Colchicine - an alternative?


I have noticed that colchicine still seems to be the chemical of choice for
inducing tetraploidy in irises. During the past few years, most lily
hybridizers have changed to using oryzalin. It is reported to be both
considerably safer and more effective.
I am enclosing a copy of a paper by Dr. Arthur Evans from a recent yearbook
of the North American Lily Society in tne hope that it may be of interest to
iris hybridizers. The techniques would require modification but it may be
worth pursuing.
This paper is copyrighted by the NALS. Permission has been granted by NALS
and the author for posting to the iris list.
--------------------------------------------------------------

Cooking Up Tetraploids in the Kitchen
Arthur Evans, Arkansas =20
(NALS Yearbook 1993 No. 46, pg 10-12)

My wife, Crow, is very good about sharing the kitchen with my
non-cooking projects.
You know, ordinary things like brewing beer, sterilizing soil in the
oven, making nutrient medium for embryo cultures, "nuking"
critter-infested bulbs in the microwave, and storing an unreasonable
number of lily bulbs in the refrigerator. She does raise an eyebrow,
however, when I start playing with poisonous chemicals in the food-prep
area. That's one reason I decided to switch from colchicine to oryzalin
for my conversion attempts as per Dr. Jaap van Tuyl's article in the
1990 NALS Yearbook. Oryzalin is alleged to be much less toxic to people
than colchicine. By the same token, it is supposedly more effective in
interrupting normal mitosis in rapidly dividing lily cells which can
result in converting a diploid lily scale into a tetraploid scale
bulblet.=20

It works. I've done it. Here's how.=20

Oryzalin is sold by Dow-Elanco as a pre-emergence herbicide under the
name Surflan AS.
It comes in one gallon jugs of 40% active ingredient at a cost of about
$100 per gallon. I got mine from Hummert Co. Inc., the well-known
horticultural supply company. Since Dr.
Van Tuyl advises the .005% strength treatment solution, I calculate that
one gallon would make a fair-sized swimming pool of treatment solution,
more than enough even for an obsessive-compulsive gardener like me. I
might even use some of it for the purpose stated on the label. Seriously
though, if my math is correct, .5 ml, of 40% oryzalin dissolved in 4
liters of water makes an approximately .005% oryzalin treatment
solution. That sounds weak, but it is strong enough. Don't be tempted to
make it stronger. In fact, next time I try a batch of conversions, I
will make it half strength. It burns some scales so severely that they
never form bulblets.=20

The oryzalin is hard to dissolve in water. I pre-dissolve .5 ml (easy to
measure with an injection syringe from the drugstore) in .5 cup of warm
isopropyl alcohol or vodka to which I have added 2 tablespoons of 99%
DMSO (dimethyl sulfoxide) from the veterinary drug shelf of my local
farmers' supply store. The DMSO is a strong organic solvent known to
enhance the absorption of drugs through skin. It smells awful, and it
couldn't be good for you, more than enough reason to wear latex rubber
examination gloves and a moisture-resistant face mask. Your family
dentist will be glad to give you plenty of these. Gradually add warm
water in increments to the oryzalin and solvent mixture in a clean milk
jug with violent agitation and incantation. The incantation is optional,
but the looks on the faces of the grandchildren are priceless. I presume
this solution is fairly stable, but I make up a new batch after treating
2 batches of scales. =20
Use scales which have started to show a tiny ridge or bump of rapidly
dividing cells or "callus" around the broken edge at the base of the
scale. If you incubate scales in the moist sphagnum in a zip-lock bag at
80=BAF it takes from 1-4 weeks to get to this stage of development, Don't
wait until the callus develops a tiny bulblet. The more tissue the drug
has to penetrate in order to reach the actively dividing meristematic
cells, the less likely you will be to get a completely convened tetra
bulblet. If a bulblet arises from a mixture of converted and
non-converted cells we call it a mixoploid. These plants revert to
diploid status since diploid cells grow and multiply faster than tetra
cells and tend to take over completely over time.=20

Now, for the actual treatment, bear in mind that the drug only works on
cells that are actively dividing, so it makes sense that you want to use
a temperature high enough to stimulate rapid cell division and a time
long enough to intercept lots of divisions. I don't know what the
optimum temperature or time is for most lilies. It may vary with the
genetic background of the cultivar you are working with. What I have
done is soak scales in converted quart jars in the hot water heater
closet which maintains about 85=BAF for 6 hours. Make sure your jars,
scales, and solution are all close to your chosen treatment temperature
before you start. Due to the amount of burning of the scales in previous
treatments, I may reduce treatment temperature to 75=BAF or 80=BAF next
time.=20

