cisgenesis and intragenesis
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Farmers fate , hungers and malnutrition are barriers in developing nations
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Breeder who facing failure to come up with potential variety
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Respect the nature Nature for our desire not for grid ………
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What is solution for all these?
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MAHESH R HAMPANNAVAR 6
Sisters in Innovative Plant BreedingCisgenesis and Intragenesis ;
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MAHESH R HAMPANNAVAR 7
Contents Introduction
Definition Pre-requisite for cis/intragenesis
Case study Comparison
Bio-safety measures Potential and disadvantage
Conclusion
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MAHESH R HAMPANNAVAR 8
• Environmentally sound and efficient
production method
• Exclusive use genetic material from same species or related species
why it require?
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MAHESH R HAMPANNAVAR 9
CISGENIC
Schouten et al. (2006)
“A cisgenic is a crop plant that has been genetically modified with one or more genes isolated from crop plant”
What is definition?
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MAHESH R HAMPANNAVAR 10
the intragenic concept as the isolation of specific genetic elements from a plant, recombination of these elements in vitro and insertion of the resulting expression cassettes into a plant belonging to the same sexual compatibility group
INTRAGENIC
Rommens et.al.(2004)5/22/2015
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MAHESH R HAMPANNAVAR 115/22/2015
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MAHESH R HAMPANNAVAR 12
(i). One or more identical
copy of the endogenous
gene including its promoter,
introns and the terminator.
(ii). No in vitro rearrangements
cisgenic can harbour-
What it contain ?
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MAHESH R HAMPANNAVAR 13
(i). Combination of genetic elements
from sexually compatible gene
pool.(ii). Coding regions of 1 gene
can combine with the promoters and terminators from different genes of the
sexually compatible gene pool.
(iii). T-DNA border sequences for Agrobacterium
mediated transformation
isolated from sexually compatible DNA pool known as P- Borders.
Intragenic can harbour-
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MAHESH R HAMPANNAVAR 14
. cisgenesis
intragenesis
Within species or related spice
Intragesis
P-DNA boarders
In vitro rearrangement
What is similarities and difference?
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MAHESH R HAMPANNAVAR 15
Variations in definitions of coding-, regulatory-, border- and vector-backbone-sequences used for intragenesis and cisgenesis
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MAHESH R HAMPANNAVAR 16
What is Sources of genes ?
l
Tertiary gene pool
Secondary gene pool
Primary gene pool
Breeder gene pool
Quaternary gene pool
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MAHESH R HAMPANNAVAR 17
What are all Pre requisites of cis/intragenic plant?
• Sequence information of plant
• The isolation and characterization of gene of interest from crossable relatives
• Transformation technique• Marker free transformation• Intragenic vectors
development5/22/2015
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MAHESH R HAMPANNAVAR 18
Web site address for data base info..
