Download - Genetic engineering for male sterility
GENETIC ENGINEERING FOR MALE STERILITY
By:
Nidhi Singh
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)
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)
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 )
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)
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)
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)
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)
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)
Mariani et al ,1992
Mariani et al ,1992
Female lines
cross to homozyg
ous maintaine
rBarN link to
herbicide resistance
Male parent line C carries
BarS Inhibit
barnase activity,res
tore
fertilty
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
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
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
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
Tehseen et al ,2010
Tehseen et al ,2010
Tehseen et al ,2010
Tehseen et al ,2010
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
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
DISCUSSION
Dreams are made real
THANKU