Cisgenics as a next generation GMO crops. This concept is new and alternative to transgenic crops...can avoid fear of transgenics w.r.t health and environment problems.

1. Welcome

2. Cisgenesis strongly improves
Introgression and Induced Translocation
breeding of plants.
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3. Introduction
Why cisgenic approach?
Prerequisites for cisgenic approach
Method to develop cisgenic plant
Conclusion
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4. Concept of cisgenesis introduced by Dutch
researchers Schouten, Krens and Jacobsen (2006)
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5. How can we go for genetic improvement?
g PLANT VARIETIES
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Modern
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insights GMO
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&techniques REGULATIONS
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biotechnology
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6. Overview of existing Technologies
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7. Key terms
• Cisgenesis is the genetic modification of a recipient plant with a
natural gene (includes its introns and is flanked by its native
promoter and terminator in the normal sense orientation) from a
crossable sexually compatible plant
• A cisgene is a natural gene, coding for an (agricultural) trait,
from the crop plant itself or from a sexually compatible donor
plant that can be used in conventional breeding.
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8. Contd...
 The gene pool for cisgenesis is identical to the gene pool available
for classical breeding.
 Cisgenic plants can harbor one or more cisgenes, do not contain any
transgenes.
 No foreign DNA, such as selection marker genes and vector-
backbone sequences, should remain in the final cisgenic plant.
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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9. Contd…
• Transgenesis is the genetic
modification of a recipient plant
with one or more genes from any
non-plant organism, or from a
donor plant that is sexually
incompatible with the recipient
plant.
• This includes gene sequences of
any origin in the anti-sense
orientation, any artificial
combination of a coding sequence
and a regulatory sequence, such as
a promoter from another gene, or
a synthetic gene.
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10. Contd…
• Traditional or conventional breeding: uses all classical and
modern insights and techniques, including those related to
biotechnology, however, without genetic modification.
• It includes a number of techniques that are not regarded as
genetic modification or that are exempted from the GMO-
legislation.
• Particularly the European Directive 2001/18/EC on the deliberate
release of GMOs into the environment
(European Parliament, 2001)
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11. Contd…
• Translocations or interchanges:
structural changes in chromosomes,
where segments of non homologous
chromosomes have exchanged
positions.
 Why interchanges important for
plant breeders, geneticists &
evolutionists?
 Interchanges bring about changes in
linkage relationship, changes in
chromosome structure & behavior
by which variability is created.
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12. Traditional breeding
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13. Why cisgenic 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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14. To appreciate cisgenesis……
1st we need to understand the problems related to…
1. Transgenic approach
2. Traditional breeding and
3. Translocation breeding.
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15. What is the problem with transgenesis?
B.t
 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.
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16. Contd…
 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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17. T
a
r
g
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t
p
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t
GENE FLOW
17 FITNESS OF THE POPULATION

18. How cisgenic plants can overcome problems of
transgenic plants?
Transgenesis
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19. 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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20. 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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21. How cisgenesis can overcome problems of
introgression breeding?
Introgression
breeding
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22. 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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23. Major problem in induced translocation breeding.
 Radiation treatment causes random chromosome breaks.
 The majority of translocations resulting from radiation
treatments were formed between non-homoeologous 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
23 The et al., 1988

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25. Is Cisgenesis efficient method in crops? Yes…
 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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26. The prerequisites for cisgenesis….
Sequence information of the plant.
The isolation and characterization of genes of interest from
crossable relatives.
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27. Web site address for data base info..
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28. 28

29. Solutions via cisgenesis
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30. Development of a cisgenic apple plant.
Thalia Vanblaere et.al.,2011
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31. Present day Apple
(Malus x domestica) cultivars
are susceptible to Apple scab which is
caused by the
fungus Venturia inaequalis
early symptoms
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32. Apple growers spray on an average 15 times in one season to control
the disease.
Spraying fungicides in Apple orchard
Patocchi et al., 2004
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33. Any natural resistance? Yes……..
 Source natural resistance to diseases is known .
 Classical breeding has developed scab resistant cultivars, mostly
by introgression of Vf resistance from Malus floribunda 821
(Lespinasse,1989; MacHardy, 1996)
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34. Several apple scab resistance genes
Identified and were mapped on different linkage
groups of the Apple genome.
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35. Impact of Vf genes in conventionally bred Apple
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36.  Only Vf locus has been positionally cloned
( Vinatzer et al., 2001)
 Proved to consists of a gene cluster with four
paralogs :
1. HcrVf1
2. HcrVf2
3. HcrVf3 and
4. HcrVf4
( Xu and Korban 2002)
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37.  Initially, only the HcrVf2 gene was tested by overexpression using
the CaMV35S promoter & also own promoter and nos terminator,in
scab susceptible Apple plants.
 Only one resistance encoding gene, HcrVf2, has been isolated and
proven functional to date in cvs. Gala and Elstar.
Belfanti et al., 2004;
Szankowski et al., 2009;
Joshi,
37 2010.

