We've had some improvements in working with primary transgenics. Several steps
are important. First, isolate a SINGLE shoot with a small ball of callus on
the bottom. (Cut this from a kan resistant shoot forming mass). Let it grow
on a plate until the SINGLE shoot is about 1 cm. Typically 3-5 days. Next,
dip the callus end in Rootone (F) so that the ball is cover with the white
powder (I got my rootone from a store like K-mart). Place this shoot/callus
structure in thoroughly wet soil. Be sure the callus ball is buried and that
the soil goes about halfway up the shoot. Put one shoot per 2 &1/2 inch pot.
Put TWO 2.5 inch pots in a bag (THE size and thickness of the plastic bag is
critical). We use 6X12 inch by 2mm bags from Consolidated Plastics (#90392).
If the bag is too big the shoots get fungus, too small they rot. Fold the bag
over and staple it close. Give low (80 uE) light at 22 C for at least 4-5 days
or until VISIBLE growth starts. DO not open the bag til the little weed starts
to grow. After 4-5 days-remove the staple in the evening. Next day-open the
bag (don't touch the plant). Two-three FURTHER days later cut 3-4 inches off
the bag. Another day, cut a few more inches. All changes should be done in
the evening. If the flowers are showing hyperstyllic development--pollinate
with a wildtype plant.
This method has worked well for us. (Forgot one thing-- before you put
the plant in the soil, make a hole in the soil and put a little fertilizer in
it. We use Peters. Be sure the soil is entirely in contact with the plant
when you place it there).
The critical point is to let the plant tell you when it's ready to come
out of culture or a bag. June Medford
Dept. of Biology
Penn State University