• Database integrating genotypic and phenotypic data useful to finding candidate genomic regions involved in agronomic traits of interest
Crop plant world wide web address
Grass http://www.gramene.org/qtl/index.html
Grape http://www.vitaceae.org
Tomato http://164.107.85.47:8004/cgi-bin/_information.plhttp://zamir.sgn.cornell.edu/Qtl/Html/home.htm
Potato http://www.scri.ac.uk/research/genetics/GeneticsAndBreeding/potatoes/mappingqtls
Cucurbitaceous www.icugi.org
Rosaceous http://www.bioinfo.wsu.edu/gdr/
Various http://www.phenome-networks.com/
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MAHESH R HAMPANNAVAR 19
General scheme for the selection, confirmation and introduction of alleles from the breeder’s gene pool
• QTL mapping • Fine mapping • LD mapping • Transient Assays(VIGS)• New alleles TILLING/eco• Translational genomics
• Phenomics • synteny QTL maps• BIBAC libraries • transformation large
fragment • Validation of standard
transfer
Confirm gene of interest
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MAHESH R HAMPANNAVAR 20
Table . Examples of Traits That Can Be Incorporated into a Plant by either Transferring or Modifying the Expression of Native Genes
(Rommens, 2004)
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MAHESH R HAMPANNAVAR 21
Table . Examples of currently available native traits
(Rommens, 2004)
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MAHESH R HAMPANNAVAR 22
Transformational technique
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MAHESH R HAMPANNAVAR 23
Methods to produce the marker free
cis/intragenesis plant
Co-transformation
Site-specific recombinase-
mediated marker
Transposaon-based expelling
systems
Intrachromosomal
recombination based excision
Transformation without marker
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MAHESH R HAMPANNAVAR 24
Co-transformation
Borys Chong.et.al5/22/2015
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MAHESH R HAMPANNAVAR 25
Site-specific recombinase-mediated marker
Targeted site Recombinase source lox Cre Bacteriophase P1
FTR FLP Saccharomyces cerevisiae
RS RS Zygosaccharomyces rouxii
Excised and lost
Borys Chong.et.al5/22/2015
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MAHESH R HAMPANNAVAR 26
Transposon-based expelling systems
Borys Chong.et.al5/22/2015
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MAHESH R HAMPANNAVAR 27
Methods used to produce marker-free intragenic/cisgenic plants
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MAHESH R HAMPANNAVAR 28
Development of Intragenic vector
• Intragenic vector system is an extension of the minimal T-DNA vector system.
Plasmid
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MAHESH R HAMPANNAVAR 29
Intragenic vector
a plant derived T-DNA like region that should contain
2 or at least 1 T-DNA border like sequences in the correct orientation.
an origin of replication(ori)
a selectable antibiotic gene
segment
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MAHESH R HAMPANNAVAR 30
The P-DNA Approach
• Rommens et al. in 2004.• A series of border specific degenerate primers, putative P-DNA’s
were isolated from pooled DNA’s of 66 genetically diverse potato accessions by PCR.
• The amplified fragment were sequenced. • And this information was used for inverse PCR with nested
primers to determine the sequence of the border like regions.
• This information allowed the identification of sequences with sufficient similarity to Agrobacterium T-DNA border sequences.
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MAHESH R HAMPANNAVAR 31
A T-DNA-like region assembled from Petunia hybrida (petunia) ESTs
Source-Euphytica (2007) 154:341–3535/22/2015
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MAHESH R HAMPANNAVAR 32
• may horizontal gene transfer from bacteria
• T-DNA border-like sequences – rice, tomato, potato, Arabidopsis
• Replace - T-DNA for transformation
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MAHESH R HAMPANNAVAR 33
Disadvantages P-DNA
• Found in some species only.
• P-DNA
• Probability of finding such features on a single relatively short fragment in a plant genome is extremely small.
• Reduced frequencies of gene transfer.(Rommens et al.2005)
Left boarder 1-2 kb apart / Restriction sites Right boarders
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MAHESH R HAMPANNAVAR 34
Origin of replication
• The smallest known prokaryotic origins of replication are
the 32-33bp
• Helper plasmid-provide specific factor for replication . • The Col E- characterized by 2 direct repeat sequences of 7-
9bp separated by 5-8bp. • BLAST searches of plant ESTs with sequences similar to
ColE2 or ColE3 identified in numerous species
Bacteria Plasmid Ori of replicationE.coli Colicin E2 ColE2Shigella sp. Colicin E3 ColE3
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MAHESH R HAMPANNAVAR 35
Selectable Marker
• Derived from plant sequences. • Mutant forms of the endogenous genes specific
herbicides resistance.• Over expression of the endogenous Atwbc19 ABC
transporter gene confers kanamycin resistance (Mentewab and Stewart 2005).
• For easy to transform crops such as potato, selectable marker genes are unnecessary (de Vetten et al.2003)
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MAHESH R HAMPANNAVAR 36
Considerations for Proper Design of Intragenic Vectors
• T-DNA not from regulatory(promoter) elements of plants
• The DNA fragment should not derived from heterochromatic
region.
