shonman Posted June 20, 2013 (edited) I appreciate all feedback, thanks, Darklight, PlantHelper, and everyone! P.H....I got a decent digital camera, I will figure out the close up manual focus.....it is a Nikon Coolpix 1000 or something like that. Darklight....thanks for the tips! No, the dark spot is not fungus, it is something else..... Perhaps I will try submerging new explants in liquidy media with PPM next time, before 'planting' in a jar with firm agar. The tape is 3M micropore tape for bandages and skin wounds...... the pores are not as big as they look, But perhaps I should cover them up with clear plastic, on top of the lid only? I ordered a few hormones, with the new Magenta brand polycarbonate 'great for photography' jars I ordered from Carol at Kitchen Culture kits....because of the info she shared with everyone online. Magenta GA7 vessel: http://www.phytotechlab.com/detail.aspx?id=216I Recently, I have thought about making my own containers, from borosilicate glass tubes. I also do occasional lampworking, being a horticulturist and artist by trade. Maybe they would be an inch across (2.5 cm for metric scientists who like logical base ten measurements) And about four or five times that tall. Another idea I had...was to make a little fishbowl container, and create a micropropagation landscape inside.... With various plants, maybe a few sculptures... But I digress....... Soon, I will post photos of all jars, with their lids individually...... for critique, commentary, educational purposes, and advice! I apologize for the photos, will work on that next time....after posting these I recently took..... Edited September 14, 2017 by shonman 1 Share this post Link to post Share on other sites
Darklight Posted June 21, 2013 Darklight....thanks for the tips! No, the dark spot is not fungus, it is something else..... Phew! Perhaps I will try submerging new explants in liquidy media with PPM next time, before 'planting' in a jar with firm agar. Don't make too much work of it at this point. You've had a few successes, keep going with them- they will breed and make enough work for you If you actually do the above tho, run a side-by-side with non PPM explants and compare data. New explants only need be submerged for a couple of days, then subculture and treat them normally The tape is 3M micropore tape for bandages and skin wounds......the pores are not as big as they look, But perhaps I should cover them up with clear plastic, on top of the lid only? Nah, that would defeat the purpose of the 3M tape and maybe the still moist air inside the glad-wrap could cause it's own issues I ordered a few hormones, with the new Magenta brand polycarbonate 'great for photography' jars I ordered from Carol at Kitchen Culture kits....because of the info she shared with everyone online. Magenta GA7 vessel: http://www.phytotechlab.com/detail.aspx?id=216 I also ordered these hormones... thidiazuron Kinetin NAA Gibberellic acid IBA Wow- more than you'll ever need, well, for a bit anyhow. Good on you for supporting Carol, she's awesome Absolutely do read the technical specs ( I don't have a link here, should be via Phytotech labs ) on preparation, storage and use of your chems. It's vital for them to be effective and consistent. It's a few pages on a spreadsheet and some of the most important info you will get on TC chems Recently, I have thought about making my own containers, from borosilicate glass tubes. I also do occasional lampworking, being a horticulturist and artist by trade. Maybe they would be an inch across (2.5 cm for metric scientists who like logical base ten measurements) And about four or five times that tall. You're making too much work of it again I doubt it will save you money. 30ml autoclaveable polycarb tubes are cheap and re-usable Another idea I had...was to make a little fishbowl container, and create a micropropagation landscape inside.... With various plants, maybe a few sculptures... I apologize for the photos, will work on that next time....after posting these I recently took..... The terrarium thing sounds sweet and cool, but do consider that your plants are getting enough light, and that if the jars don't get enough clean airflow you could be sitting them in dirtier air than desirable Nothing wrong with your pics, that's what you get taking pics of TC. Most of my pics get ditched b/c it is hard to focus and still see what's in the jar aside from contam 1 Share this post Link to post Share on other sites
shonman Posted June 22, 2013 (edited) I am going to post pictures of all current jars, with their reference/ID next to them.....so they can be referred to through time... They are being posted for comment, criticism, and suggestions, all of which I appreciate very much and do not take personally. Questions: Where would you split the plant in post number 73? Just cut the stem above the branches, or cut off each branch to get two explants? How do I start a callus culture with Mitragyna, or get it to sprout numerous shoots with hormones? It would be interesting to try several techs at once....... I read somewhere that "For micropropagation, shoot multiplication was successfully induced from the axillary buds of the regenerated plantlets in WPM supplemented with 0.1 mg/l thidiazuron." By this, do they mean multiple shoots, or just the one axillary bud becoming the new explant? How can I generate 1000 of this plant, in the most effective manner? Edited June 22, 2013 by shonman 1 Share this post Link to post Share on other sites
