This case study describes the development of cisgenic barley with improved phytase activity through Agrobacterium-mediated transformation. 72 transgenic T0 barley plants were produced by co-transforming with two vectors, one containing the HvPAPhy_a gene and the other a selectable marker. 19 plants were found to contain only the HvPAPhy_a insert through PCR analysis. Two of these plants, PAPhy05 and PAPhy07, showed increased phytase activity and a 3:1 segregation ratio in progeny, indicating single insertions of HvPAPhy_a without the selectable marker. This demonstrates the successful production and analysis of cisgenic barley

1. Department ofGenetics andPlantBreeding
FACULTYOF AGRICULTURE
BIRSAAGRICULTURALUNIVERSITY
KANKE,RANCHI–834006(JHARKHAND)
PRESENTEDBY-
ANJANI KUMAR

2. CONTENTS
What is cisgenesis
History
Why cisgenesis
Advantages
Achievements
How to produce cisgenics
Marker free cisgenesis
Case study
Limitations
Current status on regulation of cisgenic crops
Future trends
Conclusion
Key references

3. What is cisgenesis???
Cisgenic plants can harbour one or more cisgenes, but they do not contain
any parts of transgenes or inserted foreign sequences.
To produce cisgenic plants any suitable technique used for production of
transgenics may be used. Genes must be isolated, cloned or synthesized and
transferred back into a recipient where stably integrated and expressed.
Cisgenesis is also used to describe an Agrobacterium-mediated transfer of a
gene from a sexually compatible – plant where T-DNA borders may remain
after transformation. This is referred as cisgenesis with T-DNA borders.
“Cisgenesis is the genetic modification of a recipient organism with a gene from a crossable
– sexually compatible – organism (same species or closely related species). This gene
includes its introns and is flanked by its native promoter and terminator in the normal
sense orientation.”

4. Fig. Illustration of cisgene construct . The cisgene is an identical copy of a gene from the sexually compatible pool
including promoter, introns and terminator (a,b). When using Agrobacterium-mediated transformation the cisgene
is inserted within Agrobacterium-derived T-DNA borders.
Holme et al. (2013)

5. • The term “cisgenesis” was introduced by
Jochemsen and Schouten (2000) in the book –
‘Toetsen en begrenzen. Een ethische en politieke
beoordeling van de moderne biotechnologie.’
• It was made international in 2006 by Schouten,
Krens and Jacobsen.
HISTO
RY

6. Why cisgenics

7. 1. Linkage drag
2.Time-consuming
1. Presence of foreign gene
2. Presence of marker
gene and vector backbone
sequences
Linkage drag
Foreign gene
Additional sequences
 less time

8. As it provides no additional traits, so no changes in fitness occur
Carries no risks—such as effects on non-target organisms or soil
ecosystems, toxicity or a possible allergy risk for GM food or
feed
Can improve traits with limited natural allelic variation within
the sexually compatible gene pool (e.g., phytases activity in
barley, processing qualities in potatoes)
Allow rise of SMEs and small breeders
For stacking genes e.g., to develop multigenic resistance

9. Crop Disease Source of genes
Apple Apple scab Malus floribunda
Potato Late blight Solanum bulbocastanum
Jacobsen and Schouten, 2008
Gene stacking using cisgenes

10. ACHIEVEMENTS

11. CISGENIC CROPS DEVELOPED OR CURRENTLY UNDER DEVELOPMENT
CROP TYPE PROMOTER GENE TRAIT AUTHORS
RICE EXPRESSION
35S-CMV/35S-CMV
+ core promoter
DREB2A Drought tolerance Raj et al.(2015)
BRINJAL - - -
Reduced number of
trichomes
J.H.J. Van Den
Enden (2015)
CHESTNUT OVEREXPRESSION
UBQ11 + core
promoter
Laccase like
gene
Blight resistance
Newhouse et al.
(2013)
BARLEY OVEREXPRESSION GENE’S OWN HvPAPhy_a
Improved grain
phytase activity
Holme et al.
(2012)
MAIZE EXPRESSION - - Cd- accumulation
Simic et al.
(2011)
APPLE EXPRESSION GENE’S OWN HcrVf-2 Scab resistance
Vanblaere et al.
(2011)
GRAPEVINE EXPRESSION
35S-CMV/35S-CMV
+ core promoter
VVTL-1,
NtpII
Fungal disease
resistance
Dhekney et al.
(2011)
POPLAR OVEREXPRESSION
GENE’S OWN Growth
genes PAT
Different growth types Han et al. (2011)
POTATO EXPRESSION GENE’S OWN R-genes Late blight resistance
Haverkort et al.
(2009)
WHEAT EXPRESSION
GENE’S OWN
1Dy10
Improved baking
quality
Gadaleta et al.
(2008)
STRAWBERRY OVEREXPRESSION GENE’S OWN PGIP Grey mould resistance Schaart (2004)

