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 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 .

2. TRANSGENIC MALE-STERILITY
Rajesh J. Panchal
submitted to
Proff. Mithlesh Kumar
C. P.College of Agriculture
S.D.Agricultural University,SKNagar

3. 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
• 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 .

4. 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 .

5. Barnase Barstar 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 barnase /barstar system composes
about 10% of commercially cultivated crops in Canada and
is one of the few GMO cleared for agricultural use in
Europe.

6. Dominant nuclear male sterility (Barnase/Barstar
system)
• 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

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

12. Sterile and fertile anthers

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

14. 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

15. 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/-)

17. 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

18. 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)

19. 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

20. 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

21. 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

22. 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.

23. 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.

24. CASE STUDY

25. Male-sterility induction in
transgenic tobacco plants with
an unedited atp9 mitochondrial
gene from wheat

26. MATERIALS AND METHODS
Plant Material - Nicotiana tabacum cv. Petit Havana
lineSR1 was maintained as an axenic shoot culture in
hormone-free MS medium . Fully expanded leaves of
1-month-old plants were used for protoplast isolation.
Protoplast Isolation and Plant Regeneration - Leaf
mesophyll protoplast transformation and selection of
resistant colonies were performed . Regenerants were
rooted and transplanted in soil until flowering.
Phenotype and Fertility Analyses - The plant
phenotype was evaluated as the size of the plants, the
number of nodes, and the color of leaves. The size and
number of nodes were scored from soil surface to the
emergence of the inflorescence. For generative organs,
the shape and color of flowers including corolla,
anthers, and pistil were analyzed.

27. Genetic Analysis - Genetic segregation of the hygromycin
phosphotransferase (hpt) gene was determined in offspring
(200-500 seedlings). Back-crossing was performed by emas-
culation just before flower opening when anthers were still
closed. Capsules or seeds were treated as described . Seeds
were sown on MS supplemented with 40 mg of hygromycin
per liter. Seedlings were scored for hygromycin resistance 1
month after sowing.
PCR Analysis - The oligonucleotide primers used for
amplification were (i) 5'-CACTACGTCAATCTATAAG-3',
spanning codon 3-9 of the coxlV presequence; and (ii)
5'-TATGCTCAACACATGAGCG-3' located at the CaMV
terminator gene VI (45 bp upstream of the polyadenylylation
signal).

28. One microgram of DNA was amplified in a final volume of
100 ,ul by using 0.5 units of Taq I polymerase , 0.8 mM each
dNTP, and 100 pmol of each amplification primer. The
denaturation step was at 95°C for 1 min, the annealing step
was at 52°C for 2 min, and the polymerization step was at
72°C for 1 min. Twenty-five cycles were performed. Samples
were electrophoresed through 1.5% agarose gel and
transferred to Hybond-N . Filters were prehybridized at 42°C
in 50% deionized formamide containing 5x SSC (1 x = 0.15 M
NaCl/0.015 M sodium citrate, pH 7), 8x Denhardt's solution
(lx = 0.02% polyvinyl pyrrolidone/0.02% bovine serum
albumin/0.02% Ficoll), and 0.5% SDS. The blots were
hybridized with a 32P-labeled EcoRI/HindIll atp9 300-bp
coding sequence.

29. RNA Extraction and Gel Blot Analysis
RNA was extracted from leaves. Five grams of tissue was
ground with a pestle and mortar in liquid nitrogen. Frozen
powder was extracted with 5 ml of phenol/chloroform/isoamyl
alcohol, 25:24:1 (vol/vol), and 5 ml of TNES + DTT (0.1 M
NaCl/10 mM Tris-HCl, pH 7.5/1 mM EDTA/0.1% SDS
containing 2 mM dithiothreitol). The aqueous phase was
extracted twice with an equal volume of chloroform/isoamyl
alcohol, 24:1 (vol/ vol), and RNA was precipitated with an
equal volume of 4 M lithium chloride at 0°C overnight.

30. The RNAs were dissolved in diethyl pyrocarbonate-treated
water. RNA concentration was measured as A260. The
poly(A)+ RNAs were purified by oligo(dT)-cellulose affinity
chromatography (25). Twenty micrograms of total RNA and
1ug of poly(A)+ RNA were electrophoresed in
formaldehyde/formamide 1.5% agarose gels as and transferred
to nylon membranes Hybond-N+ .Hybridizations with the atp9
probe was performed .

31. Isolation of Proteins and Protein Gel Blot Analysis
A Xba I/Kpn I fragment containing the yeast coxIV codons 21-54 was
isolated from plasmid . This fragment was ligated to the plasmid pGEX-
A in frame with the gluthathione S-transferase open reading frame. The
fusion protein was induced after transformation of Escherichia coli DH5
a cells and purified .The fusion protein was used as antigen to produce
anti-COXIV antibodies in rabbits. Leaves of greenhouse-grown plants
were used for cell fractionation as . One hundred micrograms of
cytosolic and mitochondrial proteins were fractionated by
urea/SDS/PAGE . Proteins were electroblotted onto Immobion-P
(Millipore) membrane . Immunoreaction was performed by using a 1:500
dilution of anti-COXIV antiserum according to Darley-Usmar et al..
Peroxidase anti-rabbit IgG-conjugated .

32. Transformation and Culture - Plasmid constructs used for
protoplast transformation Tobacco protoplasts from leaves of the SR1
line were transformed with plasmids pHi, pH2, and pH5 by direct gene
transfer .The protoplast-derived cells were placed under selective
conditions. The frequency of regeneration of pHi, pH2, and pH5
transformant lines was generally >50%. Best growing green plants were
transferred to soil.
Phenotype Analysis of Transgenic Plants - Flowering in
Hi, H2, and H5 lines was induced 7-14 weeks after transplanting. The
flowers of transgenic plants were similar in shape and color to those of
SR1 plants, with five reddish-pink petals and five anthers in each flower.
Malesterile plants had white anthers containing some pollen grains or
none at all whereas fertile plants had yellowishwhite anthers with normal
pollen grains .

33. Fertility Analysis of Transgenic Plants
Hi and H5 transformants produced fertile plants, whereas H2
transformants exhibit fertile, semifertile, or sterile traits,
defined on the base of pollen germination or by the fluorescein
diacetate reaction . In fertile transgenic plants, pollen viability
ranged between 31% and 75%, close to the values found in the
SR1 control; in semifertile plants, pollen viability was around
10% to 20%; in male-sterile plants, the viability was generally
<2%.
Molecular Analysis of Transformants
To ascertain the presence and the transcription of the atp9
transgene, Southern and Northern blotting experiments were
performed.

34. Morphology of pollen grains from the male-sterile H2
plant (A) and the fertile H5 plant (B) under identical

36. Thank you….!