In vitro micropropigation

[caveman4.20]

i dont understand less than half the stuff but i read above my understanding hoping to understand it eventually but this is indeed intense....i would like to see some pics cuz i never have not of this.

 

[squiggly]

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

Yes for your 1M solution calculation you were correct.

0. 2mg*L^-1 is a special mathy way to say mg/L or milligrams per liter.

Also, you mentioned the fizzing--next time, add the KOH more slowly--and never add water to the powder, always powder to water.

This is an exothermic reaction and it can be bad news bears if you rush it--slow and careful = not losing your eyesight.

ALWAYS WEAR EYE PROTECTION WHEN MIXING CHEMICALS--ALWAYS!!!!!!!!!!! The glasses should specify on them somewhere "z78" compliance.

 

[rootsnshoots]

Yes for your 1M solution calculation you were correct.

0. 2mg*L^-1 is a special mathy way to say mg/L or milligrams per liter.

Also, you mentioned the fizzing--next time, add the KOH more slowly--and never add water to the powder, always powder to water.

This is an exothermic reaction and it can be bad news bears if you rush it--slow and careful = not losing your eyesight.

ALWAYS WEAR EYE PROTECTION WHEN MIXING CHEMICALS--ALWAYS!!!!!!!!!!! The glasses should specify on them somewhere "z78" compliance.

Awesome dude,

I put the water into the powder and was NOT wearing safety glasses, I was careful not to touch my eyes and wore gloves but that could have been bad and.... I'd really like to see this through...I will be sure to pick some up from the science store tomorrow before I continue anything. I should probably pick up a basic chemistry book from the library or a friend as well. The way I am doing this is pretty janky.

and for my other question about the .8% (w/v) when i was looking through plants from test tubes they have a ton of recipes for different plants a lot of them had 30 grams of sucrose and 8 grams of agar per L so im assuming thats that.

Well I am pretty confident I can mix and cook media tomorrow, Sterile sesh on Tuesday, then we wait. I'll post some pics too!

 

[squiggly]

Yeah dude, be careful.

KOH not only can, but will blind you. You should also be wearing gloves, especially when handling the dmso.

 

[Ythor]

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?

Dropp is the commercial version of TDZ; it's used to defoliate cotton in the field. If you live in cotton country, it's all over the place. Several plant growth regulators are used commercially (Liberty, for example) in large quantities in a form that is vastly less expensive than the lab counterpart.

As for TDZ in the freezer- it should store indefinitely when frozen at -20C (your typical freezer).

>and for my other question about the .8% (w/v) when i was looking through plants from test
>tubes they have a ton of recipes for different plants a lot of them had 30 grams of sucrose and 8
>grams of agar per L so im assuming thats that.

These components are often used in these concentrations. Typical for sugar is somewhere between 20 and 35 g/L (depending upon many factors, including the crop- up to 50 g/L for potato microtubers, but more is not always better!), and 8 g/L agar depends upon several factors including the gel strength, concentration of nutrients, etc. Try some batches at 8 g/L of A111 PhytoTech agar and see how well it gels; too strong isn't necessarily bad- the roots will still penetrate- but it's wasteful and agar is expensive so when you're making hundreds of liters, it's common to tickle the dragon's tail and reduce the amount of agar to the point where the gel is very weak- but the plants are still well-supported physically in this fashion.

 

[rootsnshoots]

Dropp is the commercial version of TDZ; it's used to defoliate cotton in the field. If you live in cotton country, it's all over the place. Several plant growth regulators are used commercially (Liberty, for example) in large quantities in a form that is vastly less expensive than the lab counterpart.

As for TDZ in the freezer- it should store indefinitely when frozen at -20C (your typical freezer).

>and for my other question about the .8% (w/v) when i was looking through plants from test
>tubes they have a ton of recipes for different plants a lot of them had 30 grams of sucrose and 8
>grams of agar per L so im assuming thats that.

These components are often used in these concentrations. Typical for sugar is somewhere between 20 and 35 g/L (depending upon many factors, including the crop- up to 50 g/L for potato microtubers, but more is not always better!), and 8 g/L agar depends upon several factors including the gel strength, concentration of nutrients, etc. Try some batches at 8 g/L of A111 PhytoTech agar and see how well it gels; too strong isn't necessarily bad- the roots will still penetrate- but it's wasteful and agar is expensive so when you're making hundreds of liters, it's common to tickle the dragon's tail and reduce the amount of agar to the point where the gel is very weak- but the plants are still well-supported physically in this fashion.

Awesome info thanks a million,

Nope I live out west, where hopefully hemp will be our cotton one day. I'll have to do some searching. I love cheaper alternatives.

You obviously have some good experience with micropropigation. Hopefully you can be around for a while and give me pointers.

 

[squiggly]

it's common to tickle the dragon's tail and reduce the amount of agar to the point where the gel is very weak- but the plants are still well-supported physically in this fashion.

If my biochem professor's "garden" is any indication--the plants actually prefer to be *just* supported--the less the roots have to work the better.

 

[rootsnshoots]

Lets talk culture vessels. My plan is to use my old half pint jars I have used for mycology work. I image they need gas exchange?

Do you guys think this will work? or do I need more air?

3 layers of tyvek over the holes taped down with micropore tape.

other side, never mind the injection port

agar is expensive so when you're making hundreds of liters, it's common to tickle the dragon's tail and reduce the amount of agar to the point where the gel is very weak- but the plants are still well-supported physically in this fashion.

In PFTT they show liquid media and build little bridges to hold up the cuttings for some plants. Very cool!

 

[rootsnshoots]

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.

I think this could be done on a relatively small budget. Saying this with no successful experience in TC..

