Transgenic male sterility usefulness in hybrid seed production

2. 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

3. TRANSGENIC
MALE-STERILITY

4. 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

7. 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

8. I. 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

9. 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

10. Barstar Barnase system
• Barnase (110 amino acids) is a secreted
ribonuclease from Bacillus amyloliquefaciens.
Barstar (89 amino acids) is a cytoplasmic barnase
inhibitor with which the host protects itself.
RNase is linked with bar gene (glufosinate
tolerant), 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.

11. Dominant nuclear male sterility (Barnase/Barstar
system (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

12. Induced GMS
Promoter which
induces transcription
in male reproductive
specifically
Gene which disrupts
normal function of cell
Agrobacterium-
mediated
transformation
regeneration
male-sterile
plant

17. 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

20. Sterile and fertile anthers

22. Strategies to Propagate Male-Sterile Plant
 Selection by herbicide application
 Inducible sterility
 Inducible fertility
 Two-component system

23. 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

24. 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/-)

29. 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

30. 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)

31. 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

32. 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 sterility
accumulation
in 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.

33. 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

34. 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

35. 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

36. Two-Component System
X
F1 (Bn3/-)
A (Bn5/Bn3)
A2 (Bn3/Bn3)
fertile
A1 (B5/B5)
fertile
fertile
fertile
sterile
X A2 (Bn3/Bn3)
fertile
A1 (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

37. 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

38.  Sporophytic genes controlling male sterility. Such genes cause male
sterile expression by disrupting functioning in Sporophytic tissues.
 Expected site of transcription of Gametophytic genes is in haploid
microspores following meiotic division. Ex: pollen specific gene
LAT52 in tomato.
 Anther specific gene Bcp1 shows different pattern of expression in
both diploid Tapetum and haploid microspores.
 The Gametophytic and Sporophytic activity of this gene was
observed.
Bcp1 m RNA in both diploid Tapetum and haploid microspores.
 Bcp1 expression in Tapetal cells initiated shortly after microspore
formation and continued until Tapetum degeneration.
 In unicellular microspores the activity was first detected in the late
Vacuolate stage.

39.  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.

40.  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.

41. 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.

43. 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.

44. • 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.

45. • 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.

46. 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).

47. 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.

48. 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.

49. 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.

50. • 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.

51. • 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.

52. • 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.

53. 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.

54. • 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.

55. • 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

56. • 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

57. • 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

58. 2. Jasmonic acid
Jasmonic acid (JA), synthesized from linolenic acid (LA)
through octadecanoid pathway plays an important role in anther
dehiscence and pollen maturation .
The triple fad (fad3,fad7,fad8) mutants lacking LA had
indehiscent anthers and hence showed functional male sterility.
 Ishiguru et al. (2001) linked impaired dehiscence in Defective
Anther Dehiscence I (DAD I) mutant (Sanders et al. 2000;Stintzi
and Browse 1996) to mutation in DAD I gene that encoded a
chloroplastic phospholipase AI to supply free LA at the initial
step in JA biosynthesis, they further suggested that JA also
regulated flower opening, anther dehiscence as well as pollen
maturation.
Exogenous application of JA reversed the impaired dehiscence

59.  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.

60. 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.

61. 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.

62. 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.

63. 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

64. 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.
•

65. • 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.

66. 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

67. 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.

68. Reversibility of Male Fertility
Acetoacetyl-CoA
Acetyl-CoA
Malonyl-CoA Fatty acid
Acetyl-CoA
carboxylase
Illumination
for 8 ~ 10 days
Male fertility
b-ketothiolase

69. 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.

70. 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.

71. CASE STUDY

72. In case of RAPESEED

73. 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 tapetal
cell cytoplasm at a premeiotic stage of
development were observed in all five
transfomed plants

74. Thank you….!