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shonman

Micropropagation, tropical trees, Mitragyna Speciosa

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I am in a place where it is still ok to have the Mitragyna Speciosa

....if that changes, things will change here.....I am following the local rules and regs etc.

I am very interested in using plant tissue culture to mass propagate this plant.

Can anyone point me in the right direction?

I have alot of respect for the work Darklight has done in this area....

No one owes me anything, but I would be very grateful for knowledge of how to micro propagate this plant......would be willing to share too.

Thanks!

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There are scientific publications out there which have successfully micropropagated Mitragyna species. Like a lot of tissue culture protocols these days, one of the better ones is found in a much more comprehensive paper

Google yields this:

www.znaturforsch.com/rc/s63c0691.pdf
M. speciosa seeds were collected from Hat Yai District, Songkhla, Thailand. They were surface-sterilized by rinsing with 70% (v/v) ethanol for 5 min, rinsing with 20% (v/v) Clorox (NaClO; Selangor, Malaysia) for 5 min and finally rinsing with sterile distilled water thoroughly. Sterile seeds were germinated on WPM supplemented with 1.0 mg/l BA and incubated at 25C under 16 h daily light.
Above is the tissue culture info from that paper. You pretty much won't need anything else from that paper for clonal replication. It isn't the method I used back when it was legal ( I haven't had kratom plants in my lab for seven or eight years, after repeated subculture the culture I had became weak about the time the plant became illegal and saved me the trouble of wondering whether to ditch it )
The only flaw I can find in that section is that the Chlorox concentration looks dubious. Not sure if it is a final concentration of the actives in Chlorox, or a dilution standard of Chlorox, which as a commerical formulation could change in composition over time, without reference to the paper. I used a final bleach concentration on 1.5% NaOCl and for much less than 5 minutes- the seed is tiny. The 70% ETOH/ Chlorox protocol above could work too- just don't use all your seed stock at once
How is your tissue culture technique? It could be cheaper and faster to fly me over there and contract it out to me until you have a few for replication :)
Thanks for the respect in your request mate, almost all of the requests I have had have been via PM, from n00bs with no contribution history demanding I privately supply them with my protocol.
Start there and see where it takes you
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Thanks! I have great respect for your posts on micropropagation......I actually signed up here back in 2000 or 2001, when I ordered plants from Torstein...but couldn't find my old arrangement here....So I signed up again.

I have done very well propagating this plant from two node cuttings using techniques I developed experimentally myself....

I am very in tune with plants...

I have just started tissue culture/micropropagation, have assembled the supplies except for the right hormones, etc..

I wish I could fly you over here and start and entire botanical garden and epic classical glass conservatory,

But my funds are limited.....

Perhaps thru space, the energy of your thoughs, could move my hands here, and I could return a percentage of green energy

For the spirit and energy that you put in....

I could be instructed, take digital photos, and send feedback as it went along....

Currently, I have 30 or so nice mother plants.

They are very cool...most of them have been carefully used for cuttings over a couple years.

This has made them grow low and wide.

I kept them in smaller containers, one gallon, for quite some time until transplanting for increased growth recently.

They are almost like bonsai trees!

What I would like to do, is what you have done...I am impressed....would share the results, in an arrangement we could agree on in advance, if you like....I understand and respect the work you have gone through to gain this knowledge.

I wonder if it would be possible to split the (green,new) stem at a node, and grow out both buds.....

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post-12633-0-96683700-1364075143_thumb.gif

post-12633-0-72351000-1364073285_thumb.gif

post-12633-0-78751100-1364073712_thumb.jpg

post-12633-0-96683700-1364075143_thumb.gif

Edited by shonman
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I'm happy to help when I have time and as long as you share results here so they benefit everyone. I've frequently been the beneficiary of other's expertise here- there is a fair bit around :)

You have all the TC gear, but do you have any experience with TC? I'd start by trying a few things you can afford to lose rather than subjecting your veg. clones to your learning phase. Acacia seed is a good place to start if you can easily get it

Once you've done a few runs and are comfortable with your sterile tek generally, get back to me

In the meantime, prep your parent plants. This is an incredbly important part of TC and something a lot of ppl neglect. Plants are infested with fungus and bacteria, as are we, and it's an important part of being alive.

Fresh new vegetative growth, especially when forced along with nutes, dry heat, and only watering at the base for a few weeks, is your preferred starting point as it has a lower concentration of contaminants at the actively growing sites and increases the chance you will get sterile plants

Next hint: use only 1/3 of your propagation stock per experiment. That way there is more available to refire your technique, and gives your stock plants time to recover

I haven't TC'd axillary nodes of Mitragyna, the challenges of disinfesting the tissue from nursery stock were too high, and then they became illegal and I haven't worked on them since. If you can get viable, fresh seed, as in the paper, start there

Edited by Darklight
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I wonder if it would be possible to split the (green,new) stem at a node, and grow out both buds.....

