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OfflinePsiloman
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Registered: 04/11/03
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Inducing polyploidy : A Simple Guide. (Make your own "species")
    #4233548 - 05/29/05 03:42 PM (12 years, 4 months ago)


1. The Dicitonary Definition of POLYPLOIDY

pol?y?ploid Audio pronunciation of "polyploidy" ( P ) Pronunciation Key (pl-ploid)
adj.
Having one or more extra sets of chromosomes: a polyploid species; a polyploid cell.


2. The simple result of following the document

A more fancy title would have been "Create a species of your own,well,kind of!". You think that your anandenanthera plants are special? What about your salvia? Well there are indeed special but if one is up for experimentation here is a very interesting idea.

Polyploidy: In other words increase twofold the genome of your plants! This could lead to higher alkaloid production from what i gather so far...As well as making the plant exhibit a "multiplied" (read bigger) phenotype in all its traits! The methods giver are mainly for seeds..What about plants that set not-tha-viable seeds (Read :Psychotria Viridis) or plants that never set seeds or if they do they are infertile? The method can be applied also to plantets grown on agar culture!!! Also a very good candidate to be tried on are Salvia Divinorum plantets: The reason salvia does rarely produces seeds and even more rarely produces FERTILE ones is that during the meiotic procees for making pollen and female oocytes some chromosomes cannot "pair up" and thus are not subjected to normal meiotic procedures.This happens because salvia divinorum is most propably a hybrid that its chromosomes bear little homology to one another.Maybe it would be a good idea to match up its chromosomes with some more,now lemme think where did i put my genetic automanipulator kit...Oooops,wrong century! We can suffice ourselves to doubling up the chromosomes and see what happens!

Also ,if one uses plant culture methods on antheres of plants he can develop the haploid counterpart of the plant that then he can subject to methods inducing polyploidy so he can get a plant which is homozygotic to all its traits!


Of course it goes without saying that this proccess is one for responsible adults that understant the term "safety" as well as simple instructions such as "wear your fucking gloves"

Enjoy this passage i received today which explains the procces:

Quote:

Surflan Seed Conversion Method
Kevin Vaughn Method USDA
A. Reagents

Note: ( Label all reagents with name, conc., date made, expiration date, and storage temperature. )
(1) Preparation of 10M Oryzalin solution

(i) Oryzalin -99.9% pure (Molecular Weight - 346.36)
Also known as 3,5-dinitro-N4N4-dipropyl-sulfanilamide.
Pure reagent available from Elanco Products Co., Division of Eli Lilly & Co.

(ii) Stock Solution - Using 99.9% pure Oryzalin make a 10 M solution in DMSO.
Dissolve 0.3463 grams Oryzalin in DMSO ( Dimethyl Sulfoxide ) and dilute to 100 mls with DMSO.

(iii) Working solution -10 M (10-5 M solution = 3.4636 mg/L)
Dilute 1 ml of stock solution to 100 mls in Distilled or D.I. Water. ( 1-2 mls DMSO may be added if desired.)


. (2) Preparation of 10M Oryzalin solution using Surflan AS

(i) Surflan AS - sold by Dow-Elanco as a pre-emergence herbicide under the name Surflan AS.
Purity of oryzalin in surflan AS is 40.4% , thus stock solution should be adjusted accordingly.

(ii) Stock Solution - Using 40.4% purity Oryzalin make a 10 M solution in Distilled or D.I. Water.
Dissolve 0.857 grams ( approx. 1 ml) Surflan AS in Distilled or D.I. Water and dilute to 100 mls.

(iii) Working solution -10 M (10-5 M solution = 3.4636 mg/L)
Dilute 1 ml of stock solution to 100 mls in Distilled or D.I. Water. ( 1-2 mls DMSO may be added if desired.)

B. Procedure

1) Germinate seed in a container with wet filter paper or paper towels. An alternative method under less
sterile conditions would be to germinate seeds on Pro-Mix or equivalent.

2) Germinate seeds under lights - 24 hours on.

3) Treat seeds 5-8 days from start of germination - seeds must show germination.

4) Treat germinated seeds for 24 hours in 10 M working solution of Oryzalin in the dark. Use glass
containers or glass petri dishes, plastic may absorb the oryzalin. Also do not use cheesecloth, as it may
also absorb the oryzalin.

5) After 24 hours the seedling roots and the base of the growing shoot should be swollen, as an indication
that treatment was effective.

6) Rinse seeds wrapped in cheesecloth or pantyhose for 4 hours under slowly running water.

7) Transfer to promix or similar growing medium.. Plant at surface of mix, but not too deep as seed is
prone to rot. Cover with plastic bag to prevent moisture loss.

Cool A cool 72 F dimly-lighted area ( 1/10 th full sunlight area ) works well until good growth occurs.


Subj: Preparing Solutions of Colchicine and Oryzalin / Surflan, from Rick Grazzini
Date: 10/11/99 10:19:13 AM Atlantic Standard Time
From: harryd@KREATIVE.NET (Harry Dewey, Beltsville, Maryland USA)
Sender: ALPINE-L@NIC.SURFNET.NL (Alpine-L, the Electronic Rock Garden Society [postings copyright by authors])
Reply-to: ALPINE-L@NIC.SURFNET.NL (Alpine-L, the Electronic Rock Garden Society [postings copyright by authors])
To: ALPINE-L@NIC.SURFNET.NL

Subject: Preparing Solutions of Colchicine and Oryzalin /
Surflan

Correspondence from Marc Hachdourian, seeking a web site
with procedure & methodology for inducing polyploidy by
using Surflan (reproduced at the end of this message), has
led to the following posting from Rick Grazzini, to whom we
are all indebted:

Date: Mon, 8 Nov 1999 08:57:49 -0500
From: Rick Grazzini <rickg@centrelab.com>
To: "'harryd@Kreative.net'" <harryd@Kreative.net>
CC: "'Beth and Bob Matney'" <bmatney@mail.snider.net>

Harry,
I am in the middle of all of this. Beth Matney and I have
been compiling references and correspondences re this
topic. You should now feel free to post this to Alpine-L.

