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On the use of colchicine to induce polyploidy
Colchicine has been used for many years to induce
the formation of polyploidy in orchids (and other plants).
Polyploids are plants that have multiples of genes; that is
to say, if a plant has n chromosomes, application of
colchicine may induce a plant to have 2n, or more, chromosomes.
This causes many plants to express their genes differently;
in many cases, these expressions are highly desirable.
Colchicine is a nasty chemical; a materials safety
data sheet (MSDS) reveals the following:
Highly toxic; may cause cancer; may cause heritable
genetic damage; very toxic by inhalation, skin contact, or
ingestion; possible teratogen; it targets the liver and kidneys,
and damages the bone marrow, nerves, and cardiovascular system;
mutagen; the oral human lethal dose is calculated to be 86
micrograms (thousandths of a milligram) per kilogram body weight by
one account. This is a nasty, lethal chemical with a rap sheet as long
as my arm. The list of effects with respect to exposure is impressive
to this editor (a chemist by training, geologist by insanity); it
is not suggested that colchicine should be used in the home- a
laboratory setting with proper attention to all safety recommendations
as put forth by the source of the colchicine is highly suggested.
With that in mind, here is a suggested pathway for those
who understand polyploidy, and want to utilize colchicine to
induce polyploids. Play with it at your peril.
Aaron J. Hicks, Editor
4/8/95
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Robert M. Hamilton
Subject: Polyploid orchids via colchicine
I have successfully converted many orchid species and primary crosses from
2n to 4n (amphidiploids), using a colchicine protocol I devised. Amongst
the genera converted are ada, bollea, brassia, cochlioda, dendrobium,
masdevallia, maxillaria, miltonia, odontoglossum, oncidium, rossioglossum
and zygopetalum. My method differs from perviously published methods. I will
append my protocol to this e-mail. [ed. note: it's all here!] Note, no growth
salts are used in my colchicine protocol. Conversion rates are good
(Don Wimber, personal communication).
I confirm ploidy conversions from diploid (2n) to tertaploid (4n) by chromosome
counting root tips. I avoid a simpler method touted for testing for
conversion, i.e. looking at guard cell area. In theory, guard cells increase
in area in proportion to a plants ploidy. I have not found this theory
to be true. In fact, in one plant crop I looked at, (liatrus) guard cell
areas decreased while guard cell numbers increased in plants with higher
ploidy.
I have not worked with Paphs; however, disparent crosses between Paph
subgenera are common. Such crosses have limitations for future breeding.
Converting Paph primaries to 4n could be of significant value for increasing
fertility and open new pathways in breeding.
My conversion rates are about 25%. This is hard to measure because I carefully
select the plants I replate, looking for the 4n's. I treat plants when they
are protocorms. Conversion rate seems more dependent on the stage of
development of the protocorm at the time of treatment than colchicine exposure
time.
The scientist who deserves great credit for work with orchids, chromosome
number and ploidy conversion is Dr. Don Wimber. (Wimber's work is
inspirational as is Don for his modesty). Don Wimber was recently honored
with a gold medal from the Cymbidium Society of America. Alan Moon of the
Eric Young Foundation credits Wimber with "sorting things out" at the
Foundation. Alan has put Wimber's breeding theories to practice with stunning
results. Alan has grown and bloomed a number of crosses I have colchicine
treated (CT'd). Plestead Orchids is currently doing flasking and ploidy
coversion for the Eric Young Foundation. I am flattered Ian and Janet
Plestead will be trying my protocol after reviewing results.
Bill (wgheckeroth@ccgate.hac.com) notes on a previous Orchid List:
"The only cross I have seen flower was a sibing of delenatii that had been
treated with colchicine. The flowers were much smaller than typical, the
flowers maybe slightly more intense pink, but the fragrence was much stronger
than the typical delenatii. Quite a few of the ones I saw were deformed."
Alan Moon showed me slides of Paph rothchildianum crosses he has CT'd.
He report these grow faster than the 2n's - 5 years to flowering size . I have
noticed significant differences in the growth rate and flower size of
CT'd plants. Often, converted plants are initially chimeric containing both
2n and 4n tissue resulting in irregular, distorted bulbs and leaves. Plants
usually normalize once one tissue type prevails. In time, plants often
"straighten out" to either a stable 2n or stable 4n.
Blooming results of converted plants are not always consistent. CT'd
Masdevallia Patricia Hills grew at the same rate as untreated plants.
