cisgenesis and intragenesis

5/22/2015 1MAHESH R HAMPANNAVAR
MAHESH R HAMPANNAVAR
Genetics and Plant breeding
mahi5295@gmail.com

2
Farmers fate , hungers and
malnutrition are barriers
in developing nations
5/22/2015 MAHESH R HAMPANNAVAR

3
Breeder
who
facing
failure to
come up
with
potential
variety

4
Respect the nature
Nature for our desire not
for grid ………

Sisters in Innovative Plant Breeding
Cisgenesis and Intragenesis;

Contents
 Introduction
 Definition
 Pre-requisite for cis/intragenesis
 Case study
 Comparison
 Bio-safety measures
 Potential and disadvantage
 Conclusion

• Environmentally sound and efficient
production method
• Exclusive use genetic material from
same species or related species
why it require?

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?

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)105/22/2015 MAHESH R HAMPANNAVAR

(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 ?

(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-

.
cisgenesis intragenesis
Within species
or related spice
Intragesis
P-DNA
boarders
In vitro
rearrangement
Cisgenesis
No strictly use
P-DNA
Native form of
gene induce
What is similarities and difference?

Variations in definitions of coding-, regulatory-, border- and vector-backbone-sequences used for
intragenesis and cisgenesis

What is Sources of genes ?
l
Tertiary
gene pool
Secondary
gene pool
Primary
gene pool
Breeder gene pool
Quater
nary
gene
pool

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
development

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.pl
http://zamir.sgn.cornell.edu/Qtl/Html/home.htm
Potato http://www.scri.ac.uk/research/genetics/GeneticsAndBreeding/p
otatoes/mappingqtls
Cucurbitaceous www.icugi.org
Rosaceous http://www.bioinfo.wsu.edu/gdr/
Various http://www.phenome-networks.com/

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

Table . Examples of Traits That Can Be Incorporated into a Plant by either
Transferring or Modifying the Expression of Native Genes
(Rommens, 2004)

Table . Examples of currently available native traits
(Rommens, 2004)215/22/2015 MAHESH R HAMPANNAVAR

Transformational technique

Methods to
produce
the marker
free
cis/intrage
nesis plant
Co-
transformation
Site-specific
recombinase-
mediated
marker
Transposaon-
based expelling
systems
Intrachromoso
mal
recombination
based excision
Transformation
without marker

Co-transformation
Borys Chong.et.al

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.al

Transposon-based expelling systems
Borys Chong.et.al265/22/2015 MAHESH R HAMPANNAVAR

Methods used to produce marker-free
intragenic/cisgenic plants

Development of Intragenic vector
• Intragenic vector system is an extension of the
minimal T-DNA vector system.
Plasmid

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

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.

A T-DNA-like region assembled from Petunia hybrida (petunia)
ESTs
Source-Euphytica (2007) 154:341–353

• may horizontal gene transfer from bacteria
• T-DNA border-like sequences – rice, tomato,
potato, Arabidopsis
• Replace - T-DNA for transformation

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

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 replication
E.coli Colicin E2 ColE2
Shigella sp. Colicin E3 ColE3

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)

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

•Improve the existing varieties with disease resistance
•Stacking of multiple R genes – broad spectrum resistance

Materials and Methods
• Plant materials
• Potato varieties –Atlantic , Bintje , potae9
• Five Phytophthora infestans isolates and late
blight resistance test
IPO-C 90128 EC1 Pic99189 DHD11
European American Korean 395/22/2015 MAHESH R HAMPANNAVAR

Vector construction-
• Resistance governing genes
i. Rpi-vnt 1.1- S . venturii
ii. Rpi-sto 1 - S. stoniliferum
• pBINPLUS – binary vector
• pBINAW2- modified form of pBINPLUS

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 Gene 425/22/2015 MAHESH R HAMPANNAVAR

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).

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 forward
GAAAGCTGCCTGTTCCAAAG
162 Vector backbone
reverse
GAAAGAGCCTGATGCACTCC
ColE1 forward ATAAGTGCCCTGCGGTATTG 246 Vector backbone
reverse GCAGCCCTGGTTAAAAACAA
oriV forward TGCGGCGAGCGGTATCAG 1045 Vector backbone
reverse CTTCTTGATGGAGCGCATGGG
traJ forward
ACGAAGAGCGATTGAGGAAA
260 Vector backbone
reverse
CAAGCTCGTCCTGCTTCTCT
Primers used for PCR analysis of transformants

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

• 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
containing
both Rpi-
vnt1.1 , Rpi-sto
1 gene
19 events are
vector back
bone free
transformation
14 plants were
tested for
agroinfiltration
8 plants
responded for
to both Avrvnt
1 , Avrsto1
infiltration
Cisgenic
plant

Detached leaf assays for cisgenic transformant H43-7.

WHY APPRECIATE THIS TECHNIQUE?

• Conclusion of case study
cisgenesis
C.M. ROMMENS 2007

Comparable with
traditional
introgression
resistance
breeding using
same gene pool.
Enhance the
breeding speed to
obtain durable
multigenic
resistance
linkage drag
free.
How cis/intragenic can overcome problems of
introgression breeding?
Conventional
breeding

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.
Transgenesis

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

Regulatory guidelines

•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.

• 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 2012 615/22/2015 MAHESH R HAMPANNAVAR

Global overview of consequences of different new plant breeding techniques
for the environment and for food and feed safety.

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.

Objection and clarification
Objection
I. Random insertion of gene
II. Mutation caused in plant
Clarification
I. Translocation breeding , Transposable
element
II. 2500 mutant varieties growing all around
world in different crops

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

Intragenic/cisgenic crops developed or currently under development

Field trials with intragenic/cisgenic
crops

Future trends
• generation and commercialization of
intragenic and cisgenic crops will depend on
willingness to apply less stringent regulation
to these crops worldwide

• 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
SWOT
Analysis of
Cis/intragnic

Discussion

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cisgenesis and intragenesis

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