The text and calculations have been reviewed for accuracy
and technique by two other scientist friends, one a
professional plant breeder. However, you should also feel
free to send it out for additional review if you like.

Rick Grazzini, PhD
central PA USA
USDA z5b/6a
minimum temperature: average -5 to -10 F
minimum temperature: minus 20 F (rarely)
487 Nimitz Avenue
State College, PA 16801 USA

Following is the text that Beth Matney and I have been
working on:
==============
PREPARING SOLUTIONS OF COLCHICINE AND ORYZALIN
This information is distributed to assist plant breeders,
both laypersons and professionals.

These solutions --- the final working solutions --- are
useful in attempts to induce polyploidy in plants.

My thanks to all who have helped this discussion get to
this point:
Tony Avent
Richard Craig
George Darrow (in spirit)
Haig Dermen (in spirit)
Arthur Evans
John Fiala (in spirit)
James Gossard (c/o KW)
Jim Hawes
Augie Kehr
Kay Lancaster
Beth Matney
Dan Nelson
Kevin Vaughn
Jim Waddick
Kevin Walek (KW)
John Weagle

This information has been compiled from a number of
sources, including USDA reprints, classroom notes, email
posts, and published texts. It has been supplemented by my
experience, as well as that of the contributing personnel
list above.

=================
DISCLAIMER
The following text is provided to scientists and
researchers attempting to induce polyploidy in plant
species. Both colchicine (FDA) and oryzalin
(EPA) are regulated compounds, and as such, should not be
used in ways other than those indicated on the label. Any
such non-label usage indicated below is for experimental
research purposes only.
=================

TO PREPARE WORKING SOLUTIONS OF COLCHICINE
The colchicine solutions for inducing polyploidy are
usually 0.25%, 0.5% or 1%, depending on species and tissue.
I generally start with the strongest solution strength. If
tissue (bud or seedling) kill is 100%, then decrease the
concentration. I hope that it is obvious that range-
finding trials are necessary to determine the most
effective concentrations to use. You should
NEVER treat the only plant you have. Propagate it, then
treat one of the propagules, reserving the original just in
case.

TOOLS AND SUPPLIES NEEDED:
1. laboratory (safety) glasses
2. laboratory gloves (latex or PVC or equivalent)
3. analytical balance (able to accurately measure to 0.1
g)
4. graduated cylinder (or equivalent accurate measure to
10 mL)
5. micro-spatula or small spoon (able to enter vial in
which colchicine powder is stored). Alternatively, you can
practice (using corn starch) tapping powder from a vial.
6. balance pan or balance paper (to transfer powder from
balance to solution bottle). A square of wax paper works
well for this. Even better, weigh directly into a beaker.
The little disposable medicine cups that pharmacies have
are very good for this (they also come packed with Nyquil).
7. distilled water
8. eyedropper or pasteur pipette(s)
9. small bottle with tight seal or cap (amber preferred)
10. (optional) DMSO (pure, 99.9%)
11. dust mask, disposable, discarded after use.

CAUTION: Colchicine is a HIGHLY TOXIC POISON. Please be
prepared to handle it safely, or do not handle it at all.
Wear gloves. Throw the gloves away after use. Wear safety
glasses. Wash the safety glasses after use. Be sure to
wear clean gloves when you wash the safety glasses.

Colchicine can be purchased from scientific supply houses
as a white powder. As of January 1999, 1 g of colchicine
powder sells for about $60.

Pour 10 mL of distilled water into the graduated cylinder.

Using the most accurate analytical balance you have
available, and transferring the powder using a
microspatula, measure 100 mg (0.1 g) colchicine onto a
clean piece of balance paper, or into a clean disposable
balance pan.

Transfer the colchicine powder from the paper or pan into
the small bottle.

Carefully rinse the powder dust from the paper or pan into
the bottle, using some of the previously measured distilled
water, and an eyedropper or pasteur pipette.

After rinsing the paper or pan, dispose of the paper or pan
so that it cannot be re-used.

Transfer the remainder of the distilled water into the
small bottle. Cap and shake. The colchicine powder is
highly water-soluble, and should go into solution quickly.

100 mg/10 ml is the same as 0.1 g/10 ml is the same as 1 g
in 100 ml, or 1%

Alternatively, if you're working with a good balance, tap X
amount of colchicine into the container you'll be mixing
in. Weigh it. Figure the amount of colchicine you'll need
(say you got 53 mg of colchicine into the container: add
5.3 ml water for a 1% solution). Dissolve the colchicine
In half (roughly) of the water, transfer that solution to
the storage vial, then use the rest of the water to rinse
the mixing container, adding that to the solution in the
storage vial. Stopper and shake the vial to complete
mixing.

The measurements above will give you a 1% solution.

