In planta transformation: now working!
21847LAB at MSU.EDU
Thu Jul 2 11:34:00 EST 1992
This non-tissue culture method to get transformed Arabidopsis was first
described by Chang et al. at the Arabidopsis meeting in Vienna. Ljerka Kunst
and George Haughn invested some time to get the method to work in their
respective laboratories. Then Ljerka introduced it to Chris Somerville's Lab
where it has now become the method of choice for Arabidopsis transformation.
In general, we are performing the procedure as described in Chang's poster
abstract from the meeting in Vienna, but the percentage of transformed seeds
is not as high as reported.
We grow and treat the plants in the following way:
1. We seed 10-12 Arabidopsis plants (Columbia, Landsberg and RLD have been
tested) in a 6 in. pot containing Arabidopsis soil and grow them under
continuous light, or long day photoperiod.
2. When the plants start to bolt (flowering shoot < 2 cm) we cut the flowering
shoot and all visible axillary buds with a forceps, or a razor blade. On the
wounded area we apply a droplet of overnight culture of Agrobacterium
tumefaciens (strain C58 or GV3101 containing the desired vector).
3. When the plants make secondary flowering shoots (< 2 cm) we remove them
again and infect as above. After the second infection, plants are grown to
maturity to produce seeds.
4. All the seeds from one pot are bulk harvested, or collected in 3 batches
from each pot.
5. Transformed seeds are selected on MS plates containing the appropriate
antibiotic (Kanamycin: 50 ug/ml, Hygromycin: 30 ug/ml, Chlorsulforon: 0.05
ng/ml). 2000 sterilized seeds are spread regularly per 150 mm petri dish
(10000 seeds in case of Chlorsulforon selection).
6. After 10 days to 3 weeks transformed plants are easily visible. If you have
a good seed set (> 1g seeds per pot) you can expect an average of one
transformant per pot.
Good luck Christiane (Chris Somerville's Lab)
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