At 8:32 AM 9/10/96 -0700, Carl Pike wrote:
>Does anyone have any suggestions on how to apply auxin in a class lab on
>apical dominance (peas)? We use the standard method of mixing the auxin
>(ethanol solution) in hot lanolin. How stable is auxin in lanolin? I
>worry about loss of hormone in the heated lanolin, and the inability to be
>sure a consistent amount has been applied to the stumps. Does it help to
>recut the stump after a few days and make a fresh application? Is there
>some way to apply the auxin in liquid form?
Carl,
I do this exercise frequently, so it's an old shoe for me.
I use 5000 ppm IBA for routine non-major classes. The
compound is fairly stable to heat...so you can be casual.
I generally weigh the container, add the necessary IBA,
dissolve it in a few drops of acetone, then add the necessary
amount of cold lanolin by observation with a top-loading
balance. The goopy lanolin sticks to the sides and so on
at this point and that's OK. Then I warm the container
just until the lanolin melts...I wouldn't call it HOT! All
I want to do is get it to a transparent liquid state for
adequate mixing. Then I mix and use. I store it in the
refrigerator, and have found it quite active for several
years! I would be very cautious with IAA...it is both
temperature and light sensitive. NAA and 2,4-D are very
stable and lower concentrations are probably effective
in supressing lateral bud growth. BTW the appearance of
any pinkish cast to the lanolin is an indication of
considerable breakdown. If it is pink, throw it out.
5000 ppm is a very high concentration. I generally use
beans (the students find the morphology easier to understand
and I think it takes more IBA than peas need to respond).
I decapitate just below the first trifoliate so the plants
have the two simple leaves and a stump. This concentration
IS effective in reducing branch formation. You do not need
to reapply or recut, and, in fact, in our greenhouse we
get a very nice bonus. The stem stump forms a good callus,
and in our humid greenhouses, we can usually get a nice
crop of roots on that callus too...usually in one week...
surely in two!
With biology majors I use 10-fold dilutions of this stock
lanolin to show a dose response. The responses are dose-
dependent. 500 ppm is marginally effective. 50 ppm is
not different from controls. The concentration is only
one part of the situation, though. The amount applied
has to be considered. I am informal about that too. I tell
students to apply an amount of cold lanolin about the size
of the head of a wooden match...less than a pea, more than a
radish seed. I remind them to try to put the SAME amount on
all the stumps. If students weigh a sample of their "dollop",
they can estimate the amount of IBA actually applied to the
plant. 5000 ppm sounds like a whopping amount until you do
the calculations.
A few students are clumsy enough to get some of the IBA lanolin
on the petioles of the simple leaf. They end up with an
interesting swelling that correlates with the curvature of that
petiole. It makes tropisms easier to understand.
Beans are a bit slower to respond than pea, but obvious results are
obtained in one week even with bean.
To be precise for a moment, I don't think there is a decent way
to quantify what is going on when lanolin gets in the picture.
You know how much you put in it, but how much is held up in the
lanolin matrix? How much decays in light? How much actually
gets taken up by the cells at the stump surface? How much is
transported away and metabolized in one of several directions?
These and others are good ones to expect students to come up
with. Probably someone in the old days determined where some
isotope went from a lanolin application. I'd be willing to bet
though that there isn't a decent study to determine whether that
isotope was still IAA (or whatever). So I guess I'm headed back
to blissful ignorance of what is a whole list of very good
questions!
By the way, in my OLD plant phys course we did the project
with peas and we used microdrops (old-fashioned microliter
pipettes in those days--heavy glass by mouth...:-O). I don't
now remember the volume used (25 uL?) but the drops were
applied between the two stipules at the base of the stump and
the base of the highest remaining compound leaf. The solution
was aqueous and contained some Tween 20 to cut the cuticle.
Years later I went to eppendorff pipette tips...easier and
safer but no better. You still don't know how much gets in,
etc. Also the drops rolled between the stipules and down
the internodes too frequently. Drops ended up on the soil,
on lower leaves, etc. etc. Frustration was HIGH!
Sometimes I think the good-old lanolin and toothpick routine
works best. If you think qualitative rather than quantitative
you can sleep at night with this exercise.
ross
______________________________________________________________
|
Ross Koning | Koning at ecsu.ctstateu.edu
Biology Department | http://koning.ecsu.ctstateu.edu/
Eastern CT State University | Phone: 860-465-5327
Willimantic, CT 06226 USA | Fax: 860-465-5213
____________________________|_________________________________
Plant Physiology is Phun!
/\|___/\ //\______COOH NH-CH2-CH=C-CH2OH \/OH
| | | | | | || //\___ \CH3 /\|/\\/\\COOH
\/ \/|\/| \\/ \ / N || N | |
/\ | |__|= NH | || || //\//\
| COOH \\ /\ / O
COOH H2C=CH2 N NH
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