To make solutions of different concentrations, use these
amounts:
0.5% solution: 50 mg (0.05g) colchicine, 10 mL water
0.5% solution: 500 mg (0.5 g) colchicine, 100 mL water
0.5% solution: 5 mL 1% solution, 5 mL water

0.25% solution: 25 mg (0.025 g) colchicine, 10 mL water
0.25% solution: 250 mg (0.25 g) colchicine, 100 mL water
0.25% solution: 5 mL 0.5% solution, 5 mL water
0.25% solution: 5 mL 1% solution, 15 mL water

To enhance penetration of the colchicine solution into
plant tissue, you can add 1 or 2 drops of pure DMSO to the
solution. No more is necessary.

BE CAREFUL. DMSO enhances penetration through HUMAN tissue
as well as plant tissue. If you are using a DMSO-enhanced
solution, you should use DOUBLE gloves (wear TWO pairs).
DMSO, if it gets into your system, will produce a
garlic-ey taste in the back of your throat. By itself,
that's not a problem: people rub it into their skin as a
home arthritis treatment (a practice which is NOT to be
recommended, by the way). But if you taste garlic when you
are using DMSO-spiked colchicine solutions, you have a
problem.

The bottom line is BE CAREFUL. If you are not familiar
with how to safely handle poisons --- DON'T DO IT!

The question frequently arises re the use of colchicine
gout pills as a preweighed source of colchicine. These
will work; however, the colchicine gout pills listed in the
PDR as a Merck product are a combo probenecid-colchicine
pill. Colchicine content is 0.5 mg. Bill Nash (Guelph,
Ontario, Canada) tells me his pharmacist stocks 1 mg
colchicine tabs. I have no information re the interaction
of probenecid and plants. But 0.5 mg is a pretty low
dosage to be using for this purpose. For instance, to make
a 0.5% solution, you would have to dissolve one hundred
0.5 mg pills in 10 mL of water. That's a lot of tablets to
dissolve in a small amount of water.

===================
SURFLAN / ORYZALIN CONCENTRATION CALCULATIONS
Surflan AS is sold as a 40.4% solution of oryzalin (the
active ingredient, or AI). The AI oryzalin is only
slightly water-soluble (2.6 mg/L). The formulation
contains carriers (solvents) that will increase this
solubility. As a result, I would not expect DMSO to be
needed if you are beginning from a Surflan formulation.
The formulation should already be compounded to enhance
penetration of the oryzalin through plant tissues ---
Surflan IS an herbicide, remember.

There is a very wide range of Surflan concentrations which
are in use throughout the plant breeding community. Tony
Avent (Plant Delights, NC) uses either a 1:100 or a 1:200
dilution on hostas (among other species). Kevin Vaughn
(USDA, MS) uses a 10 uM (micromolar) solution on daylilies.
Arthur Evans (AR) uses a 0.005% oryzalin solution on
oriental and Asian lilies.

How do these different ways of expressing calculations
compare? This can get very confusing.

0.005% is a 1:20,000 dilution. 0.005% is 0.00005 as a
decimal. 1 /0.00005 equals 20,000. This is the dilution
IF you base the calculation on the FORMULATION, i.e. 0.005%
Surflan. This is very different from a 0.005% oryzalin,
which if you start from the 40.4% Surflan AS formulation,
is actually a 1:8,000 dilution (or 0.5 mL Surflan AS in 4L
water, as Arthur Evans describes it). This second
perspective (1:8,000) is BASED ON THE ACTIVE INGREDIENT
ORYZALIN.

In any case, 0.005% oryzalin is a 1:8,000 dilution of
Surflan AS. This is 80X more dilute than 1:100. Yet, both
concentrations are effective. There are two possible
reasons: 1) more is NOT necessarily better. Oryzalin
Is barely water-soluble: throwing more in the bucket does
not mean that more will dissolve; and 2) oryzalin may have
a broad effective range, and although more may not be
better, it may also not be worse.

Surflan AS is only 40.4% oryzalin. Therefore, 1 kg of
Surflan AS contains 40.4% oryzalin, or 404 g oryzalin. The
remainder is solvent. I estimate that the density of
Surflan AS is about 1.2 (meaning 1 mL Surflan would
Have a mass of 1.2 g). One mL of Surflan AS is about 1.2 g
of formulation, and would contain 40.4% x 1.2, or 0.48 g
oryzalin. 0.48 g equals 480 mg (actually 484.8 mg). One
mL Surflan would contain 484.8 mg oryzalin.

0.5 mL Surflan AS would contain half of this, or 242.4 mg
oryzalin. Dissolving 242.4 mg in 4 L (or 4,000 mL) would
give you a solution of about 0.000175 M. Or 174.96
micromolar. This is about 20X the amount of oryzalin that
will normally dissolve in water. The carrier solvents help
keep the oryzalin in solution.

[NOTE: If my estimated density is in error, and it might
be, the rest of the calculation will change, too. However,
this will have no impact on the preparation of the working
solutions.]

A 1:100 dilution would be 10 mL Surflan AS in 1 L water.
10 mL Surflan contains 1.4 g or 1,400 mg oryzalin.
Dissolving 1.4 g oryzalin into 1 L of water would make a
0.004 M solution, which is a 4,000 uM solution. This is
just about 23X more concentrated than a 175 uM solution.

A 10 uM solution (just a little bit over the solubility of
pure oryzalin in water) requires 3.46 mg of oryzalin
dissolved in 1 L of water.

