Simple tissue culture help

Linkedlight · Feb 25, 2013

5 out of 6 bottles contam'd out, but the 6th, of, the 6th...

It's DJ Shorts True Blueberry and the 2-3cm cuttings are callused and rooting. It has been 13 days. I figure another 2 weeks and they should be nicely rooted and happily ready to go into a hardening chamber.

I have a load of other work to do, but I'm hoping to do more jars and keep them to 5 cuttings each. I need to get the contam issue under control.

Linkedlight · Feb 25, 2013

5 out of 6 bottles contam'd out, but the 6th, of, the 6th...

It's DJ Shorts True Blueberry and the 2-3cm cuttings are callused and rooting. It has been 13 days. I figure another 2 weeks and they should be nicely rooted and happily ready to go into a hardening chamber.

I have a load of other work to do, but I'm hoping to do more jars and keep them to 5 cuttings each. I need to get the contam issue under control.

BadMojo · Feb 26, 2013

Probably contaminated plant tissue. The pest way to reduce that is use Trichlor(sodium dichloro-s-triazinetrione) as a disinfectant. Only thing is you have to get a fair size sample of plant tissue to start the culture with. After you disinfect it you cut off the pieces that are damaged by the Trichlor. Yes it is some toxic shit but it is equal opportunity toxic. You can look up the concentration of trichlor to use in sterilization techniques as it was fairly commonly used at one time. Oh and I recommend using Tween(Polysorbate 20) as a wetting agent to reduce surface tension. It can be purchased quite cheaply on ebay.

Nunsacred · Feb 26, 2013

BadMojo said:
The biggest problem for amateurs conducting tissue culture is not the culture itself or the contamination. It is the moisture super saturation itself. .

Are you sure its not the tricky business of getting enzymes/hormones to suit the species in the exact right concentrations?

They're expensive and easily ruined by temperature, UV, or even oxygen

BadMojo · Feb 26, 2013

Yes, quite sure. A person will never reach the point to develop the chemical cocktails if contamination is not brought under control. It is the single most important act we can make in tissue culture. It effectively instantly wins us half the battle.

Nunsacred · Feb 26, 2013

Ok.

The coconut water supplying auxins & gibbers sounds too good to be true

Do you test it for contamination? I'm imagining a streak test it's been so long since I worked at a hood.

ClackamasCootz · Feb 27, 2013

Linkedlight said:
For tissue culture gear, I'd suggest phytotechlab.com in the US, not sure about Europe or use himedialabs.com

Linkedlight

Unrelated to tissue culture propagation I have been looking for a realistic price on freeze-dried coconut water. The prices at PhytoTech Labs are definitely realistic.

Thanks!

CC

Linkedlight · Feb 27, 2013

@ BadMojo-I'm going to limit the cuttings and keep it the same way. I don't like really nasty stuff

@Nunsacred-Coconut water is wonderful and it gets sterilized in the PC.

I can't wait to get to use the 1.5ppm IBA and .5ppm NAA for TC. I use it for the first 24 hours of regular cuttings and it's fantastic stuff for my really hard to clone varieties. IBA becomes too disgusting after 24 hours at room temp in regular conditions.

I'm hoping to get some pics up soon. to show more of what I want to do.

BadMojo · Feb 27, 2013

Nunsacred said:
Ok.

The coconut water supplying auxins & gibbers sounds too good to be true
Do you test it for contamination? I'm imagining a streak test it's been so long since I worked at a hood.

Never made a point to test it. Let me just say I hardly call it magical. My preferred Cytokinin is 6-Benzylaminopurine or Bap 6. My preferred Auxin is IBA though I am not above NAA or even IAA when it is available.

(Found that the different auxins can have radical effects on root formation of different plants. Also cocktails of two or all can have differing effects depending on the ratios.)

