In vitro micropropigation

[swisscheese]

Awesome thread roots now put that beer down and get this going the canna community has been waiting for a thorough thread like this. I hope you'll throw some pics up for us too. I've got a flow hood already and have been waiting for someone to copy! Wishing the best of luck.

 

[squiggly]

I think you'll have better results if you wait for the DMSO (if this is what was suggested you use).

DMSO and KOH solution couldn't be any different as solvents go if they tried to be.

 

[rootsnshoots]

Squiggly, on the site is says soluble with dmso. But under the product information it says that it can be dissolved in koh according to a different site but they have not personally tried it. I have no chemistry education. I may take up your offer as chemistry kid for hire and have some questions for you.

 

[squiggly]

For sure, I'm here to help--you wouldn't be the first person who wanted to pick my brain :)

DMSO has very special activity with biological membranes--in terms of modulating their permeability to substances like hormones and the like. The stuff may dissolve in KOH--but it will have a very different activity with DMSO. I'd go ask those Ph.D dudes what they think the ticket is.

 

[rootsnshoots]

thats what the guy at the science store said. he said he worked with it in school and it will absorb into your skin. i looked some stuff up on it and they used it in pharmas and horses or some shit amd if i remember right it said you will taste garlic if a lot is ingested.. any ways..

I did get a hold of Thidiazuron-influenced high frequency direct shoot organogenesis that thor posted when i started the thread. Im kind of wanting to go off of their hormone ratios for my first run. most other tissue culture stuff ive researched have used a mg to liter ratio. theirs is measured 0.05–5.0 μM thidiazuron.
I have no idea what μM means. (or how to get the font back):confused:

Also to make a koh solution? it says add a few drops of 1 N NaOH (or KOH) to dissolve the hormones then add water. once again i have no idea what 1 N is? I have a printed copy of the tdz article at work ill bring home and go over as well as plants from test tubes and ill have some more questions for you. your help is appreciated and i want to do this and share it with all the farm because i think it kicks ass but i know i need some help.

 

[Pancho-N-Lefty]

Way way over me old cabesa but im super interested to follow along and see what enlightenment you find roots! great stuff guys...

 

[squiggly]

thats what the guy at the science store said. he said he worked with it in school and it will absorb into your skin. i looked some stuff up on it and they used it in pharmas and horses or some shit amd if i remember right it said you will taste garlic if a lot is ingested.. any ways..

I did get a hold of Thidiazuron-influenced high frequency direct shoot organogenesis that thor posted when i started the thread. Im kind of wanting to go off of their hormone ratios for my first run. most other tissue culture stuff ive researched have used a mg to liter ratio. theirs is measured 0.05–5.0 μM thidiazuron.
I have no idea what μM means. (or how to get the font back):confused:

Also to make a koh solution? it says add a few drops of 1 N NaOH (or KOH) to dissolve the hormones then add water. once again i have no idea what 1 N is? I have a printed copy of the tdz article at work ill bring home and go over as well as plants from test tubes and ill have some more questions for you. your help is appreciated and i want to do this and share it with all the farm because i think it kicks ass but i know i need some help.

0.05–5.0 μM refers to micromolarity. The mu ( μ ) is for micro as in μL (microliters). A microliter is one millionth of a liter. Micromolar is one millionth of a molarity.

Molarity is measured in moles per liter ( mol/L). A mole is the amount of substance which contains 6.022x10^23 molecules (Avogadro's number) of that substance--and it is related to its molecular weight.

The following is a sample calculation (BUT PLEASE BEAR IN MIND THAT YOUR REAGENT MAY COME WITH BOUND WATER WHICH CAN CHANGE ITS MOLECULAR WEIGHT).

Thiadiazuron has a molecular weight of 220.25 grams per mole (g/mol). To make a 1M (1 molar) solution of Thiadiazuron you would add 220.25 grams of thiadiazuron to 1L water.

To bring this from 1M to 5μM we would then take 5μL of the 1M solution( a total of 5μmoles of substance is contained in this aliquot--because the original solution has a concentration of 1 mole/liter) and dilute it to 1L total volume.

Presto change-oh--you have a 5 micromolar solution.


In the lab we wouldn't do it this way, beause 5 mircoliters is difficult to deliver--even with a pipetteman. Instead we would use what is known as serial dilution (dilute 1 molar to 0.5 molar, to 0.05 molar to 0.005 molar to 0.0005 all the way down to 0.000005 molar, which is what we want).

