Induction and Recognition of Polyploidy - by Wright
The Induction of Polyploids
- by Watts
Colchicine Information
Posted by PAXCO on March 21, 1999 at 11:41:04:
Polyploidy (favorable traits in Cannabis) has not been
shown to occur naturally in Cannabis; however, it
may be induced artificially with colchicine treatments.
Colchicine is a poi- sonous compound extracted
from the roots of certain Colchicum species; it inhibits
chromosome segregation to daughter cells and
cell wall formation, resulting in larger than average
daughter cells with multiple chromosome sets. The
studies of H. E. Warmke et al. (1942-1944) seem to indicate
that colchicine raised drug levels in
Cannabis. It is unfortunate that Warmke was unaware of
the actual psychoactive ingredients of Cannabis
and was therefore unable to extract THC. His crude acetone
extract and archaic techniques of bioassay
using killifish and small freshwater crustaceans are far
from conclusive. He was, however, able to produce
both triploid and tetraploid strains of Cannabis with
up to twice the potency of dip- bid strains (in their
ability to kill small aquatic organisms). The aim of his
research was to "produce a strain of hemp with
materially reduced marijuana content" and his results
indicated that polyploidy raised the potency of
Cannabis without any apparent increase in fiber quality
or yield. Warmke's work with polyploids shed
light on the nature of sexual determination in Cannabis.
He also illus- trated that potency is genetically
determined by creating a lower potency strain of hemp
through selective breeding with low potency
parents. More recent research by A. I. Zhatov (1979) with
fiber Cannabis showed that some
economically valuable traits such as fiber quantity may
be improved through polyploidy. Polyploids
require more water and are usually more sensitive to changes
in environment. Vegetative growth cycles
are extended by up to 30-40% in polyploids. An extended
vegetative period could delay the flowering of
polyploid drug strains and interfere with the formation
of floral clusters. It would be difficult to determine if
canna- binoid levels had been raised by polyploidy if
polyploid plants were not able to mature fully in the
favorable part of the season when cannabinoid production
is promoted by plentiful light and warm
temperatures. Greenhouses and artificial lighting can
be used to extend the season and test polyploid
strains. The height of tetraploid (4n) Cannabis in these
exper- iments often exceeded the height of the
original diploid plants by 25-30%. Tetraploids were intensely
colored, with dark green leaves and stems
and a well developed gross phenotype. Increased height
and vigorous growth, as a rule, vanish in
subsequent generations. Tetraploid plants often revert
back to the diploid condition, making it diffi- cult to
support tetraploid populations. Frequent tests are performed
to determine if ploidy is changing. Triploid
(3n) strains were formed with great difficulty by crossing
artificially created tetraploids (4n) with dip- bids
(2n). Triploids proved to be inferior to both diploids
and tetraploids in many cases. De Pasquale et al.
(1979) conducted experiments with Cannabis which was treated
with 0.25% and 0.50% solu- tions of
colchicine at the primary meristem seven days after generation.
Treated plants were slightly taller and
possessed slightly larger leaves than the controls, Anoma-
lies in leaf growth occurred in 20% and 39%,
respectively, of the surviving treated plants. In the
first group (0.25%) cannabinoid levels were highest in
the plants without anomalies, and in the second group
(0.50%) cannabinoid levels were highest in plants
with anomalies, Overall, treated plants showed a 166-250%
increase in THC with respect to controls and
a decrease of CBD (30-33%) and CBN (39-65%). CBD (cannabidiol)
and CBN (cannabinol) are
cannabinoids involved in the biosynthesis and degrada-
tion of THC. THC levels in the control plants were
very low (less than 1%). Possibly colchicine or the resulting
polyploidy interferes with cannabinoid
biogenesis to favor THC. In treated plants with deformed
leaf lamina, 90% of the cells are tetraploid (4n
40) and 10% diploid (2n 20). In treated plants without
deformed lamina a few cells are tetraploid and the
remainder are triploid or diploid. The transformation
of diploid plants to the tetraploid level inevitably
results in the formation of a few plants with an unbalanced
set of chromosomes (2n + 1, 2n - 1, etc.).
These plants are called aneuploids. Aneuploids are inferior
to polyploids in every economic respect.