The flowers were significantly SMALLER. CT'd Masd. igneas had much fatter
roots and smaller leaves but similar flower size between 2n and 4n. CT'd Masd.
tovorensis had short round leaves, huge flowers but lower flower count. On
average, polyploid oncidinae grow MUCH FASTER than diploid. Flowers are bigger,
rounder, fatter and darker. I have a group of Odm hallii side-by-side and the
4n's outpreform the 2n in growth and shorter flowering times. Milt
phalaenopsis are stunning once converted. 4n Disas are nothing short of
incredible. Their stem are MANY many times thicker and about half the height.
The flowers are bigger and MUCH rounder. The leaves look like a succulent
rather than a Disa and form a symetrical crown. Dendrobium cuthbertsonii I
treated have taken several awards. (Regretably, these plants were shown and
not labeled as CT'd plants by the grower . At present, there is currently no
way to delineate 2n vs. 4n species in orchid nomenclature so natural
cuthbertsonii will be compared by judges against man-made mutants).
I propose we capitalize species which have been converted; Dend cuthbertsonii
will be written Dend Cuthbertsonii after colchicine treatment.
As for handling and safety, colchicine is ranked a mutagen and highly
toxic by ingestion. It is not ranked as a carcinogen. [** ed. note: this
is still under dispute; although in a day and age when anything on the
supermarket shelves is likely to kill you eventually, it's still not a
good idea to snort colchicine, and then go back to the company and complain
when you get lung cancer. It has shown to cause genetic damage to fruit
flies according to the EPA (1988), as well as mammalian micronuclei, and
mouse sperm morphology (ibid). **] Colchicine should be worked with in a
fume hood. Colchicine has been used to treat gout. Doctor friends tell
me alternative therapies are now in use. Colchicine is degraded by
exposure to light. Reports that colchicine is heat labile at standard
autoclave temperatures are not confirmed by several different analytical
tests. Conclusions that colchicine activity decreases after autoclaving may
be in error because of poorly understood reactions between colchicine and
media salts, i.e. artifact of experimental protocol.
My research is self-funded. Regretably, full time employement makes for
little spare time (try no spare time) so I do not do flasking or plant
tissue culture for others and do not sell plants.
Here is my colchicine treatment protocol for those interested:
Protocol For Colchicine Treating Protocorms
Make a stock solution of .5% colchicine weight by volume by adding 1 gram
of colchicine to 200 ml. of distilled water. This stock solution should be
cold filtered or autoclaved and stored in an amber bottle protected from
light. I keep my solution refrigerated. Colchicine stock solutions are
subject to contamination by fungi. (Note: colchicine is ranked highly toxic.)
Make water/agar media by adding 10 grams of agar to 1 liter of water
(note: no growth salts or sugar is used). Fill pint jars with 30 ml
of water/agar media. Autoclave and allow to partially cool under a laminar
flow hood. Before the water/agar gels (~ 40C), add 3 ml of colchicine stock
solution cold filtered through a .2u nylon syringe filter, into each jar.
Final colchicine concentration will be ~.05% w/v. Cap with a solid lid.
Cover jars with aluminum foil to protect from light.
Choose protocorms for conversion at a development stage when they have
chloroplasts (i.e. greened up). For Odonts, diameters are typically
2-3 mm with the signs of a leaf primodia just beginning to develop.
Treat protocorms by placing them on this colchicine substrate for 3-5 days.
After treatment, remove and spread onto replate media. A simple hook is
all that is needed to remove the protocorms from the colchicine media
which has been made stiff on purpose. I do not rinse the protocorms off.
I "plow" protocorms into the surface of the replate media to make
sure protocorms are in close contact with the media.
There is a long recovery period for treated protocorms before they resume
growing. Some or many will die (sometimes all them die). After growing
on I select plants for final replate. When replating, I search for plants
with the bluntest leaves (rounded ends versus pointed ends) for final replates.
Polyploid Odonts will typically grow as fast or faster than diploid plants.
I mention this fast growth because it came as a surprise to me. Precedent
growers told me to always save the slower growing seedlings because,
"they're the tetraploids" nonsense. Conversion rates are around 25%.
