Biotechnology applications in male

sterility and hybrid production

Anilkumar C

MSc (GPB)

PALM 3001

Several forms of pollination control

1. Manual emasculation

2. Use of male sterility

3. Use of self-incompatibility alleles

4. Use of male gametocides

5. Use of genetically engineered “pollen killer” genetic system

Male-sterile

Plant that do not produce viable, functional pollen grainsAn inability to produce or to release functional pollen as a result of

failure of formation or development of functional stamens, microspores or gametes

Three types of sterility:

1. “Pollen sterility” in which male sterile individuals differ from normal only in the absence or extreme scarcity of functional pollen grains (the most common and the only one that has played a major role in plant breeding)

2. “Structural or staminal male sterility” in which male flowers or stamen are malformed and non functional or completely absent

3. “Functional male sterility” in which perfectly good and viable pollen is trapped in indehiscent anther and thus prevented from functioning

Type of Male-sterile

Based on its inheritance or origin

Cytoplasmic male sterility (CMS) = sterile cytoplasm (S)

Male sterility 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)

Cytoplasmic male-sterile

Stamen (anther and filament) and pollen grains are affected It is divided into:

a. AutoplasmicCMS has arisen within a species as a result of spontaneous

mutational changes in the cytoplasm, most likely in the mitochondrial genome

b. AlloplasmicCMS 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 organellargenome anotherCMS 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 sterile2. Difficulties in restoration system3. Difficulties with seed production4. Undesirable pleitropic effect

CMS Utilization

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

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

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

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

Simple hybrid with cms and

restoration

Maintainer line (B-line)

N, rfrfN1

C1

Large amounts

of CMS line

xCMS line (A-line)

CMS, rfrfN1

C1

N1

C1C2

xN2

Male line (C-line)

N and RfRf

C1

Fertile F1 hybrid

CMS, Rfrf

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

Nuclear male sterility

Biochemical means of producing male

sterile plants

Feminizing hormones

Inhibitors of anther or pollen development

a. acting on sporophytic tissue

b. acting on gametophytic tissue (gametocides)

Inhibitors of pollen fertility

Chemical hybridizing agent (CHA)

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

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

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

MALE-STERILITY THROUGH

RECOMBINANT DNA TECHNOLOGY

I. Dominant Male-Sterility Genes

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 disfunction, a general phenotype observed in many CMS

Fertility restoration

Restorer gene (RF) must be devised that can suppress the

action of the male sterility gene (Barstar)

1. a specific inhibitor of barnase

2. Also derived from B. amyloliquefaciens

3. Served to protect the bacterium from its own RNAse activity by

forming a diffusion-dependent, extreemely one to one complex

which is devoid of residual RNase activity

The use of similar promoter to ensure that it would be

activated in tapetal cells at the same time and to

maximize the chance that barstar molecule would

accumulate in amounts at least equal to barnase

Inhibiting the male sterility gene by antisense. But in the

cases where the male sterility gene is itself antisense,

designing a restorer counterpart is more problematic

Production of 100% male sterile

population

When using a dominant GMS gene, a means to

produce 100% male sterile population is required in

order to produce a practical pollination control system

Linkage to a selectable marker

Use of a dominant selectable marker gene (bar) that confers

tolerance to glufosinate herbicide

Treatment at an early stage with glufosinate during female

parent increase and hybrid seed production phases eliminates

50% sensitive plants

Pollen lethality

add a second locus to female parent lines consisting of an RF

gene linked to a pollen lethality gene (expressing with a pollen

specific promoter)

Induced GMS

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 propagatemale-sterile plants?

How to restorefertility?

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)

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

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.

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

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.

Research articles on this topic

Transgenic Male Sterlity For Hybrid Seed Production

In Vegetables -A Review

M.Ananthi, P.Selvaraju And P.Srimathi

Weekly Science Research Journal

Vol-1, Issue-16, 7 November 2013

They illustrated several approaches for inducing male

sterility. like-

1 cell cytotoxicity

2 male sterility through hormonal engineering

3 pollen self-destructive engineered male

sterility

4 leading to male sterility by early degrading

callose

5 male sterility through modification of

biochemical pathways

6 engineering cytoplasmic male sterility via the

chloroplast genome

They also explained maintenance and restoration of genetic

engineering male sterility.

They gave more emphasis to barnase and barstar system of

male sterility induction and restoration.

Engineering genic male sterility using Barnase

Hybrid seed Production

Restoring male fertility using Barstar

Transgenic Studies on the Involvement of Cytokinin

and

Gibberellin in Male Development

Shihshieh Huang, R. Eric Cerny, Youlin Qi, Deepti Bhat, Carrie M. Aydt,

Doris D. Hanson,

Kathleen P. Malloy, and Linda A. Ness

Plant Physiol. Vol. 131, 2003

They used tissue-specific promoters expressing CKX1 and gai, genes

involved in oxidative cytokinin degradation and gibberellin (GA)

signal transduction, respectively, to study the roles of cytokinin

and GA in male organ development.

Accumulation of CKX1 in reproductive tissues of transgenic maize

(Zea mays) resulted in male-sterile plants.

The male development of these plants was restored by applications

of kinetin and thidiazuron.

Figure: R0 transgenic plants of pMON51827 (S16400 and

S16409) and pMON51826 (S16802, S16825, and S16832).

The sizes of the sterile tassels on the transgenic plants

were variable and noticeably smaller than wild-type

tassels. All of the transgenic sterile tassels in the figure

are enlarged approximately 2- to 3-fold.

CKX1-Transformed Maize Plants Display

Male-Sterile Phenotype

Questions…………..?