12. FIELD TRIALS WITH CISGENIC
CROPS
COMPANY/INSTITUTE STATE
NOT.
NUMBER
YEARS PLANT TRAIT GENE
Plant Research
International
(Wageningen University)
NL
B/NL/07/01
B/NL/09/02
B/NL/10/06
2007-2012
2010-2020
2011-2021 Potato
Late blight
resistance
R-genes
BE B/BE/10/V1 2011-2121
IE B/IE/12/01 2012-2016
Plant Research
International
(Wageningen University)
NL B/NL/10/05 2011-2021 Apple Scab HcrVf2
Aarhus University DK B/DK/12/01 2012-2016 Barley
Improved
grain
phytase
activity
HvPAPhy_a
Holme et al.(2013)

13. Cisgenic Arctic™ “Golden Delicious” and
“Granny Smith” apples (Okanagan
Specialty Fruits Inc., Summerland, BC,
Canada) and a cisgenic alfalfa with altered
lignin production (Monsanto) are currently
under cultivation for commercial purposes.
Pastoral Genomics in New Zealand has
registered the trademark Cisgenics® and
uses this trademark for their future
genetically modified ryegrass .
Lombardo et al. (2016)

14. cisgenic plant
regenerated from a
single transformed cell
transformed cell
Gene inserted
into plasmid
Cells screened
for cisgenes
Gold particles
coated with DNA
Cells shot with gene gun and
DNA incorporated into plant
cell chromosome
Gene replicationBacterium mixed
with plant cells
Plasmid moves to
insert DNA into
plant chromosome
A
Agrobacterium
B
Gene gun
C
Screening of cells
with cisgenes
Cisgene identified
and isolated

15. The other very important step in the development of cisgenic
plants is the production of plants devoid of the foreign DNA
from marker genes, T-DNA border sequences and vector-
backbone sequences. Several standard methods for the
generation of marker-free crops are available. Most of these
methods are protected by patents, which limits freedom to
operate (Holme et al., 2013).

16. METHODS USED TO PRODUCE
MARKER-FREE CISGENIC
PLANTS
1. Co-transformation of two T-DNA molecules (Multiple T-
DNA approach)
2. Site Specific Recombination
3. Transposition Mediated Repositioning
4. Intra Chromosomal Recombination

17. METHODS USED TO PRODUCE MARKER-
FREE CISGENIC PLANTS
CROP GENE
METHOD OF
DELIVERY
MARKER FREE METHOD
P-DNA
BORD
ERS
VECTOR-
BACKBONE
DETECTION
A Potato R-genes Agrobacterium
Transformation without
marker gene
No
PCR and
Southern blot
B Potato StAs1, StAs1 Agrobacterium
Ipt-gene in vector-
backbone
Yes
Ipt in vector
backbone
C Alfalfa Comt Agrobacterium
Transformation without
marker gene
Yes PCR
D Strawberry PGIP Agrobacterium
Site-specific recombination
(R/Rs)
No
PCR and
Southern blot
E Apple HcVf2 Agrobacterium
Cotransformation with
vector with marker gene
No
PCR, flanking
sequence
analysis
F Wheat 1Dy10 Biolistic
Cotransformation of linear
fragments with GOI and
marker gene
No
Not
applicable
Holme et al.(2013)

18. A CASE
STUDY

19. OBJECTI
VES
• Co-transformation efficiency
• Increased phytase activities in the grain
• Integration of the antibiotic resistance gene of the
vector-backbone, and
• Segregation between the HvPAPhy_a insert and the
antibiotic resistance gene
To analyze-

20. MATERI
ALS
Genomic HvPAPhy_a-gene (a gene that encodes a
phytase enzyme, identified and isolated from
genomic barley library (Stratagene, Cedar Creek,
TX)
Vector pairs- pClean-G185 and pClean-S166
Agrobacterium strain AGL0
Spring cultivar “Golden Promise”

21. METHO
DS
Co-transformation of Agrobacterium with both vectors;
Transformation of barley embryos with Agrobacterium;
PCR analysis of T0 and T1-plants with four primer pairs
(to identify transformants and marker free lines);
Phytase activity analysis assay;
Genomic DNA gel blot analysis of marker free plant lines

