shonman Posted July 18, 2013 (edited) I think this might be the end of this thread... I will continue to experiment with plant tissue culture. However, I have no doubt, for anyone interested in propagating this plant, That literally hundreds of cuttings could be generated the conventional way, While attempting to T.C. Experiments Edited August 3, 2013 by shonman 1 Share this post Link to post Share on other sites
Darklight Posted August 8, 2013 Thanks, Darklight! I appreciate your insights....could you please outline how to decide what technique to use, When attempting to micropropagation new plants? The tek you are using is fine. No hormones is better than hormones for long term proliferation, to avoid potential mutation or ( bloody hell, can't remember what the technical term is, hang on ) hormone habituation. ( You can always choose to add hormones to media if your hormone-free stock start to decline in performance re. proliferation numbers The media I used is markedly dissimilar to the one outlined in the paper I mentioned. However that can be common with TC. Sometimes it is an effect caused by the fact that diverse parent populations can have very different media requirements. And sometimes it is caused by the fact that regardless of plant parentage, the species is very tolerant of a wide range of conditions In the case of Mitragyna I suspect the latter Unfortunately, as the initial supply of genetic material was really limited, I wasn't in a position to play around with lots of different kinds of media at the start, and was simply lucky the best guess worked. And once something works, why fiddle with it? The media itself was fairly simple. When the plants started to 'fail' in culture- it does happen in a lot of species after multiple generations of transfer- changing media wasn't helpful. Probably too late. 'Fail' was characterised by browning and wilting in vascular tissues, with progressively larger numbers of individuals succumbing per subculture batch. It's started to happen in a related species after 12 years, now *that's* a good long run That said I think I got about 4-5 years of solid multigenerational transfers from my kratom TC How's your TC going? I'm sorry I don't get to reply to these as often as I should, it requires that I take time out to really focus on providing good quality replies and some of these have taken several hours because I often need to correct my post for clarity 1 Share this post Link to post Share on other sites
shonman Posted August 9, 2013 (edited) Thank you, Darklight, for all of your insights and time spent helping myself and others in their first encounters with T.C. I seem to be getting some results, with an unexpected addition the the media....At first, I thought what I saw we're roots growing upward, and turning green... Then, when I transferred a culture, a piece broke off... The 'roots' were growing from chunks of callus, and seem to be differentiating a bit at the top, To the familiar 'bishops mitre' shape associated with Mitragyna.... Will know more for sure, after they develop a bit. I could be mistaken, but these are a lot greener than roots, grow upward, and are on a piece of callus that developed and grew From the base of an older surviving cutting....... Will take pictures when I can.... Somehow, the 'great for photography' magenta vessels have condensation all over the sides too! The chunk of callus, is separate from the original plant now.. How can I proliferate and grow that chunk of callus....and generate more plants...or is that something that is worthy or attempting? If I mash the end of new explants, will it promote callus development, or just more phenolic leeching? Edited August 9, 2013 by shonman Share this post Link to post Share on other sites
Darklight Posted August 9, 2013 seem to be getting some results, with an unexpected addition the the media....At first, I thought what I saw we're roots growing upward, and turning green... Then, when I transferred a culture, a piece broke off... The 'roots' were growing from chunks of callus, and seem to be differentiating a bit at the top, To the familiar 'bishops mitre' shape associated with Mitragyna.... Will know more for sure, after they develop a bit. I could be mistaken, but these are a lot greener than roots, grow upward, and are on a piece of callus that developed and grew From the base of an older surviving cutting....... Cool huh Thanks for keeping us informed Will take pictures when I can.... Somehow, the 'great for photography' magenta vessels have condensation all over the sides too! Yep that happens, especially if you need to keep the temps a bit tropical Tap the container a little on a benchtop before you take the pic. That's OK with Magenta vessels, if you're working with callus in petri dishes I've been warned against it as it can drop sudden amounts of condensation on your work to potential affect. But with explants in Magenta vessels it prolly won't matter The chunk of callus, is separate from the original plant now.. How can I proliferate and grow that chunk of callus....and generate more plants...or is that something that is worthy or attempting? If I mash the end of new explants, will it promote callus development, or just more phenolic leeching? OK, I haven't had that result, how cool for you! First- if the clump is large enough, split it in two. Have two media ready, keep one in the original media. Try the other one in 1/2 strength media of whatever you used, and half strength sugar, but full strength agar. This is often used in your first step to lengthening and root establishment If you mash the end of new explants, either is possible. Give it a try if you have enough so you can subculture some normally so you have some explants for more replication 1 Share this post Link to post Share on other sites
