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Presented By,

Prudhviraj Vennela

PG-14045

Dept. of Genetics and Plant Breeding

IAS, Varanasi.

Flow of presentation

What is engineered male sterility

Genes and expression system

Transgenic genetic male

sterilty Transgene – a gene introduced into

genome of an organism by rDNA or

G.E.

Many transgene have been shown to

produce GMS.

These genes are dominant to fertility. (Mariani et al , 1990 )

ENGINEERING MALE

STERILITY a. Anther development

i. Tapetum–stomium/circular cell cluster–microspores are the

major targeting sites for manipulation

ii. Tapetum involved in microspore maturation.

iii. Stomium/ccc involved in dehiscence of pollen grains

b. Two phases of development

i. Phase 1 : Histo-differentiation of various anther cell types

ii. Phase 2 : Cell degeneration and dehiscence (programmed

destruction of CCC/connective and stomium leading to pollen

release).

(Goldberg et al. 1993)

Why Engineering of male

sterility? breakdown of male sterility

chlorosis

abnormalities in petals, poor nectary function.

lack of appropriate restorer lines.

Poor availability of agronomicallysuitable CMS/restorer system

Absence of marker genes in GMS does not permit the sorting of male sterile or fertile plants in the progeny.

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

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

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

Selected transgenes used for

production of male sterility

(B.D.Singh, 2008)

Approaches for Development of

Male Sterility

Dominant Nuclear Male Sterility (Barnase-Barstar

System). The FLP/FRT recombinase system of yeast is

used to regulate expression of the barnase and barstar

genes.

Male Sterility through Hormone Engineering ;

(Sawhney 1997 )

Pollen Self-Destructive Engineered Male Sterility;

McCormick et al. (1989)( Mohammad Mehdi et al,2009)

Transgenic induction of mitochondrial

rearrangements for Cytoplasmic male sterility in crop

plants; Ajay et al. (2007)

Engineering Cytoplasmic Male Sterility via the

Chloroplast Genome ; Ruiz and Daniel (2005) , reported

the first engineered cytoplasmic male sterility system

in plants( Mohammad Mehdi et al, 2009)

Barnase/barstar system for

engineered male sterility 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

Cross male sterile (barnase) with male fertile (barstar) to

get hybrid seed

(Mariani et al,1990)

Mariani et al ,1992

Mariani et al ,1992

Female lines cross to homozygous maintainer

BarN link to herbicide resistance

Male parent line C carries BarS

Inhibit barnase activity,restore

fertilty

Selection by Herbicide

Application

TA29 Banase NOS-T

TA29 Ba1rstar NOS-T Gene for a RNase from B. amyloliqefaciens

Tapetum-specitic

promoter

35S PAT NOS-T

Gene for glufosinateresistance from S.

hygroscopicus

Gene for inhibitor of barnase from

B. amyloliqefaciens

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/-)

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1. Male sterility through hormonal engineering

Drastic changes in endogenous levels of auxins

have been demonstrated to cause male sterility in

tomato

Induction of male sterility by manipulating

endogenous hormone levels was reported in

transformed tobacco plants having the “rol c”

Done by using Agrobacterium rhizogenes under

the control of 35S CaMV promoter and flanked with

a marker gene

Other systems of male sterility engineering

It is feasible to genetically engineer plants having

altered endogenous auxins indole acetic acid (IAA)

levels with pollen exhibiting self-destructive

mechanisms

Transformed plants with a chimeric gene consisting

of pollen-specific promoter (LAT59) and a gene

(fins2) that converts indole acetamide (IAM) into

IAA

plants carrying the LAT59-fins2 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

2.Pollen Self-Destructive Engineered Male

Sterility

3. Male Sterility Using Patho genesis-Related

Protein Genes

Specific cell wall made of callase, a â-1,3-linked glucan between cellulose cell wall and plasma membrane and tetrads synthesized by microsporocyte. The â-1, 3 glucanase(callase)

Secreted by the tapetum helps to release free microspores into locularspace by breaking down the callasewall.

The genetic alteration of this mechanism in plants caused male sterility.

Other approaches

Antisense rna or RNAi to silence relevant

gene expression of pollen development

Male sterility by early degrading callose

Male sterility through modification of

biochemical pathways (altering flavonoids,

jasmonic acid and carbohydrates)

Transgenic induction of mitochondrial

rearrangements for cytoplasmic male

sterility in crop plants

Fusing the specific promoter with a toxinic

gene of chemical-inducible expression by

simulating chemical hybridizing to

transform plants

Obtaining male sterile lines through double

transgenic lines hybridization

Transpose on mutation

Maintenance and restoration of

genetic engineering male sterility In transgenic plants, the sterility gene and the

herbicide-resistant gene are closely linked which

enable us to selectively kill the male fertile plants

with herbicides and maintain the sterile plants

The maintainer genes have been constructed

which exist as the allelic genes for lethal genes of

pollen

Transferring the genes into plants can produce

engineered maintainer lines

Approaches to restore

engineered male sterility The first approach is using a gene of

inhibitor protein.,

The restorer line for TA29-barnase male sterile line can be obtained by transferring barstar gene and barstar is th intracellular inhibitor protein of the barnase Rnase

The second approach is using antisense RNA to inhibit the expression of male sterility gene.

rol C gene, its restorer line can be obtained through transformation of other cultivars with the antisense gene. Then the fertility can be restored through hybridization.

Continue..

The third approach is using site-specific recombination system that generally contains a recombinase and its specific recognition sequence.

• Common site-specific recombination system includes Cre/loxp and FLP/FRT.

The fourth approach is using exogenous substances.

Inhibition of relevant gene expression of pollen development and cause the reduction of substances needed to regulate development and finally lead to male sterility.

Such kind of sterility can be remedied using exogenous substances.

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