Hmm. I guess that didn't work so well. Here's another
attempt at posting that carrot recipe.
There have been enough requests for the recipe for
getting cultures from carrot that I have decided to
post the protocol. It is based on Yeoman and
Macleod, Tissue (Callus) Cultures--Techniques, pp.
31-59 in Plant Tissue and Cell Culture, Street HE,
ed., Botanical Monographs 11, Blackwell 1977. I
must have picked it up from another book, but I
can't place it right now. Obtain good carrots from
the grocery, at least 200 mm long and 40 mm in
diameter, and trim down to the middle 100 mm.
The surfaces are cleaned and scrubbed and steri-
lized with hypochlorite. Using sterile techique, the
carrots are rinsed thoroughly to remove the
hypochlorite and slized with a razor to about 1 mm
thick. From these transverse sections, explants 4
mm x 4 mm square are cut across the cambium,
and the pieces placed (apical side down) on agar
slants containing the medium described below, two
pieces per tube. Each group should do several
tubes, so that contaminated ones can be discarded
and good tissues share with groups that don't have
any. They are incubated in the dark at 25 oC.
After 4-5 weeks, in my experience 80% of the
cultures which are not contaminated will have
grown sufficiently to be subdivided and recultured.
4-5 weeks later, there should be enough to do an
organogenesis experiment. (In a 15 week semester,
and a class that starts with cellular stuff and moves
up to organismal physiology, this makes for a good
schedule if tissue culturing is the first lab.) The
cultures can be subdivided again and cultured on
the same medium, but with 0.1 to 3.0 mg/l of IAA,
and 0.1 to 3.0 mg/l of kinetin. Here they are
cultured on Petri dishes instead of on slants to
improve viewing. Within about 4 weeks, roots
and/or shoots will form, and the students can draw
their own conclusions about the significance of
ratios of the hormones. (On bad semesters, this may
mean showing them the results at the final exam
time!) If students are having trouble identifying
roots vs. shoots (gravitropic preferences is a good
way to do it), cultures which have initiated organs
can be transferred to the light, when shoots will
green up and, in the notes of one previous TA, are
"quite cute". Culturing them without hormones at
all can cause embryos to form. I generally have
good luck with this procedure; the hardest part is
getting the students to have good sterile technique
throughout. I generally write out a lot of detail
about this, and also make sure the TAs are good
and alert.
M-DAUC
mg/l
CaCl2.2H20 440
FESO4.H2O 16.9
KH2PO4 170
KNO3 1900
MgSO4.7H2O 370
Na2EDTA 37.3
NH4NO3 1650
CoCl2.6H2O 0.025
CuSO4.5H2O 0.025
H3BO3 6.2
KI 0.83
MnSO4.4H2O 22.3
NaMoO4.2H2O 0.25
ZnSO4.7H2O 8.6
Nicotinic acid 0.50
Pyridoxine HCl 0.123
Thiamine HCl 0.1
Glycine 3.0
2-4 D 2.5 x 10^-5
g/l
Sucrose 20
Agar 8
