TC of mitragyna speciosa

[Jojobafruit]

Not sure if this should go here or where.

I have just bought a nice flowhood and tc tubes and a pressure cooker and i am working on getting a controlled enviroment for the cultures to grow in. I have learned of deflasking and introducing them when they are ready. I know one could use agar. but i dont know what types of nutes or ratio's to add to support root growth. also would one use a leaf or node or stem peices for the tc. ? maybe there is a tek for doing tc of mitragyna out there. ?? Im willing to componsate rather nicely. I figure this is the best place to ask. this is where every other board has sent me.

If anyone can help lemm know. I also plan on doing tc's on diplopterys since it is sooo hard to root cuttings from..

[Darklight]

Jojobafruit:

Not sure if this should go here or where.

As far as I know its not a native species here

I have just bought a nice flowhood

Ooooh! Whadja get?

The TC protocol for Mitragyna speciosa was developed here and is awaiting publication.

Do you have any experience with plant tissue culture, orchid deflasking or mycological transfers? If not it might- no it definitely would be- best to get a good background in the practicalities and practices of aseptic culture before you start throwing genetic material around.

Even after yeeears of working fulltime in TC I still get horrible stagefright when confronted with a new species and a limited amount of material ( that's OK, everyone does ) and the risk of contamination from outside is huge.

[Stonehenge]

I asked about the same thing a while back and got a crash course from darklight and Torsten which will help a lot when I get back into it. There is a thread or two on the subject if you search back a bit.

The flow hood will help a bunch but there are so many gremlins that no one is guaranteed of success. Even the big companys with research labs and PHD's running them sometimes have to stop production and decontam everything in site. Usually they have it set up so it's not a big deal. I had done shrooms and thought I had a fairly good grounding in sterile procedures. It turns out that when you go to something else you find new obstacles that you didn't face previously. I had lots of probs with contams. After talking it over here, I came to the conclusion that it was likely dust mites or paper mites causing the probs or at least many of them. I had baby food jars with neoprene lids made for this work. I'll swap you some if you like. I pressure cooked them and let them sit and every single one eventually cam down with contams including a dozen or so I never opened after pc'ing. That's what lead me to the thought that it was a mobile contam like a mite bringing in fungus spores. You need sticky traps around all entrances or around your jars or plates to keep them out.

Torsten has been there and done that with Mit. S. I wish I could do it myself and may get back into it one of these days. One of the probs is it's hard to decontam living plant tissue. Read the ghetto tc website. After you get your tissue decontamed and in the culture medium then all you have to worry about is proper temps, proper nutes, proper hormones, proper ph and a few other things.

Stoney

[Darklight]

Stonehenge:

There is a thread or two on the subject if you search back a bit.

Thanks stoney I appreciate you remembering this cos I forgot. Sometimes I worry I sound like some bizarre den mother repeating the basics over and over again

It turns out that when you go to something else you find new obstacles that you didn't face previously.

That's the one I said to jojoba- the difference is you don't contaminate your mycelial work with plant material too often, but it does happen the other way around I think Rev said that initially and its so true.

I pressure cooked them and let them sit and every single one eventually cam down with contams including a dozen or so I never opened after pc'ing.

Did you ever get around to getting that lovely breathing sealing tape? Austraseal. Love it. Or even the nonporus stuff, what's it called? Hang on I'll check... Nescofilm ( I prefer it to Parafilm as it doesn't stick to everything else so readily ) Don't use 3M tape, no matter what you read on the web, its paper and goes fungussy, releasing spores around you when you open it

G'wan, give it another bash stoney...

I wish I could do it myself and may get back into it one of these days.

I wish you could too. One of the reasons I harp on about the basics ( this doesn't apply to you stoney ) is that so many people ask me these questions so early in the peace, and ignore the answers. There is the deathly silence of a hundred unopened laminar flow cabinets out there simply because people got demoralised once they realised how much work and thought there is in this process

Frankly, if you're not prepared to *dedicate* six months of your life to getting started up and your first species in tissue culture- don't bother. And if you have the six months, be prepared to spend it working hard for long hours with the odd stint of sitting round waiting for something to happen. You might strike it lucky in six weeks, and your chances with a new species are as good as a lab full of PhD students- but more often than not science doesn't work

One of the probs is it's hard to decontam living plant tissue.

Even when you do two weeks proper pre-prep first and have a decontamination protocol already which straddles the fine line of successful decontamination without actually killing your material- its still difficult.

Read the ghetto tc website.

What bloody ghetto TC website? Arghhh now I'm curious...stoney?

You can succeed jojoba, but like I said, you have to give yourself every chance. Once your groundwork and your practice are underway though, you are set for life. TC isn't rocket scince, but it is picky, detailed and often very very dull, except when you get it right- then its WUNNERFUL!

[Darklight]

Darklight:

Frankly, if you're not prepared to *dedicate* six months of your life to getting started up and your first species in tissue culture- don't bother.

OK I exaggerate a bit. You can put the same amount of time in over a longer period, and make it work for you, by getting good training, background and practice. Which of course saves time in the long run!

[Darklight]

One of the main differences between plant and mycelial culture is timing. You can leave a plate of mycelia in suspension for months- years maybe. In one sterile form or another- spores, plates etc

But once you have an actively growing culture you have to keep at it, unless you have a long term maintenance protocol for it to allow it to grow slowly. Think between two to five weeks for the average subculture period. The plants set your schedule- not you. Forever

Every three to five weeks you either have to have a successful protocol to transfer to, or work one out ( saving enough material back to retry in two weeks should it fail ). Which means you have to have a sterile space, sterile culture vessels full of the right medium etc. Then you go through it all again to obtain a rooting protocol. And again for deflasking.

So for a new species you have intitated successfully in sterile culture but not obtained a protocol for, and are running four or five experiments on simultaneously, that's a lot of firing pressure cookers up and keeping stuff separate so you don't get your experiments mixed up, and obtaining different materials and researching and keeping records for and washing bloody jars out. Etc

This isn't to put anyone off. Having experience in mycelial culture, or better still orchid culture, will make it *much* easier to learn. At the very least plant TC is a rewarding, if time hungry hobby and can stay that way for yeeeears without necessarily overrunning your life. If you don't have the time, stick to orchids and fungus for just as much fun and reward

[puffinstuf87]

 

quote:

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The TC protocol for Mitragyna speciosa was developed here and is awaiting publication.

 

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Do you have any idea when it will be published? Thanks

[Jojobafruit]

Yes that would be a publication i wouldnt mind getting. .

[jokerb]

any news about the TC protocol for Mitragyna? Was it already released on the net?

thanx for your help,

cheers

[teonanacatl]

is propagation by cuttings not viable for you johoba? or do you want to do it via TC? i can't wait to get into some TC :D

[ 26. July 2005, 14:12: Message edited by: teonanacatl ]

[Benzito]

I believe the Mitragyna TC protocol was developed by Torsten, Darklight, and some others from the SAB camp.

Back before it became illegal.

So, whenever they get time to publish, I guess.

[Guest d0tb0y]

Although i have said it before, (procrastination, study and other commitments) i am working at getting a flowhood and have most of the gear for TC ready to go.

Perhaps if torsten was nice enough he could maybe open a forum for TC'ers or maybe even a page we can post on, so we can post protocols and tips for each of the ethneogens/plants we are tc'ing at the time.