SUMMARY TABLE:

molar (M) percent (%)
dilution*
0.005% oryzalin 175 uM 0.005%
1:8,000
1:100 Surflan dilution 4,000 uM 1%
1:100
10 micromolar sol'n 10 uM 0.0003%
1:140,000

* This is dilution from Surflan AS @ 40.4% AI.

[Note: I re-formatted the table to e-mail a little easier:
[table begins]
0.005% oryzalin equals
175 uM (molar) equals
0.005% (percent) equals
1:8,000 (dilution)

1:100 Surflan dilution equals
4,000 uM (molar) equals
1% (percent) equals
1:100 (dilution)

10 micromolar sol'n equals
10 uM (molar) solution equals
0.0003% (percent) equals
1:140,000 (dilution)
[table ends]

>From this information, I am guessing that the most likely
effective range is going to be between 10 uM and 100 uM.
Preparation of working solutions for both of these
concentrations is given below, and using relatively easy-
to-locate measuring tools.

=================

TO PREPARE WORKING SOLUTIONS OF SURFLAN
This assumes that you start with the Surflan AS
formulation, with a concentration of 40.4% AI (oryzalin).
Surflan AS sells for about $100 per gallon.

CAUTION: Although Surflan is more safe than colchicine, it
is a HIGHLY TOXIC POISON. Please be prepared to handle it
safely, or do not handle it at all. Wear gloves. Throw
the gloves away after use. Wear safety glasses.
Wash the safety glasses after use. Wear clean gloves to
wash safety glasses.

TOOLS AND SUPPLIES NEEDED:
1. laboratory (safety) glasses
2. laboratory gloves (latex or PVC or equivalent)
3. (optional) analytical balance (able to accurately
measure to 0.1 g)
4. graduated cylinder (or equivalent accurate measure to
10 mL)
5. (optional) disposable balance pan
6. distilled water
7. eyedropper or pasteur pipette(s)
8. small bottle with tight seal or cap (amber preferred)
9. large bottle (1 L to 4 L, or 1 qt to 1 gal) with a
tightly fitting cap
10. a metric measuring cup so you can measure 1 L
11. dust mask, disposable, discarded after use.
=================

TO MAKE A STOCK SOLUTION (about 100 mM, or 0.1 M)
Measure 9 mL water into the graduated cylinder. Leave this
solution in the graduated cylinder.

Using an eye dropper or small pipette, add Surflan AS to
the graduated cylinder until a total of 10 mL are contained
in the cylinder. You will be adding formulation until the
total reaches the 10 mL line (9 + 1 = 10).
The graduated cylinder should now contain 9 mL water plus 1
mL Surflan AS formulation.

[NOTE: You can also use an analytical balance to measure
the formulation. Place the small bottle (or a disposable
pan) on the balance. Tare the bottle. Measure 0.857 g of
the Surflan AS formulation into the bottle (or pan). If
you use a disposable pan, be sure to rinse it into the
bottle with some of the water from the 9 ml in the
graduated cylinder. ]

Pour the contents of the cylinder into the small bottle.
Cap (close) the bottle, and shake vigorously.

Slowly uncap the bottle, and pour the solution back into
the graduated cylinder. Repeat transfer from cylinder to
bottle and back again three or four times.

Cap the bottle of stock solution. The concentration of
this stock solution is 100 millimolar (or 0.1 M). Each mL
of stock solution contains 0.034636 g oryzalin, or 34.64 mg
oryzalin.

Label the bottle of stock solution: "STOCK SOLUTION - 100
millimolar (100 mM). DILUTE 1,000X to 10,000X BEFORE USE".
=================

TO MAKE WORKING SOLUTIONS (10 uM and 100 uM)
The problem is this: you have a stock solution of 0.1 M
(100 mM). How do you make a working solution of 100 uM?
Expressed in another way, you want to dilute your 0.1 M
solution down to 0.0001 M (or 0.1 mM).

There are some equivalences here that are important:
1 M = 1,000 mM = 1,000,000 uM.
Therefore, 0.1 M = 100 mM = 100,000 uM.
And thus, 0.0001 M = 0.1 mM = 100 uM.

To go from 0.1 M down to 0.0001 M (which is from 100 mM
down to 100 uM), you have to make a 1:1,000 dilution.

TO MAKE 1 LITER (1L = 1,000 mL) OF A 100 uM WORKING
SOLUTION:
Measure 1 L of water into the large bottle. Put a mark
(use a permanent marker) on the side of the large bottle to
indicate the fill level for 1 L of fluid. Empty the large
bottle, which you have just turned into a 1 L graduated
bottle.

Measure 9 mL water into the graduated cylinder. Leave the
water in the cylinder.

Use an eyedropper or pipette to add 1 mL of the 0.1M STOCK
solution to the graduated cylinder. You will be adding
STOCK solution until the total in the cylinder reaches the
10 mL line (9 + 1 = 10).

Pour the cylinder into the 1 L large container. Rinse the
cylinder into the 1 L container two or three times. Then
add sufficient water to bring the total fluid level up to 1
L. Cap and shake. Label this container: "100 MICROMOLAR
(100 uM) WORKING SOLUTION".
=================

TO MAKE 10 uM WORKING SOLUTION:
You need to make a 1:10 dilution of the 100 uM working
solution.

Measure 9 mL water into the graduated cylinder. Leave the
water in the cylinder.

Use an eyedropper or pipette to add 1 mL of the 100 uM
WORKING solution to the graduated cylinder. You will be
adding 100 uM WORKING solution until the total in the
cylinder reaches the 10 mL line (9 + 1 = 10).