Where Coconut water becomes useful is that say you want to encourage say 80 percent root growth and 20 percent top growth. Well you would use an auxin namely a synthetic one like IBA or NAA(coconut milk has natural IAA) you would reduce the amount of auxin slightly and add coconut milk.

Now overall this will slow the culture slightly but it will make it a bit more shock resistant when you transplant it to a higher cytokinin media when enough roots have grown.

One would use the coconut milk in the Cytokinin media much the same way we did the Auxin media. You want to slow root growth but not stop it more or less.

Another cool thing is that on some plants just agar and coconut milk can make for radical amounts of callus formation.

Think of Coconut milk as being a moderating agent. The goal is not to get faster cultures. It is to get more cultures to survive.

Javadog · Feb 27, 2013

Hello all,

I am not that close to trying this, but am looking to start gathering information.

I am an experienced mushroom cultivator, and so have a laminar flow hood, PC,
and all the other materials related to the sterile work that mycology requires.

It seems that there is no perfect book (think "Growing Gourmet and Medicinal Mushrooms")
for getting started with this topic.

So, let me ask whether there is a links page associated with this topic?

Please post any good resources for the new to follow.

Thank you!

JD

BadMojo · Feb 27, 2013

Hey Javadog. Sounds like you are already pretty close to full prep. Tissue culture is easy to do with the right equipment. As far as I know there is no link page.

My primary suppliers are Caissonlabs.com(Serious) and Phytotechlabs.com(has some unusual chems)

Javadog · Feb 27, 2013

Thanks for that BM. I have bookmarked them for getting supplies.

Take care,

JD

Linkedlight · Feb 28, 2013

[FONT=Arial, Helvetica, sans-serif]@ClackamasCootz-Check Asian grocery stores for coconut water. It what the tissue people in my neck of the woods do. The cost savings are substantial. I'm lucky that almost all of my TC equipment and chemicals are cheap as chips.

@BadMojo- You seem to know your stuff. Do you think that rooting needs different cultivars may require different concentrations. I'm keen to experiment, but I'm busy atm. How much have you tried so far?

@Javadog-Check out himedialabs.com as well. Indians are a pleasure to work with in my experience, but YMMV. Since you are into mushrooms and have good sterile technique I'm guessing, then you're a hop, skip and jump away from TC. The problem being there is no good book on the subject as of yet with conscise easy to follow directions for technicians. You'll find that you have to trawl the web and books to get all the information that you need.

Here's my tek: To be called LinkedLight's tek and make me rich and famous in the future. :biggrin:

)
150ml of coconut water
5g agar(Asian grocery store)
1.5ppm IBA(1ml of 1.5g of IBA dissolved in a few drops of 1M KOH solution* and then fill up to 1L of water and keep covered in a cold fridge)
.5ppm NAA[/FONT][FONT=Arial, Helvetica, sans-serif](1ml of .5g of NAA dissolved in a few drops of 1M KOH solution* and then fill up to 1L of water and keep covered in a cold fridge)
1L worths of MS nutes+vitamins
20g Sucrose

*1M of KOH solution is done as follows. 5.6 g of KOH and 100ml of water. That will last you a long time in your chem fridge. You can also buy the salts of both IBA and NAA with the salts already added such as Na-NAA and that dissolves just in water.

I have to PC that for 1 hour due to the nature of my PC, but have no problems with caramelization. I make 6 jars and then have 2 more jars filled with plain water for my clean water for a total of 8 jars. I'll also throw in my special tweezers and paper towels wrapped in aluminum foil and bagged.

I cut 2-3 cm cuttings off of the plant, then do the following
*10 minutes 1% bleach solution with 2 drops of tween 20 on stir plate(1ml of bleach to 99ml of water)
*Rinse in distilled water(I use the water I PC'd since distilled would have to be done by myself)
*5 seconds in 3% H2O2(Hydrogen Peroxide) from a pharmacy is fine.
[/FONT][FONT=Arial, Helvetica, sans-serif][FONT=Arial, Helvetica, sans-serif]*Rinse in distilled water
*To that initial 1% bleach solution I add 9ml of bleach and then soak the plants in it for 15 minutes.

