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John P. Helgeson
USDA/ARS, Dept of Plant Pathology
University of Wisconsin - Madison
Protoplast Preparation and
Somatic Hybrids
• In many cases the somatic hybrids can be
crossed back to one or the other of the two
parents.
• In that way the DNA of a wild species can
be incorporated into a breeding line of a
crop species.
• Our work has been done primarily with
potato and its wild relatives.
Somatic Hybridization
Using somatic hybrids to introgress important genes into potato
Potato plants growing in a
test tube
Somatic Hybridization
Potato leaves suspended in wall-dissolving enzyme solution
Protoplasts collected in
a Babcock bottle
Somatic Hybridization
Protoplasts float to top in
sugar solution
Somatic Hybridization
Potato leaf protoplasts immediately
after digestion of cell walls
Close up of protoplasts - green discs are
chloroplasts
Two potato leaf protoplasts about to fuse
Fusion products begin to divide on
nutrient medium
Further cell divisions yield masses of
cells called “Calli.”
The calli turn green in light
If the conditions are right, small
shoots emerge from the green calli
The shoots on calli will develop into
new plants
One
often
gets
many
shoots
from
each of
the calli.
• The shoots can be excised from
the calli and rooted on nutrient
media in a test tube. This will
result in a whole new potato
plant.
• The new potato plant can be
grown in a test tube
Somatic Hybridization
A form of DNA
fingerprinting
called RFLP
analysis can be
used to
demonstrate
that one really
does have a
somatic hybrid.
Note that the somatic hybrids have the DNA
bands from S. bulbocastanum and potato
PI203900, the parents of the hybrids.
• Characteristics of both of the
combined species can be expressed
in the somatic hybrids.
• Note the purple stems vs the green
stems and compound vs simple
leaves stems in the following slide
Somatic Hybridization
S.
bulbocastanum
Potato
Somatic
hybrid
S.
bulbocastanum
Potato
Somatic
hybrid
We have been using this procedure
to capture disease resistances
from wild potato species that can’t
be crossed with the domestic
potato
Somatic Hybridization
Somatic hybrids obtained in
the Helgeson Lab
S. brevidens PL 218228 (2x) + S. tuberosum (4x)
S. brevidens PL 218228 (2x) + Russet Burbank
(4x)
S. brevidens PL 218228 (2x) + S. tuberosum (2x)
S. bulbocastanum PL 245763 (2x) + S. tuberosum (4x)
S. bulbocastanum PL 245310 (2x) + S. tuberosum (4x)
S. cardiophyllum PL 279272 (2x) + S. brevidens
(2x)
S. commersonii PL 320266 (2x) + S. tuberosum (4x)
S. commersonii PL 320266 (2x) + S. tuberosum (2x)
S. etuberosum PL 245939 (2x) + S. tuberosum (4x)
S. etuberosum PL 245939 (2x) + W730 x S. Ber. (2x)
S. polyadenium PL 230480 (2x) + S. tuberosum (2x)
Some new disease resistances
from somatic hybrids
Disease/pathogen Somatic
hybrid
Late blight
Early blight
M. Chitwoodii
Erwinia Soft
rot
Bacterial wilt
PVY
PLRV
S. bulbocastanum + S. tuberosum
S. bulbocastanum + S. tuberosum
S. bulbocastanum + S. tuberosum
S. brevidens + S. tuberosum
S. commersonii + S. tuberosum
S. etuberosum + S. tuberosum
S. brevidens + S. tuberosum
We have been able to capture late
blight resistance from the wild
potato species, Solanum
bulbocastanum and, using sexual
crosses of the somatic hybrid we
have developed a number of
highly resistant potato breeding
lines.
Somatic Hybridization
Tubers of Solanum bulbocastanum,
source of the “RB” gene
S. bulbcastanum in the field
at Hancock, Wisconsin
View of test field - Hancock Research
Station. The green plants are those
with the new resistance gene
Backcross 1
line (J101- K27)
in field at
Hancock, WI
The field
received no
fungicide and
the dead plants
are from the
cultivar Russet
Burbank, killed
by late blight.
BC1 line J138A12 in Toluca, Mexico.
The arrow points to what is left of a
plant of the susceptible cultivar
“Alpha.”
The resistance to late
blight is effective in
Toluca Mexico, where
the variation in the late
blight organisms is the
greatest in the world.
By somatic hybridizations such as
those of potato and S.
bulbocastanum we hope to
introduce new genes into potato so
that pesticide use can be
decreased substantially.
