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Testing Micropropagation of Mitragyna Speciosa VIA Tissue Culture

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Hello, I've been doing a lot of research and would love to get any feedback on my first attempt at a Mitragyna Speciosa tissue culture (which is legal plant/tree in my location).  I know there are a few threads on here that have addressed this (shonman, Darklight), and I'm not sure if I should post on there and resurrect, or just start a new topic/thread; don't want to hijack shonman's thread, so I'm starting this as a new thread.

 

I appreciate the time, energy, and resources that the members here have put into their practice, and would love to politely pick their brains; I understand that experience was hard-won, so I don't want to offend and ask for "info-freebies". My first hurdle is that I have limited mother plant material, and would really like to have at least one successful replication before I run out of plant. I've been working on this for a few months and have not had one yet; many were lost to contamination.

A little about my method:

-Working under plastic bin hood

- WPM at label suggested strength

- SIDNC (Milton Tablets) at 5000ppm for 15 minutes, but I wonder if my tissue samples are too large? Anyone tried 300-400ppm for 24 or so hours?

-I also mix the SIDNC into the medium at 0.032%, but having a hard time adding it at the right time (ie. low enough temp to keep avail chlorine) without the medium ending up a little too soft. It's almost as if I need to heat/microwave the medium again, but that would destroy the SIDNC protection, right? For medium prep via microwave, pre SIDNC, how long do you let it boil?

- 10% v/v coconut water

- 4% Honey

- 0.5 mg/l TDZ (this strength was at suggestion of fellow practitioner, open to suggestions on strength or using it at all?) Best way to dissolve TDZ powder?

 

I've heard that petioles are best with this species (can anyone confirm that; goal is micropropagation) (I knocked off a leaf, so I decided to try that also, anyone had success with leaves of this plant?) What's the best way to position the sample on/in the medium? Size and cut of sample?

 

Has anyone been able to micropropagate this with only two phases of medium (#1 medium=produce shoots AND roots, then #2 medium=hardening), OR does it need three phases of medium (#1=shoots, #2=roots, #3=hardening), OR four phases of medium (#1=callus, #2=shoots, #3=roots, #4=hardening) OR different combo...? Any thoughts on hormone use for these phases, I know 2,4 D is good for this particular plant to produce callus, but callus is not my end goal, Im not sure how to get callus to reorganize to shoots & roots. 

 

Air supply (hole in lid), I've read both nays and yays as to whether or not it needs it, right now, no holes, too worried about dust mites, etc getting in.

 

I saw a thread post on here from a few years ago saying there might be an article/paper/protocol put out or published on this by Darklight and a few others, did that ever happen? I searched and searched, but could not find anything; I wish there were a manual for this;)  

 

After jumping in and doing a few trial runs, I can deeply appreciate how challenging and rewarding this can be. I wholeheartedly appreciate any and all feedback and tips! And I hope I've followed proper protocol for rules and etiquette on this post!  I normally keep to myself, but after lack of success I realized I needed to reach out for help on this one.

 

I only have pics of my most recent batch. The last one (#5) must be contaminated, can anyone "Name That Contam"?

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Hello, I've been doing a lot of research and would love to get any feedback on my first attempt at a Mitragyna Speciosa tissue culture (which is legal plant/tree in my location). 

 

I appreciate the time, energy, and resources that the members here have put into their practice, and would love to politely pick their brains; I understand that experience was hard-won, so I don't want to offend and ask for "info-freebies". My first hurdle is that I have limited mother plant material, and would really like to have at least one successful replication before I run out of plant. I've been working on this for a few months and have not had one yet; many were lost to contamination.

A little about my method:

-Working under plastic bin hood

- WPM at label suggested strength

- SIDNC (Milton Tablets) at 5000ppm for 15 minutes, but I wonder if my tissue samples are too large? Anyone tried 300-400ppm for 24 or so hours?

-I also mix the SIDNC into the medium at 0.032%, but having a hard time adding it at the right time (ie. low enough temp to keep avail chlorine) without the medium ending up a little too soft. It's almost as if I need to heat/microwave the medium again, but that would destroy the SIDNC protection, right? For medium prep via microwave, pre SIDNC, how long do you let it boil?