shonman Posted August 11, 2013 I am still not entirely sure about these recently developed 'plantlets'... There may be contamination, or they might be mutating in a way that is not productive..... I may be wrong entirely in identifying them as productive, new explants.... Will get a photo when I can. Darklight, is there anything you can tell us about your experiences with this plant? Did you multiply it strictly by dividing explants, Or did you get it to generate multiple plantlets? Can you tell me anything about media, methods, or results that you experienced? I am not asking for a step by step, or exact formula....just whatever you are comfortable sharing...that might be helpful. I have been working on this for a while now, and any suggestions of any nature are always appreciated. I understand if you don't want to describe the exact process.... Share this post Link to post Share on other sites
shonman Posted August 12, 2013 Several Kratom explants seem to have made it, and are still uncontaminated as far as I can tell. I will keep dividing these. I am still nmot sure about these green plantlike or rootlike thigs, they seem to have characteristics of both...although, i am starting to wonder if they are just roots. Here are some photos... I placed the cultures in aquariums, with acetate on the open space, just to keep things out better. 1 Share this post Link to post Share on other sites
shonman Posted August 12, 2013 Here is one of the explants in question...the small growths can be seen coming out of the agar. Also shown....closeups and view from under these roots/plantlets..... The closeup is a different piece than the small group of roots/plantlets shown, but it is from the same culture/ plant. Notice the difference between the obvious roots, and the possible plantlet..... Some that i thought were plantlets, became a bit fuzzy at the top, like a root would....... not quite sure what to think..... 1 Share this post Link to post Share on other sites
shonman Posted September 1, 2013 Here is a closer up picture of the unknown growth(s) from the explants... Do you think these are roots, or plants? Unfortunately, these got contaminated by the time I took this picture... 1 Share this post Link to post Share on other sites
shonman Posted September 19, 2013 I have planted the first rooted explants propagated entirely by micropropagation. There are more which are 'functionally sterile', from which I will continue to propagate this plant. 2 Share this post Link to post Share on other sites
shonman Posted September 19, 2013 Well rooted! I added a different ingredient to my medium, and it promoted radical rooting! 2 Share this post Link to post Share on other sites
shonman Posted September 19, 2013 planted explant 1 Share this post Link to post Share on other sites
Darklight Posted September 19, 2013 Here is a closer up picture of the unknown growth(s) from the explants... Do you think these are roots, or plants? Post #108 YES! That's IT! I think you've done it :D Congratulations! Sorry, I was away and didn't see this post until now Congratulations! How pleased are you? I would be :D 1 Share this post Link to post Share on other sites
Darklight Posted September 19, 2013 And thank you for your continued posting of the process all the way through to the finish, it does mean a lot to me that your learning was done publicly and for the benefit of other members as well and that you followed it with a conclusion rather than tapering off into invisibility leaving newbies wondering whether it worked or not ( this often happens but not in your case ). I hope your work inspires others Sorry I wasn't always there to answer on the spot as you posted, I've been away for a few weeks and often busy at other times. Sometimes I've been overwhelmed by the sheer number of experiments you started out with ( experiments will always multiply like ducks, start with two and they will reproduce ) and felt unsure as to where you were travelling You just put a smile on my face for the rest of the morning, thank you 3 Share this post Link to post Share on other sites
Darklight Posted September 19, 2013 I am getting all sooky and maternal just looking at the pics of yr babies I remember those days I do like your culture chamber, it seems to give good access to light and is easy to monitor and clean The reason I didn't give you the exact formula I used is that i) the paper I read which was written much later than my work used fewer hormones and was successful anyhow and ii) it is entirely possible that my use of hormones was responsible for the long term loss of viability in culture ( though other factors may have been responsible also, long term viability of replicable tissue is an issue in many species ) Remember: just because a scientific protocol works doesn't mean that there isn't a better protocol out there. The incentive to find a better TC protocol is not usually driven by the presence of an existing successful protocol unless financial factors are involved, an ingredient becomes unreliable or unavailable. Like everyone else, scientists tend to stick with what works 1 Share this post Link to post Share on other sites
planthelper Posted September 20, 2013 (edited) nice and good quality pic's, great progress, shonman! and thx Dl for all your input! Edited September 20, 2013 by planthelper 1 Share this post Link to post Share on other sites
shonman Posted September 21, 2013 Thanks Darklight, Planthelper, and everyone! Share this post Link to post Share on other sites