Bob Hamilton - Nov 94
Updated June 1995
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Following added on 7/23/96
From markdim@azstarnet.com Wed Jul 17 23:10:54 1996
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To: Aaron Hicks <ahicks@nmt.edu>
Subject: Re: Colchicine
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Aaron,
Attached is a file (POLYPLOI.ASC) describing a colchicine protocol I found
in the nurseryman's journal. The method avoids treating the roots, which
are apparently more easily killed than the apical meristem. The method is
for seedlings, but I heard of a Bougainvillea breeder in New Orleans who
uses it on cuttings with success.
Mark
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Excerpt from: Kehr, August E. Woody plant polyploidy. American
Nurseryman, Feb. 1, 1996, pp 38-43.
This method avoids getting colchicine on the roots, which are
much more sensitive than the apical meristem. This sensitivity
is why the traditional method of soaking seedlings germinated on
culture plates results in such high mortality.
Polyploidy step-by-step
1. Make a 0.5% stock solution by dissolving 500 mg colchicine in
100 ml water. By making this stock solution using the entire
colchicine bottle, there is no need for an analytical scale to
weigh out tiny amounts. The stock solution will keep for years
in a refrigerator. To prevent mold growth, place a tiny crystal
of paradichlorobenzene (moth ball) in the solution.
2. Dilute 20 ml stock solution in 180 ml water to make a 0.25%
[sic] solution of colchicine. [If my math is correct, this would
make a 0.05%???] To this add 1 ml dimethyl sulfoxide (DMSO) and
0.1 drop of surfactant (dilute 1 part liquid detergent in 9 parts
water and add 1 drop of this to the colchicine solution).
The DMSO and surfactant both help the colchicine to
penetrate the cells.
3. Plant seeds in the normal manner. As soon as cotyledons begin
to expand and before true leaves are visible, fine-mist with the
colchicine solution. Spray until a small droplet collects
between the cotyledons.
4. Mist seedlings of small seeds twice daily for 7-10 days, until
true leaves appear. Mist those of larger seeds for 2-3 days.
This works best when the foliage doesn't dry out; the
seedlings should be wet most of the day. Therefore keep the
humidity as high as possible.
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Mark Dimmitt
Tucson, Arizona USA
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Date: Fri, 24 Oct 1997 16:39:28 -0700 (PDT)
From: "Robert M. Hamilton" <bob@argon.eecs.berkeley.edu>
Subject: [16739] Colchicine/Phrags
>Date: Thu, 23 Oct 1997 18:23:23 -0600 (MDT)
>From: Knight Tony <aknight@vagus.vth.colostate.edu>
>Subject: [16721] Colchicine &Phrags
>Has anyone used colchicine in phragmepediums with success and would like
>to share their methods? I would appreciate any help you can give me.
Dr. Don Wimber, the man who has done the significant work with colchicine
and phrags passed away a few days ago in Sydney, Australia. I do not know
enough of the details of Don Wimber's life to sufficiently eulogize Don.
Don was an unassuming scientist of great skill who made seminal
contributions to orchids and orchid hybridizing, particularly cymbidiums.
His latest work was with phrags at the Eric Young Foundation.
I met Don Wimber a few years ago in Australia and he was helpful in
guiding me with my work on colchicine and odontoglossums. We corresponded
via e-mail. I saved every one of his replies and treasure his lucid
guidance. Don was very patient with me -- a not so scientific engineer.
In response to your question:
I have done a large amount of colchicine conversions. My protocol has been
published via the OLD. Here are additional iterations. Please note that
colchicine is considered VERY TOXIC:
I use cold filtered (.2u) colchicine .05% weight/volume in water. I
add a small amount of ethanol to dissolve the colchicine from the
container and then add this to deionized water.
I treat protocorms after they have reached spheres about 2-3 mm in
diameter. I prefer to wait until I see a leaf primodia begin to
emerge from a "ballast" volume of cells.
Time: 4-7 days
In the light of my flask shelves (300fc)
Conc: .05% w/v in di water
Methods that have worked:
1) Backfill a mother bottle with 2-3x the depth of agar with .05%
Wait five days.
Decant off colchicine
Rinse protocorms with sterile di water in a sterile stainless sieve
Place on replate media
2) Make a stock solution .5% w/v
Make a 10g/l water/agar media (no salts)
Place about 50 ml in a mason jar, autoclave
While cooling under HEPA add 5 ml of stock solution
Place protocorms on top of this media for 5 days
Replate onto replate media
3) Place protocorms in a sterile jar.
Cover with .05% c in water
Wait 5 days
Rinse with sterile di water
Place on replate media
Bob Hamilton
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