Pour the cylinder into a small bottle. Cap and shake.
Slowly uncap the bottle, and pour the solution back into
the graduated cylinder. Repeat transfer from cylinder to
bottle and back again three or four times.
Make the last transfer into the small bottle, and cap
securely.

Label this container: "10 MICROMOLAR (10 uM) WORKING
SOLUTION".
=================

There is an easier alternative, if you are able to
accurately measure 0.1 mL:
stock (0.1 M)
dilute to:
100 uM working: 0.1 mL 100 mL
10 uM working: 0.1 mL 1,000 mL

In words (rather than a table):
To make a 100 uM working solution, dilute 0.1 mL of a 0.1M
stock solution to 100 mL.

To make a 10 uM working solution, dilute 0.1 mL of a 0.1 M
stock solution to 1,000 mL.

=================

The Web address of my page on inducing tetraploidy is:
http://members.tripod.com/~h_syriacus/tetraploidy.htm

I am glad to see that Rick posted his calculation methodology as I have
been very remiss in updating the page.




Edited by Psiloman (12/25/05 09:15 AM)


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Offlinefelixhigh
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Re: Inducing polyploidy : A Simple Guide. (Make your own "species") [Re: Psiloman]
    #4233664 - 05/29/05 04:34 PM (12 years, 4 months ago)

very interesting!!!


FH


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OfflinePsiloman
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Registered: 04/11/03
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Re: Inducing polyploidy : A Simple Guide. (Make your own "species") [Re: felixhigh] * 1
    #4233946 - 05/29/05 06:35 PM (12 years, 4 months ago)

Now watch this ,its at least entertaining: Im going to attack myself!

"But colchicine is a TOXIC POISON!"

Simple answer:

Yes it is ,in the right dosage which happens to be low hence it is a poison. For the procedures mentioned it can be used safely by people who adhere on safety rules or by people who have OCD with a focus to hygiene! Compared to colchicine Surflan is safe (it is toxic in the right dosage though!) and it could be used as an alternative

More in depth answers for those who like Real Answers:

Colchicine and Surflan inhibit microtubule formation in the metaphase/anaphase of the mitotic and meiotic cell cycle.Surflan is more selective to plants ,hence it does not harm animal cells.


Lets talk about the most toxic of the two:
Colchicine is also a pharmaceutical drug taken orally! Check here: http://www.rxlist.com/cgi/generic2/colch.htm .It is used in 0.5 and 0.6 mg per pill (see,here is the toxicity) but also it can be found in 1mg pills. In laymans terms "people get percribed this stuff and eat it".If you are carefull with it ,wear gloves,dont eat it ,clean yourself immediatelly if it spillson you,wash away any spillage in the area you work on with an OCD devotion and keep it away from food you have nothing to be afraid of...

So why is it "toxic" and "extremelly poisonous"? Because Homo Sapiens is known NOT to follow instructions and NOT to follow regulations.How many people have heard of the ills of Heroin and Opium abuse but choose to disregard them? How many people know of the dangers of driving inebriated but still do it? How many people actively seek ways to override perscription mechanisms in order to acquire pills with abuse/addictive potential and MANAGE to get addicted? See? We simply cant follow regulations! So if you think colchicine ,PLEASE FOLLOW THE FREAKING SAFETY REGULATIONS!!!!! I will stop here on this issue before it tends to be more of a "Philosophy ans Spirituality" thread.

So one might say "Hey smart-ass ,im going to apply it on the plantets or seeds,if later on i eat the plant wont i suffer bad effects"?

Ingenious question! Lets take the most toxic of the two colchicine (it doesnt even compare to toxicity with the other substance).Please bear with me :Lets say you use a 0.5% solution to treat your seeds which is 5mg colchicine per ml and that you use 1 ml of solution for 10 plantets of Datura Inoxia.Lets also assume that by the help of the little naughty gnomes when you sleep all 5mg of colchicine enter the only seed that sprouts and that you happen to plant in your garden.After some months your Datura plant has 10 leaves and you choose to smoke one.For insanity's continuation lets say that the colchicine in the plant did not get metabolised and by some wicked force that wants to harm you spreads evenly only on the leaves of the plant.

Ok,where am i going at? Smoking that leaf and assuming that all its colchicine enters your body you will have just received 0.5mg of colchicine.Excuse me for saying so but i consider this ammount of colchicine safer than ingesting a mystery pill labeled as "MDMA" by a dealer. Now take into account that little gnomes that help 5mg of colchicine enter a single seed as you sleep DONT exist and you can figure the rest yourselves.Take also into account that Furlan is innocuous compared to colchicine and gives better results at tetraploidy experiments (hint hint!)

So it all boils down to "Be Reasonable,Follow Safety procedures".If you can do that then you can experiment safely with polyploidy. If one wants more info on why to "mess with polyploidy" fire away and i will answer :laugh:


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Offlinepod3
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- [Re: Psiloman]
    #4236387 - 05/30/05 01:15 PM (12 years, 4 months ago)

-


Edited by pod3 (10/26/06 01:17 PM)


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OfflinePsiloman
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Re: Inducing polyploidy : A Simple Guide. (Make your own "species") [Re: pod3]
    #4236490 - 05/30/05 01:54 PM (12 years, 4 months ago)

Polyploid by grafting? I lost you here...
Can you explain please?


