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1. Durable multigenic resistance for new apple
varieties through cisgenesis
Ton P.M. den Nijs and Henk J. Schouten,
Wageningen UR Plant Breeding
Bolzano (It), 17 November 2012

2. Resistance is best for crop protection
 Appel scab: requires 20-25 fungicide applications in
North-West Europe
 Environmental and economical threat
 Breeding for resistance since over 60 years!
 Single source of resistance: Malus floribunda 821

3. Classical breeding for scab resistance in apple
 1914: Vf-resistance
discovered in crab apple M.floribunda 821 X X Rome Beauty
M.floribunda 821 Rome Beauty
Malus floribunda 821 in
USA. 9433-2-2 X 9433-2-8
 World wide used source
? X F2-26829-2-2
? X ResistantResistant selection
? X selection
 80+ years later Vf-varieties ? X Resistant selection
with good quality
Elstar X Priscilla NL
 Santana
Santana

4. Scab resistance continued
 Seventh backcross
generation 1972-010-033 X Priscilla NL
 Strong focus on fruit quality
1980-015-047
X Golden Delicious X Elise
1990-045-133 1990-022-159
 SQ- 159
 SQ- 133
Natyra

5. Resistance management
 Crop protection evolves into gene protection
 We strongly advise to keep orchards with
Vf- varieties clean
 One to two spraying at asco-spore release peak

6. R gene resistance breeding: durable protection?
 Many successes but also leading to “man-guided
evolution”
 Multiple gene resistance presents higher barrier
 Pyramiding of different – if available- genes needed

7. M.micromalus extra source of resistance
Origin of Resistance
Cox's
Cox's Dr.Oldenburg M.micromalus Elstar
238 - Vm
Clivia Golden_Delicious
Gala 1993-209-015
238 - Vm
Pinova
Pinova 1998-002-021
238 - Vm
2004-002-012
2004-002-012
U family
U family
238, Vm

8. Pyramiding genes for resistance
 Combination breeding through cross-breeding
and MAB (Molecular marker Assisted Breeding)
 Long-lasting process because of long juvenile
period and linkage drag.
 Possible solutions:
 Shorten juvenile period
 Prevent linkage drag  CISGENESIS

9. Definition of Cisgenesis
 A cisgenic plant is genetically modified with one or
more natural genes from a crossable donor plant.
 The gene is under control of its native promoter,
contains its introns and terminator.
 No foreign genes, such as bacterial genes
 Same genes as in classical breeding
 No linkage drag

10. Cisgenesis - Transgenesis
 Cisgene (= gene from breeder’s germplasm) -
transgene (= foreign gene)
 Cisgenic – transgenic
Intragenesis: only native DNA. New combinations of
promoter and coding region allowed.
Cisgenesis is a strict case of intragenesis.

11. Cisgenesis: steps in the process
Steps:
1. Isolate target gene from donor plant
2. Bring this gene into a high quality cultivar (genetic
modification)
3. Evaluate the cisgenic plants for trueness to type
and target character, select out somaclonal
variants

12. Cisgenesis in apple in practice
 We developed pMF1 for marker free transformation
 ETH (Zürich) isolated HcrVf2
 We inserted it into susceptible cv. ‘Gala’
 We micrografted the cisgenic plants on M9

13. Micrografting
cisgenic plantlet
rootstock

14. Cisgenic shoot

15. Sporulation on Vf2 transformants
Sporulation (6 plants per event, leaf 1 to 4)
5 ef
4.5
cdef cdef cdef
4
3.5
Sporulation
3
2.5 abc
2
1.5
ab
1 ab ab ab ab
ab ab
0.5 a
a a
0
LPVf2-1
LPVf2-4
SPVf2-1
SPVf2-2
SPVf2-11
SPVf2-15
LPVf2-16
RbcVf2-1
RbcVf2-2
RbcVf2-3
RbcVf2-10
RbcVf2-11
RbcVf2-12
Gala
Santana
Transgenic lines

16. First cisgenic test orchard (Vf2) in Wageningen

17. First cisgenic test orchard (Vf2) in Wageningen

18. How to turn this into durable resistance ?
Pyramiding different genes.
We intend to pyramid three resistance genes
 Vf2 gene from Malus floribunda
 Additional resistance genes from apple
(V25, Vr2)
 We isolated already Vr2.
 We are testing candidate gene for V25.

19. Ex: Multigenic resistance to late blight in potato

20. Graphical view of binary vector pBINPLUS:
Rpi-blb3+Rpi-vnt1.1+Rpi-sto1

21. Multiple resistant transformants and control

22. Why cisgenesis?
Pyramiding via cisgenesis is feasible.
Classical breeding requires many
generations and years, even with MAB
Cisgenesis ~7 years
One-step introgression without linkage drag

23. Why cisgenesis?
High quality apple cultivar maintained
Genetic make-up of the original cultivar is preserved.
One or a few genes added.
Especially important for outbreeding, vegetatively
propagated plants (apple, potato, banana, grape, etc.)

24. Why cisgenesis?
As safe as conventional breeding
 A cultivar with a ‘history of safe use’ is used.
 Only well known genes from classical breeding are added
 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.
 EFSA Journal 2012, (10) 2561. “Cisgenesis is as safe as conventional
breeding”

25. Why cisgenesis?
Consumers generally prefer cisgenesis to
transgenesis ( Eurobarometer)
Cisgenesis respects natural crossing borders
Remains within the order of the creation
“No strange genes in my food”.

26. support for transgenic and cisgenic apples
From:
George Gaskell et al.,
Europeans and biotechnology in
2010, Winds of change?
A report to the European
Commission’s DG Research
October 2010

27. Conclusions
 Why cisgenesis?
- Durable disease resistance due to pyramiding of R genes
- Gain of time
- Specific; only wanted alleles inserted - High-quality
cultivar maintained
- Preferred by consumers compared to transgenesis;
natural genes from the crop species itself
- Reduces genetic vulnerability due to single gene
resistance

28. Acknowledgements
 Suxian Zhu (Wag UR Plant Breeding) for
unpublished results on Phytophthora-potato
 Cesare Gessler’s group, ETH, Zürich
 Inovafruit, funding
Thank you for your attention!!
Henk.schouten@wur.nl
Ton.dennijs@wur.nl