transgenic male sterility

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TRANSGENIC MALE-STERILITY- ALTERANATIVE TOOL FOR
HYBRID SEED PRODUCTION IN PLANTS
Bharat taindu jain 2013A58M 25/10/2014
http//biology.mcgill.ca/faculty/brown/male_steril
ity
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Male sterility

• the failure of plants to produce
  functional anthers, pollen, or male gametes (
  Rick 1944)
• 1763--Kölreuter observed anther abortion in some
  plant species and some inter-specific hybrids
  (Darwin, 1876)
• the first male sterility system (CGMS) was
  developed in onion in 1943 (Jones, 1943)
• The CMS in carrot by Welch and Grimball (1947)
• Induced CMS in pearl millet by Ethidium bromide
  (Burton and hanna, 1976)
• More prevalent than female sterility
• Male sporophyte and gametophyte less protected
  from environment than ovule and embryo sac.
• Easy to detect male sterility, because a large
  number of pollen for study available.
• Easy to assay male sterility staining technique
  (caramine, lactophenol or iodine)

Mariani et al., 1990
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CAUSES OF MALE STERILITY

• Absence or malformation of male organs (stamens)
  in bisexual plants or no male flowers in
  dioecious plants
• Failure to develop normal anther
• Abnormal pollen maturation inability to
  germinate on compatible stigma
• Non dehiscent anthers but viable pollen
• Barriers other than incompatibility preventing
  pollen from reaching ovule

(Kaul ,1988)
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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

(Kaul , 1988)
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Constraints of genetic and cytoplasmic male
sterility

• Absence of marker genes in GMS does not permit
  the sorting of male sterile or fertile plants in
  the progeny.
• Undesirable effect of the cytoplasm
• Unsatisfactory fertility restoration
• CGMS only available to limited crop
• Unsatisfactory pollination
• Male sterility brake down due to environmental
  effect
• No availability of suitable restorer line

(Banga and Banga,1998)
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Transgenic genetic male sterilty

• Transgene a gene introduced into genome of an
  organism by recombinant DNA or genetic
  engineering.
• The organism that develop after transformation
  are known as transgenic
• First transgenic plants (1983) Tobacco line
  resistance to kanamycin
• In 2013 , global area of GM crop 175.2 million
  hectare and in india is 11 million hactare

(www.isaaa.org)
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• Transgenic genic male sterility
• First transgenic genetic male sterile devised
  Mariani et al(1992) by barnase/barstar sysyem
  (Ananthi et al ., 2013)
• The SeedLink Invigor has successfully
  introduced a GE canola hybrid, using
  the barnase/barstar gene system in
  1996(Department of Agronomy, Iowa State
  University)
• Canola was originally naturally bred from
  rapeseed in Canada by Keith and Baldur in the
  early 1970s
• The term canola refers to those varieties of B.
  napus that meet specific standards on the levels
  of erucic acid (below 2)and glucosinolates( 30
  µmoles/g(CODEX 1999), and are often referred to
  as double low varieties.
• Indian canola varieties - Low erucic (lt 2)and
  low glucosinolate
• B.napus (gobhi sarson) (00)- Hyola-401
  (Hybrid) , GSC-5
• B. juncea(0) - Pusa Karishma , Pusa Mustard
  -22

)
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• Area of transgenic canola mustard is about 8.16
  million hactare in 2013 mainly in North America
  and Australia (www.isaaa.org)

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Approach for development of transgenic male
sterility

• Barnase/Barstar system for engineered male
  sterility
• (Mariani et al .,1992)
• 2. RNA interfernece for male sterility (
  Tehseen et al.,2010)
• 3. Maize 32138 SPT maintainer used in the
  PIONEER seed production technology (SPT) process
  (reference)
• 4. Male sterility through modification of
  biochemical pathways (Marc et al.,2000 )
• 5. Transgenic Reversible male sterility in
  plants by expression of cytokinin oxidase (
  Huang et al.,2011)

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1.Targeting the tapetum

• A specialized anther tissue the tapetum , play an
  important part in pollen develpoment
• The tapetum surrounds the pollen sac early in
  anther development, degenerates during the latter
  stages of development
• Tobacco TA29 gene expression in anther tapetum
  and that related gene exist in many other plant
  such as tomato , oilseed , rape, lettuce and
  alfaalfa (Seurinck et al.,1990 )
• TA29 tobacco tapetum specific gene can activate
  the ribonuclease genes Rnase T1 from Aspergillus
  oryzae (Mariani et al.,1990) and Barnase from
  Bacillus amyloliquefaciens within tapetal cell of
  tobacco and Brassica napus.
• Expression of chimaric Rnase genes slectively
  destroy the tapetum during anther delopment