Aneu- ploid Cannabis is characterized by extremely small
seeds. The weight of 1,000 seeds ranges from
7 to 9 grams (1/4 to 1/3 ounce). Under natural conditions
diploid plants do not have such small seeds
and average 14-19 grams (1/2- 2/3 ounce) per 1,000 (Zhatov
1979). Once again, little emphasis has
been placed on the relationship between flower or resin
production and poly- ploidy. Further research to
determine the effect of poly- ploidy on these and other
economically valuable traits of Cannabis is
needed.
Colchicine is sold by laboratory supply houses, and breeders
have used it to induce polyploldy in
Cannabis. However, colchicine is poisonous, so special
care is exer- cised by the breeder in any use of
it. Many clandestine cultivators have started polyploid
strains with colchicine. Except for changes in leaf
shape and phyllotaxy, no out- standing characteristics
have developed in these strains and potency
seems unaffected. However, none of the strains have been
examined to determine if they are actually
poly ploid or if they were merely treated with colchicine
to no effect. Seed treatment is the most effective
and safest way to apply colchicine. * In this way, the
entire plant growing from a colchicine-treated seed
could be polyploid and if any colchicine exists at the
end of the growing season the amount would be
infinitesimal. Colchicine is nearly always lethal to Cannabis
seeds, and in the treatment there is a very
fine line between polyploidy and death. In other words,
if 100 viable seeds are treated with colchicine and
40 of them germinate it is unlikely that the treatment
in- duced polyploidy in any of the survivors. On the
other hand, if 1,000 viable treated seeds give rise to
3 seedlings, the chances are better that they are
polyploid since the treatment killed all of the seeds
but those three. It is still necessary to determine if
the offspring are actually poly- ploid by microscopic
examination. The work of Menzel (1964) presents
us with a crude map of the chromosomes of Cannabis, Chromosomes
2-6 and 9 are distinguished by the
length of each arm. Chromo- some 1 is distinguished by
a large knob on one end and a dark chromomere
1 micron from the knob. Chromosome 7 is extremely short
and dense, and chromosome 8 is assumed to
be the sex chromosome. In the future, chromosome *The
word "safest" is used here as a relative term.
Coichicine has received recent media attention as a dangerous
poison and while these accounts are
probably a bit too lurid, the real dangers of expo- iure
to coichicine have not been fully researched. The
possibility of bodily harm exists and this is multiplied
when breeders inexperi- enced in handling toxins
use colchicine. Seed treatment might be safer than spraying
a grown plant but the safest method of all is
to not use colchicine. mapping will enable us to picture
the location of the genes influencing the
phenotype of Cannabis. This will enable geneticists to
determine and manipulate the important
characteristics contained in the gene pool. For each trait
the number of genes in control will be known,
which chromosomes carry them, and where they are located
along those chromosomes.
PAXCO
Author: ike loan
Subject: Colchicine Information
Description:
good post, cant remember where i got mine from, i tried it about 10 years
ago, its available in a pill
used to treat gout. thats what i used and i treated over 200 good seed
with it. i got 15 or so to sprout
and was real happy about that. but as they grew they were horrible looking
mutants. one had leaves
growing almost sideways and most were twisting and contorting. i think
i wound up with 3 that didnt
die. after 3 months of growing them i got rid of them, i didnt have any
that had the favorable
characteristics i had read about, and since its so poisonous i was a little
worried about an adverse
reaction in some way... im sure if i had continued i would have came up
with some with super growth
and yield. the possibilites exist to mutate the common dipliod cannabis
and develop a super
strain.that might grow very fast produce more branchs, and much higher
yeilds and the plant might
be indistinguishable from the normal looking cannabis. thanks and keep
up the good work... ike
Author: the Fool
Subject: Colchicine Information
Description:
About 25 years ago, when "head shops" were the rage, a bud and I bought
a book about growing. We
set up a couple of shop floros in a closet over 3 gallon buckets of SuperSoil
as suggested in the
book. Lights were left on 24/7 and we smoked the leaves. The book never
even explained how to
flower. The first batch was like hay. The book also talked about soaking
seeds in Colchicine derived
from the Autumn Crocus. It's readily available at nurseries in the
month of September. We bought a
bulb, squeezed it in a garlic press, added a little water and soaked the
seeds. The result was the
best smoke that he and I ever remember. And it was just leaves! Cloned
a closet full of the best plant for
each of us and kept the plants going for at least a year. We are convinced
that the difference was the
Colchicine but it could have just been an excellent seed to start
with. Wish I knew then what I know
now so I could report on the flowers but I can't.