Transfer to hood onto a clean paper towel with good sterile technique, then open a bottle and put your cuttings in. I then cap them up, put them in a bag that I seal, date and mark the strain/pheno and then put them under T5's till roots appear.

Let me know if you have any advice or questions. I'll reread it later to make sure I haven't missed anything.
[/FONT][/FONT]

Javadog · Feb 28, 2013

What a TC write up! Thank you for taking the time.

(that was the text from my Reputation thingy, and I figured why not copy-pasta ;0)

Seriously, I have a small list of chemicals to get, and this may take a while, but it
started here. LOL.

Take care,

JD

P.S. If you ever want to figure out growing fruits, then let me know:

Linkedlight · Feb 28, 2013

Things I remembered:
Agar-Oh how I hate it
When adding agar, make sure the water is boiling. If using a heated stir plate which makes everything easy, just sprinkle it in. The water will cloud up, keep it stirred and wait for it to go clear. It must be clear, not cloudy at all. I tend to let it boil for 5-10 minutes with the agar in. Cloudy agar=fail, adding it to non-boiling water=clumps.

pH should be 5.7 thereabouts.

When adding the 9ml of bleach to make a 1% 100ml bleach solution become a 10% solution, make sure the cuttings are being stirred.

Trim the cuttings up some, it's an art not a science.

When I get pm priviliges, I'll be pm'ing you(Javadog) loads to help me out with mushrooms. We need them for medicine here.

Hardest/dangerous chemical to get would be KOH. Everything else is easy. Try to get a heated stir plate, using a stove and spoon to make the agar sucks.

You can make a stir plate and there are some good teks how to do it cheaply, even for heated ones.

With the IBA and NAA I make a nice solution with both and keep it in the fridge. I then use it for the TC and as clone water for the first 24 hours after taking the cuttings. Waste not, want not.

Javadog · Feb 28, 2013

Yeah, I take a do-no-harm approach and have one online identity. (and avatar, but I cannot
get it to show up here)

LOL, I cook my agar on the stove, like I am making a soup or something.

I do have stir plates, but will start watching for one that it heated. That would indeed
be a cool tool to add to the mix.

Take care,

JD

BadMojo · Mar 1, 2013

@BadMojo- You seem to know your stuff. Do you think that rooting needs different cultivars may require different concentrations. I'm keen to experiment, but I'm busy atm. How much have you tried so far?

Sorry about the delay. Had to find my notes. My lab got hit a few years back and lost a lot of stuff.

Yes absolutely. I have found that variations occur with all varieties including individual plants of a single variety. Though the more stable the line the easier it is to isolate what the line likes. Take Herijuana for instance. It takes quite well to the formula below for root formation. All the Herijuanas I tried liked it but some of them were dialed in better than others.

Unless otherwise noted all ingredients are lab grade reagents.

200 ml of coconut water(Freshest Coconuts I could find)
4.33 grams of MS mac and mic nutes
500mg of Casein hydrolysate
800mg of Mannitol
100 mg of Insositol
30 grams of Sucrose
2 grams of Gelrite
.8 mg of NAA
make up the balance with distilled water only. Total batch size 1 litre.

Bio boy · Mar 1, 2013

any 1 in the uk give me a supplyer i can use?

BadMojo · Mar 1, 2013

Will have to do a little checking but there are UK suppliers or at least EU ones. There is a source for PPM in Australia too.

Bud-Boy · Mar 2, 2013

Sub'd

Tall tale in these parts of some of the old growers using tissue culture

Sorry,
Idoubt it after researching

But i know its possible

So good luck friends

---- had a lam flow hood from my fungus days
Loved that thing
The filter was about $400 20 years ago. The set up, about 6 bills.
I use syrynges now

Simple tissue culture help

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