shonman Posted March 10, 2014 Ok, I now have about 160 functionally sterile mitragyne specious explants. I have heard that using PPM continuously, weakens the ex plants eventually. So I may be switching to a form of bleach used to sterilize baby bottles, in the medium for the plants. I will separate off about 64 plants, and add a hormone to get each explant, To sprout multiple plants. Later these will be separated, left to root, etc. I am realizing it is somewhat expensive and perhaps not entirely necessary To keep all the mother plants all winter long under HID lighting. Can you suggest logistics, for propagating ALOT of plants all winter, Then hardening off and growing out in summer? I would like to keep the mother plants in test tubes or magenta containers, Propagate all winter, not use Hid lights, and then grow out lots in spring..... 1 Share this post Link to post Share on other sites
Darklight Posted March 11, 2014 Ok, I now have about 160 functionally sterile mitragyne specious explants. Hey that's great I have heard that using PPM continuously, weakens the ex plants eventually. So I may be switching to a form of bleach used to sterilize baby bottles, in the medium for the plants. OK that's interesting, I haven't heard that Would love to see the data. Subculturing some species down multiple generations can weaken them and cause them to be unable to proliferate further so you need to start again. The time period differs for each species, and some media can shorten this length. The timeframe for many species aren't known- but anecdotally I have heard from reliable banana TC people that you shouldn't go past five generations in a standard replication media or you lose productivity. In contrast, Mitragyna survived about 20 generations here until it became non-viable and Yohimbe has survived nearly 60 generations but is starting to slow What's the baby-bleach sterilisation thing? I haven't heard that either. If I were you I'd separate a small group, say 20 explants and run them on trial amounts of that compound for three generations against your main batch of PPM containing media. Let us know the results. You really can't be too careful. It's an interesting idea, but some of the dumbest things I ever found didn't work I learned about on the internets. Do you have a reference I love how you've only been doing this a short time but can now teach me stuff. That's what this is about I will separate off about 64 plants, and add a hormone to get each explant, To sprout multiple plants. Later these will be separated, left to root, etc. I am realizing it is somewhat expensive and perhaps not entirely necessary To keep all the mother plants all winter long under HID lighting. Can you suggest logistics, for propagating ALOT of plants all winter, Then hardening off and growing out in summer? I would like to keep the mother plants in test tubes or magenta containers, Propagate all winter, not use Hid lights, and then grow out lots in spring..... Do you have heating to your normal grow temps in your grow area? Why are you using HID lights for all your plants? When you refer to mother plants, are these the multi generation stock you are going to use for the next generation? Or the individual you started from in the beginning? 1 Share this post Link to post Share on other sites
shonman Posted March 12, 2014 The lights, etc are for conventional mother plants. I would rather just maintain plants In test tubes or Jars, grow those out etc. The larger plants are from the 'old days' of taking cuttings the regular way. Which in fact, is my own unique tech too. They have the capacity to generate 100 clones per week plus. The lighting costs are kind of significant in winter though. And they can get bugs too. Next post i will go into detail about the chlorine tech to prevent contamination without antibiotics. The source of that and the questions about PPM were from a micro propagation email list I am on. Share this post Link to post Share on other sites
shonman Posted March 12, 2014 Here is what so done involved in TC said.... "> > anti biotics affect the plasid and mitochondrial genome and can induce > > somatic mutations.Eventually the microbes could overcome the antibiotics > > effect and suddenly sprung up and destroy the cultures.My advice is to > > raise and maintain the axenic cultures to as many passages as one could.And > > throw away once they start showing contamination.The starter cultures if > > indexed for bacteria and fungus can be multiplied for pretty long time > > axenically. Share this post Link to post Share on other sites
shonman Posted March 12, 2014 Chlorine as antibiotic, no mutations.... http://www.saps.org.uk/attachments/article/843/SAPS-%20STEM%20Careers%20-%20Using%20tissue%20culture%20and%20%27cloning%27%20for%20rare%20plant%20conservation.pdf 1 Share this post Link to post Share on other sites
Darklight Posted March 14, 2014 OMG, that is hot! Thank you- I'll try it From the article: The technique is based on a protocol developed by plant scientists at the Royal Botanic Gardens, Kew, and is used in their conservation programmes, allowing critically endangered plant material to be cloned in the field, rather than waiting until a ‘clean lab’ can be found to rescue plant material 1 Share this post Link to post Share on other sites
shonman Posted March 16, 2014 (edited) Dark light...... I am now in between worlds.......of regular propagation, and micro propagation. I would like to make the switch to maintaining plants and propagating by micro propagation.... Eliminating the need to maintain alot of mother plants under HID lighting in winter. (As when propagating the 'regular' way) Can you detail the logistics, in maintaining and propagating plant stock by micro propagation only? Ie....maintain some stock. Multiply over winter, root, and have ready to go into greenhouse when warm, then sell? I am impressed with this one man show....he does not maintain plants under HID lighting in winter..... Hmm.....image does not show up..... Edited July 1, 2014 by shonman Share this post Link to post Share on other sites
