breakdown in the

tapetum by the β-glucuronidase enzyme.

• The transgenic plants not sprayed with protoxins

remain fertile, so a fertility restoration system like

TA29-barstar is not required.

• Shihshieh et al (2003) used tissue-specific

promoters expressing CKX1 and gai , genes

involved in oxidative cytokinin degradation and

gibberellins (GA) signal transduction, respectively,

to study the roles of cytokinin and GA in male

organ development.

Male Sterility through Hormone Engineering.

Drastic changes in endogenous levels of auxins have

been demonstrated to cause male sterility in tomato

(Sawhney 1997) and several other crops.

Induction of male sterility by manipulating

endogenous hormone levels was reported in

transformed tobacco plants having the “rol C” gene

of Agrobacterium rhizogenes under the control of 35

S CaMV promoter and flanked with a marker gene

(Spena et al. 1987).

Such male sterile can be maintained by linking

the gene for herbicide resistance to the male

sterilizing “rol c”.

Male fertile segregants can be chemically

rouged out by selective elimination in

maintainer population.

In another experiment, Spena et al. (1992)

used the tissue specific promoter “tap 1” and

found that the inserted gene “rol b” from A.

rhizogenes greatly impaired the flower

development of transgenic tobacco, due to an

increase in the levels of indole acetic acid and

decreased levels of gibberillin in anthers.

Male Sterility Using Pathogenesis-Related Protein Genes

• Pathogenesis-related (PR) protein β-1,3 glucanase

(callase) is known to dissolve specific cell wall

made of callase, a β-1,3-linked glucan between

cellulose cell wall and plasma membrane and tetrads

synthesized by microsporocyte.

• The β-1, 3 glucanase (callase)secreted by the

tapetum helps to release free microspores into

locular space by breaking down the callase wall.

• The genetic alteration of this mechanism in plants

caused male sterility.

• Callase appearance and distribution was

normal in male sterile transgenic plants up to

prophasI, whereupon callase was prematurely

degraded.

• Electron microscopy revealed a thick callase

wall surrounding each microspore of the tetrad

in fertile anthers, whereas it was clearly absent

in sterile microspores.

• The premature dissolution of callase indicated that the

modified glucanase is secreted from the tapetum and is

active within the anther locule.

• Transgenic tobacco plants expressing the β-1, 3 glucanase

under the control of CaMV35S promoter showed normal

fertility despite the expression of modified glucanase.

• This was attributed to either lower expression of the

modified gene in the tapetum or inappropriate timing of the

modified callase synthesis during the process of

microsporogenesis, stronger (Rf-WA-1) being located on

chromosome 10.

• It has also been possible to identify 6 RAPD

markers linked to another gene for fertility

restoration (Rf-WA-3), three of these mapped

on chromosome 1.

• These marker-aided selections are for fertility-

restoring ability.

Male Sterility through Modification of Biochemical Pathways

1. Flavonoids

Among the three major flower pigments,

(flavonoids, carotenoids, and betalains), the

flavonoids are the most common and most

important.

Apart from their role in color development,

flavonoids are important in plant reproduction and

defence-related mechanisms.

Vander Meer et al. (1992) induced male sterility by

antisense inhibition of flavonoid biosynthesis.

• A large number of genes involved in the

biochemical pathway of flavonoids synthesis have

been cloned and characterized (Forkmann 1991).

• However, it was not concluded whether the anther

box on its own directs the expression in the

tapetum cells.

• It was argued that modules that confer organ

specificity to the CaMV35S promoter may act in

concern with anther box.

• About 14% of transgenic plants showed

a clear reduction of anther and pollen

pigmentation, which confirmed the

involvement of anther box in tapetum-

specific expression.

• In Maize and Petunia the pollen lacking

flavonoids are unable to produce

functional pollen tubes

• Flavonoids are essential for normal pollen

development and function and flavonoids

deficiency prevents pollen maturation

• Chalcone synthase is a key enzyme of

Flavonoid biosynthesis

• In petunia, antisense construct of the gene

encoding CHS has been transferred and

transgenic plants carrying this construct have

been regenerated

• These plants shows negligible chalcone

synthase activity white flowers and non-

functional pollen

• However this pollengrains become

functional when certain flavonoids are

either applied to the stigmas or mixed

with the pollen

• Antisense approach has also been used to

restore fertility in the case of MS induced

by the role of C gene of A.rhizogenes

2. Jasmonic acid

Jasmonic acid (JA), synthesized from linolenic acid (LA)through octadecanoid pathway plays an important role in antherdehiscence and pollen maturation .