Well, yes. But that will happen anyhow once you take the top node off each plant for your TC experiments.

PS. I really didnt expect you to fly me over. But it doesn't hurt to ask :)

Edited by Darklight

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TC is cool and all, but if you have 30 mothers and have successful protocols for regular vegetative propagation is that not a more practical option since it's legal where you are?

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Oh and DL if you'll work for travel and accommodation expenses, I'll shoot you a PM when I have lab access again mmk? ;)

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Darklight, I will do as you suggested....

The plants began the prepping process three weeks ago...

They have many growing tips...I prune in a way so as to get two new tips for each one I cut.

Did you suggest starting from seed to obtain a sterile culture , due to challenges of using buds from plants?

It seemed like if I were to just grow out these new tips...With PPM to kill fungus, bacteria...Using each one just as a basis for a new plant...

That I could get five times as many starts as I do now...

One for the top,

Four total......from the two nodes.....then stack these on my shelves with floursecent lights,

or under 400w HID light....far enough away....they could just grow out in little containers by the hundreds.

At least in my imagination.

Endorfinder....I will definately be continuing to root from cuttings in the meantime....but want to do these en masse, efficiently...I am absolutely fascinated my micropropagation....

"Plants from test tubes" was where it began, then collecting the equipment a bit at a time.....getting started now...

Edited by shonman

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hey if you want to start with microprop mitragyna is a great place to do it :)

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.....use the leafvein of one of the seedlings grown in sterile conditions, to generate plantlets?......

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TC is cool and all, but if you have 30 mothers and have successful protocols for regular vegetative propagation is that not a more practical option since it's legal where you are?

YES!

This. Above all this. Veg propagation will usually yield quicker until a sterile culture and reliable protocol has been established. Which can take a long time ( or you can get lucky ). What tissue culture does best is:

i) bulk numbers up exponentially once you have a sterile culture and working protocol, which can give you a competitive advantage

ii) offer another method to propagate species otherwise recalcitrant to conventional nursery propagation and

iii) cloning multiple individuals of a species under pressure can create a temporary genetic ark for them at several locations until they are grown out and genetic diversity maintained via cross pollination

TC is only ever faster if you have a sterile plantlet and a functional protocol. Up til then it's just research and hacking at your precious babies

hey if you want to start with microprop mitragyna is a great place to do it :)

No it's not. Really it's not. Unless you have extensive experience with sterile tek, especially with starting from ex-vitro nursery stock, you risk wasting plant material. Start with Acacia seed. There are millions of them and they'll teach you a lot. Once you've aseptically germinated and propagated an Acacia species then you can try something more complex from ex-vitro stock. *Then* start being profligate with your favourite parent plant material

.....use the leafvein of one of the seedlings grown in sterile conditions, to generate plantlets?......

Well, yes, technically it's possible. All plant cells are held to be totipotent ( or last time I checked they were ) and theoretically, given the right media, any plant cell can be induced to divide and differentiate into any other part. But in practice it can take years to get the media right for this. And of course, you could be lucky. If you do this then you'll learn some cool stuff either way. Let us know how it goes and send pics

When plants are being fussy they can be really fussy. In some species, different plant parts require very different media to achieve the same outcome. When plants are being *really* fussy, individuals from different populations can require very different protocols to get the same result from the same plant part. I'm thinking grapes here, specifically, but I've seen this happen in other species too, like iboga

Oh and DL if you'll work for travel and accommodation expenses, I'll shoot you a PM when I have lab access again mmk? ;)

Ooh cool. I can be bribed :) Especially if it's an interesting species. And especially if it's legal where you are. You mightn't need a lab tho, there are ghetto teks which work for inducing cultures, as long as you show up with sterile media ready and have pretreated the parent as far as possible, and you can take the cultures you get that day back to a grow space for further work. Shoot me a PM anyhow

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Darklight, I do see what you mean..

I am continuing to feed the plants well, cutting back on water a bit....

Can you elaborate on dry heat and how this plant responds to it?

So far I have mostly worked with relatively high humidity,

It seems to help promote new growth....

I am very interested in the ghetto culture/ kitchen culture tech for micropropagation.

Like art, though, you cant just dive right in and create a perfect masterpiece,

There have to be a bunch of scribbly crayon drawings first!

Hopefully some will be good enough to put the picture up, as it were!