38.  In Transgenic Apple, for successful transformation marker gene
nptII & other regulators added.
Joshi, (2010)
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39. But people don’t need Transgenic Apple……..
 A large proportion of the consumers in Europe view genetically
modified foods as a risk to both health and the environment
(Gaskell et al., 2000)
CISGENIC
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40. Cisgenic plants are produced by the same transformation
techniques as transgenic plants.
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41. Construction of vector
17434 bp
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42.  In order to develop marker-free plants the
chemically inducible recombinase system reported in
strawberry was applied to apple.
Schaart et al., (2004)
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43. Method to develop cisgenic 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)
43 Schaart et.al.,2004

44. Clean vector system
T-DNA insert in transgenic line
RB RS R-LBD Recombinase CodA-NptII fusion RS LB
prom HcrVf2 term
Recombination
RB RS
R-LBD Recombinase CodA-NptII fusion RS LB
prom HcrVf2 term
T-DNA insert after cisgenic line
RS LB
RB
prom term
HcrVf2
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45. 10 transgenic lines were
regenerated through
selection on Kanamycin medium
8 out of 10 lines have backbone integration (nptII)
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46. ‘Gala’ scab susceptible cultivar
HcrVf2 Florina,classical bred cv. Vf resistance
3 cisgenic lines are derived
CodA marker gene
pMF vector
nptII (backbone)
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47. Gene expression analysis by PCR of cDNA
Transgenic Transgenic
Cisgenic lines Cisgenic lines
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48. Copy number by southern Blot
1 copy of nptII in all transgenic lines
1 copy of HcrVf2 in C7.1.49 & C 11.1.53
2 copies of HcrVf2 in T12.1 (C12.1.49)
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50. A cisgenic GM strategy for durable
resistance based on
R and Avr genes
The new concept : HEALTHY POTATO
Haverkort, A. J. et al.,2007
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51. Late blight of potato deadly disease in potato.
Phytophthora resistance in potato is easily broken.
A better strategy is needded for Sustainable resistance
Many R-genes are available in crossable wild species, enabling
the development of a resistance strategy
With useful molecular knowledge Avr-genes from the pathogen.
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53. List of R genes and Avr genes
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54. Approach:
Cloning of R genes
Marker free transformation
Cisgene field tests
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56. Endogenous Fungal Resistance Genes
Plants have been genetically engineered with
either:
 Endogenous gene 1: V. vinifera thaumatin-like protein
gene (vvtl-1) for fungal disease resistance or
 Endogenous gene 2: 2S albumin gene (alb) from
grapevine
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57. Genes are expressed via bidirectional promoter system
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59. Cisgenic Approach for Improving the Bioavailability
of Phosphate in the Barley Grain.
Holme I B et al.,2012
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60. leading to water pollution…….
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61. PAPhy (purple acid phosphatase)
 Two types: HvPAPhy_a and HvPAPhy_b.
 HvPAPhy_a is preferentially synthesized during seed
development and stored as preformed phytase in the
mature grain,
 HvPAPhy_b is preferentially synthesized during
germination.
Therefore ,the HvPAPhy_a gene as a candidate for our
cisgenic approach.
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63. Phytase Activities in Cisgenic Barley Seeds
Revealed a
positive
correlation
between gene
dosage and gene
expression of the
phytase gene.
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64. Modifying Plant Growth using GA-associated cisgenes.
POPLAR TRE
ES
Han K et.al.,2010
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65.  The transfer of entire native genes that play roles in
biosynthesis or signaling of gibberellic acids (GAs),
including their 5’ and 3’ proximal regulatory regions,
impart changes in growth rate and stature in poplar
trees.
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66.  Five different cisgenes :
GA20ox7,GA2ox2, GAI1, RGL 1_1, and RGL 1_2 .
 GA20oxidase catalyzes the penultimate step in the biosynthetic
GA pathway, in turn promote cell division and elongation.
 The other genes tend to repress or attenuate active GA
actions.
a) GA degradation by GA2ox2.
b) DELLA domain proteins that attenuate GA signals.
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67.  Plants transformed with GA20ox7 cisgene had a higher rate and
frequency of regeneration of transgenic shoots during antibiotic
selection.
 The average stem volume of the GA20ox7 transformed plants
increased by 40% compared with the transgenic (empty vector)
controls.
 GA20ox7 gene expression was also statistically associated with
the growth enhancement.
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70. Thank you