Author: cowboy
Subject: Introduction
Description:
hi guys,i was once 13 and first trying my hand at growing,but that
was more than 25 years ago. and i
must add im slightly burnt but still smokin,ha ha.you guys just hang
in there were your at and youll do
fine.dont take any b.s. about being to young to do what your doing,age
is a frame of mind.i for one am
glad to see some young dudes in here.and the comment about the older angry
guys gave me a
pretty good chuckle.you young dudes seem to have hit the nail right on
the head(mine included).in the
days when i started, my first grow manual was a hand me down from one of
my brothers,it was
basically in cartoon form and was one that dealt with colchicine and grafting
MJ onto hops,things
have come a long way,and i believe with young and old blood mixing here
things will go a lot farther than
i could have ever imagined.i guess they already have.well this ramble was
caused by the good
feeling i got when i read you young dudes post.if i can ever help
just let me know.thanks for the
memories. later cowboy
Author: ike loan
Subject: Getting supplies?
Description:
never needed em but, you can make your own colchicine . like this, colchicine
is derived from
bulbs of the winter or autum crocus[colchicum]. mash the bulbs and
add an equal part of water.
strain through coffee filters or paper towels. soak seeds in the solution
and plant when they start to
germinate.cultivate as usual. only some of the seeds will become polyploid.
polyploids generally have
thicker stems, and the leaves are often unusually shaped, with uneven sized
blades or more blades
than usual. as the plant grows leaves should return to normal but still
have larger and with more blades..
the above was copied from the book mj growers guide by frank . it also
says it cannot guaratee one
can safely smoke colchicine treated plants as it is highly poisnous.
as for g.a. i read you could get that
stuff at a nursery. ive never used it though its poison too.. hope someone
else can help you with it.
p.s i think you can get the col. at nurseries too.
peace
Cochesene toxicality
Posted by ChronX on December 19, 1998 at 16:39:05:
i have heard that the use of the chemical "colchesene"in the forks
betwen leaves and soaking seeds in it gives the
plant a poly-ploid structure instead of bi-ploid.the catch
is that the 1st generation is toxic. i think that the 2nd is
too but i cant remember. anybody know?
Re: colchicine toxicity by Kohala
on December 19, 1998 at 18:23:14
Colchicine itself is extremely toxic, but it does not make
the plant treated toxic. There is no literature available that
I am aware of that describes work done with this chemical
on cannabis. Colchicine is used to create polyploidy in
many other plants and the result is often bigger yield,
and bigger fruit and flowers.
Re: Cochesene toxicality by loki
on December 19, 1998 at 17:52:09
The substance I believe you are reffering to is an alkaloid
called colchicine.Colchicine is extracted from the autumn crocus and is
approximately 5 times as toxic as potassium cyanide.
One of the more interesting sidelines of this substance is that
it serves as a mutagen when applied to cannabis seeds.
The mutation usually attributed to colchicine treatment is
a marked increase in potency. Whether this is true or not is yet to be
conclusivley proven.
The story goes, the U.S govt. was experimenting with improving
known hemp strains when they noticed THC. present in plants treated with
colchicine!This might just be folk lore but this is what I've read.
What I do Know is that colchicine is EXTEMELY toxic and not worth
screwing with unless you really know what you are doing. There are already
a ton of good kickass strains available don't fix 'em if they ain't broke.
Re: Cochesene toxicality by Chronic Cough
on December 19, 1998 at 22:45:48
Polyploidy in marijauna plants is not stable. Polyploidy
is when the cells contain extra chromosomes. Diploid is
the normal state that occurs in marijuana plants(one set
of chromosomes from the male pollen and one set from the
female pistil). Colchicine is poisonous and should be
handled carefully. It will cost $35/gram. If you treat 1000
seeds with colchicine and 10 germinate, they will probably
be polyploid. In other words, this substance is more
likely to kill your seeds than to make them any better.
Polyploid plants may or may not pass on this trait. I have
read many sources that say seeds from these plants will
be normal, but many say they will be like the mother
plant. These plants will often have extremely abnormal
leaf formation and will have thicker stems. As to making
the plant more potent...who knows. Look on the net. Search
"Colchicine" and "Marijuana"- you can view the
OPINIONS of other people.
Also, colchicine is a treatment of gout(in small amounts).
In concentration it would be a good idea to wear latex
gloves to prevent absorption through the skin. Do I have
to say birth defects?
Dont high with poisons play when!!
