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CISGENESIS AND
INTRAGENESIS :NEW TOOLS
IN CROP IMPROVEMENT
Presented By:
SAURABH PANDEY
PALB-3252
Sr. M.Sc.(Ag.)

Seminar Flow
Introduction
Why Cisgenesis/Intragenesis
Pre-requisites of Cisgenesis/Intragenesis approach
Method to develop Cisgenic/Intragenic plant
Crops and traits currently modified by the
Cisgenesis/Intragenesis
Case study
Conclusion

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Introduction
A concept named cisgenesis was introduced by
Jochemsen and Schouten in 2000 in the book
Toetsen en begrenzen. Eenethische en
politieke beoordeling van de moderne
biotechnologie.
Cisgenesis is the introduction of isolated
genes with their native promoters from
crossable species or from the crop plant itself
(Schouten, H.J. et al., 2006).

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Intragenesis

By Rommens in 2004.
allows for the design of cassettes combining
specific genetic elements from plants belonging to
the same sexually compatible gene pool.
Intragenesis refers to GMOs where the
introduced intragene also originates from the
same species or a crossable species, but in
contrast to cisgenes, intragenes are hybrid
genes, which can have genetic elements from
dierent genes and loci (Rommens et al.,2007).
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Holme et al., (2013)

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Different gene pools for plant improvement

(Michelmore,2003)

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Cisgenesis/Intragenesis as Alternative approach

Some plant spp. difficult to breed by classical method
e.g. woody plants- dont flower for many years, intolerant
to inbreeding, highly heterozygous.

Some plant spp. are naturally sterile / are part of a highly

desired and commercially widespread clone whose genotype
needs to remain intact.
e.g. potato, apple, grape, and banana.
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To appreciate cisgenesis/intragenesis

1st we need to understand the

problems related to
1. Transgenic approach
2. Traditional breeding and
3. Translocation breeding.

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What is the problem with transgenesis?

Transferred
gene
usually
derives from an alien species.

B.t

Extends the gene pool of the

recipient species.
Such a novel gene might provide
the target plant with a new
trait that neither occurs in the
recipient species in nature nor
can be introduced through
traditional breeding.
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Bacter
ia

Anima
ls

Plant
kingdo
m

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Viruse
s
Etc.

In recipient species fitness may change in

various ways:
Through gene flow between a GM crop and
its wild relatives potentially creating shifts
in natural vegetation.

The generation of these new unnatural

gene combinations is regarded as both
unethical and having potential long-term
risks

for

health

and

environment.(non-

targeted organisms/soil ecosystems)

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Den Nijs et.al., 2004

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T
a
r
g
e
t
p
l
a
n
t

Gene flow
population.

fitness of
Animals

Bacteria

Other
sources

Plant
kingdom

Local
population

GENE FLOW
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FITNESS OF THE POPULATION

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Concerns
Insect resistance
Super weeds
Pollen drift
Harm to wildlife
Harm to soil
Harm to human health
Hidden allergens
Religious and moral
considerations
Antibiotic resistance

GE is unfair to farmers
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How cisgenic plants can overcome problems

of transgenic plants?
Cisgenesis Transgenesis

No
additional
traits in
recipient
spp.

No alter
in gene
pool

No
change
in
fitness
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No risk-on
non target
org.,
ecosystem

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What is the problem with introgression breeding?

High-quality genotype (cultivar) X wild plant
(gene of interest).
The wild plant, passes genes of interest to the
progeny, but also deleterious genes.
This linkage drag tremendously slow down the
breeding process, esp. if the gene of interest
is genetically tightly linked to one or more
deleterious genes.
Quality of crop is ruined.
To reduce linkage drag, need successive
generations of recurrent backcrossing with the
cultivated plant and simultaneous selection for
the trait.
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Contd..

As apple cultivars are self-incompatible and highly heterozygous,

the phenotype of a cultivar is unique and breeding produces
genotypes with new and distinct characteristics.
Limited Popularity of the new cultivars carrying disease resistance
genes since originality of cultivar lost.

(Gardiner et al., 2007)

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How cisgenesis can overcome problems of introgression

breeding?

Cisgenesis Introgression
breeding

Enhance the
breeding
speed to
obtain
durable
Comparable with multigenic
traditional
resistance
introgression
resistance
breeding using
same gene pool.
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Single step
insertion &
domestication of
natural R genes
linkage drag
free.

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Induced Translocation breeding

Chromosome pairing and recombination in common wheat is largely

governed by the gene Phi, located on the long arm of chromosome 5B,
which ensures that only homologous chromosomes can pair and
recombine.
Sears (1956) used ionizing radiation treatment to induce chromosome
breaks and transferred a gene conditioning resistance to:
leaf rust caused by Puccinia recondita f. sp. tritici from

Ae. umbellulata Zhuk. to wheat.

