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Envenomated agar? Any one try this?

Not that i have immediate plans for this knowledge but I've been reading all over about agar work and one bizare thing I came across was utilizing envenomated agar! That is, adding a percentage of powdered snake venom to your agar mix to help with the sprouting of hard to sprout seeds/spores and making invitro mushroom mycelium hybrids by the venom weakening the cell walls of whatever is placed on it but I assume it's easy to overdo it and kill the culture.

Unfortunately no ratios were given as to how much is used in a liter of agar solution. Sigma chemical sells a variety of powdered snake venoms but in microgram increments, it ain't cheap, so I assume not much is required.

How about adding powdered activated carbon in your agar to get fewer contaminations? Phyto Tek Labs even sells orchid agar mixes with activated carbon already added, is it worth it? Of course you must use sterile technique with agar but any extra preventitive is a plus in my book.
 
My cohort who does TC uses the activated carbon in some flasks with good results. There are some plants (like P. filifolia) that seem to produce a toxin that builds up in the agar, using the carbon can extend the period of time the plants can stay in flask without being transfered to new media. But it is not a fail-safe under those circumstances, you still have to attend to them regularly.
 
Thanks for the input Pyro, I suppose rain/watering washes those self-produced waste chemicals away whereas on agar they just sit there and build up. Good to know! On average how often does your pal do transfers? Monthly, bi-monthly, quarterly?

I got excited over the idea of less contams getting a foothold and bought a screw top 900 g container of activated charcoal and powdered it in a coffee grinder, at 1 - 5 g per liter I'll probably never need another box.
 
Could you kindly reference this article? It seems difficult to believe that cytotoxic/hemotoxic snake venom would be effective against plants. Animals cells have membranes which certain snake toxins indeed do destroy, but plants have cell walls. It is almost unfathomable that mother nature would endow snakes with enzymes targeting plant cell walls since they are not a food target and require no immobilization for an animal such as a snake to consume.
Thanks.
 
Snakes don't need venom to immobilize a plant, but use it instead as a salad dressing.
 
I have no idea where the original study is or who performed it but it was being discussed in another online forum in the context of agar work with creating mushroom hybrids. Someone must have a use for it since you can buy a huge variety of venoms from here just search "powdered snake venom":
http://www.sigmaaldrich.com/homepage/Site_level_pages/CatalogHome.html

If you were interested enough you could contact Sigma and see what uses they sell it for, maybe they can give you some reference, I just thought it was weird! :-))
 
Hey Swords,

Yeah, in nature the plant toxins are washed away but in flask they just continue to accumulate till they reach self toxic levels.

AS for schedule of transferring, it depends on the plant. With those that produce a toxin it is usually obvious when it needs to be done because the media goes from clear to straw to yellow to orange to brown. The orange/brown level is where toxic effects begin to be noticed. If the plants do not produce toxins then it is dependent on the growth of the plant. Something that divides prolifically (like D. falconeri) can be thinned and transfered every 3-4 weeks. Other things that go slow can stay in flask for months on end.

Dnieter,

Just because the venom of snakes was evolved to effect does not mean it can not effect plants. Many proteins have multiple effects aside from their principle one. On the topic of venom there are spiders in Australia that are only hyper-toxic to humans and other primates. Just a bizarre side effect of them evolving venom to subdue their insect prey.

And based on what Swords said, it was principally used on mushrooms and the cells of fungi are more like those of an animal than that of a plant so the venom is more likely to have an effect on them.
 
Thanks Pyro, do the plants toxin colors show up on the agar containing activated charcoal? I've only seen one pic of charcoaled agar - it was quite dark. That's the main drawback I can see with it, is not knowing whether something like that is happening.

I guess that's what experimenting is for! :)
 
AS for schedule of transferring, it depends on the plant. With those that produce a toxin it is usually obvious when it needs to be done because the media goes from clear to straw to yellow to orange to brown. The orange/brown level is where toxic effects begin to be noticed.

VERY interesting! Thanks for sharing this. I had a culture of D. Capensis seedlings that I just let do their own thing for about half a year (or more) after taking what I wanted from the culture.

