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second try at carrot info

Wed Sep 27 07:15:51 EST 1995

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.



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


Sucrose	20
Agar	  8	

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