This document provides an overview of cisgenesis, which involves genetically modifying a plant with a natural gene from a sexually compatible plant species. It defines cisgenesis and provides examples of cisgenic crops. It discusses the limitations of traditional breeding and transgenesis, and how cisgenesis addresses some of these limitations. The document outlines the process for developing cisgenic plants, including gene isolation, vector construction, transformation and selection methods. It summarizes a case study on developing cisgenic apple with scab resistance. Finally, it discusses opportunities and challenges for the future of cisgenesis.

1. DEPARTMENT OF GENETICS AND PLANT BREEDING
RAJASTHAN COLLEGE OF AGRICULTURE
MPUAT, UDAIPUR (313 001)
Presented By:
Rajwanti Saran
M.Sc. (Ag.)
GPB
Major Advisor
Sh. P.P. Sharma
Associate Professor
Department of Genetics
and Plant Breeding

2. • Introduction
• Cisgenesis
• Why cisgenic approach
• Prerequisites for cisgenic approach
• Methods to develop cisgenic plant
• Case study
• Limitations
• Future trends
• Conclusion

3. Concept of cisgenesis introduced by
Dutch researchers Schouten, Krens and
Jacobsen (2006).
According to this concept, genetic
material transferred to the plant should
originate from the plant itself or from
closely related species capable of sexual
hybridization.
The gene pool for cisgenesis is identical
to the gene pool available for classical
breeding.

4. Definition: 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.
(Schouten et al. 2006).
Examples:
Cisgenic apple which confer scab
resistance (Vanblaere et al., 2011)
 Cisgenic barley with improved phytase
activity (Holme et al., 2012)

5. Cont….
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.

6. 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.

7. Cont…
Escape of foreign genes via pollen flow to
natural vegetation can be a problem for
transgenesis. However, in case of
cisgenesis the genes are taken from wild
relatives.
“Cisgenesis is as safer as conventional
breeding” (EFSA journal 2012, (10) 2561.)
To overcome the problem of linkage drag
Genetic make-up of the original cultivar is
preserved. Only one or few genes added.

8. 1st we need to understand the problems
related to…
1. Transgenic approach
2. Traditional breeding

9.  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.
Plants
Bacteria
Animals
viruses

10. Cont…
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

11. CISGENESIS TRANSGENESIS

12.  Time taking
 ‘linkage drag’ tremendously slow down the breeding
process, esp. if the gene of interest is genetically tightly
linked to one or more deleterious genes.
 Plants contain undesired genetic elements, which is
modified by classical breeding. eg.- Breeding of apple for
scab resistance took 40 years.
 Change vigour, generating a change in the natural
vegetation.

13. CISGENESIS TRADITIONAL BREEDING

15. Sequence information of the plant.
The isolation and characterization of genes of
interest from crossable relatives.

16. Clean vector technology aims to produce
GM plants with only the gene-of-
interest as newly introduced gene
function without any regulatory gene
sequences.
Primarily, the goal is to avoid the use or
the continued presence of antibiotic
resistance genes as selectable markers.

17. A. Co-transformation :Based on
Agrobacterium- or biolistic mediated
transformation in which a SMG and gene
of interest are on separate construct .
B. Site-specific recombination :It takes
place only between defined excision
sites in the phage and in the bacterial
chromosome.
C. Transposon-based marker method.
D. chloroplast marker gene.

19. Apple
Potato
Grapes
• Disease resistance
Barley
• Bioavailability of
phosphate
Poplar tree
• Modifying growth

20. Apple Cultivar Gala is susceptible to Apple scab
which is caused by the fungus Venturia inaequalis .
→
Vanblaere et al.,2011, Journal of Biotechnology

21. Classical breeding has developed scab resistant
cultivars, mostly by introgression of Vf
resistance from Malus floribunda
821(Lespinasse,1989; MacHardy, 1996).

22. 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 ( Dexamethosone treatment)
↓
Selection for marker free plants using negative
selection (codA) on 5-Fluro cytosine (toxic 5-Fluro
uracil)
Schaart et.al.,2004

24. 10 transgenic lines were regenerated through selection
on Kanamycin medium.
Analysis of genomic DNA (presence/ absence of gene):
8 out of 10 lines have backbone integration (nptIII)
210bp
Fig: Backbone integration, PCR analysis using primers specific for
nptIII to detect backbone integration

25. 3 transgenic lines 2
lines without backbon
integration and 1 line
with backbone
integration as control .
3 derived cislines
Florina, classical bred
Vf cutivar Gala, scab
susceptible cultivar
856bp
HcrVf2
226
bp
Cod A
marke
r gene
210 bp
Npt III

26. 856bp
HcrVf
2
226 bp
Cod A
marker
gene

27. Characters outside the sexually
compatible gene pool cannot be
introduced.
Sequence information of the plant.
Low transformation efficiencies to
create large number of transformants.
The production of marker free plants
usually requires the development of
innovative protocols, since such
protocols may not be readily available
for the crop.

28. Future developments regarding the
generation and commercialization of
intragenic and cisgenic crops will depend
on application of less stringent
regulation to these crops worldwide.
Cisgenic crops are acceptable to more
number of people than transgenic crops

29. STRENGTH
Manipulate crop within
gene of same species.
Overcome existing
methods in some
aspects
WEAKNESSES
Low transformation
Efficiency.
• Detail sequence study
of crops
OPPORTUNITIES
Transgenic
opposition
•consumer
preference
THREATS
Use of biotechnical
Tools & questionable
biosafety measures
SWOT
ANALYSIS

30. Despite success, the Classical methods of alien
gene transfer have disadvantages and
difficulties, particularly linkage drag, that
require time-consuming backcrosses and
simultaneous selection steps.
Cisgenesis is a powerful alternative:
It is a single-step gene transfer without linkage
drag
Specific: only desirable alleles inserted
Stacking of (resistance) genes is more feasible
Existing varieties can be improved directly using
genes from the gene pool of breeders
Preferred by consumers compared to transgenics