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Re: Inducing polyploidy : A Simple Guide. (Make your own "species") [Re: pod3]
    #4236494 - 05/30/05 01:55 PM (12 years, 4 months ago)

This idea has been around a long long time. From what I hear, it never lived up to it's potential. Pot growers were encouraged to try it going back 20 years or more. If it worked and was easy, don't you think commercial growers would be all over it? The stuff is poisonous and I don't recommend fooling with it. I've never heard of anyone getting good results from it.


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- [Re: Psiloman]
    #4237406 - 05/30/05 06:44 PM (12 years, 4 months ago)

-


Edited by pod3 (10/26/06 01:16 PM)


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Re: Inducing polyploidy : A Simple Guide. (Make your own "species") [Re: Stonehenge]
    #4237487 - 05/30/05 07:19 PM (12 years, 4 months ago)

Quote:

Stonehenge said:
This idea has been around a long long time. From what I hear, it never lived up to it's potential. Pot growers were encouraged to try it going back 20 years or more. If it worked and was easy, don't you think commercial growers would be all over it? The stuff is poisonous and I don't recommend fooling with it. I've never heard of anyone getting good results from it.




Oh commercial growers are indeed all over it! Polyploid magnolias in the market are made using mostly Sulfran! Same holds truee for many brugmansia species and it has worked on tomatoes,maze and other plants! Simply most people dont bother with it,a pot grower would just buy skunk seeds and thats the end of the story.He plants them,gets his bud,sells it,smokes it and doesnt bother alot with it. Keep in mind though that Skunk variety IS POLYPLOID,arising either from natural polyploidy (very rare) or those colchicine experiments!

Colchicine isnt that good of a choice,it effects animal cells as well as plant cells ,plus it has a lower rate of success and higher rate of lethal effects on seeds.

Sulfran on the other hand:

It works (for a fact),its medium difficult but it requires an adult.When i  say adult i mean adult in brains not in age.Certainly i would not urge people to fool with it (No room for fools here sorry,thats why i wrote that huge essay attacking myself).I would urge though careful ,responsible,sane,uninebriated at the time of the procedure ADULTS to give it a go. If one of the shroomerites wants to try it i urge him to read thrice (3) my "attack on myself" until it sinks in :laugh: Certainly its not for individuals that will "fool around" ,smoke after applying this chemical without washing hands,eating while applying the solution (!),having sex while applying the solution (!!) ,rubing their eyes ,nose mouth >> , trying to taste the solution,putting it in places where a sudden elbow move can spill it on the sofa and so on and so forth....

Pod3:

Grafts are not considered Chimeras..A Chimera is a)either a plant that is made by joining two different species by method of PROTOPLAS FUSION or a "freak accident" of fertilisation that wasnt supposed to happen b)an organism having throughout its body mixed cells usually reffered to as a mosaic. Chimeras/mosaics are some lab rats that come from....4 parents! Well,its not the effect of a rat gangbang,byt the effect of putting together two blastomeres of zygotes that come from two different SETS of parents (Breed mouse A with mouse B,Breed mouse C with mouse D ,get a cell from each zygote,put it together and you receive a mouse whose cells will be a mixture of A and B recombination plus C and D recombination)

Grafting a plant on another ,thus is not called a chimera and does not cause poliploidy.If the peyote you graft on a Trichocereus is diploid ,it will continue to be diploid no matter what!


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Re: Inducing polyploidy : A Simple Guide. (Make your own "species") [Re: Psiloman] * 1
    #4237524 - 05/30/05 07:35 PM (12 years, 4 months ago)

Oh ,and once more for those who failed to get it so far:

Between colchicine and Surflan choose Sulfran.

Basic extra paranoid safety tips for Simple Minds:

1)Wear Gloves
2)Make them double!
3)Where disposable masks like the one dentists use!
4)Wash your hands after use!
5)Choose to work with solutions rather with straight Oryzaline powder.Powder can spread in the air the solution cant!
6) Keep your workbench always clear ,wipe thrice after each experiment
7)Store all your supplies in containers,solutions must be well sealed
8)Clearly label them as poisonous....You never know when your granny will be thirsty in the middle of the night
9)Any accidental spillage in your area should be cleaned thrice with ample water!
10) Any accidental spillage on you should be cleaned thrice with ample water
11)Any accidental spillage on politicians should not be cleaned,let it soak in.Respill if needed
12)Please : Dont eat it ,Dont Smoke it ,Dont Inject it,Dont Flirt it,Dont smell it ,DOnt play with it,Dont parade the chemicals in a room full of stoners,Dont brag about them to your girlfriend/boyfriend/pet just stick to the rules
13)It goes without saying that the area of the experiments SHOULD NOT BE THE KITCHEN!
14)Any glassware,equipment,spatulas ,forceps,razors,dildos used in the procedures should not be used for anything else but this procedure.


Ok i have been clear,and quite humorous.If one cannnot follow these simple rules,is not of an adult mentality but tries the above method then certainly he will be a victim of natural selection.Sue Darwin.


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Re: Inducing polyploidy : A Simple Guide. (Make your own "species") [Re: Psiloman]
    #4237761 - 05/30/05 08:52 PM (12 years, 4 months ago)

This is similar to making mutations. The likelyhood of success is about as great, close to zero. Sure, it has worked and given useful varieties but nothing major in all the years it's been used. It's interesting to think about but using poisonous junk hoping for some miracle is not worthwhile, imho. What exactly are you guys thinking will happen? What is the most likely thing you expect and what do you think you have even a 1 in 1000 chance of achieving?