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Riley & Chapman,1958;

Sears & Okamoto, 1958;
Sears, 1976;
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Major problem in induced translocation breeding.

Radiation treatment causes random chromosome breaks.
The majority of translocations resulting from radiation
treatments were formed between non-homologous chromosome
arms.
These non-compensating translocations are genetically
unbalanced, and lead to reduced agronomic performance.
The radiation-induced Sr26 transfer, derived from A.

elongatum, the translocation causes a reduction of about 10% in

yield
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Importance of Cisgenesis

Particularly

efficient

method

for

cross-fertilizing

heterozygous plants that propagate vegetatively, such as

potato, apple and banana.
Cisgenesis might also supplement classical breeding for
improving traits with. limited natural allelic variation in
cultivars and wild species
E.g. expression of an endogenous phytase gene in barley
through the insertion of extra gene copies of the
endogenous phytase gene isolated from barley itself
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Prerequisite of Cisgenic/Intragenic approach

Sequence information of the plant.
Sequence information of the plant.
The isolation and characterization of genes of
The isolation and characterization of genes of
interest from crossable relatives.
interest from crossable relatives.
Intragenic vectors
Intragenic vectors
P-DNA
P-DNA
Chimeric right T-DNA border
Chimeric right T-DNA border
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Web site address for data base info..

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Intragenic vectors
Minimum requirements are:
plant-derived T-DNA-like region containing border
sequence and a multiple cloning site
Origin of replication
Selectable marker
(maintenance of vector in E. coli and Agrobacterium)
(Conner et al., 2007)

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P-DNA

Plant-derived T-DNA-like region,

referred to as P-DNA.
Identification of a 391 bp
fragment flanked by sequences in
potato which shows sufficient
similarity to Agrobacterium TDNA border sequences.

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Chimeric right T-DNA border

Only the first 34 nucleotides of the right border
need to be of plant origin, whereas the remainder of
the right border can be made identical to the
authentic Agrobacterium borders
Arabidopsis genomic DNA which contains a single TDNA like border sequence
additional 23 nucleotides of the authentic right
border of pTiT37 was fused and Ligated into the
backbone of a bacterial binary vector pART37
(Conner et al., Department
2004) of Plant Biotechnology

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Whole plant derived vectors

Plant derived bacterial ori:
The smallest known prokaryotic origins of replication are the
3233 bp ColE2 and ColE3 from the Colicin E2 and E3 plasmids
found in E.coli and Shigella sp.
Plant derived selectable elements:
The smallest bacterial selection marker is based on a short lacoperator sequence which is part of the operator repression
titration system of E. coli.

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Fig: Vector pSIM1256 for plant transformation with a silencing construct

targeting the potato asparagine synthatase genes( StAs1 and StAs2).

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Considerations for proper design of intragenic vectors

Not contain regulatory elements such as promoters
Not be derived from the heterochromatic regions
recommended that a significant length of 1-2kb of
intragenic DNA occurs outside the left border
should comprise of a minimal number of genetic elements:
mimic naturally occurring DNA rearrangements in plant
genomes.
selectable marker genes: Plant endogenous genes conferring
resistance to herbicides or antibiotics
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Tec hniq ues used i n gen erati on of Cisg enic /Intra genic plan t

Tec hni q ues us ed i n gene rati o n of Ci sg eni c/I ntrag eni c pl ant

Marker free transformation

Using super virulent strains of Agrobacterium:
(High amylopectin potato de Vetten et al., 2003)

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 Co-transformation
(Seed expression of barley phytase gene Holme et al., 2012)

Ebinuma et al.,(2001) Plant cell reports

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 Recombinase induced excision

(Apple scab resistance Joshi et al., 2011)

Excision by homologous recombination

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 Transposan based exicision

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Examples of marker free method used

in cisgenesis

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Method to develop Cisgenic/ Intragenic plants

Technique used: 2 independent regeneration step with 1
binary vector
Transformation with stable integration using positive selection e.g. on
kanamycin (nptII)

Removal of marker by chemical induction of Recombinase R activity

( Decamethosone treatment)

Selection for marker free plants using negative selection (codA) on 5-Fluro
cytosine (toxic 5-Fluro uracil)
Schaart et.al.,2004
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Cisgenic plants are produced by the same transformation

techniques as transgenic plants.