=========================================================================
Time 15:03, 3 Apr 1998
From Mr. Two-Footer (mrtwofooter@yahoo.com)
Does anyone know: Is it worth poisoning my
first generation with colcichine (to
disable the meieotic spindle apparatus) to
create polyploid plants (sometimes called
"Gold" varieties) or is this just a waste
of a generation (since you can't smoke
colcichine-poisoned plants)?If anyone has
tried this I would be very interested to
know their results.
========================================================================
Time 10:24, 5 Apr 1998
From insider
Can anybody here relate to 25 mg of colchocine
in 100 cc water, soaking a seed that
had sprouted the previous day, and soaking
for about 6 hours.If memory serves me
right, that produced good crop of what I think
was a potent polyploid that was some
of Konas finest in the mid 70's.I'm back,
and interested in swaping botantical info
and getting in tune with what is going in
in genetics.
Time 11:47, 5 Apr 1998
From insider
uncle Joe:
Colchicine is a chenmical that inhibits cell
division, but allows chromososmes to
multipily.Orchid growers in the past, when
I grew, used it to play with genetics, it is
like throwing the dice, one never knows what
will result.
Years ago I had heard that some growers were
painting colchicine in matu5e
flowering plants, and then not using that
plant, but letting it go to seed and planting the
seeds.
I knew no one with real botanical knowledge,
purchased some colchicine under
disguise as orchid grower, and tried the (if
memory serves me right) the 25 mg in 100
cc of water for about 6 hours, diring which
cell devision is haulted, but chromosomes
will divide.
The produce was killer, had about half dozen
plants, and it was one hit la la land.I
just for friends, lived in Kona, non commercial.I
moved and have not been able to
grow for many years, am now interested in
using, I am in need of medical use, so am
joining revolution. I would guess colchocine
is a controlled substance, but available
through chemical wearhouse and school labs.Cost
in 70's was abut $40 a gram.
Research in any university libraty will yiels
all necessary information, I wlll search on
net and get back, no library here.
Also, know of any chemical that will keep deer
from a pach?I have heard pool
chlorine, mothballs, any info appreciated.
Time 13:05, 5 Apr 1998
From Occam's Razor
In 1993 Colchicine was available for $6.80/100mg.
at my favourite
micropropagation supply house, however access
was limited to professionals, and
occasionally students and others who had legitimate
uses for it.
You can make your own colchicine by crushing
the bulbs of Colchicum
autumnale,(the plant that the chemical is
obtained from) commonly known as the
autumn crocus or meadow saffron, in a garlic
press and collecting the juice. The % of
colchicine will vary with the time of year,
plant genetics, and environmental factors,
but diluting one part bulb juice to three
parts distilled water should get a solution
strength of ~.05%, about ideal for use as
a mutating agent.
Colchicine is VERY toxic, wisdom should govern
it's use, WEAR RUBBER
GLOVES WHEN HANDLING!
grovver
posted September 05, 1999
08:31 AM
Does anybody know anything about KOLCHICIN, it is a poison. But applyed
on seeds in some ratio in water
for few hours makes them to mutate.
The result is a very potent plant, more likely the first generation is
used as a seed producer, the second
generation is prodicing smokeable budz, though the first generation is
SAFE to smoke....
Another sign of mutated seeds many bladed leafs, up to 17 blades!!!
Vic High
posted September 05, 1999 09:57
AM
Easiest way is to plant some fall purple crocus bulbs. Then squash the
bulbs and soak your seeds in the
juice. All natuaral colchicine!! Or you can pretend you have gout and get
a prescription from the doc.
But only do this with seeds you don't need, to be effective, the cochicine
is reported to need to be strong
enough to kill most of the seeds. Something like 1 in a 100 or 1000 will
survive. Of the survivers, it's a crap
shoot whether or not the mutation was a good one.
If you want more, check out the weedbase search that is featured here.
Try various spelling for cochicine
though, we all seem to spell it differently cholchicine, colchicine,
cholcine, kolchicine, etc, haha
iibro
posted September 10, 1999
01:21 AM
there is some good reading on this subject in a book i bought quite a while
back called the cultivator's
handbook of marijuana by bill drake. he goes right into this treatment
method. he explains three different
methods to treat marijuana with colchicine. in seed and as seedlings and
clones.
just so you know...... the colchicine extract is derived from the bulb
of meadow saffron which is a member
of the crocus family. the botanical name of meadow saffron is colchicum
autumnale. this is not a popular
plant but they should be in most nursery catalogues and no one should have
any trouble ordering
them.....hope this helps.