 

[rootsnshoots]

thanks squig you are my ultimate weapon,
that could possibly pin point me to some things later. the reason why i wanted to figure that out is because those guys in that article had and average of 13 shoots per explant! using nodal segments. other hemp articles use leaf explants and get less but use mg to liter ratios. im excited to get my hands dirty or i guess super clean but you know what i mean, Any ways to make this easy for a rookie like me

i have 100 mg of TDZ. if i dissolve that then add 1000 ml of water, i will have a .1 mg to 1ml of stock solution correct?

same for the IBA and that gibber dude?

in plants for test tubes it says " using a dropper, slowly add, while stirring with a spatula, several drops of 1M KOH until crystals are dissolved". How do i make 1M of KOH?

after i have the stocks im going to just play with different hormone ratios to shoot and different explant types ( new leaf, the inside of nodes, roots?( aero cloner and then run h202 through it for an hour or so?nice little easy pre clean. also growing just roots may give me a chance to play with some myco's in the future? :D ) young leaf tips, chime in guys..)
then move on to roots....>> rootsnshoots<<--->> shootsnroots? what the fuck is going on here?

 

[rootsnshoots]

also you say i should use the dmso to dissolve the tdz on the site it just says for TDZ

"Properties

Form: Powder
Appearance: White to Off-white Powder
Application: Cytokinin

Solubility: DMSO, 1 N NaOH/KOH (See Application Note below)

Storage Temp: -20 to 0° C
Storage Temp of Stock Solution-20 to 0° C

Other Notes: TDZ is a substituted urea compound that elicits cytokinin activity in several plant systems, sometimes at 1/10th to 1/100th the concentration of N6-substituted adenine-based cytokinins [Yip and Yang (1976)].Plant Tissue Culture Tested

Application Notes

a 10 mM stock solution can be prepared by dissolving 22 mg of
TDZ in a few drops of 1 N NaOH (or KOH) with vortexing. Bringing the volume up to 10 mL will produce
a 10 mM solution. This protocol has not been confirmed

For additional information concerning TDZ and its cytokinin activity see Yip and Yang (1986) and
Thomas and Katterman (1986). Many other references are available by searching the internet.



The DMSO is like fucking glass or ice in a plastic bottle, i have no idea how to make the shit to dissolve the shit

 

[purpleberry]

I grow some ornimental plants and thought about doing this for some of the rare stuff. What would it cost to set up, just good enough to mess around with? I could build some stuff. Im just looking for a ballpark number, so i can figure out if its worth starting.

 

[squiggly]

The DMSO is like fucking glass or ice in a plastic bottle, i have no idea how to make the shit to dissolve the shit

Need some additional information from you.

Go ahead and report the following for each and every bottle/substance that you have--including your KOH/NaOH:

1. Molecular Weight (may be abbreviated on the bottle Mol. Wt., MW, or molar mass).

2. The full formula as written on the bottle (especially including H2O where it is denoted).


This will allow me to help you a bit more directly, instead of assuming what numbers you're working with I can just do the calculations for you.

In general, for KOH you'll weight out 1 mole of KOH (however many grams the molecular weight of the material is) and add that to 1 liter of water.

Remember, molecular weight = grams per mole
Molarity = moles per liter

As for the DMSO, it's melting point is ~66 degrees F, so you may just need to LIGHTLY heat it to get it into liquid form.

 

[rootsnshoots]

Yep my house was 66 degrees! lol

here is whats on the bottles,

Potassium Hydroxide Flake: KOH, CS-p1028, F.W. 56.11, 500g, Assay: 90.0%, CAS#: 1310-53-3

Dimethyl Sulfoxide: ACS reagent, minimum 99%, storage room temp, formula: (CH3)2SO, F.W. 78.13, CAS: 67-68-5, UN: N/A, HC: 2930.90.9065

Thidiazuron: purity: minimum 95%, 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea; TDZ, Storage temp -20 to 0 degrees C, Formula C9H8N40S, F.W. 220.3, CAS 51707-55-2, UN: N/A, HC: 2934.99.0000

Indole-3-butyric acid: IBA; 4-[3-indolyl]butyric acid, srorage temp 2 to 6 degreed C, formula C12h13n02, F.W. 203.24, CAS: 133-32-4, UN N/A, HC 2933.998350

Gibberellic Acid: GA3, Gibberellin A3, Storage temp 2 to 6 C, Formula C19H2206 F.W. 346.41, CAS 77-06-5, HC 2923.29.6000

the rest of the stuff is agar, ms medium with gamborg vitimans and charcoal.

Do i just use the dmso strait out the bottle in a dropper to dissolve?