I have a great little digital camera, will post my experiments as available....

I think I will begin with Ma Huang seeds, I have alot of them...

One thing I would like to do, is to get the correct media for working with the Mitragyna,

or at least the right base components for it..

I want to begin experimenting with that plant from the start, not en masse,

just a few and see how it goes as I go along..

Can you suggest any components for the media, or the media I should get?

Is it better to PM you about is?

Edited by shonman
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Oh, the dry heat is only to prevent a favourable environment for bacterial and fungal infection, to give a lower level of these prior to TC excision and sterilisation. Same deal with bottom- or surface- only watering.

Ephedra seed sounds excellent to start, great idea. Just make sure you subculture the plant away from the seed coat as soon as it emerges, from memory there were some nasties that came out of the coat soon after the seedling emerged in TC

What was the media in the paper above? You could try that. I haven't done Mitragyna from cuttings. Woody Plant Media. You can buy it from any tissue culture suppliers. I'd also go for some PPM, available at the same places ( do a search for it on the forums here ). And use best grade white kitchen sugar, that'll be fine. And you can probably bodge it with store bought agar, Mitragyna was pretty tolerant there back when I was working with it- the fact that they got results from a different media to me is sometimes indicative that the plant will cope

Don't bother with hormones just yet, just get a sterile culture going and share the pics, then we can worry about hormones. BAP will prolly be the one you try first, but if you're putting in an order and want to save on freight by doing a single order, get Kinetin, IBA and NAA as well- in solid form pref as solutions degrade over time, and check the manufacturer's recommendations for storage and dilution- they're usually round on their website somewhere

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if you're getting several hormones it's probably worth grabbing some ga3 too :)

edit: just to have in general.

Edited by endorfinder
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I have started my first experimental micropropagation attempt.

It has been most revealing to me.

Sterile culture is the opposite of the natural synergies of soil and microorganisms,

decay, creatures, etc...'Filth culture'.

Micropropagation is changing my bachelor pad too.

Suddenly cleaning has meaning.

Tiny little plant pieces....

I considered taking step by step pix,

but the pressure cooker and little plant pieces didnt look extremely exciting.

When something starts to happen in the jars, I will take pix.

Basicly, this run was to familiarize myself, I don't expect alot.

However, since I did use PPm, there is a chance something might grow without getting contaminated.

I made an improvised clean box with PVC pipe and plastic, sprayed alcohol and sterilized.

I took small cuttings from several plants, and also put in some Ma Huang seeds.

They were all sterilized in isopropyl, for a couple minutes

then 10% bleach solution for ten minutes.

Later rinsed in clean box in sterile water.

I did seventeen jars, with a combination of different pieces of:

Ma Hung...seeds, several jars ,

Live stems of Ma Huang in another jar

Sinuichi...top, and nodes

Kratom, nodes, top,

Ps. Carthaginensis....leaf, whole, and cut

Calea Z 'Dream Herb'...nodes with growth

B. caapi......nodes, top, maybe a leaf

Ps. poepiggiana......leaf

T. Peruvianus. ......one little chunk with some spines on it

Just to see what it all does in the jars.....maybe some will make it.

I tried to do Nam Wah miniature Banana, but that plant needs to grow back more..

I thought I had the meristem, at the very base kf the plant, but it later unravelled and was just very new leaves....

Media was homemade, and I did also include coconut water.

The agar was from the grocery store, I think I made it too hard, but hopefully it will work...Using a Homemade TC media

...

 

Edited by shonman
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I laughed at your cleaning comment. Mycology results in my keeping a place cleaner too. I end up thinking of everything as being contaminated with mould, spores and other nasties so I have to thoroughly clean a place. Chicks dig it!

Keep us updated on your work. Don't forget to take pics too!

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Re: cleaning....I will never look at carpeting in the same way again.

Credit to 'Plants from Test Tubes' for the homemade TC recipe above.

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Now that I am starting to work with sterile procedure,

Carpeting seems like a disgusting germ filled dishrag that I can't throw away

(I am renting here)

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I have started my first experimental micropropagation attempt....

\

Will see what happens,,,,have to start somewhere!

Hey cheers, that's great, you put in a good long day or two from the sounds of it

Good write up as well, those details will help other people and hopefully will inspire a few

Let us know how it goes, I haven't any experience with that media recipe but it seems thorough and I'd love to hear more

LOL at the carpet thing, I know what you mean. Back when I started I was really scrupulous about everything in the lab. Now I know where my problem sources are I've been getting um... relaxed. But I'm not ready to carpet :)

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Thanks!