shonman Posted June 22, 2013 (edited) OK, here are almost all of the jars I have going right now that are worth looking at. I promise to figure out the manual close focus for the next series of photos. These jars can be referenced by their file names. The file name is revealed without downloading when you move the cursor over the image. The green blob is a Ps. Carthaginsensis leaf that began to root in another jar.... I took the rooting sections and replanted them. The rest are generally Mitragyna Speciosa.... except for 'K-2-Caapi' which is the very tip of a B. Caapi vine...very small too...hard to see. Not entirely sure that one will make it, but maybe. Any feedback and or suggestions on these is much appreciated. Thanks! Edited June 22, 2013 by shonman Share this post Link to post Share on other sites
shonman Posted June 27, 2013 (edited) How can I best achieve 'shoot multiplication" ? Would that be the optimum way to produce ALOT of this plant? Edited June 27, 2013 by shonman Share this post Link to post Share on other sites
Darklight Posted June 29, 2013 (edited) Where would you split the plant in post number 73? If you do it ( you can still wait for a little more growth too, that's just as good an option ) I'd do it just above the bottom node and split it in two, replant. How do I start a callus culture with Mitragyna, or get it to sprout numerous shoots with hormones? Somewhere I wrote that it is easy to start a callus culture- but what you want is callus of a quality which will re-organise. This is harder It would be interesting to try several techs at once....... It will drive you nuts at this stage. IMO concentrate on one thing at a time at this stage. Once you have a lot of sterile plantlets and an established protocol you will have more material to play with for callus culture I read somewhere that "For micropropagation, shoot multiplication was successfully induced from the axillary buds of the regenerated plantlets in WPM supplemented with 0.1 mg/l thidiazuron." By this, do they mean multiple shoots, or just the one axillary bud becoming the new explant? TDZ? Hardcore and exxy. You mightn't need it They mean one or more shoots from the upper part where the petiole joins the stem- each shoot of these becomes a new explant How can I generate 1000 of this plant, in the most effective manner? OK, here is the part of the experiment which concerns good design It looks as though you have established sterile culture and a few options around how to increase that number Yes, you may be able to increase multiplication rates using callus culture, but with minimal parent stock you risk using it all up in this process- wait until you have about 100 individual plantlets, a replication protocol giving you at least 2:1 per subculture and sacrifice no more than 30% of these numbers trying to find a callus media per subculture. That way you maintain a good population of replicants and use the rest in things which probably won't work ( as in all science ) Remember, replication is exponential. So if you have a replication rate of 2 explants per parent plant, the numbers will grow 1,2,4,8,16,32,64, 128, 256. If you subculture once a month in 12 months you will have approx 1000, if you start with one sterile explant and don't sacrifice any to callus culture experiments on the way. Nice thing about this is that it is a stable protocol and won't result in too much variation or poor quality explants over time ( high hormone regimes can cause problems over time in some species or populations But on the way you will find a bottleneck. I don't know where this will be for you, often it is something simple like time ( how long will it take you to subculture 500 plantlets? will that leave you time to undertake and monitor callus culture experiments? ) or grow shelf space, or deflasking area and time demands, or even the number of jars you will need to own and keep clean ( you will need at least 2x the number of jars you will end up using, one to have plantlets in, another to have clean and ready and full of media to transfer into. I prefer 3x jars ) If you have a protocol which gives you 4 explants per parent plant then it will only take you 6 months to get 1000 plantlets. Awesome, but you will encounter these bottlenecks sooner and will have to act faster. No biggie if you are prepared and know it's coming up Calculate this *now*. What will your bottleneck be? Can you work around it? Working around it you will still find another bottleneck, that's normal, but you need to know it. Knowing this will save you heaps of time later on I've seen a lot of ppl start protocols and then the very day they have to get something done as part of the upscale go ZOMG, why didn't I think, I need an extra 5m of grow shelf and I'm out of jars and the deflask space can only hold 100 plants HELP MEEE! Very common, very easy to learn to avoid tho OK, here are almost all of the jars I have going right now that are worth looking at. I promise to figure out the manual close focus for the next series of photos. I took the rooting sections and replanted them. The rest are generally Mitragyna Speciosa.... except for 'K-2-Caapi' which is the very tip of a B. Caapi vine...very small too...hard to see. Not entirely sure that one will make it, but maybe. I did a basic check of them all but have no real comments beyond *congratulations!