Thanks for all your help squig i appreciate it

 

[squiggly]

Yep my house was 66 degrees! lol

here is whats on the bottles,

Potassium Hydroxide Flake: KOH, CS-p1028, F.W. 56.11, 500g, Assay: 90.0%, CAS#: 1310-53-3

Dimethyl Sulfoxide: ACS reagent, minimum 99%, storage room temp, formula: (CH3)2SO, F.W. 78.13, CAS: 67-68-5, UN: N/A, HC: 2930.90.9065

Thidiazuron: purity: minimum 95%, 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea; TDZ, Storage temp -20 to 0 degrees C, Formula C9H8N40S, F.W. 220.3, CAS 51707-55-2, UN: N/A, HC: 2934.99.0000

Indole-3-butyric acid: IBA; 4-[3-indolyl]butyric acid, srorage temp 2 to 6 degreed C, formula C12h13n02, F.W. 203.24, CAS: 133-32-4, UN N/A, HC 2933.998350

Gibberellic Acid: GA3, Gibberellin A3, Storage temp 2 to 6 C, Formula C19H2206 F.W. 346.41, CAS 77-06-5, HC 2923.29.6000

The molecular weight you are looking for is denoted by F.W (or formula weight) on your bottles. The figures are in grams/mol.

Be sure to follow the storage advice on those bottles--this is very very very important.

Do i just use the dmso strait out the bottle in a dropper to dissolve?

Thanks for all your help squig i appreciate it

That's what I'd do. Keep in mind that I'm not experienced with micropropagation so you might want to look up an alternate procedure to confirm that.

It's not a problem dude--I dig helping people with chemistry shit.

 

[rootsnshoots]

ok so for KOH i need to mix 56.11 grams into a liter of water and walla i got a 1M solution? awesome dude stock solutions will be made tonight. I may have a few more qs for the media prep.

also the storage advice on the bottles i did realy good cause every thing needed to be in the fridge except for tdz which is in the freezer. i DID hower let the TDZ sit in the fridge for a COUPLE WEEKS before looking at it and putting it in the freezer!!! you think im fucked?

 

[rootsnshoots]

1M KOH solution!

All mixed up, fizzed a lot. tsshhhhh!

I made half as much 28 grams to 500ml of distilled water. Ok to do right?

 

[Ythor]

Also note for TDZ that the source matters. TDZ is chiral; the stuff you get from tissue culture suppliers should be optically pure, while Dropp (the commercial product) is racemic- a mixture of both optical enantiomers. So, you can use Dropp, but you have to use twice as much to get the same effect.

When I dissolve down TDZ, I just add it to a 50 mL centrifuge tube, add some distilled water, and then add KOH solution dropwise until it dissolves; I then add more distilled water to final volume. Sterile filter, aliquot into sterile centrifuge tubes (usually a bit more than 1.0 mL, like 1.050 because you can't always recover every last tiny bit from a centrifuge tube), and freeze.

 

[rootsnshoots]

SQUIG!

In this article http://www.pakbs.org/pjbot/PDFs/41(2)/PJB41(2)603.pdf

it says in the abstract "MS medium containing 0.2 mg·L-1TDZ, 0.1 mg·L-1NAA supported the maximal auxiliary bud multiplication rate of 3.22 per shoot tip.

Whats 0.2 mg·L-1

 

[rootsnshoots]

Also note for TDZ that the source matters. TDZ is chiral; the stuff you get from tissue culture suppliers should be optically pure, while Dropp (the commercial product) is racemic- a mixture of both optical enantiomers. So, you can use Dropp, but you have to use twice as much to get the same effect.

When I dissolve down TDZ, I just add it to a 50 mL centrifuge tube, add some distilled water, and then add KOH solution dropwise until it dissolves; I then add more distilled water to final volume. Sterile filter, aliquot into sterile centrifuge tubes (usually a bit more than 1.0 mL, like 1.050 because you can't always recover every last tiny bit from a centrifuge tube), and freeze.

So what is, or where are you getting Dropp? Do you purify it?

If I make a 1L stock solution of TDZ how long does it store for in the freezer?

 

[rootsnshoots]

Turns out a TDZ stock will expand greatly in the freezer. I am storing it in a 1 quart small mouth mason jar. I filled it up too the 800 ml mark and it must have been a hair to much. The jar popped and cracked but it didnt get all over the freezer! high five?!? girlfriend says.. "no."

 

[rootsnshoots]

The stock solutions are done. I am ready for media prep.

Can you help me out Squig? lets just figure I am making 1 Liter of media. In the TDZ article it says Disinfected explants were inoculated on MS medium containing 3% (w/v) sucrose, 0.8% (w/v) agar and various concentrations of cytokinins.

How do i figure .8% (w/v) I know these questions seem stupid to you guys, so I apologize. also what the -1 in 0.2 mg·L-1 means.