22. RESU
LTS
Out of 1500 Agrobacterium infected plants, only 72 T0
plants survived;
Based on the PCR analysis, all 72 T0-plants obtained in this
study contained the antibiotic resistance gene;
The PCR analysis revealed that 73.6% of the
transformants also contained PAPhy_a insert(s)

23. In the total material of 72 T0-lines, we obtained 19 plants that
were co-transformed with both T-DNAs, expressed the
PAPhy_a insert and did not show the PCR product of the
antibiotic resistance gene from the vector-backbone of
pClean-G185-PAPhy_a.
Based on the 60% unlinked integration frequency obtained,
we can predict that it should be possible to select 11
potentially cisgenic T1-lines out of the 72 T0-lines obtained.

24. • The results after determining phytase activity in seeds of
transformed T0 plants, were divided into three categories-
(i) phytase activities in seeds of plants where the PCR
product of the PE- and TE-primer pairs were not
detected
(ii) phytase activities in seeds of plants only showing the
PCR product of the TE-primer pairs and
(iii) phytase activities in seeds of plants showing the PCR
product of both the PE- and TE-primers pairs

25. Fig1. Phytase activities in seeds of T0-plants. The T0-plants were divided into three groups: (a) T0-plants not showing the
PCR products of PAPhy_a insert, (b) T0-plants showing only the PCR product of the terminator-end (TE) primer pairs at the
right T-DNA border of PAPhy_a inserts, (c) T0-plants showing the PCR products of both the TE and promoter-end (PE)
primer pairs of PAPhy_a inserts. FTU: phytase units. The first column of each figure represents the phytase activity of non-
transformed Golden Promise seeds. Plants not showing the PCR product of the kanamycin resistance gene of the
pClean-G185 vector-backbone. Plants showing the PCR product of the kanamycin resistance gene of the pClean-G185
vector-backbone. Asterisks indicate the two plants PAPhy05 and PAPhy07 from which marker-free progeny were later
identified.

26. PAPhy05 and PAPhy07 both almost
exactly followed the 3 : 1 segregation
for a single PAPhy_a insert, indicating
a single PAPhy_a insert in both plants.
PAPhy05 and PAPhy07 also lacked the
kanamycin resistance gene of the pClean
G185- PAPhy_a vector backbone as
judged by the PCR analysis.
The segregation study was performed in the progeny of five T0-plants obtained from the
first transformation experiment. The plants were named PAPhy01, PAPhy02, PAPhy03,
PAPhy05 and PAPhy07
Table: Segregation between the PAPhy_a inserts and the hygromycin resistance
inserts in progeny of five T0-plants
Observed segregation
Transformants No. of progeny H+P+* H+P- H-P+ H-P-
PAPhy01 50 34 14 0 2
PAPhy02 29 28 0 0 1
PAPhy03 25 17 3 5 0
PAPhy05 40 24 9 5 2
PAPhy07 56 41 13 2 0
*P: PAPhy insert H: hygromycin resistant gene

27. The phytase activity in the seeds of the hemizygous and
homozygous plants of PAPhy05 and PAPhy07 was almost the
same.
The 1 : 2 : 1 mixture of the PAPhy_a segregating seeds from the
hemizygous plants showed a two-fold increase in phytase
activity.
Seeds from the homozygous plants of PAPhy05 and PAPhy07
showed a 2.6- and 2.8-fold increase in phytase activity,
respectively.

28. Fig2. Phytase activities in seeds of plants segregating for the PAPhy_a
insert. The phytase activities were measured in seeds derived from (a)
PAPhy05 marker-free plants and (b) PAPhy07 marker-free plants
without the PAPhy_a insert (1), hemizygous for the insert (2) and
homozygous for the insert (3). FTU: phytase units.

29. FINAL
OUTCOME
• The genomic clone proved to be fully functional when inserted
into the barley genome.
• Transformed T0-plants showed up to six fold increase in grain
phytase activity as compared to the wild type.
• The increase in phytase activity monitored in two marker-free
plant lines with a single-copy PAPhy_a insert was found to be
stable over the three generations analysed.
• The marker-free transformed plant line PAPhy07 can be
classified as a cisgenic plant line according to the definitions of
Schouten et al. (2006).