* Many of them look highly viable. You may have no idea how awesome this is, especially for a first timer learning by wire as it were How can I best achieve 'shoot multiplication" ? Would that be the optimum way to produce ALOT of this plant? Yes, at this point. Don't underestimate the power of a stable protocol to get things done faster Because we are all going through the research phase together, you need to come back here with at least 3 proposed scenarios/ protocols on this so you learn the way to make decisions for this as a part of normal research processes. Then I'll kick in again. I have no idea what may work for you, but you are doing so well Remember, always keep a significant portion of your population in your standard ( in this case, your starter media )- that way you will always have more explants to play with if your experiments fail Also, wait a while before trying callus- you will have more numbers to play with and that can provide statistical validity for whatever callus protocol you develop Edited June 29, 2013 by Darklight 1 Share this post Link to post Share on other sites
Darklight Posted June 30, 2013 Autoclave/ pressure cooker size and cooldown times are a classic bottleneck too If your pressure cooker only holds 10 jars and you have 4 plants per jar, then you'll be working in batches of 40 plants at a time ( about 1L media for babyfood jars ) You'll need to allow time between pressure cooking for the media to cool down- usually at least 4 hours, more like 8 if you have a bigger pressure cooker How much time does it take to setup and run ( and watch! ) each batch if you have a manual pressure cooker? How does this fit in with your work schedule both outside and inside the TC lab etc 1 Share this post Link to post Share on other sites
shonman Posted June 30, 2013 Thanks, Darkllight! Planning ahead is a very good idea. I can imagine needing another several hundred containers, etc. Fortunately, I have acquired four large stainless steel five shelf units, (four foot long/ 1.3 meter shelves) in advance And purchased enough fluorescent lights for all of them. Around $1000. US just in shelves and lighting! I am broke now. I will have to start getting containers in advance. Would you put four plantlets per jar (after they are clean, not contaminated cultures) As you suggested, I will begin dividing plantlets, and transfer them. My Mom gave me the pressure cooker (no, I do not live in her basement, and am grown up!) Which is ever so greatly appreciated....but it only does maybe five or six jars at a time. It can do them fairly quickly, though, as it has some sort of LCD computer control. Of course it still takes at least 15 minutes, then a couple minutes for pressure to come down, so I can open it, etc. I am going to just plan on taking all day or more to do media, etc...but will add up the a,punt of time first. Three scenarios will be posted soon, for discussion. Already, I am considering: A) Just splitting sterile explants, ad infinitum .1 mg/ liter TDZ, in WPM, and transferring sterile explants from existing cultures, To see if the stem/ buds / petiole multiplies into multiple plantlets C) meristem culture.......just disinfecting and growing out nodes from the (non sterile) motherplants. Will go into more detail soon, on these....... 'Plants from Test tubes' said that 'sterilize' means kill all living things, 'disinfect' means kill germs and fungi, bacteria, etc... So, would 'sterile explant' be an incorrect term...? I am going to take more explants, from the motherplants, too...... I would like to start sub culturing with 20 plants, if possible...... But will start with what I have now. Share this post Link to post Share on other sites
shonman Posted June 30, 2013 Darkllight....would you transfer several 'clean' explants, free of contamination, into the same jar, then? As you mentioned, maybe four per jar? That would save 4x the shelf space, and media! I have those new Magenta 'great for photography' polycarbonate jars now, am using them and some test tubes next batch. Also, Carol of Kitchen Culture Kits, is connecting me with someone who perhaps has interest in doing a project! I have 2 acres, and a 20 x 40 ft greenhouse on the Northern Oregon Coast.........would love to have something happen there. I told Carol she could take it over, because, yes, she is absolutely awesome.....but she has her own agenda. She lives not terrible far from there. If anyone here, reading this, wants perhaps be involved, somehow, or has a project of their own...PM me! Carol also put out to TC listserv email group.....to email any suggestions for custom made borosilicate tissue culture vessels or tools, etc I do glass work, too.......perhaps some Pasteur looking stuff.....if anyone had any suggestions for this, too, let me know! A bit off topic, here....back to the the issues at hand! I will post more photos, soon....the plants have changed already! 1 Share this post Link to post Share on other sites
