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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
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
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
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
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
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
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
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
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
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.
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
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
Micrografting
                     cisgenic plantlet




         rootstock
Cisgenic shoot
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
First cisgenic test orchard (Vf2) in Wageningen
First cisgenic test orchard (Vf2) in Wageningen
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.
Ex: Multigenic resistance to late blight in potato
Graphical view of binary vector pBINPLUS:
      Rpi-blb3+Rpi-vnt1.1+Rpi-sto1
Multiple resistant transformants and control
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
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.)
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”
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”.
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
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
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

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Convegno la mela nel mondo interpoma bz - 17-11-2012 1 - ton den nijs

  • 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
  • 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
  • 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