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HYB: Chromosome counting - one more time

From: "Rodney Barton" <rbarton@hsc.unt.edu>

I see by the archive that this arrived without returns so here is a more readable version:

Here are the instructions for making chromosome counts as was
used on Christy's ensibs.  They are probably not complete
enough for someone who is a stranger to the lab, but they will
give you an idea of what is involved.


Instructions for making Chromosome Counts in Iris

Plants must be making active growth when material for
chromosome count is taken.  It is good to take 3 or 4 root
tips as some will not have dividing cells.  Cut at least 1/4
inches off the root tip for use.

Pretreatment:  Place root tip in colchicine solution (0.05 to
0.1% in water) for 6-12 hours. (colchicine is poisonous, wear
disposable gloves when handling it and promptly wash your
hands after any contact.)  

Fixative: Remove root tips from colchicine, blot off any
moisture and place in Farmers fixative for 1 to 24 hr.
(Farmers fixative: 3 parts anhydrous ethyl alcohol to 1 part
glacial acetic acid)

Stain:  Remove  from fixative and place in stain for 24 hrs.
(Stain: 2 drops of 2% orcein stain in glacial acetic acid
(heat to dissolve and filter) and 3 drops of 0.3 N
hydrochloric acid.)

Slides:  Remove from stain and place on a clean microscope
slide.  Some roots will have a root cap which will not squash
and must be removed. Cut 1/2 to 1 mm from the remainder of the
root tip and discard the rest.

Squash:  Put a small drop of stain on the root tip. Place 
cover glass over the root and use the lead of the pencil to
tap gently on the cover glass.  Allow the stain to flow back
around the root between taps.  The cells from the root tip
should spread out from 1/4 to 1/2 inches until there is a
single layer of cells.  Ideally the cells should not be

Completing squash: Examine the squash (under the microscope ?)
If the cells are spread is satisfactory use the pencil eraser
to further flatten the cells.  Be careful not to move or slide
the cover glass.  Some material makes a better slide when heat
is applied to help flatten and stick the cells to the slide. 
(Shockey says to pass the slide over a candle flame to heat.)

Clearing:  When stain prevents clear view of the chromosomes,
remove the cover glass and examine which, slide or glass, has
the most cells attached.  Drop 4 drops of alcohol on the
squash, one at a time to wash off excess stain.  Allow to dry. 
Place a drop of immersion oil on the squash, and place on a
clean slide or cover with a clean cover glass which ever is

Examining silde: Scan the slide using the 40X objective to find
a good cell.  Place a drop of immersion oil on the cover glass
and rotate the 100 power objective to do the counting.

My comments:

I have yet to try this so I can't warn you of other pitfalls.

A reasonably good light microscope with a 100x oil immersion
objective is necessary.

You may find it difficult to purchase the necessary chemicals.
Chemicals suppliers generally will not sell to individuals. 
Colchicine can be purchased from some orchid flasking supply

I do suggest you read Shockley's ASI article.  The basic
procedure is similar but more complex, and he give some hits
that should help with this procedure. 

Counting the chromosomes is not a simple as it sounds. As
Christy says, it takes some experience to know what it is you
are seeing.  Also, it is difficult to find cells where the
chromosomes are separated well enough to count. The larger the
number of chromosomes, the harder this task becomes. To get a
true count you need count a statistically significant number of
cells. In human diagnostic work they will count as many as 50
cells to insure an accurate count!

Rodney Barton
Hickory Creek (North Central) Texas, USA

North American Native Iris web site:

Rodney Barton
Hickory Creek (North Central) Texas, USA

North American Native Iris web site:

Rodney Barton
Hickory Creek (North Central) Texas, USA

North American Native Iris web site:
http://molly.hsc.unt.edu/~rbarton/Iris/NANI.html                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             !


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