Vic High
posted September 11, 1999 11:03
PM
Hey Libro, good timing on your post. I was just reading today that I was
spreading a little mis-information
about using the fall purple crocus for getting colchicine. I was just repeating
what I was told, hehe. You
were closer to the truth, but also with a small mistake By coincidence
today, I picked up the September
issue of a local magazine called The Coastal Grower. In it, there is an
article on Colchicum, or the meadow
saffron as it's also called, and it's use in gardening. This is the plant
who's bulb contains colchicine.
"Botanically colchicums reside in the lily family (Liliaceae) like tulips
and lilies, not the iris family
(Iridaceae)as do crocuses."
iibro
posted September 12, 1999 09:52
AM
you are right vic. the meadow saffron is a plant from the lily family. thanx for the correction, my mistake.
Author: webfish179
Subject: MrSoul Colchicine?
Description:
MrSoul im sure you have read the works of
HEwarnike and understand it but could you explain it
to me ?The article is on the use of Colchicine to
create triploid and tetratriploid polyploid versions of
the diploid marijuana plant doubling the potency
what is Colchicine ?how is it applied?Is any of this
true or just crap?
Author: MrSoul
Subject: MrSoul Colchicine?
Description:
That's H.E. Warmke. He claimed to have doubled the
potency (measured by it's toxicity to fish) but his
methodology seems flawed to me. The colchicine
(EXTREMELY poisonous stuff) was more likely the
cause of the fish deaths than increased levels of
THC; which isn't very toxic even in very high
concentrations.My take on polyploidy is that it's
best left to research projects. I think the
uncertainty of positive results and possibilty of
poisoning oneself with colchicine make it a
dangerous waste of time for most growers. The art
of breeding cannabis is sufficiently complex without
the added complications of polyploidy. Read pages
70 - 75 of Tom Flowers' "Marijuana Flower Forcing"
and form your own opinion. I feel there is no need to
play games with polyploidy when there are so many
fine, potent strains available.
Author: A-1
Subject: MrSoul Colchicine?
Description:
Mrsoul already knows this, most plants we have are
poly's already! It happened in the early 80's. Some
breeders all read that book, baby. Including me.
Author: crane
Subject: MrSoul Colchicine?
Description:
No offense A-1, but I don't know if that's right. I've
never messed wih colchicine, but apparently even if
you overcome the difficulty of creating a tetraploid
plant in the first place, it's very difficult to keep it
that way. It's supposed to be a very difficult thing
to pass on to the next generation, and polyploid
plants even revert back to a diploid state all on their
own, so it would be difficult to propagate even with
cloning.My catalog from nirvana claims their master
kush is tetraploid, but it doesn't seem very likely to
me. Everything I've heard about polys sound like
they're unpredictable, plagued by abnormal growth,
slow to flower etc. Not ideal stuff for the home
grower.Do correct me if you have contrary info, I'd
be interested if a more up to date study has been
done than the ones I've seen.
Author: Kumquat
Subject: Bugs - FrankenQuat
Description:
Very interesting! I wonder if alcohol is penetrating
the tissues? (my guess is yes). Then this solvent
may be a very good carrier for the colchicine tabs i
have. I'm gonna have to do some 'speriments now!
Nurse, voltage please! But seriously, there is about
a truckload of things a person can do to mess with
plant genes, everything from alternating magnetic
fields to chemicals to ionizing radiation. Did you
know that Coleman gas light mantles are emmiters of
a fair amount of alpha particles? Not exactly
ionizing, but prolly the closesst i'll come to my dream
of cobalt-60 for quite some time LOL! But UV is
ionizing and it's easy to do a timed exposure
titration of pollen prior to pollenation. (try saying
that 3 times real fast!)I haven't put Richards seeds
in just yet. They will have to wait until later this fall.
On your advice of low germ rate, I think i'll add a
spritz of gibb acid to the soak water as that is
claimed to enhance germ rates of marginal
seeds.Good seeing you here and other places,
Jacker.
Author: jackerspackle
Subject: Bugs - FrankenQuat
Description:
I'd be concerned about Colemen's mantles modifying
MY genes!I think X-rays can be included in your
list...Clarke says that the key with colchicine is to
kill off most of the seeds - that way, the ones that
sprout are more likely to be mutated.. Seems like a
good approach with ANY mutagen, no?