shonman Posted June 30, 2013 "Plants from test tubes" said the principles for plant, and animal, tissue culture are very similar...... So, could I grow and replicate mammals, or reptiles and amphibians, or insects, this way, too? In liquid media perhaps? Predatory mites seem to be worth more than gold be weight....... Or, imagine replicating show quality Koi...... Share this post Link to post Share on other sites
Darklight Posted June 30, 2013 Thanks, Darkllight! Planning ahead is a very good idea. I can imagine needing another several hundred containers, etc. Fortunately, I have acquired four large stainless steel five shelf units, (four foot long/ 1.3 meter shelves) in advance And purchased enough fluorescent lights for all of them. Around $1000. US just in shelves and lighting! I am broke now. I will have to start getting containers in advance. Whoa mule! I mean that's excellent and all, but I was just trying to get you to think about what you might need first up, not send you broke! I should have added " Do you need to produce 1000 plantlets all at once and have them of the same age? ". I thought that was implied in my previous response, but obviously not With a large exxy setup like that you will have enough to produce clones for years! Which isn't a good thing if you were only looking for a hobby or a single run I was envisioning say, once you got to 60 jars of 4 plants each, deflask 30 jars and subculture the other 30, so you would have 120 plantlets in the deflask area and another 120 ready to subculture to 240, repeat until you have the numbers If you are subculturing once a month, how many jars will you need ( for whatever future species you work on ) to get numbers for your proliferation plan? That's your shelf space for that species. A 5" shelf on one of my units holds 125 jars approx, easy and without crowding. 125 jars x 4 explants per jar ( for one particular species, some are many more, some less ). 4 shelves per unit = 2400 plantlets at the end of each cycle. If I don't have a market for that species I need to tone the numbers down, switch off a couple of the lights until I'm not running them without cause. So that's how I work my numbers. YMMV Please avoid one of the common pitfalls of both noobs and old hands alike- don't buy too many toys until you are certain there is no other way! I'm as guilty of this as anyone- not so much these days but... Would you put four plantlets per jar (after they are clean, not contaminated cultures) Yep. Saves you shelf space and work as I said As you suggested, I will begin dividing plantlets, and transfer them. My Mom gave me the pressure cooker (no, I do not live in her basement, and am grown up!) Which is ever so greatly appreciated....but it only does maybe five or six jars at a time. It can do them fairly quickly, though, as it has some sort of LCD computer control. Those pressure cookers are great! If I were starting again I would def get one instead of an autoclave, they're way cheaper and IMO safer. Your mum rocks Just check the manual to make sure it is being cleaned properly every so often ( every 3 months is good to start ). Not so much clean media, but food prep and sterilising things like soils can build up salts and other compounds on the pressure sensors and change results or increase wear and tear. I'm not saying this will happen to you, but do check the manual ( online if you don't have one ) for cleaning requirements Three scenarios will be posted soon, for discussion. Already, I am considering: A) Just splitting sterile explants, ad infinitum .1 mg/ liter TDZ, in WPM, and transferring sterile explants from existing cultures, To see if the stem/ buds / petiole multiplies into multiple plantlets C) meristem culture.......just disinfecting and growing out nodes from the (non sterile) motherplants. Sounds a good place to start. Remember, don't run too many experiments at once. Three is a good number, especially since option A is the control Will go into more detail soon, on these....... 'Plants from Test tubes' said that 'sterilize' means kill all living things, 'disinfect' means kill germs and fungi, bacteria, etc... So, would 'sterile explant' be an incorrect term...? You got me there, spot on Sometimes I use the term 'functionally sterile' instead of sterile, or disinfest ( which is what it really is, as you said ). Functionally sterile works in a variety of fields including mycology. To me it means 'sufficiently clean for the purposes of our experiment'. The material can still contain contaminants, but not of they type or at a level where they will affect our work ( ie, if you were working with tomato plants as a control and didn't need them to be virus free, the explants could still be disinfested and the virus wouldn't affect the outcomes required of the experiments ) I'm splitting hairs here, basically