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Construction of vector

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Clean vector system

T-DNA insert in transgenic line

RS R-LBD Recombinase CodA-NptII fusion RS LB

RB
prom HcrVf2

term

Recombination
RS

RB
prom

HcrVf2

R-LBD Recombinase CodA-NptII fusion RS LB

term

T-DNA insert after cisgenic line

RB
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RS LB
prom

HcrVf2

term

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Crops and traits currently modified by the

Cisgenesis/Intragenesis

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Cisgenesis
Type

Gene

Trait

Author

Potato

Expression

R-genes

Late blight
resistance

Haverkort et al.,
(2009)

Apple

Expression

HcrVf2

Scab resistance

Vanblaere et al.,
(2011)

Grapevine

Expression

VVTL-1, NtpII

Fungal disease
resistance

Dhekney et al.,
(2011)

Poplar

Overexpression

Genes involved
in growth, PAT

Different growth
types

Han et al., (2011)

Barley

Overexpression

HvPAPhya

Improved grain
phytase activity

Holme et al.,
(2012)

Durum wheat

Expression

1Dy10

Improved baking
quality

Gadaleta et al.,
(2008)
Holme et al., 2013

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Intragenesis
Type

Gene
GBSS

Trait

Author

High amylopectin

de Vetten et al., (2003)

Potato

Silencing

Potato

Silencing

Ppo

Preventing
black spot bruise

Potato

Silencing

StAs1

Limit acrylamide in
French Fries

Chawla et al., (2012)

Strawberr
y

Overexpression

PGIP

Gray mould
resistance

Schaart,(2004)

Alfalfa

Silencing

Comt

Reduced levels of
lignin

Weeks et al., (2008)

Perennial
ryegrass

Overexpression

Lpvp1

Drought tolerance

Bajaj et al., (2008)

Apple

Expression

HcrVf2

Scab resistance

Joshi et al., (2011)

Rommens et al., (2004)

Holme et al., 2013

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Field trials with Intragenic/Cisgenic crops

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Case Study

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Introduction
Genetic disease resistance is an effective tool for
sustainable management of late blight, caused by
Phytophthora infestans, which is economically the most
important disease of potato.
Introduction of two broad spectrum potato late blight R
genes, Rpi-sto1 and Rpi-vnt1.1 from the crossable species
Solanum stoloniferum and Solanum venturii.
This is the first scientific report on the production and
functional evaluation of cisgenic R gene stacking in
different potato varieties.

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Materials and Methods

Potato Varieties: Atlantic, Bintje, Potae9
Phytophthora infestans and late blight resistance
tests:
Five P. infestans isolates were used-the European
isolates IPO-C and 90128; The American isolates,
EC1 and pic99189; and The Korean isolate DHD11.
Detached Leaf Assays were used
Introduction method: Transformation by marker-free
binary vectors(pBINAW2) and subsequent
regeneration in medium without selective
antibiotics
followed by PCR-based selection of transformation
events.
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 Agrobacterium tumefaciens strain used: AGL1+ virG

T-DNA size: 11 kb of Rpi-vnt 1:Rpi-sto 1
Potato transformation: Marker assisted
transformation(kanamycin mediated)
Marker-free transformation
( Removal of a selectable marker gene: by site specific
recombination)
Functional tests of resistance(R) genes: Agroinfiltration
DNA extraction and PCR: Genomic DNA isolation by following
Fulton et al., method.(1995)
PCR reaction: 940C for 60s followed by 30 cycles of 94 0C for 30s,
580C for60s, 720C for 90s and a final extension time of 5 min at
720C.
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AGROINFILTRATION
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Results and Discussion:

Selection of two cisgenic events in Atlantic, five in
Bintje and one in Potae9 containing and functionally
expressing a stack of two late blight R genes.
Average marker free transformation frequency: 1.3%
Marker-assisted transformation frequency: 10-71%
In terms of vector backbone integration, marker-free
transformation apparently produces a lower
percentage (24%) compared to marker-assisted
transformation(40-50%).
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Table:Phenotypic characterization of vector backbone free(cisgenic) events in different potato

varieties carrying the Rpi-vnt1 and Rpi-sto 1 genes

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Conclusion
Development of a marker-free transformation
pipeline to select potato plants functionally
expressing a stack of late blight R genes by
cisgenesis.
Marker-free transformation is less genotype
dependent and less prone to vector backbone
integration as compared to marker-assisted
transformation.
Thereby, this study provides an important tool for
the successful deployment of R genes in agriculture
and contributes to the production of potentially
durable late blight resistant potatoes.
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CHALLENGES AND LIMITATIONS OF CISGENESIS AND

INTRAGENESIS

Only those of the traits in sexually compatible gene

pool can be transferred to a crop.
The gene of interest or fragments of genes may not
be readily available but need to be isolated from the
sexually compatible gene pool
Development of marker and vector backbone free
plants.
Position effect
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Regulatory Issues
According to EFSA Panel on GMOs(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 of food and feed products
needs to be developed for plants derived from these 2 concepts.

EFSA Journal, 2012.

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Summary
Cisgenesis and intragenesis have created an opportunity to
discuss about a new group of genetically modified crops which
are consumer friendly.
The author of Invasion of Genes - Genetic Heritage of India
Dr B. S. Ahloowalia said:
Cisgenics removes all fears associated with transgenic
crops.

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P
o
t
e
n
t

Thank you

i
a
l

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Cisgenic
approach

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