Author: Kohjala
Subject: crocus bulbs/colchicine
Description:
Fall crocus bulbs are a natural source of the mitotic
agent colchicine, but a purer source is medical
grade colchicine. Colchicine is a drug used in the
treatment of Gout, and is available at low cost in 5
mg. tablets from any pharmacy. It does require a
physicians prescription, or the address of a mail
order pharmacy in mexico in mexico.You do havce a
lot of questions, and a trip to the local library to
look up books on introductory genetics would be
helpful.
Author: big man
Subject: Colchicine is highly toxic. It can be used in
Description:
plants to disable the meieotic spindle apparatus and
create a ployploid plant. You need to soak several
seeds tho, because some will die. If you search
"colchicine" on the www you might find a pamphlet
detailing how to use colchicine. I read it a couple
of years ago. The guy who wrote it was of the
opinion that the plant you used it on should not be
smoked (I tend to agree with Kohala) and also
stated that all future generations would be
polyploid. Polyploid plants are used in commercial ag
as well. I believe triticale, or winter wheat, is a
hexaploid plant, with 8 pairs of chromosomes,
making it exceptionally hardy.
Author: MrSoul
Subject: colchicine = Polyploids
Description:
Colchicine is a powerful chemical that can actually
affect the chromosomes. Seeds soaked in a DILUTE
solution of colchicine will MOSTLY die from it, but
the few survivors will have a conditon known as
"Polyploidy", which means that instead of the normal
TWO sets of 10 chromosomes, the plants could have
3, 4, or another number. The chromosomal
re-arrangement often causes strange traits like
gigantic plants, oddly coloured plants or highly
potent ones as well as usually causing such plants
to be sterile. The germination rate of reated seeds is
EXTREMELY low(~5% survivors). Not too many
people see a future in using colchicine in MJ
breeding. Experiments already done by Warmke, et
al. were inconclusive. There are many POTENT
strains available already and colchicine is VERY
DANGEROUS to humans...the risks outweigh any
slight possibility of creating a special plant are not
worth it. This substance should be left to the
scientists to play with, it's not for amateur pot
breeders IMO.
Author: Moonbeam
Subject: colchicine
Description:
MrSoul pretty much has it right concerning
colchicine, I seen the same episode on Quincy a
few years back...where kids were dying smoking the
pot sprayed w/ colch.? Kinda HOLLYWOOD, but I
have read extensively about plant mutations & when
you treat the seeds it is not always potency that is
affected...poly=several, and when you start playing
with the chromosome to mutate, nothing is
exact...another method that has been used to
polyploid is radiation, simply X-ray your seeds! I
ordered a book on plant mutations from the
inter-library loan while I was in prison & of te
different methods used, this seemed the safest &
actually the most beneficial for the benefits we wish
to achieve as MJ cultivators. I would be real
interested in hearing from anyone who try's
this...once again the chromo. will go poly. making
the signal to produce, 4 to 5 times the normal
ratio...could have huge plants could have extremely
potent, could have wierd-looking hermaphrodites
An excerpt from:
Wright JW. 1976. Introduction to Forest Genetics. Academic Press, New York. pages 415-417
Induction by Colchicine and Podophyllin
These chemicals are the most common means of inducing polyploidy.
They do not affect chromosome division as much as they inhibit the
spindle fiber mechanism by which divided chromosomes are drawn to
the poles. Thus the chromosomes divide but do not pass poleward and
a 2n cell becomes 4n.
Of the two chemicals, colchicine is used most commonly. It is usually
applied as a weak (0.1 to 0.5%) aqueous solution for a period of several
hours or several days. It is effective on any actively growing tissue,
such as a root tip or germinating pollen grain. However, the goal is
a tree or branch capable of producing 4n flowers. To achieve that goal
it must be applied to an actively growing apical meristem, such as
in
a germinating seed or an actively growing bud. Germinating seeds can
be soaked in a colchicine solution. Growing buds can be surrounded
by colchicine-soaked cotton. Einspahr (1965) obtained 50 4n aspen seed-
lings by soaking entire female catkins in a 0.3% solution for 1 day
at
a time when the fertilized embryos consisted of a few cells. At the
Hessian Forest Research Institute, good results have been obtained
by
treating young seedlings having only one leaf with a 0.3 to 0.4% colchicine
solution for 72 hr (Anonymous, 1975). -The third year after treatment
approximately 40% of the seedlings were polyploid or mixoploid.