The staples.
Pressure cooker. I scored an 21.5 quart all american a couple years ago for 100 bucks on craigslist. but they retail for around $300 i think.
air purifier. you can get a hepa room filter for like $80 at walmartinez , then tape a clear trash bag to it and do your sterile work in the bag. I have done plate work like this for years with minimal contams, it takes practice though.
Then i guess your down to vessels, cleaning supplies, media, measuring thangs, which adds up! SAFTEY GLASSES!!! my guess ball park $800 would get you a nice hobby set up. You should do it for sure!!

After I get to rockin here ill do some step by step posts with the deets. ornamentals would be fun there is soo much info on them you could probably pick it up pretty quick id imagine. with my experience in mycology its patience, being anal as all hell, clean clean clean, and going through gracefully and efficiently. It takes practice and persistence. So fun when it goes though..

 

[Ythor]

Lets talk culture vessels. My plan is to use my old half pint jars I have used for mycology work. I image they need gas exchange?


In PFTT they show liquid media and build little bridges to hold up the cuttings for some plants. Very cool!

The containers normally employed in tissue culture (Magenta boxes, Magenta B-caps for baby food jars) allow sufficient gas exchange when the containers are sealed with Micropore tape.

Also note that rockwool cubes can be autoclaved along with liquid media- no agar needed.

 

[rootsnshoots]

sorry i didnt know how to upload pics

 

[rootsnshoots]

OK I mixed up some media a while ago. I hadn't really thought it through and winged it. I messed up on my TDZ measurements. Anyways I ended up with a .25mg to ml ratio on 4 jars and a .45 mg to ml on 4 and then 8 jars with really strong amounts which i have not yet inoculated. And i added 2mg of Gibberellec acid to all of them.

Also apparently my old roommate sold our flowhood on fucking craigslist. So i'm going old school and using a SHMUV till i build another.

Here is the SHMUV set up. I have used this system for a long time but still stress about contamination every time. I have had it to 100% success rates but also lost everything. The filter I am using in the photo is NOT a true HEPA it is a HEPA like filter, but this should get me through the experimental stages. As well as show you folks at home how to set up your own lab in your kitchen and put this into practice on a budget.


Here are the explants after a 15% bleach wash and two sterile distilled water washes


Inoculated jars under fluorescent


5 Days after inoc. I took this cutting from the leaf at the very tip of a vegetative plant. It was pretty small, maybe 2cm long. The leaf grew huge fast!


well have to see if these shoot. I want to make up more jars with a little more TDZ. i might as well try the really strong ones too. Also i will definitely be using less media in the jars, and pick up some jars that are easier to see into.

 

[squiggly]

Very cool--keep the updates coming

 

[Ythor]

OK I mixed up some media a while ago. I hadn't really thought it through and winged it. I messed up on my TDZ measurements. Anyways I ended up with a .25mg to ml ratio on 4 jars and a .45 mg to ml on 4 and then 8 jars with really strong amounts which i have not yet inoculated. And i added 2mg of Gibberellec acid to all of them.

0.25 mg/mL TDZ is way too high. Do you mean a final concentration of 0.25 mg per liter?

 

[rootsnshoots]

0.25 mg/mL TDZ is way too high. Do you mean a final concentration of 0.25 mg per liter?

Oops yep .25mg/L. My stock solution isn't even that strong. Good eye!

 

[YarraSparra]

Thanks for the love yarra,

Out of curiousity what was the company you applied for? I have heard a lady on a video cinfidently say " you can turn one plant into a million in under ten months" which by no means I have that type of facility/dollars/reason to do numbers like that, but I believe it is possible no doubt.

I have heard of in vitro breeding, but currently am not trying to accomplish or even think about (though as soon as i heard it i was like WAAAA!!!!) that untill I have the basics down to a science. I will definitley be taking things slow in this operation, experiment by experiment. I have a major issue with trying to jump forward too fast and try new things before I even know the basics. ( even my first grow many many years ago was a very complicated aeroponics system, which even now as a pretty experienced grower would have a hard time keeping up with) so i will be keeping the horse in front of the cart on this one. Meaning this thread should be around for a long time to come.

If there is any information you know of please share it with me, I am far from a scientist, but maybe one at heart.

much love,
rns

I can't remember the name of it. Too much smoking. :D
IIRC it was a company that dealt almost exclusively with roses, which would have been dull.
Would have loved to have learned the skills.

Have to say, things are coming along really well!
Pro looking setup you've got. The inoculated jars look good.
Any updates?

YS

 

[rootsnshoots]

Thanks Yarra
I love roses that shit woulda been tight!

Unfortunately the only update I have is...CONTAMINATION! I was expecting it. I didn't flame sterilize my tweezers and was sloppy on the transfer. I also don't think I cleaned the explants throughly enough. I think I had a little beginners luck with one though..
View attachment 274277

It's hard to see in the jar sorry

Also I have made up 24 jars with .1 , .2 , .3 , .4 mg/L (6 of each) the prep went much better! Yay math. And I am excited to go again with another sterile sesh. Now that I know what to expect it should go smoother.

Edit. Sorry if that attachment doesn't work. I tried to upload it off my phone. I'll take some pics and upload tonight on my comp.

 

[rootsnshoots]

This one is shootin nice. Kind of a yellowing and getting pale though. It's hard to see through the jar but I got better ones. This was a nodal segment explant in what I thought was way too strong of a tdz solution.





^^ all the same jar

24 ready.


And introducing, the all new high visibility, super kerr vessel! ;)


Mondays are busy for me so ill hopefully be able to prep water and tools teusday and giver a run on wed.

 

[monkeymun]

Great thread! Are you forming a callous first and then going the way of roots and shoots (no pun intended!)?

 

[carBon.14]

wow that is some crazy shit! you're like a mad scientist! so cool...