somatic

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

16. One
often
gets
many
shoots
from
each of
the calli.

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

20. S.
bulbocastanum
Potato
Somatic
hybrid

21. S.
bulbocastanum
Potato
Somatic
hybrid

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