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