That exact effect started happening. The media began to haze and spread as you indicated along with the color changing. I figured it was just contaminated but still it was different, the spread was -through- the agar, not mold puffs or anything growing -on- the agar. The darker stages began to kill the plants in the culture.

I was a bit fuzzy for the need of doing sub cultures. Transferring the plant into the exact same media wasn't making a lot of sense to me. But that toxin info was new to me!

Nate
 
  • #10
@ Swords:

You can see the colour change in charcoal containing media but it is more difficult. I find that in charcoal media the charcoal will settle, not a huge surprise as the agar is still a "liquid". As such there is usually a "clear" zone that is about the first 1-3mm of depth. You have to view it at an oblique angle but you can see it.

I have a few cultures that tox'd out, I'll try to photo them so you can see what it looks like.

An alternative that may work for you is to make an expendable culture in plain media that you can watch for colour change and when it reaches the yellow stage then switch all the flasks.

Nate,

Never heard of capensis being one of the toxin makers, course I have never heard of anyone TCing capensis so that may be why. But in my experience no Drosera does the tox thing.

The hazing sounds more like a bacterial swarm to me but I could be wrong. There is also a strange algae that can swim through the media as well.

The transfer thing is to keep the plants happy. They do deplete the resources in the agar with time so you need to transfer for that reason alone. And some plants just divide like gang busters even in basic growth media (N. rajah is notorious for this, which is why Rob has 20,000 of them...) so you need to just divide and divide and divide.
 
  • #11
Never heard of capensis being one of the toxin makers, course I have never heard of anyone TCing capensis so that may be why. But in my experience no Drosera does the tox thing.

The hazing sounds more like a bacterial swarm to me but I could be wrong. There is also a strange algae that can swim through the media as well.

The transfer thing is to keep the plants happy. They do deplete the resources in the agar with time so you need to transfer for that reason alone. And some plants just divide like gang busters even in basic growth media (N. rajah is notorious for this, which is why Rob has 20,000 of them...) so you need to just divide and divide and divide.

Pyro, I'm sure it could have been a zillion different thing, and without actually examining the culture in person, or visual photos at the least I'm sure it'd be very difficult to find out what happened to it. I think that if anything you've given me some more insight to some of the 'other' reasons cultures go bad, and it seems worth a shot at least to post failed cultures for the experts out there that might be able to give me a better idea of why it crashed.

And yes I do get snickers when I talk about culturing capes, but it's not that I want to raise a genetic army of D. capensis for a lifetime of profit... I want to learn about the process, the procedures, and tools needed in order to do TC properly. Capes are cheap (free!), seeds seem to always be viable, and I won't be heart broken if they go bad. Before I want to begin laying waste to Nepenthes seed, I want to feel a lot more comfortable with experience. 8)

Nathan
 
  • #12
actually capensis seems like a good one to practice on to me.....if they dont germinate you KNOW its something you did, no second guessing weither it was the source material that was the actual problem........
 
  • #13
I'm gonna be starting my TCing with droseras too (not capes specifically) but mainly cos they sell the seed on ebay by the lb. But i want t\o do all kinds of things just figure dews are easier going.

Has anyone tried using a drosera leaf soaked in hydrogen perxiode then placed on agar to start droseras invitro that don't have seed available? I don't mind a big failure rate on such a procedure since it's only a hobby but if one piece stays clean and grows that one vial is all that's needed. I have started sundews on peat this way but not sure if it could be done clean enough to do invitro?
 
  • #14
Swords,

I'll double check with my buddy but I think you need something stronger than H2O2 for TC sterilization. IIRC he used a bleach solution but I can not recall the %. It worked for both Drosera leaves/tubers/gemmae and Ping leaves
 
  • #15
Okay, sorry it took so long, had a million other things on my plate

Here are some pics for you all

Toxin accumulation in agar
P2280001.jpg


Charcoal agar, note how there is separation
P2280002.jpg



And to give you an idea of how different tranfer times can be...

This flask was transfered into on 5/07
P2280003.jpg


And this one on 4/07
P2280004.jpg


So obviously different growth rates make for different frequency of transfer
 
  • #16
Neat stuff thanks for showing the pics!

I suggested Peroxide cos I've heard bleach was sometimes too strong for seeds and merstems.
 
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