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Re: Inducing polyploidy : A Simple Guide. (Make your own "species") [Re: Stonehenge] * 1
    #4238890 - 05/31/05 05:17 AM (12 years, 4 months ago)

Quote:

Stonehenge said:
This is similar to making mutations. The likelyhood of success is about as great, close to zero. Sure, it has worked and given useful varieties but nothing major in all the years it's been used. It's interesting to think about but using poisonous junk hoping for some miracle is not worthwhile, imho. What exactly are you guys thinking will happen? What is the most likely thing you expect and what do you think you have even a 1 in 1000 chance of achieving?




Note that many commercial plants with big fruits are a result of polyploidy even through breeding that went astray or by use of such agents.Sure, they miss the label "New and Improved,a polyploid product made by Microtubulin Inhibitors" but those products and trees are around us,nothing "novel" about it.

This is quite dissimilar to making mutations using a mutagen and the succes rate is not close to zero if one performs the procedure as given...

Lets have a closer look to it,to demystify it shall we? Maybe this will help us not imagine it being handled by geniuses in secret labs wearing full body protective suits!

If one uses a mutagen (Such as DNA base analogs,Ethidium Bromide,acrylamide) etc one RANDOMLY MUTATES the DNA of a plant.Now ,the most "innocent " mutation could be a point mutation in the DNA,that is one base (Lets say Adenine) getting "transformed" to another ,Lets say cytosin.Now if this is in a protein coding region of the enzyme it could a)increase potency of the enzyme/protein b)decrease it c)do nothing at all....If the mutation is on a promoter or another binding sequence of the DNA the mutation might a)cause transcription enzymes/signaling molecules to bind with greater affinity b)destroy the affinity or reduce it so the gene following wont be expressed or c)have no effect at all...Now this was for point mutations,inversions etc etc.If one causes a mutation adding 2 bases 4 bases or so or a translocation of such an amount of bases (multiple of 2) the reading frame changes completely and ...well in layman's terms you are fucked mate! You just destroyed the plant!

Dont mutations of this kind look "random as shit" even to the untrained eye? Yes they do,and guess what! They are! Chances of somethng usefull being made if you spill some mutagen on the plant is...less likely to happen that having sex with Einstein in a Burrito place.

Microtubulin inhibitors on the other hand do NOT cause mutation. Lets start with a diploid plant (2n ,where n number of chromosomes ,diploid means it hgas two copies of each gene). In the S (synthesis) phase of the cell cycle the DNA gets doubled (4n) and during anaphase of Mitosis the DNA is equaly split to each of the two daughter cells arising giving rise to two cells with 2n nuclei.This is done by the help of microtubules,which are made of a protein named tubulin that polymerises and pulls the chromosomes in each daughter cell.

A microtubulin inhibitor just wont let the microtubules polymerise! So there is a GREAT chance that when daughter cells form by cell division to have a daughter cell with no DNA and a daughter cell with 4n DNA.The DNAless cell die ,the 4n cell continues as normal.Keep in mind that no mutation occurs.

Twice the DNA means on a molecular level : In laymans terms "twice of everything".Twice of enzymes,twice of proteins,twice of amount of signaling molecules and...twice of any secondary metabolist present!

On a phenotypical level it means : Bigger plants (about twice in height girth),bigger leaves etc etc.

Its not a "random proccess" it has been tested,it works and you dont need a full body protection suit ,nor the brain of a genius,nor geeky glasses for it to work.Just follow safety and instructions.

So far so good...I could be talking though out of my ass but what we could consider "scientists" (you know those geeky people that spill numbers and weird theories) have tested this theory [url=http://www.taylorandfrancis.metapress.com/]www.taylorandfrancis.metapress.com/index/K2HFKUQ2YVNFC9HB.pdf
(If link unclicable copy paste)
1.86 ,1.65 and 1.96 times more alkaloids in the species tested. It worked,almost the double!

What i want to outline is that this is a viable procecedure,and relatively safe for one who can adhere to the rules.


Edited by Psiloman (05/31/05 05:29 AM)


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Re: Inducing polyploidy : A Simple Guide. (Make your own "species") [Re: Psiloman]
    #4238973 - 05/31/05 06:30 AM (12 years, 4 months ago)

Well, the substances we are interestet in are secondary metabolites. Not the primary. There probably will be an increase in primary as these are always expressed and mostly mediated by enzymes. Secondary metabolites often include non enzymatic steps like oxidation.

It has been tryed before with for example artemisia annua, to increase production of artemisine (a secondary metabolite) without succes.

If you want bigger plants with more lignin and other cell wall materials it can be usefull but for these substances its not an option.


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Re: Inducing polyploidy : A Simple Guide. (Make your own "species") [Re: darkice]
    #4239054 - 05/31/05 08:11 AM (12 years, 4 months ago)

Erm...Do you guys read my posts?

I will quote myself :

Quote:

So far so good...I could be talking though out of my ass but what we could consider "scientists" (you know those geeky people that spill numbers and weird theories) have tested this theory taylorandfrancis.metapress.com/index/K2HFKUQ2YVNFC9HB.pdf
(If link unclicable copy paste)
1.86 ,1.65 and 1.96 times more alkaloids in the species tested. It worked,almost the double!





Atropine and scopolamine are alkaloids,secondary metabolites.If only primary metabolites were increased then this doesnt explain the 1.86 times higher alkaloid content!

Quote:

It has been tryed before with for example artemisia annua, to increase production of artemisine (a secondary metabolite) without succes.