• Significant length of 1-2kb of intragenic DNA occurs outside the
left border.
• Small number of DNA fragments
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MAHESH R HAMPANNAVAR 375/22/2015
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MAHESH R HAMPANNAVAR 38
Objectives•Improve the existing varieties with disease resistance•Stacking of multiple R genes – broad spectrum resistance
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MAHESH R HAMPANNAVAR 39
Materials and Methods
• Plant materials• Potato varieties –Atlantic , Bintje , potae9• Five Phytophthora infestans isolates and late
blight resistance test
IPO-C 90128EC1 Pic99189 DHD11
European American Korean 5/22/2015
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MAHESH R HAMPANNAVAR 40
Vector construction-• Resistance governing genesi. Rpi-vnt 1.1- S . venturiiii. Rpi-sto 1 - S. stoniliferum• pBINPLUS – binary vector• pBINAW2- modified form of pBINPLUS
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MAHESH R HAMPANNAVAR 415/22/2015
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MAHESH R HAMPANNAVAR 42
Potato transformation Explants – 4 week-old in vitro grown plants
Pre cultured on R3B medium supplemented with PACM- for two days
Explants were inoculated with agrobacterium strain AGL1+VirG+Binary plasmid –Two days
Explants transferred to GCVK medium for shoot regeneration
Shoot were transferred to CK medium for root regeneration
Three week later rooted plantlets were analyzed through PCR for desired R Gene5/22/2015
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MAHESH R HAMPANNAVAR 43
DNA extraction and polymerase chain reaction
• Total genomic DNA was isolated from young leaves
• PCR positive for both R genes , PCR negative for back bone integration
• Reaction performed using DreamTaqTM
• standard PCR program (94°C for 60 s followed by 30 cycles of 94°C for 30 s, 58°C for 60 s, 72°C for 90 s and a final extension time of 5 min at 72°C).
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MAHESH R HAMPANNAVAR 44
Gene ID Sequences (5'-3')Fragment size (bp) Detection of
Rpi-vnt1.1 forward ATGAATTATTGTGTTTACAAGACTTG 1100 T-DNA
reverse AGCATTGGCCCAATTATCATTAAC
Rpi-sto1 forward ACCAAGGCCACAAGATTCTC 890 T-DNA
reverse CCTGCGGTTCGGTTAATACA
tetA forward CTGCTAGGTAGCCCGATACG 396 Vector backbone
reverse CCGAGAACATTGGTTCCTGT
trfA forward CGTCAACAAGGACGTGAAGA 146 Vector backbone
reverse CCTGGCAAAGCTCGTAGAAC
NPTIII forwardGAAAGCTGCCTGTTCCAAAG
162 Vector backbone
reverseGAAAGAGCCTGATGCACTCC
ColE1 forward ATAAGTGCCCTGCGGTATTG 246 Vector backbone
reverse GCAGCCCTGGTTAAAAACAA
oriV forward TGCGGCGAGCGGTATCAG 1045 Vector backbone
reverse CTTCTTGATGGAGCGCATGGG
traJ forwardACGAAGAGCGATTGAGGAAA
260 Vector backbone
reverseCAAGCTCGTCCTGCTTCTCT
Primers used for PCR analysis of transformants
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MAHESH R HAMPANNAVAR 45
Results
Var/ strains IPO-C 90128 EC1 pic99189 DHD11
Atlantic S S S S S
Bintje S S S S S
potae9 S R R S S
Dethatched leaf assay conducted for testing varieties
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MAHESH R HAMPANNAVAR 46
• Selection and validation of cisgenic potato plant with two late blight R genes
200 stem
explants from each
variety
1515 shoots were
collected and
screened by PCR with
Rpi-vnt1.1 , Rpi-sto 1 primers
27 PCR positive
But 25 containin
g both Rpi-
vnt1.1 , Rpi-sto 1
gene
19 events are
vector back
bone free transformation
14 plants were
tested for agroinfiltr
ation
8 plants responded for to both Avrvnt 1 , Avrsto1
infiltration
Cisgenic plant
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MAHESH R HAMPANNAVAR 475/22/2015
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MAHESH R HAMPANNAVAR 48
Detached leaf assays for cisgenic transformant H43-7.
