LifeSynergy started following Testing Micropropagation of Mitragyna Speciosa VIA Tissue Culture
LifeSynergy posted a topic in EthnobotanyHello, 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"?