I would be interested to see the source.Do you have a citation?

Quote:

There probably will be an increase in primary as these are always expressed and mostly mediated by enzymes. Secondary metabolites often include non enzymatic steps like oxidation.





Secondary metabolites are heavily relying on enzymes as well.The oxidation you mention within the controled enviroment in the cell when needed happens mostly with enzymes. Dont forget that many conversions that take place for secondary metabolites to apperas happens in the endoplasmatic reticulum ,via enzymes. DMT synthesis happens this way for example as well as harmala alkaloid formation . DMT and harmala alkaloids are interesting compounds no? Same with mescaline as well...

Keep in mind that polyploidy is being used for production of medicinally important alkaloids...That surely says that it is an option,and a pretty viable one if i may add!


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Re: Inducing polyploidy : A Simple Guide. (Make your own "species") [Re: Psiloman]
    #4239093 - 05/31/05 08:43 AM (12 years, 4 months ago)

I found it, ok, the alkoloids production was enhanced. But the plants stayed smaller and had less leaf production so there was no netto gain.
http://www.ncbi.nlm.nih.gov/entrez/query...&query_hl=3


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Re: Inducing polyploidy : A Simple Guide. (Make your own "species") [Re: Psiloman]
    #4239128 - 05/31/05 09:10 AM (12 years, 4 months ago)

Ah preety nice article!!! I missed that completely on pubmed.Yes slower growing plants can be developed in tetraploidy.Here i wonder if some selective breeding at the top of that can speed up the growth! I wonder if the problem lies in roots :maybe tetraploid roots have some problem absorbing elemtns from the soil (thats a wild guess guys) which could be solved if only the apical stem (tip of the plant) is converted.so you have a mixaploid plant, that could show increased growth,or even compensate for the 25% loss in the mass.... Heh,if one doesnt run dry of ideas everything can be achieved!


Another interesting idea,leaving alkaloids on the self for the moment,is restoration of fertility in hybrids
http://www.sna.org/research/03proceedings/03proceedingspdfs/section12.pdf

In your acrodat choose "Search" and input "Oryzalin".

Now that could be used on salvia to get some fertile pollen,maybe after some tweaking we can have Salvia divinorum that reproduces through seeds reliably! (For those who want to know more about salvia and why i reffer it as a hybrid check here http://www.sabia.com/salvia/ and check the barrier to fertility )

All in all polyploidy in hands of a responsible adult can be a very usefull tool.


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- [Re: Psiloman]
    #4239258 - 05/31/05 10:29 AM (12 years, 4 months ago)

-


Edited by pod3 (10/26/06 01:15 PM)


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Re: Inducing polyploidy : A Simple Guide. (Make your own "species") [Re: pod3]
    #4240114 - 05/31/05 03:28 PM (12 years, 4 months ago)

"the same grafts produce a different result every time."

Ah the magic of genetics coupled with enviromental factors!!! A genotype can give many phenotypes under different enviromental conditions.

"Speaking of Salvia divinorum, it was supposedly the hybrid of two different coleuses (crosspollination?), and apparently neither contined salvinorin. It would be interesting to see what interspecies chimera could produce."

Same here! Another method on salvia would be to use the anther part where the gametic cells are aploid and cultivate them as aploid plantets on agar with different rations of auxins and cytokinins to initiate shoot and root formation in the callus.These plantets would not be fertile but one could convert them to diploids by Surflan.

The advantages are

1)The resulting plant will be diploid (that is if you want it diploid and you dont suffice yourself to a tetraploid)
2)The plant will be homozygotic for all its traits..I dont know though the effect on salvinrin production...


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- [Re: Psiloman]
    #4264208 - 06/06/05 05:07 PM (12 years, 4 months ago)

-


Edited by pod3 (10/26/06 01:14 PM)


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Re: Inducing polyploidy : A Simple Guide. (Make your own "species") [Re: pod3]
    #4264335 - 06/06/05 05:50 PM (12 years, 4 months ago)

If you tissue culture two species together from different points of the petri dish the cultures will just meet at a point,or if you culture them from the same point they will just grow together along. If you are thinking of "mixing species" this way,i am afraid it wont work.For this kind of hybridisation you are propably looking at protoplast fusion.

Steps of protoplast fusion:

1)Take cells from donors (leaves are good)
2)Digest their cellwalls using enzymes at right temperature for some times (Kits for that can be foun online)
3)What you are left with is protoplasts.Naked plant cells.Their membranes are charged negatively so they repel eachother. In order for them to fuse you either add PEG (polyethylene glycol ,easy to find) or use electrophoresis
4)Watch under microscope which protoplasts duse and choose them.
5)Cultivate on agar on different concnentration of auxins and cytokinins to obrain plantets!

Also the plant you may receive might not be fertile! A trick to try and restore fertility is polyploidy (yes ,again the same with my hypothesis on salvia divinorum!) but if you start the protoplast business with two diploid cells you are already gonna have a tetraploid.If you subject it to polyploidy you will have at least an octaploid which is generally bad news...Slow growth malformed!

One though could overcome this by selecting the starting material for the fusion to be anthers..I think they are haploid so by fusion they will give you a diploid hybrid!

THey did that with Datura inoxia and Datura stramonium and produced a hybrid that actually broke the "incobatibility factor" between those two plants and gave way for new hybrids!


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- [Re: Psiloman]
    #4265715 - 06/06/05 11:37 PM (12 years, 4 months ago)

-


Edited by pod3 (10/26/06 01:13 PM)


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