30. • Seeds of plants homozygous for the single-copy PAPhy_a
insert showed 2.6- to 2.8-fold increases in phytase activity,
revealing a positive correlation between gene dosage and
gene expression.
• It is not possible with the method used in the present study
to generate plants that are totally devoid of foreign DNA.
In PAPhy07, a total of 19 T-DNA border nucleotides and 36
synthetic nucleotides were integrated into genome. Current
progress in genome sequencing will enable the detection
and thus facilitate the elimination of such foreign
sequences.
FINAL
OUTCOME

31. Random insertions;
Mutation at insertion site;
Donor sequence does not replace an allelic
sequence, but is added to the recipient
species’ genome;
Somaclonal variation;
Formation of new ORF;
Labelling requirement;
Seeks expertise and time
LIMITATIONS OF
CISGENICS

32. • The ease, timeframe and cost of approval of cisgenic crops under
development will depend on the future regulations of these crops.
• Release of cisgenic crops currently falls under the same regulatory
guidelines as transgenic crops.
• Less stringent regulations of these crops has been within EU, the USA
and New Zealand. The European Commission (EC) set up a New
Techniques Working Group (NTWG). Their study showed that with
respect to the number of recent scientific publications and filed patents
cisgenesis ranked 2nd amongst the seven NPBTs (Holme et al.,2013).
• USA has exempt cisgenics from GMO regulations, when used for pest
protection. (Philip Hunter, 2013)
CURRENT STATUS ON THE REGULATION
OF CISGENIC CROPS

33. It carries a high potential for generating plants with
environmental, economic and health benefits that may be
essential for meeting the global need for a more efficient
and sustainable crop production.
The development of cisgenic crop plants based on the latest
genome editing techniques(such as the CRISPR-Cas9
system), which replace genes in the same genomic
locations, instead of simply adding on/off target changes,
are expected to revolutionize plant improvement in
agricultural production systems.
(Kushalappa et al., 2016)
FUTURE
TRENDS

35. • It is important to identify three main arguments-ecological
argument, public acceptance argument, competition
argument in relation to cisgenics (Pavone et al., 2015).
• Whether this technique will develop into a powerful new tool
strongly depends on several factors: how cisgenic plants are
treated by existing legal framewok; consumer acceptance of
such products; whether these plants and any products derived
from them must be labelled as GM; and intellectual property
rights on GM technologies and genes.
CONCLUS
ION

36. If genetically modified crops were to be modified only by
inserting genes proceeding from wild or crossable varieties of
the same species, would they cease to be GMOs?
What differentiates a genetically modified plant
from a natural one?
How phylogenetically distant have to be these plants from the
origin of the inserted genes to be considered “transgenic”?
How do cisgenics impact existing boundaries between
traditional and innovative, natural and artificial?
How do scientists researching in the field construct, frame,
define and promote cisgenics?
What are the implications for science, regulation and society?

37. The number of availablegenes is increasing exponentially
The techniques are available
The majority of the consumers are positive
The main bottleneck is the GMO regulation
If cisgenicplants are not regarded asGMOs in the regulation, then I see a
bright future !!!

38. KEY
REFERENC
ES• Holme I.B., Dionisio G., Pedersen H.B., Wendt T., Madsen C.K., Vincze E. and Holm P.B.
(2012). Cisgenic barley with improved phytase activity. Plant Biotechnology.10: 237–247;
• Jacobsen E. and Schouten H.J. (2009). Cisgenesis: An important sub-invention for
traditional plant breeding companies. Euphytica. 170: 235–247;
• Schouten H.J., Krens F.A. and Jacobsen E. (2006). Cisgenic plants are similar to
traditionally bred plants. Science and Society. 7:750-753;
• Holme I.B., Wendt T. and Holm P. B. (2013). Intragenesis and cisgenesis as alternatives to
transgenic. Plant Biotechnology Journal. 11: 395–407;
• Lombardo L. and Zelasco S. (2016). Biotech Approaches to Overcome the Limitations of
Using Transgenic Plants in Organic Farming. Sustainability. 8:497;
• Hunter P. (2014). “Genetically Modified Lite” placates public but not activists. EMBO
Reports. 15:2;
• Kushalappa A.C., Yogendra K.N., Sarkar K., Kage U.K. and Karre S. (2016). Gene
discovery and genome editing to develop cisgenic crops with improved resistance against
pathogen infection. Canadian Journal of Plant Pathology;
• Pavone V. and Martinelli L. (2015). Cisgenics as emerging bio-objects: Bio-objectification
and bio-identification in agrobiotech innovation. New Genetics and Society.