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Mariani et al.,1990
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BARNASE/BARSTAR SYSTEM FOR ENGINEERED MALE
STERILITY

• Barnase is extracellular RNase barstar is
  inhibitor of barnase (both from bacterium
  Bacillus amyloliquefaciens which uses barnase for
  protection from microbial predators and barstar
  to protect itself from barnase
• Fuse the barnase and barstar genes to TA29
  promoterTA29 is a plant gene that has tapetum
  specific expression.
• Both gene link to bar gene (Murakami et al.,
  1986 ) which confers resistance to the herbicide
  PPT.
• Brassica napus cv. Darkar containing the
  TA29barnase construct are male sterile those
  with TA29barstar are not affected by the
  transgene ( Male fertile )
• Cross male sterile (barnase) with male fertile
  (barstar) to get hybrid seed, which now has both
  barnase and barstar expressed in tapetum and,
  hence, is fully fertile. barstar is dominant over
  barnase
• 
  
  (Mariani et al., 1992)
• 
  

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Martin et al.,1993
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Hybrid seed production in Brassica napus
http//www.yourarticlelibrary.com
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• Development of male sterile lines in B. juncea
  found that tissue-specific expression of the
  barnase gene was deregulated under the influence
  of a strong constitutive promoter (CaMV35S) used
  for expression of the marker gene bar affected
  several agronomically important traits (Jagannath
  et al ., 2001 )
• Spacer DNA fragment as an effective insulator to
  protect tissue-specific expression of the
  barnase gene which significantly enhanced
  recovery of agronomically viable male sterile
  lines in B. juncea (Jagannath et al ., 2001 )
  and three agronomically suitable male sterile
  lines (namely bn2.2, bn3.6 and bn3.4)
• B. juncea lines containing the wild type or a
  modified sequence of the barstar gene
• 3 male sterile barnase lines and 14 barstar lines
  (12 wild type barstar lines and 2 modified
  barstar lines), only one combination was found to
  restore complete male fertility in F1 progeny

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GM Events with Male sterility
Event Name Trade Name Trade Name Gene(s) Introduced Gene(s) Introduced
Argentine Canola - Brassica napus  Argentine Canola - Brassica napus  Argentine Canola - Brassica napus  Argentine Canola - Brassica napus  Argentine Canola - Brassica napus 
Name MS1 (B91-4) Name MS1 (B91-4) InVigor Canola InVigor Canola barnase
Name MS1 x RF1 (PGS1) Name MS1 x RF1 (PGS1) InVigor Canola InVigor Canola barnase
Name MS1 x RF2 (PGS2) Name MS1 x RF2 (PGS2) InVigor Canola InVigor Canola barnase
Name MS1 x RF3 Name MS1 x RF3 Invigor Canola Invigor Canola barnase
Name MS8 Name MS8 InVigor Canola InVigor Canola barnase
Name MS8 x RF3 Name MS8 x RF3 InVigor Canola InVigor Canola barnase
Name MS8 x RF3 x GT73 (RT73) Name MS8 x RF3 x GT73 (RT73) not available not available barnase
Name PHY14 Name PHY14 not available not available barnase
Name PHY23 Name PHY23 not available not available barnase
Name PHY35 Name PHY35 not available not available barnase
Name PHY36 Name PHY36 not available not available barnase
Chicory - Cichorium intybus  Chicory - Cichorium intybus  Chicory - Cichorium intybus  Chicory - Cichorium intybus  Chicory - Cichorium intybus 
Name RM3-3 Name RM3-3 Seed Link Seed Link barnase
Name RM3-4 Name RM3-4 Seed Link Seed Link barnase
Name RM3-6 Name RM3-6 Seed Link Seed Link barnase
Maize - Zea mays L. Maize - Zea mays L. Maize - Zea mays L. Maize - Zea mays L. Maize - Zea mays L.
Name 32138 Name 32138 32138 SPT 32138 SPT zm-aa1
Name 676 Name 676 not available not available dam
Name 678 Name 678 not available not available dam
Name 680 Name 680 not available not available dam
Name MS3 Name MS3 InVigor Maize InVigor Maize barnase
Name MS6 Name MS6 InVigor Maize InVigor Maize barnase
www.isaaa.org
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2. RNA interfernece for male sterility

• A phenomenon in which the introduction of double
  stranded RNA (dsRNA) into a diverse range of
  organisms and cell types causes transcriptional
  and post-transcriptional silencing of gene.
• RNAi was preceded first by observations of
  transcriptional inhibition by antisense RNA
  expressed in petunia flower (Ecker et al ., 1986)

http//www.genetiks.org/genomics419.html
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General mechanism of RNAi -
http//lc-molecular.wikispaces.com
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• BCP1 gene show anther specific expression on
  arbidopsis ( Xu et al , 1995 )
• Bcp1 gene active in both diploid tapetum and
  haploid microspore (Xu et al , 1995 )
• Downregulation of anther pecific gene Bcp1 of
  arbidopsis by RNA interfernce.