For more information contact John Helgeson,
JPH@plantpath.wisc.edu
Protoplast Preparation and
Somatic Hybrids

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helgesonsomatic.ppt

  • 1. John P. Helgeson USDA/ARS, Dept of Plant Pathology University of Wisconsin - Madison Protoplast Preparation and Somatic Hybrids
  • 2. • In many cases the somatic hybrids can be crossed back to one or the other of the two parents. • In that way the DNA of a wild species can be incorporated into a breeding line of a crop species. • Our work has been done primarily with potato and its wild relatives. Somatic Hybridization
  • 3. Using somatic hybrids to introgress important genes into potato
  • 4. Potato plants growing in a test tube Somatic Hybridization
  • 5. Potato leaves suspended in wall-dissolving enzyme solution
  • 6. Protoplasts collected in a Babcock bottle Somatic Hybridization
  • 7. Protoplasts float to top in sugar solution Somatic Hybridization
  • 8. Potato leaf protoplasts immediately after digestion of cell walls
  • 9. Close up of protoplasts - green discs are chloroplasts
  • 10. Two potato leaf protoplasts about to fuse
  • 11. Fusion products begin to divide on nutrient medium
  • 12. Further cell divisions yield masses of cells called “Calli.”
  • 13. The calli turn green in light
  • 14. If the conditions are right, small shoots emerge from the green calli
  • 15. The shoots on calli will develop into new plants
  • 17. • The shoots can be excised from the calli and rooted on nutrient media in a test tube. This will result in a whole new potato plant. • The new potato plant can be grown in a test tube Somatic Hybridization
  • 18. A form of DNA fingerprinting called RFLP analysis can be used to demonstrate that one really does have a somatic hybrid. Note that the somatic hybrids have the DNA bands from S. bulbocastanum and potato PI203900, the parents of the hybrids.
  • 19. • Characteristics of both of the combined species can be expressed in the somatic hybrids. • Note the purple stems vs the green stems and compound vs simple leaves stems in the following slide Somatic Hybridization
  • 22. We have been using this procedure to capture disease resistances from wild potato species that can’t be crossed with the domestic potato Somatic Hybridization
  • 23. Somatic hybrids obtained in the Helgeson Lab S. brevidens PL 218228 (2x) + S. tuberosum (4x) S. brevidens PL 218228 (2x) + Russet Burbank (4x) S. brevidens PL 218228 (2x) + S. tuberosum (2x) S. bulbocastanum PL 245763 (2x) + S. tuberosum (4x) S. bulbocastanum PL 245310 (2x) + S. tuberosum (4x) S. cardiophyllum PL 279272 (2x) + S. brevidens (2x) S. commersonii PL 320266 (2x) + S. tuberosum (4x) S. commersonii PL 320266 (2x) + S. tuberosum (2x) S. etuberosum PL 245939 (2x) + S. tuberosum (4x) S. etuberosum PL 245939 (2x) + W730 x S. Ber. (2x) S. polyadenium PL 230480 (2x) + S. tuberosum (2x)
  • 24. Some new disease resistances from somatic hybrids Disease/pathogen Somatic hybrid Late blight Early blight M. Chitwoodii Erwinia Soft rot Bacterial wilt PVY PLRV S. bulbocastanum + S. tuberosum S. bulbocastanum + S. tuberosum S. bulbocastanum + S. tuberosum S. brevidens + S. tuberosum S. commersonii + S. tuberosum S. etuberosum + S. tuberosum S. brevidens + S. tuberosum
  • 25. We have been able to capture late blight resistance from the wild potato species, Solanum bulbocastanum and, using sexual crosses of the somatic hybrid we have developed a number of highly resistant potato breeding lines. Somatic Hybridization
  • 26. Tubers of Solanum bulbocastanum, source of the “RB” gene
  • 27. S. bulbcastanum in the field at Hancock, Wisconsin
  • 28. View of test field - Hancock Research Station. The green plants are those with the new resistance gene
  • 29. Backcross 1 line (J101- K27) in field at Hancock, WI The field received no fungicide and the dead plants are from the cultivar Russet Burbank, killed by late blight.
  • 30. BC1 line J138A12 in Toluca, Mexico. The arrow points to what is left of a plant of the susceptible cultivar “Alpha.” The resistance to late blight is effective in Toluca Mexico, where the variation in the late blight organisms is the greatest in the world.
  • 31. By somatic hybridizations such as those of potato and S. bulbocastanum we hope to introduce new genes into potato so that pesticide use can be decreased substantially. For more information contact John Helgeson, JPH@plantpath.wisc.edu Protoplast Preparation and Somatic Hybrids