you are right. I'll try to correct my terms here "Plants from test tubes" said the principles for plant, and animal, tissue culture are very similar...... So, could I grow and replicate mammals, or reptiles and amphibians, or insects, this way, too? In liquid media perhaps? Predatory mites seem to be worth more than gold be weight....... Or, imagine replicating show quality Koi...... Well, yes, in theory. But in practice getting cells to differentiate into functional organs in insect and animal tissue is difficult right now. Do some reading on the topics to look at the issues in more depth I for one would welcome a zip drive liver, for those heavy days and nights. If you make one I'll test it for you ( probably to destruction ;) ) We won't see that kind of tek available for a while, and maybe it will raise ethical issues we aren't ready to deal with yet. If you ever get the differentiation triggers down for functional organ culture you'll be up for a Nobel Prize I reckon In the meantime it is great that you are thinking of other uses for your setup in case you need to change focus. Predatory mites could be a great idea. I'm sure there are others I have those new Magenta 'great for photography' polycarbonate jars now, am using them and some test tubes next batch. Another thing Magenta are great for in other species is keeping larger plants in aseptic tek- did you get any couplers? Joining 2 together and using them for older plants in TC ( you don't need to do this now, just keep it in mind for other species in future )- the way they split apart when you open them in the flow hood- gives you easy access to all parts of the taller parent plant if you need to keep some back for some reason You can't do that with the taller culture tubes of the same volume, with these you risk contamination even in the flow hood because you have to stick your forceps all the way down the tube to get to the root base or media. I'm probably not making sense now, you'll get it when you come to it And I'm digressing too, sorry if you're using Magenta vessels, how much media are you putting in each jar? You can prolly whack a few more plants in there, at least 5, up to 12 depending on how that layout affects your future work. It's a tradeoff- you will save time but may have to disentangle the leaves of the grown explants in kratom and that can lead to tissue damage or increased contam risk 1 Share this post Link to post Share on other sites
shonman Posted July 1, 2013 (edited) Thank you for sharing these insights, Darklight...... I have use for the shelves, and big plans regarding various plants. There is a huge market for Chinese medicinal herbs. In Appalachia, USA, ginseng and goldenseal are big. There is a protocol for Ginseng micropropagation in ' Plants from test tubes'. These, and goldenseal can be planted out in the woods, the end result bring 'wild grown' as opposed to 'farm grown'. Wild grown fetches a much higher price. The plantlets themselves could be sold, or, planted then harvested much later. I am also interested in aeroponic culture of some root crops such as these. Also, don't worry.....I didn't now run out and buy up all this stuff because of your post... Two sets of shelves are already alost totally filled with cuttings in various stages of rooting, And also the micropropagation jars ..... I have been planning this for some time now, and gathering supplies....am broke in an ongoing way, due to buying equipment, etc. When I can...... This is what I plant(t) to do .........grow Chinese medicinal herbs, and interesting, collectible, marketable plants. Besides, you really can't go wrong with chrome plated utility shelves, they are SO useful..... I will acquire the connectors you mentioned for the Magenta vessels.... Edited July 1, 2013 by shonman 1 Share this post Link to post Share on other sites
shonman Posted July 2, 2013 (edited) Update, and a couple questions..... Here are a couple of the jars, file name will identify them in relation to earlier photos...... They have grown a bit! First, here is one of my shelf setups......definately will use all four shelving units! Edited July 2, 2013 by shonman Share this post Link to post Share on other sites
shonman Posted July 2, 2013 The last batch, taken a couple weeks ago, are doing nicely! The HPS light which reaches them maybe every ten minutes or so for a little while when the light mover passes near the shelf...does seem to speed up rooting and growth. Mitragyna loves light, humidity, and heat. Sorry again about photo quality. Share this post Link to post Share on other sites
shonman Posted July 2, 2013 This one is also doing ok..... Share this post Link to post Share on other sites
shonman Posted July 2, 2013 The explants I took last time, were done with triple branching in mind...it is working well! Lots of condensation on this jar...I am looking forward to using the new magenta vessels next... Share this post Link to post Share on other sites
shonman Posted July 2, 2013 (edited) is this yellowish stuff...contamination....or just stuff from inside the plant? Probably hard to tell from the picture, but the plant seems healthy... It is the same one shown in the circular photo above... Edited July 2, 2013 by shonman Share this post Link to post Share on other sites