This first run was really just to see how things are done a bit,

Using various plant material available at the time.

I do see the advantage of starting things initially using seeds to establish the sterile culture.

Pictures of anything exciting that happens will be taken, for sure!

The recipe I used is from a book called "Plants from testubes'"

Next batch will be MS woody plant medium.

I also got some BAP.

I think I made the agar too dense/hard...

If I may ask......

What is the proper consistency?

Like jello?

How hard should it be to push things into?

Also,

If I used PPM again,

Is it possible a couple cuttings just stuck in agar in the jar would root?

The way I root them now, there is littke to eat in the medium I use for rooting.

Unlike the agar, which has nutrients for contaminates as well as plants.

Edited by shonman

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The recipe I used is from a book called "Plants from testubes'"

 

That's a good book hey. It's not exactly easy to follow as it isn't step by step, but it's an excellent reference and good on you for getting this far

>Next batch will be MS woody plant medium.

Erp, don't get the media mixed up, WPM isn't MS, it's Lloyd & McCown’s. You can buy both pre-made, and I'd advise that for beginners.

You can also buy different components of both media and add them in different arrangements- the micronutrients of one with the vitamins of another. So check your catalogue and make sure you're getting the ones you need

I think I made the agar too dense/hard...What is the proper consistency? Like jello? How hard should it be to push things into?

Agar is one of the things which affects nutrient uptake. *Most* but ny no means all media are hard set enough so they don't wobble round when you handle the container ( this is a guide, not the reasoning behind the proportion used )

8g/L at pH 5.8 ( pH below 5.5 can result in media which isn't gelled properly ) is pretty standard, but other values are OK too for different species. For example I use 1.5x the recommended gelling amount for shipping plants so they don't damage in transit when the parcel gets shaken round

 

Also, If I used PPM again, Is it possible a couple cuttings just stuck in agar in the jar would root?

 

Um, you mean non-sterile plants? Depends on whether there is a carbon source like sugar in the media or not. If there is, PPM will exhaust rapidly, even more so if there is a *lot* of contamination on your plant- sterilised or not

>

 

The way I root them now, there is littke to eat in the medium I use for rooting.

 

Unlike the agar, which has nutrients for contaminates as well as plants.

 

Keeping the nutes low is often used to generate rooting in in-vitro plants. Rule of thumb for inducing rooting in sterile TC, at least as a starting point, is 1/2 strength media and 1/2 strength sugar, and maybe an auxin

Used at standard concentrations, agar is a negligible nutrient source for your purposes. If you were doing pure academic research you'd switch to Phytagel ( which is exxy and can cause hyperhydricity in some spp, but is a standard gelling mixture and almost zero available nutrient )

Edited by Darklight

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I very much appreciate your insights in these matters, Darklight.

The media I have for next time is Lloyd & McCowans, I had to check.

I figured there would be things I had wished I would have done differently later,

After this first attempt.

That is one reason I used the home made formula.

To see how it goes, and save the stuff I bought for the next couple runs

When I have a better idea what I am doing.

I also noticed some flaws in my sterile technique,

Where there might be opportunity for contamination.

I am using kitchen agar from an Asian market.

In the proportions of that recipe mentioned earlier.

When you made jars with agar, yourself...

Do you add a measured bit of agar to each jar,

Or mix it all up at once, then add to jars?

Followed by some time in the pressure cooker....

Edited by shonman
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Do you add a measured bit of agar to each jar,

Or mix it all up at once, then add to jars?

I usually mix it up all at once. Sometimes I use gelling agents which can interfere with pH so I need to take it into account

But you can add it to the jars before autoclaving too. I don't think agar messes with the pH of the media too much

I figured there would be things I had wished I would have done differently later,

After this first attempt.

That is one reason I used the home made formula.

To see how it goes, and save the stuff I bought for the next couple runs

... I also noticed some flaws in my sterile technique,

Where there might be opportunity for contamination.

I really admire the way you are going about this and I'm happy to help

There will often be something you wished you had done differently, it still happens to me, as long as you keep notes on everything, then anything you do will give you a result.

If I think I have contaminated a jar I put a special symbol on it so I can keep a close eye on it if it develops ( it doesn't always )

Edited by Darklight

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I will start keeping better notes.

It is annoying to have one particular technique work out months later,

Then not remember what you did with that one!

A couple things I was wondering about......

1) how important is the dark cycle for the plants in jars when starting out?

Is it alot better than 24 hour?

2) are plants grown sterile in Tissue Culture/ jars..

Which almost by definition would be disease and pest free

Be easier to export or import between countries where of course, they are legal plants?

Thanks!

Edited by shonman
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