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Procedure -

• 163bp region of Bcp1 gene cloned in both sense
  and antisense orientation in pFGC5941 vector
  .
• the constructs were transformed into the A.
  tumefaciens strain LBA4404 by electroporation.
• The Agrobacterium culture was confirmed with PCR
  amplification.
• The construct was transformed in Arabidopsis
  using leaf disc method.
• Three batches of explants were selected on
  herbicide glufosinate ammonium.

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Hormone inducible male sterility based on Bcp1 in
brassicae

• Anther-specific gene, Bcp1, isolated from
  Brassica campestris  shows a unique pattern of
  expression in the diploid tapetum and haploid
  microspores and its expression in both cell types
  is essential for production of functional pollen
  (Xu et al 1995).
• This gene is conserved in members of the
  family Brassicaceae including Arabidopsis 73
  sequence identity at the amino acid level was
  found between Brassica and Arabidopsis c DNA
  clones (Theerakulpisut et al 1991).
• The transformation of Brassica plants with
  LAT52-Bcp1 antisense construct resulted in 100
  pollen sterility in transgenic plants (Bhalla et
  al )

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• Based on the antisense construct of B.compestris
  gene Bcp1 driven by LAT52 promoter linked with a
  hormone inducible enhancer sequence this system
  is used in hybrid seed production in B.oleracea(
  Banga and Banga , 1998 ) .

http//www.yourarticlelibrary.com
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3. Maize 32138 SPT maintainer used in the PIONEER
seed production technology (SPT) process

• The SPT system of Dupont/Pioneer is an
  alternative to the CMS system for producing
  hybrid seed corn without detasseling
• Pioneers SPT process is based on a transgenic
  DP-32138-1, referred to as 32138 SPT maintainer
• The transgenic 32138 SPT maintainer is currently
  grown under USDA APHIS permits on company
  controlled parent seed production fields .
• The 32138 SPT maintainer is used as a pollinator
  to propagate the seed of male sterile female
  inbred parent lines.
• Female inbred parent seed producing using does
  not contain the 32138 SPT insertion so hybrid
  seed produced are non transgenic for SPT

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• 32138 SPT maintainer -
• The 32138 SPT maintainer was generated by
  agrobacterium mediated transformation of a
  genetically male sterile (ms45/ms45) maize line
  with a plasmid designated PHP24597
• Plasmid contains three gene expression cassettes

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• The 32138 SPT maintainer is homozygous for the
  endogenous ms45 recessive mutant allele and
  hemizygous for the SPT insertion (i.e.,
  ms45/ms45 SPT/-) .
• Expression of a single copy of Ms45 gene in the
  ms45/ms45 genetic background restores
  male-fertility and enables pollen production in
  the 32138 SPT maintainer.
• The 32138 SPT maintainer sheds two different
  types of pollen in a 11 ratio
• a) fertile, does not contain SPT insertion
• b) infertile, does contain 32138 SPT insertion
  (due to the action of ZM-AA1
  a-amylase protein)
• 32138 SPT maintainer seed is replicated via
  self-pollination
• Upon self-pollination, 32138 SPT maintainer
  produces two different types of seed (yellow
  pinkish red) in a 11 ratio .

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Maintainer seed replication
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Male-Sterile Female Inbred Parent Seed Increase

• The A-line and B-line are planted in strips in 4
  2
• Half of the B-line pollen has the genotype F-Ms-A
  ms and is not viable because it has the A gene
  that destroys the starch.
• The other half of the pollen with the genotype
  f-ms-a ms is viable and fertilizes the A-line
  whose eggs have the genotype f-ms-a ms.
• As a result, all of the seed of the A line lacks
  the transgene and is homozygous for the ms/ms
  allele.
• When the A-line seed is planted as the female
  parent in a hybrid seed production field, all of
  the plants will be male sterile.

