This document discusses genetic engineering techniques for inducing male sterility in plants. It begins by defining male sterility and describing its classification, including genetic, cytoplasmic, and transgenic types. It then focuses on techniques for transgenic genetic male sterility, listing several genes that have been used to induce sterility when introduced into plants. The document proceeds to discuss approaches for engineering male sterility by targeting anther and pollen development. It provides details on the barnase/barstar system and describes its use to create a male sterile line. Finally, it summarizes work using RNA interference to silence the Bcp1 gene and induce male sterility in Arabidopsis thaliana.

1. GENETIC ENGINEERING FOR MALE
STERILITY
By:
Nidhi Singh

2. MALE STERILITY
 Failure of plants to produce functional
anthers, pollen, or male gametes.
 1763--Kölreuter observed anther abortion within
species and species hybrids.
 More prevalent than female sterility.
 Arises spontaneously via mutations in nuclear
and/or cytoplasmic genes.
(Kaul , 1988)

3. CLASSIFICATION OF MALE STERILITY
 Genetic male sterility
 Temperature -sensitive genetic male sterility
 Photoperiod-sensitive genetic male sterility
 Transgenic Genetic male sterility
 Cytoplasmic male sterility
 Cytoplasmic - genetic male sterility
 Chemically induced male sterility
(Mariani et al , 1990)

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. SELECTED TRANSGENES USED FOR
PRODUCTION OF MALE STERILITY
TRANSGENE SOURCE TRANSGENIC
PLANT
rolC A.rhizogenes Tobacco,potato,
DTA Diptheria pathogen Tobacco
barnase Bacillus
amyloliquefaciens
Tobacco, B.napus
Rnase T1 Aspergillus oryzae Tobacco,oilseed,rape
Chalcone synthase
gene antisense
construct
Petunia Petunia ,tobacco
Chalcone synthase
cDNA
Petunia petunia
rolB A.rhizogenes Tobacco
Bcp1 Brassica campestris B.oleracea
(B.D.Singh, 2005)

6. 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: Histodifferentiation 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)

7. APPROACHES FOR DEVELOPMENT OF MALE
STERILITY
 Dominant Nuclear Male Sterility (Barnase-Barstar System)
 Male Sterility through Hormone Engineering ; (Sawhney 1997
)
 Pollen Self-Destructive Engineered Male Sterility; McCormick
et al. (1989)
( Mohammad Mehdi et al,2009)

8.  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)

9. 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)

10. Mariani et al ,1992

11. 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

13.  use of RNA interference (RNAi) technology to silence a male
specific gene, Bcp1 in the model host Arabidopsis thaliana
 Bcp1 is active in both diploid tapetum and haploid
microspores.
 Three batches of explants (A. thaliana) were selected on
herbicide glufosinate ammonium and putative transgenes
were confirmed through PCR and Southern hybridization.
 The present study resulted in developing male sterile A.
thaliana (Eco. Columbia) line through genetic engineering

14. WORK DONE
Silencing of Bcp1 gene ,587bp in
size,responsible for fertility
• They targeted 0.77kb regulatory region of Bcp1
gene via antisense
Expression of both sense and antisense fragment
separated by an intron , yields more efficient
silencing than only antisense
•Targeting coding sequence of Bcp1 using
RNAi leads to male sterility
Bcp1 gene can be divided in two parts
• 163bp non conserved region
• 372bp conserved region

15. Bgp1,female fertility gene
• 87 % homology with conserved region
• To avoid silencing of female part only non
conserved region is targeted
Primers design w.r.t dsRNA binary vector
pFGC5941
• It has two mcs ,bar gene and 35S promoter
within left and right borders
• Two mcs flanked by intron of 1.364kb
•
163bp region of Bcp1 gene cloned in both sense
and antisense orientation in pFGC5941
• Cloning of gene in sense and antisense
orientation in same vector produce dsRNA
inverted repeat molecule which induce PTGS
in plant cells

16. Construct was transformed in A.tumefaciens strain
LBA4404 by electroporation
• Agrobacterium culture was confirmed with PCR
amplification
• The construct was transformed in Arabidopsis
using leaf disc method
3 batches of explants were selected on herbicide
glufosinate ammonium
• Putative transgenic plants confirmed by PCR
using bar gene specific primers and southern
hybridisation
• It gives amplification along with positive and
negative controls
Formation of sharp bands confirm the presence of
transgenes
• Transcribed mRNA of RNAi construct will result in
a dsRNA with a hairpin loop
• Resultant dsRNA triggered on the RNAi machinery

17. Tehseen et al ,2010

18. Tehseen et al ,2010

19. Tehseen et al ,2010

20. Tehseen et al ,2010

21. CONCLUSION
 dsRNA interfere the Bcp1 gene function in the transgenic
plant and thus male sterile plants obtained
 Transgenic plants phenotypically indistinguishable from non –
transgenic plants except for aborted or malfunctioning pollen
grains
 Transgenic plants used as female for crossing with wild type
non-transgenic plants to produce hybrid seeds

22.  By using same strategy it will be easy to produce
hybrid seeds on large scale for higher yield and
quality of the crop.
 Another study was made to observe conservation
of Bcp1 gene in high value crops like
cotton,squash,chillies and tomato

23. DISCUSSION

24. Dreams are made real
THANKU