An actively growing apical meristem in a germinating seed or bud
consists of two self-perpetuating tissues, the outer tunica and the
inner
corpus. These in turn differentiate into other meristems, such as leaf
primordia, flower primordia, and cambium. The exact meristems derived
from the tunica and corpus, respectively, vary among groups of trees.
Both the tunica and the corpus consist of several dividing cells.
It is relatively easy to find a concentration of colchicine which can
cause chromosome doubling in a few cells of the tunica, but it is difficult
to obtain a concentration that will cause simultaneous chromosome dou-
bling in all cells of both layers. Trial and error is the best method.
The best concentrations and times are those which result in some mor-
tality and in a high percentage of seedlings or shoots with gross abnor-
malities. The best results have been obtained with small-seeded species,
such as the poplars, willows, ehns, birches, and alders. In these species
it has been easiest to obtain good penetration into both tunica and
corpus.
Recognition and Treatment of Polyploids after Colchicine Treatment
The poisonous effects of colchicine seem to last for several cell
generations, and newly induced 4n tissue seems to grow more slowly
than surrounding 2n tissue. As colchicine rarely acts on all cells
in a
growing point, artificial induction usually results in mixtures (called
mixoploids or chimeras) of 2n and 4n tissue. These mixtures may be
of
various types, i.e., one branch 4n and another branch 2n, top of leaf
2n
and bottom of leaf 4n. If part of the top of a leaf is 4n and the bottom
is faster growing 2n, the leaf curls upward; if the reverse is true
the leaf
curls downward. Thus, misshapen leaves, crooked branches, and general
growth abnormalities are the first indications of successful colchicine
treatment. The abnormalities gradually lessen and totally disappear
as a
branch or tree becomes wholly 4n or 2n.
After the disappearance of such abnormalities, 4n cells usually have
twice the volume and 20 to 25% greater length or width than 2n cells.
Such increases in cell size are detected most easily in stomatal guard
cells, which can be measured at magnffications of about 100 X with
a light-reflecting microscope. Stomatal measurement is a quick way
to
determine whether all or most of the leaves on a branch are polyploid.
Leaf size is also a convenient way to determine approximate chromo-
some number.'ln conifers generally, polyploidy is usually accompanied
by a decrease in rate of cell division so that leaves are apt to be
small in spite of the increase in cell size. In many hardwoods there
is no change in rate of cell division so that leaves and other organs
are apt to be larger than normal.
However, since the tunica and corpus give rise to different organs,
the presence of 4n leaves on a branch is no guarantee that the branch
will produce 4n flowers. Thus final proof must come at time of flowering.
This can be done by examination of pollen grains (4n grains are usually
larger than 2n grains), by actual chromosome counts in dividing gameto-
phytic cells, or by actual chromosome counts of seedlings produced
by the branch.
As already noted, there is a tendency for newly produced 4n tissue
to grow more slowly than surrounding 2n tissue. Therefore complete
reversion to the 2n condition is common. To overcome this tendency
it is advisable to observe treated seedlings at frequent intervals,
identify
2n and 4n sectors quickly, and remove 2n buds or branches.
Anonymous. 1975. "Hessische Forstliche Versuchsanstalt, Jahresbericht
1974." Hann.-Munden.
Einspahr DW. 1965. Colchicine treatment of newly formed embryos of
quaking aspen. Forest Sci. 11,456-459.
An excerpt from:
Watts, Leslie. 1980. Flower & Vegetable Plant Breeding Grower Books,
London
The artificial induction of polypioids as recommended under (f) above
can be
practised in a number of ways, using colchicine at a concentration
of 0.5 to 1.0
per cent. At these concentrations, the length and method of exposure
to
colchicine are critical and can only be satisfactorily decided by experimentation.
Since actively growing plant tissue must be treated, the most common
methods
of using colchicine are;
(a) by applying it to seed which has already imbibed enough water for
germination - by transferring seed from a water-soaked to a colchicine-soaked
filter paper for approximately 24 hours.
(b) by applying droplets of solution to the growing points of seedlings.
(c) by applying colchicine in lanolin paste to growing points of seedlings.
(d) by applying droplets as in (b) or (c) to active buds on mature plants
- but
removing all untreated bud growth.
Mixed tissues (diploid as well as polypioid) may arise after treatment
and for
successful results it is important to remove all shoots of the original
non-
polypioid, more vigorous type.