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Hybrid seed production

• The A-line is pollinated by an R-line. Both the
  A- and R-lines are non-transgenic.
• The A-line is homozygous and homogeneous for a
  nuclear ms gene that makes it male sterile and
  the R-line is homozygous and homogeneous for the
  wild-type MS allele.
• The F1 seed sold to farmers will be male fertile
  and lack the F-Ms-A transgene.

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Advantages of the Pioneer SPT Process

• The SPT system of Dupont/Pioneer is an
  alternative to the CMS system for producing
  hybrid seed corn without detasseling
• The need to remove pollen bearing tassels from
  female inbred parent lines by hand or mechanical
  detasseling is eliminated resulting in
• higher quality seed products for growers
• increased female inbred parent seed yields
• increased production efficiency and reduced
  costs
• increased worker safety
• The process works across all germplasm types so
  it can be used to produce any maize hybrid.
• Increasing male-sterile female inbred lines is a
  simple process achieved via cross-pollination
  with a transgenic 32138 SPT maintainer.
•  

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4. MALE STERILITY THROUGH MODIFICATION OF
BIOCHEMICAL PATHWAYS

• 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
• The extracellular invertase Nin 88 of tobacco
  shows expression pattern in developing anther
• The tissue specific antisense repression of nin88
  under the control of Nin88 promoter in plant
  caused male sterility .
• Exogenous supply of carbohydrates able to
  partially overcome blocking of pollen development
  so it maintaining the male sterility.
• Resotoration by crossing this GMS system with
  plants expressing distantly relate invertase
• 
  (Marc et
  al.,2000)

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5. Transgenic Reversible male sterility in plants
by expression of cytokinin oxidase ( Huang et
al.,2011) Patent no. 7951997 b2 Monsanto pvt .
Ltd.

• Reversible male sterility (RMS), in which the
  action of the cytotoxic gene used to introduce
  male sterility is suppressed by the application
  of a chemical to the plant.
• Chemically induce male sterility with the
  application of phytohormones ( Ebninck et al.,
  1978 )
• Transgenic plants are produced by introducing
  polynucleotide construct encoding Zea mays
  cytokinin oxidase 1 (CKX1) which decrese the
  level of cytokinin by increase in cytokinin
  oxidase degradation result in failure of normal
  pollen development (Huang et al.,)
• The pollen specific promoter pZmg13(Hansen et al
  ., 1989)
• Transfer of gene by agrobacterium mediated
  transformation
• Fertility can be restored in male sterile lines
  by the restoration of normal cytokinin levels
  achieved by application of kinetin , zeatin ,
  cytokinin oxidase inhibitor thidiazuran ( Hare
  et al., 2001)

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Hybrid seed production in maize

• CKX linked to a anther specific promoter pZmg13
  expressed in the anther CKX act as a dominant
  male sterility gene
• Homozygous male sterile female line can be
  maintained by use of chemical
• Male sterile line cross wild type plant to
  produce hemizygous male sterile female line
• The hemizygous line is crossed with male line to
  produce F1 hybrid seed.
• 50 of plants will be male sterile which is
  recover by 50 male fertile plant which produce
  an excess of pollen

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Conclusion

• Genetically engineered male sterility provides
  tremendous opportunities to the breeders for
  enforcing pollination control in hybrid seed
  production systems
• On the other hand these systems have some
  disadvantages like
• availability of efficient gene construct,
• possible dispersion of transgene to other related
  species,
• availability of efficient transformation
  technique and very high initial investment.
• Apart from barnase-barstar system, no other
  system has reached the commercial stage.
• Reversion of the sterility allows the RMS parent
  to be self-fertilized, a step which overcomes the
  need to remove fertile sib plants prior to making
  the hybrid cross
• With the further researches on
• Molecular biology of pollen development and
  improvement of biotechnology, the approaches
  creating male sterile lines using genetic
  engineering will become simpler, faster and more
  effective.

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THANK YOU
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Spacer DNA are regions of non-transcribed DNA
between tandemly repeated genes, such as
ribosomal RNA genes in eukaryotes. Its function
most likely involves ensuring the high rates of
transcription associated with these genes. In
bacteria, spacer DNA sequences are only a few
nucleotides long. In eukaryotes, they can be
extensive and include repetitive DNA, comprising
the majority of the DNA of the genome. The term
is used particularly for the spacer DNA between
the many tandemly repeated copies of the
ribosomal RNA genes. the International Service
for the Acquisition of Agri-biotech Applications
(ISAAA) spacer DNA are regions of
non-transcribed DNA between tandemly
repeated genes, such as ribosomal RNA genes
in eukaryotes. Its function most likely involves
ensuring the high rates of transcription
associated with these genes.