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)
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)
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/-)
19
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.
