This document presents a seminar on cisgenesis and intragenesis as new tools in crop improvement. It begins with introductions to cisgenesis and intragenesis, noting they allow for the introduction of isolated genes from crossable species or the crop itself. It then discusses why cisgenesis/intragenesis are important alternatives to issues with transgenesis, traditional breeding, and translocation breeding. Methods for developing cisgenic/intragenic plants including vector design and transformation techniques are covered. Examples of crops modified with cisgenesis/intragenesis including late blight resistant potato and apple scab resistant apple are provided. A case study on stacking two late blight resistance genes in potato cisgenically is also summarized

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

3. 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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4. 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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5. 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
different genes and loci” (Rommens et al.,2007).
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6. Holme et al., (2013)
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8. Different gene pools for plant improvement
(Michelmore,2003)
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9. Cisgenesis/Intragenesis as Alternative approach
• Some plant spp. difficult to breed by classical method
e.g. woody plants- don’t 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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10. 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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11. What is the problem with transgenesis?
Transferred gene usually
derives from an alien species.
• 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.
Animals
Plant
kingdom
B.t
Bacteria
Viruses
Etc.
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12. 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)
Den Nijs et.al., 2004
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13. 11-09-2014
Gene flow fitness of
population
Animals
Plant
kingdom
Bacteria
Other
sources
GENE FLOW
FITNESS OF THE POPULATION
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T
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14. 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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15. How cisgenic plants can overcome problems
of transgenic plants?
No
change
in
fitness
Transgenesis
No risk-on
non target
org.,
ecosystem
No alter
in gene
pool
No
additional
traits in
recipient
spp.
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16. 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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17. 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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18. How cisgenesis can overcome problems of introgression
Introgression
breeding
Comparable with
traditional
introgression
resistance
breeding using
same gene pool.
breeding?
Enhance the
breeding
speed to
obtain
durable
multigenic
resistance
Single step
insertion &
domestication of
natural R genes
linkage drag
free.
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19. 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.
Riley & Chapman,1958;
Sears & Okamoto, 1958;
Sears, 1976;
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20. 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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22. 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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23. Prerequisite of Cisgenic/Intragenic approach
Sequence information of the plant.
The isolation and characterization of genes of
interest from crossable relatives.
Intragenic vectors
P-DNA
Chimeric right T-DNA border
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24. Web site address for data base info..
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26. 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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27. 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 T-DNA
border sequences.
(Rommens et al.,2004)
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28. Chimeric right T-DNA border
• Only the first 3–4 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 T-DNA
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., 2004)
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29. Whole plant derived vectors
• Plant derived bacterial ori:
The smallest known prokaryotic origins of replication are the
32–33 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 lac-operator
sequence which is part of the operator repression
titration system of E. coli.
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30. Fig: Vector pSIM1256 for plant transformation with a silencing construct
targeting the potato asparagine synthatase genes(StAs1 and StAs2).
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31. 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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32. Techniques used in generation of
Cisgenic/Intragenic plant
• Marker free transformation
Using super virulent strains of Agrobacterium:
(High amylopectin potato – de Vetten et al., 2003)
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33. • Co-transformation
(Seed expression of barley phytase gene – Holme et al., 2012)
Ebinuma et al.,(2001) Plant cell reports
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34. • Recombinase induced excision
(Apple scab resistance– Joshi et al., 2011)
• Excision by homologous recombination
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35. • Transposan based exicision
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36. Examples of marker free method used
in cisgenesis
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37. 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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38. Cisgenic plants are produced by the same transformation
techniques as transgenic plants.
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39. Construction of vector
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40. Clean vector system
T-DNA insert in transgenic line
RB
RS
RS
term
term
T-DNA insert after cisgenic line
RB
prom
Recombination
RB
LB
LB
CodA-NptII fusion
CodA-NptII fusion
LB
HcrVf2
R-LBD Recombinase
R-LBD Recombinase
HcrVf2
HcrVf2
RS
RS
RS
prom
prom
term
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41. Crops and traits currently modified by the
Cisgenesis/Intragenesis
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42. 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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43. Intragenesis
Type Gene Trait Author
Potato Silencing GBSS High amylopectin de Vetten et al., (2003)
Potato Silencing Ppo Preventing
black spot bruise
Rommens et al., (2004)
Potato Silencing StAs1 Limit acrylamide in
French Fries
Chawla et al., (2012)
Strawberry 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)
Holme et al., 2013
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44. Field trials with Intragenic/Cisgenic crops
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45. Case Study
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46. 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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47. 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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48. • 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 940C for
30s, 580C for60s, 720C for 90s and a final extension time of
5 min at 720C.
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49. AGROINFILTRATION
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50. 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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55. 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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57. 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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58. 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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59. Regulatory Issues
According to EFSA Panel on GMO’s-
(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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60. 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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61. Thank you
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