- 10% v/v coconut water

- 4% Honey

- 0.5 mg/l TDZ (this strength was at suggestion of fellow practitioner, open to suggestions on strength or using it at all?) Best way to dissolve TDZ powder?

 

I've heard that petioles are best with this species (can anyone confirm that; goal is micropropagation) (I knocked off a leaf, so I decided to try that also, anyone had success with leaves of this plant?) What's the best way to position the sample on/in the medium? Size and cut of sample?

 

Has anyone been able to micropropagate this with only two phases of medium (#1 medium=produce shoots AND roots, then #2 medium=hardening), OR does it need three phases of medium (#1=shoots, #2=roots, #3=hardening), OR four phases of medium (#1=callus, #2=shoots, #3=roots, #4=hardening) OR different combo...? Any thoughts on hormone use for these phases, I know 2,4 D is good for this particular plant to produce callus, but callus is not my end goal, Im not sure how to get callus to reorganize to shoots & roots. 

 

Air supply (hole in lid), I've read both nays and yays as to whether or not it needs it, right now, no holes, too worried about dust mites, etc getting in.

 

I saw a thread post on here from a few years ago saying there might be an article/paper/protocol put out or published on this by Darklight and a few others, did that ever happen? I searched and searched, but could not find anything; I wish there were a manual for this;)  

 

After jumping in and doing a few trial runs, I can deeply appreciate how challenging and rewarding this can be. I wholeheartedly appreciate any and all feedback and tips! And I hope I've followed proper protocol for rules and etiquette on this post!  I normally keep to myself, but after lack of success I realized I needed to reach out for help on this one.

 

I only have pics of my most recent batch. The last one (#5) must be contaminated, can anyone "Name That Contam"?

 

 

Hey sorry, I only just saw this today. Congratulations on both starting and keeping going! Is hard yakka but I expect you will get it eventually and learn heaps in the meantime

 

I never ended up publishing my media and methods but several other publications have come out on M. speciosa TC in the last few years- I think I linked to them in the thread where shonman and I talked it over.

 

First critique ( please don't take this personally, this stuff happens to everyone especially this early in the learning curve )- you're throwing too much Milton at your tender babies. They only need sterilising for a few min, 1 tablet in 100ml water will do it, start with 10 minutes and work more/ less time into it depending on symptoms.

 

Pretreatment of your parent plant is mission critical- make sure it only gets watered at soil level for a week or so beforehand. Harvest plant parts for TC on a warm sunny morning. This really does make a huge difference

 

Now I have absolutely no experience with microwave sterilisation so can't speak to that. But next time you do a run keep a jar uninoculated and unopened to make sure it works for you and doesn't contaminate. Don't whack any Milton into it at all. Overkill, and some plants don't like it

 

Have had no success with leaf culture, which by no means makes it impossible, keep trying.

 

Ended up after a few generations with a single stage protocol- I could have produced more plants faster with hormones, but the market for them didn't warrant the haste and the added risk of producing off-types over multiple generations

 

From the pics it looks like you have been overcutting your explants ( I could be wrong )- you do need more than just a stem. Two nodes minimum, and don't cut the petioles all the way back to the stem- leave 4-5mm on them, the extra lengths on them make them useful handles to avoid bruising the stem during transfers. Bah, I misread, sorry. Use the stem section. At least two nodes. If you don't have that spare on your plant, pinch out the main growing tip and make more stems. Always keep at least one active growing stem on the parent plant so you make more parent material if you ever need it

 

Early stage your main priority is to get it clean, they will live without hormones for a while, and even grow. Once you have a few clean stems you can worry about hormones.

 

Oi @shonman, how'd your cultured clones go? Any further hints?

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Those jars and lids you are using are exactly the right ones. No need to stick holes in them, kratom clones will survive and thrive in them for several weeks

 

No idea what your contam is, pic is unclear. If it's fuzzy it's fungal, if it's slimy it's bacterial, if it's weird it's probably a yeast

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