After treatment, rinse scales with several changes of fresh water to get
rid of as much of the drug as possible. Handle treated scales only with
gloved hands. Be a fanatic about cleaning all the equipment and food
preparation surfaces as soon as you have placed scales into labelled
plastic bags of slightly moist sphagnum or vermiculite. I use a Sharpie
pen to label the bag with the cultivar name, date of scaling, date of
oryzalin treatment, and any variation from the standard treatment. This
year I used Zip-lock vegetable bags with ventilation holes. All bags
then go into a garbage bag with loosely closed top to keep the small
bags from drying out. The big bag then gets incubated at 75=BAF to 80=BAF
from October or November until bulblets are well-formed, usually about
the end of February or March. At that time the bulblets are vernalized,
simulating winter chill, for 3 months in a refrigerator set at 35=BAF.=20

When the vernalization is complete, I inspect the bulblets individually,
looking for physical characteristics which may indicate conversions.
Most bulblets will look quite ordinary because they are still
unconverted. If you said the aforementioned incantation correctly,
however, you may find a few (5%-10% at best) bulblets which have
obviously shorter, thicker scales which stand apart from each other at
90 degrees or more. I plant these bulblets in large pots and protect
them from all harm. A high percentage of these odd-looking bulblets will
soon show the large stomates (.005 inches long) on the underside of the
leaf which indicate conversion to tetraploid status. Ordinary looking
bulblets are planted in rows in the garden. Their stomates are measured
in mid-summer to pick out any tetras that slipped by in earlier
inspection. I don't have much space so I don't grow the diploids to
maturity. When large enough to bloom, test the converted tetras both
ways in crosses with fertile seed-grown tetras. There will be variations
in the fertility between different conversions of the same cultivar. Due
to the low number of successful conversions in each batch, I would
certainly advise that you start with at least 100 clean, healthy scales
of each cultivar. If you have large, blooming-size bulbs to start with,
you can usually harvest 10-20 usable scales from each one. Scaled cores
can be replanted to grow again.=20

In dozens of attempts I was only able to convert one cultivar, old 1b
pink 'Gypsy' with colchicine. In the fall of 1993 I attempted to convert
8 cultivars with oryzalin and I believe I have succeeded in converting 4
of them. They are 'Yellow Star', 'Connecticut Star', 'Shirley', and
'Nutmegger'. These have not been tested for fertility with tetras or
chromosome-counted, so I will have to wait for these tests to confirm
what my stomate measurements and observations of other plant
characteristics lead me to believe.=20

In the fall of 1994 I treated about 20 cultivars and seedlings. The
scales are producing=20
fair crop of bulblets, a few of which look like valuable tetraploid
breeding prospects. I would be interested in corresponding with and
assisting anyone who wants to experiment
with oryzalin conversion of lilies. No doubt the method I use can be
improved. If we pool our data, we might learn a lot.=20

POSTSCRIPT.

As of 12/95 few of the bulblets from the conversion attempts of last
fall have sprouted, probably due to insufficient vernalization time. I
will chill bulblets for 3 months next time. Of the cultivars which did
sprout, stomate measurements of 'Allegra', 'Nepal' and 'Henry VIII'
indicate conversions. Hopefully, more cultivars from the 1994 attempts
will sprout in the spring of 1996 and show large stomates as well.=20

One of the 1993 conversions, 'Connecticut Star', did bloom this summer
Its pollen was used on several tetra Asiatics, including tetra
'Nutmegger' and some of my own seedlings. All produced good seed,
another indication of conversion. =20

I believe there will always be a place for excellent diploid lilies.
Many of the best  garden genotypes are still in the diploid gene pool.
Getting those superior genes into the polyploid gene pool is a
worthwhile goal for amateurs and pros alike. Some of these
diploids can be used with tetras in 4 X 2 crosses if they produce
significant amounts of pollen. Others may produce a few triploid embryos
when pollinated by tetras in 2 X 4
crosses. Many diploids however, refuse to participate in such liberal
nonsense as  crossing with polyploids. In such a predicament, the only
solution is to convert the diploid to tetraploid status. There is much
to do, and though it is time-consuming, it is not too difficult for
kitchen technology. Hopefully, your spouse will be as understanding as
mine has been.

--------------------------------------
Postscript from JM
I have the citations for the original papers by Dr. Jaap van Tuyl and will
make them available to anyone who is interested.


John Montgomery
monashee@bcgrizzly.com
Vernon, BC      Where we had 8" of snow yesterday and that is more than the
total                 for March for any of the past 20 years. As of now I
have put my                 snow shovel in storage.



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