This document discusses engineering male sterility in plants through genetic modification. It describes several approaches for inducing male sterility, including using genes that encode cytotoxic proteins like barnase under anther-specific promoters. The barnase/barstar system uses barnase to induce sterility, which can be restored by co-expressing the barnase inhibitor barstar. Other approaches discussed include altering hormone levels, using genes that induce pollen self-destruction, and modifying biochemical pathways. The document also discusses methods for maintaining sterile lines and restoring fertility, such as linking sterility genes to herbicide resistance genes.

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2. Presented By,
Prudhviraj Vennela
PG-14045
Dept. of Genetics and Plant Breeding
IAS, Varanasi.

3. Flow of presentation
 What is engineered male sterility
 Genes and expression system

4. 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 )

5. 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)

6. Why Engineering of male
sterility?
 breakdown of male sterility
 chlorosis
 abnormalities in petals, poor nectary
function.
 lack of appropriate restorer lines.
 Poor availability of agronomically
suitable CMS/restorer system
 Absence of marker genes in GMS does
not permit the sorting of male sterile or
fertile plants in the progeny.

7. 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

8. 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

9. 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)

10. Selected transgenes used for
production of male sterility
(B.D.Singh, 2008)

11. 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)

12.  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)

13. 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)

14. Mariani et al ,1992

17. 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

18. Selection by Herbicide
Application
TA29 Banase NOS-T
TA29 Ba1rstar NOS-TGene 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

19. 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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20. 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

21.  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

22. 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 locular
space by breaking down the callase
wall.
 The genetic alteration of this
mechanism in plants caused male

23. 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

24.  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

25. 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

26. 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.

27. 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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