shonman Posted July 2, 2013 (edited) This color, is really faily light tan.....hard to get it right (for me) in the photos.... A few questions...... can you tell, if this is contamination, or just stuff from inside the plant? If there are two plants, in one jar...and only one has a slightly off color..... should I throw both out, or subculture the one without discoloration.. or subculture both, seperately? What if mold shows up later in the day, but was not there in the morning....on agar next to an explant.... And there is another explant in the jar with it....would you toss them both, or try to save both...or save one? Also, I am sorry I doubted you (Darklight) when You said the micropore tape looked like the pores were not micro enough...I thought the photo made it look different, but on closer observation,,,, Those pores could allow things to get in. I suspect it is how mold got into the one I was asking about above. How do I know I have the right micropore tape? Also, one one explant, which was next to the mold.....it looked like it had grown extra shoots, More than just from auxiliary buds...interesting..... I had to transplant those quickly, using agar in jars from the batch with not enough water.... How long do jars with PPM, agar, woody plant medium, etc stay "good" for, before losing the PPM or not being suitable for use? Uploading all slightly yellowish/ light tan (very light) photos, at once here..... (Plz reference by filename, if commenting on a photo) Edited July 3, 2013 by shonman 1 Share this post Link to post Share on other sites
shonman Posted July 3, 2013 (edited) #%!!***~!!! That was definitely the wrong tape....my fault, but I will curse Walmart anyway for telling me that was the stuff.... I did get the right kind, finally.......hope my cultures are not ruined......most look ok, except for some pale discoloration near some explants...are they doomed? How can I sterilize them again, when replanting? Should I transfer them to liquid media, with PPM, for a while? I have grown somewhat attached to several of them.....many still SEEM ok, for now.... Making more media today or to tomorrow..... Micropropagation has actually caused me to vacuum,clean the kitchen and do dishes!! Edited July 3, 2013 by shonman 1 Share this post Link to post Share on other sites
Darklight Posted July 6, 2013 These, and goldenseal can be planted out in the woods, the end result bring 'wild grown' as opposed to 'farm grown'. Great idea First, here is one of my shelf setups......definately will use all four shelving units!chromeshelfnet.png I love those shelves too. You are only using the HPS light for the parent plants, right? Sorry again about photo quality. Don't worry about it, TC pics are hard is this yellowish stuff...contamination....or just stuff from inside the plant? It * looks* like contam but is a little hard to tell, is it fluffy? If not, does it look like it has a surface edge and is a different texture from the media? The contam source looks like it comes from the actual explants, maybe a late stage infection or a problem with non-sterile tools? But it is also possible it came in via the wrong tape Sub any plants in the jar which aren't touching it ASAP. Sometimes these can be saved. Throw the contam ones out, it will be quicker and easier to start from new parent plants, seriously, and you will have fewer worries Sorry mate, I can't always reply to your posts in the timeframe you need But if you do start over with proper venting, do remember that you have done so excellently well you are on your way- stuff like this happens to all of us and I def wouldn't let it stop you at all 1 Share this post Link to post Share on other sites
shonman Posted July 7, 2013 (edited) Yes, I am only using the HPS lot on the motherplants, but the Tc jars on the edge if the shelf, get exposed to it a bit, too.... The yellowish stuff does not seem to be fluffy at all, will check the surface edge. Not all the plants have the yellowish..... It ok, that you don't always respond immediately, I just appreciate all input in general, when possible...Thanks! I made another batch of media today, put in test tubes, and in seven Magenta containers........ I am going to submerge the test tube explants with PPm for a while.... I think it looks like some contamination may have come from my tools not being sterile..... Several jars still seem ok,moment went bad, some are still good, and I transferred them. Cut them in to several parts when transferring Questions: 1)If I do not vent the test tubes and Magenta containers, will they still grow explants as well as if they were vented? I just put media into the containers, did not add a vent yet.... If I make jars with media in advance, how long will it last and be good to use, Until the PPM or other ingredients "go bad"? 2)Do they last indefinitely until used for explants? Is it a good idea to have some jars or containers with media ready in advance? 3)Also, could you please post some photos, or describe.....the difference between contamination, And the slightly tan stuff that comes from the plant itself after it is cut and placed in agar? How do I recognize contaminans, as opposed to the stuff that occasionally exudes from the plant itself? 4) what are the advantages of micropropagation, as opposed to taking cuttings the regular way.. for reproducing large amounts of plants .....please list.... Edited July 9, 2013 by shonman 1 Share this post Link to post Share on other sites