The triple fad (fad3,fad7,fad8) mutants lacking LA hadindehiscent anthers and hence showed functional male sterility.

Ishiguru et al. (2001) linked impaired dehiscence in DefectiveAnther Dehiscence I (DAD I) mutant (Sanders et al. 2000;Stintziand Browse 1996) to mutation in DAD I gene that encoded achloroplastic phospholipase AI to supply free LA at the initialstep in JA biosynthesis, they further suggested that JA alsoregulated flower opening, anther dehiscence as well as pollenmaturation.

Exogenous application of JA reversed the impaired dehiscence

The male sterile phenotype could be reversed by application of JA

(0.5 μm) as well as LA.

Genetic male sterility based on DAD I gene has many advantages;

most important being the ability to induce normal pollen production

and dehiscence.

This is critical for developing lines homozygous for the male

sterilizing allele.

Moreover, this system involves suppression of a single gene and floral

morphology is not adversely affected.

The stability of expression over a range of environmental conditions,

however, remains to be investigated.

3. Carbohydrates

Carbohydrates play a critical role in the anther and

pollen development by sustaining growth as well as

signal pathways.

Their transportation from photo synthetically active

source tissues to developing sinks is regulated by

extra cellular invertase.

This class of invertase is encoded by small gene

familiar with differential regulation and expression

patterns.

The extra cellular invertase Nin 88 of tobacco

shows specific temporal and spatial expression

patterns in developing anthers.

At early stages of flower development,

the invertase is present exclusively in the

tapetum cell layers of the anthers

followed by a distinct expression pattern

during pollen development.

The tissue specific antisense repression

of Nin endothecium, and vacuolation

decreased the inner space of the locule,

affected pollen grain, and resulted in the

irregular shape or collapsed phenotype.

Reversibility of the male sterile phenotype

was observed under continuous illumination,

resulting in viable Pollen and a copious

amount of seeds.

This study offers a new tool for transgene

containment for both nuclear and organelle

genomes and provides an expedient

mechanism for F1 hybrid seed production.

Why would mitochondrial dis-unction

specifically affect pollen development

• Mitochondrial gene functions are essential to

all cells-electron transport, ATP formation, and

translocation of mitochondrial Mzna

• Interuption of any of these functions would be

expected to be lethal

• Confers both male sterility and disease

resistance

Christine D. Chase• plant mitochondrial genomes are not currently

amenable to genetic transformation, but genetic

manipulation of the plastid genome allows

engineering of maternally inherited traits in some

species.

• A recent study has shown that the Acinetobacter β-

ketothiolase gene, expressed in the Nicotiana

tabacum plastid, conditions maternally inherited

male sterility, laying the groundwork for new

approaches to control pollen fertility in crop plants.

•

• We constructed two chimaeric ribonuclease

genes that are specifically expressed in anthers

of tobacco and rapeseed plants. The expression

of these genes affects the production of

functional and viable pollen yielding plants

that are male sterile. This dominant gene for

nuclear male sterility should facilitate the

production of hybrid seed in various crops.

Induction of male sterility in plants by a chimaeric ribonuclease gene

Nature 347, 737-741 (25 October 1990)

Chimaeric ribonuclease genes that are expressed in the anthers of transformed tobacco and

oilseed rape plants were constructed. Chimaeric ribonuclease gene expression within the

anther selectively destroys the tapetal cell layer that surrounds the pollen sac, prevents

pollen formation, and leads to male sterility. These nuclear male sterility genes should

facilitate the production of hybrid seed in various crop plants.

Isoltaion of a tapetum-specific gene, TA29, facilitated tapetum specific expression of

RNase gene

Tetrad stage: maturation of microspores depends on tapetal cells

SMC= spore mother cells

V= vascular bundle

Fi= filament

w= anther inner wall

t= tapetum

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

b-ketothiolase

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.

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.

CASE STUDY

In case of RAPESEED

Characterization of transgenic male

sterility in alfalfa

Alfalfa plants obtained by genetic transformation with a construct containing the barnase gene under control of a tobacco anther tapetum specific promoter were studied.

Vacuolization and degeneration of the tapetalcell cytoplasm at a premeiotic stage of development were observed in all five transfomed plants

Thank you….!