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MAHESH R HAMPANNAVAR 505/22/2015
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MAHESH R HAMPANNAVAR 53
WHY APPRECIATE THIS TECHNIQUE?
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MAHESH R HAMPANNAVAR 54
• Conclusion of case study
cisgenesis
C.M. ROMMENS 20075/22/2015
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MAHESH R HAMPANNAVAR 55
Comparable with traditional introgression resistance breeding using same gene pool.
Enhance the breeding speed to obtain durable multigenic resistance
How cis/intragenic can overcome problems of introgression breeding?
Cis/intrgenic
Conventional breeding
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MAHESH R HAMPANNAVAR 56
How cis/intragenic plants can overcome problems of transgenic plants?
No change in fitness
No need sequence information of other species
No alter in gene pool
No additional traits in recipient spp.
Cisgenesis/ intragenic
Transgenesis
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MAHESH R HAMPANNAVAR 575/22/2015
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MAHESH R HAMPANNAVAR 58
Potential of these concept
• Improving traits with limited natural allelic variation
• Higher expression level of a trait• Hybrid gene & silencing constructs.• US and Europe, acceptable to a greater
number of people than transgenic crops
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MAHESH R HAMPANNAVAR 59
Regulatory guidelines
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MAHESH R HAMPANNAVAR 60
•Comparison hazards with other technique ( conventional, transgenic)
• The Panel concluded that-(i). Similar hazards can be associated with cisgenic and
conventionally bred plants while(ii). Novel hazards can be associated with intragenic and
transgenic plants.
(iii). No new guidelines for risk assessment.
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MAHESH R HAMPANNAVAR 61
• All of these breeding methods can produce - unintended effects.
• Unintended effect assessed case by case
• The risk to human and animal health and the environment will depend on exposure factor(cultivation and consumption)
• For cis and intragenesis less event-specific data are needed for the risk assessment
EFSA journal 20125/22/2015
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MAHESH R HAMPANNAVAR 62
Global overview of consequences of different new plant breeding techniques for the environment and for food and
feed safety.
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MAHESH R HAMPANNAVAR 635/22/2015
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MAHESH R HAMPANNAVAR 64
Limitation of the two concepts
• Traits outside the sexually compatible gene pool cannot be introduced.
• Additional expertise and time • Not clearly define• Less transformation efficiency.
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MAHESH R HAMPANNAVAR 65
Objection and clarification
ObjectionI. Random insertion of geneII. Mutation caused in plant
ClarificationIII. Translocation breeding , Transposable
elementIV. 2500 mutant varieties growing all around
world in different crops
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MAHESH R HAMPANNAVAR 66
Current status of cis/intragenic crops
• In most countries, the release of cisgenic or intragenic crops currently falls under the same regulatory guidelines as transgenic crops
• The greatest expression of interest for less stringent regulations of these crops has been within the EU, the USA and New Zealand
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MAHESH R HAMPANNAVAR 67
Intragenic/cisgenic crops developed or currently under development
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MAHESH R HAMPANNAVAR 685/22/2015
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Field trials with intragenic/cisgenic crops
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Future trends
• generation and commercialization of intragenic and cisgenic crops will depend on willingness to apply less stringent regulation to these crops worldwide
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• Manipulate crop within gene of same species
• Overcome existing methods in some aspects
Strengths
• Low transformation efficiency
• Detail sequence study of crops
Weaknesses
• Transgenic opposition• consumer preference
Opportunities
• Use of biotechnical tools
• questionable biosafety measures
Threats
SWOTAnalysis of
Cis/intragnic
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CONCLUSION
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Discussion
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