shonman Posted July 13, 2013 Also 5) Darklight, can you please tell me a story, about what tech you used to multiply and propagate Mitragyna Speciosa? Share this post Link to post Share on other sites
shonman Posted July 15, 2013 I apologize if this thread has taken up too much time.... I do not want to take for granted, the energy it takes to respond to these kind of things continuosly. I suppose we could let it end here, and just say that I am going to divide explants to eternity..... let me know your thoughts on this! Share this post Link to post Share on other sites
Darklight Posted July 16, 2013 Yes, I am only using the HPS lot on the motherplants, but the Tc jars on the edge if the shelf, get exposed to it a bit, too.... That sounds great I made another batch of media today, put in test tubes, and in seven Magenta containers........ Sounds good too I am going to submerge the test tube explants with PPm for a while.... Really, I wouldn't waste the PPM on contaminated plants. General rule of thumb- if you can see contamination it's going to be almost impossible to wipe out. Out of the hundreds of visibly contaminated explants I've tried to re-sterilise with PPM and other methods very very few have survived, and if you have healthy parent plants available to restart from it is always quicker to do that Questions: 1)If I do not vent the test tubes and Magenta containers, will they still grow explants as well as if they were vented? Yes, if you subculture them regularly If I make jars with media in advance, how long will it last and be good to use, Until the PPM or other ingredients "go bad"? 2)Do they last indefinitely until used for explants? That is a seriously good question. Some hormones ( IAA for example ) will go off really quickly and shouldn't be stored for long at room temp in the dark. Other hormones are more stable in media and don't start degrading until they hit light or are reacting with explants If you have a sterile media with no hormones it can be functional ( but prolly not equal to fresh ) for up to a fortnight- in my experience only- as long as you have sealed the lid on the jars and no contaminants can get in I have worked in labs where working media is stored for up to a fortnight at 4C in the dark ( fridge ) but you do need to seal the jars properly, and have a really good quality fridge with not a lot of temp variation as that's what causes air to be sucked into and out of containers, bringing contamination At this point in your career I wouldn't store made-up media ready for more than a few days. Make up twice what you need for any one session and store 1/2 in the freezer, carefully labelled, in liquid form so you can thaw it to autoclave it when you need it next Is it a good idea to have some jars or containers with media ready in advance? Up to a point- see above. You risk contamination and loss of activity 3)Also, could you please post some photos, or describe.....the difference between contamination, And the slightly tan stuff that comes from the plant itself after it is cut and placed in agar? Not right now mate, sorry, do a web search and come back to me with your results and we will discuss How do I recognize contaminans, as opposed to the stuff that occasionally exudes from the plant itself? Once you get a general idea you will be fine. Experience with a particular species does help tho, sometimes it is an eyeball thing with spp which exude latex for example. Or wait a few days. Contam grows really fast. However large amounts of exudates usually means that the explant needs to be moved onto fresh media 4) what are the advantages of micropropagation, as opposed to taking cuttings the regular way.. for reproducing large amounts of plants .....please list.... I've written about this here before, if not on this thread then on the forums somewhere I'm sure. UTSE and if you can't find it, repost your question I did say previously you need to do some research on possible protocols for Mitragyna reproduction and get back to me so we can discuss the decision making processes you will go through when considering protocols for any new species. Have you done that yet and I missed it? There really is no need for me to spoonfeed you information as you are learning really well- I'd rather assist your progress via furthering your good work habits around research and decision making. Sorry, been really crook the last week or so and unable to reply Share this post Link to post Share on other sites
shonman Posted July 16, 2013 (edited) Thanks, Darklight! I appreciate your insights....could you please outline how to decide what technique to use, When attempting to micropropagation new plants? Edited July 24, 2013 by shonman Share this post Link to post Share on other sites
