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inski
Cortinariologist



Registered: 02/28/06
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Micropropagation 7
#25793452 - 02/05/19 06:26 PM (4 years, 11 months ago) |
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Echeveria elegans micropropagation.Cultures next to fresh culture tubes with sterile multiplication media ready to receive subcultured propagules in the Laminar Flow Hood Tools set up in the Laminar Flow Hood Close up images of in-vitro plants on multiplication media 
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Solipsis
m̶a̶d̶ disappointed scientist



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Re: Micropropagation [Re: inski]
#25798682 - 02/08/19 05:27 AM (4 years, 11 months ago) |
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Respect!
Could you shine a light on how an explant is best sterilized?
I would be interested in growing Strombocactus on agar and have a flowhood now and should be getting MS salts, but the seeds are so incredibly tiny (and explants can be as well).
(Would also like to clone my Salvia as it is doing badly due to desiccating conditions of my apartment during winter - i believe - and there isn't quite enough to just make cuttings)
For the seeds would it be smart to rinse in a bleach solution and then with distilled water a number of times, using a coffee filter which would just as the rest would have to be autoclaved first? Then finally you place them on the medium by hand?
Also most people are not too eager to share medium recipes but are there good points of departure just based on very rough classification of cactus / other succulents / woody plants / leafy plants ?
P.S. a propagule would be an explant that has meristematic tissue correct?
Edited by Solipsis (02/08/19 05:30 AM)
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Rik
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Re: Micropropagation [Re: Solipsis]
#25899946 - 03/27/19 11:31 AM (4 years, 9 months ago) |
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beautiful!
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Solipsis
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Re: Micropropagation [Re: inski]
#26005598 - 05/21/19 01:50 PM (4 years, 8 months ago) |
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How important are the vitamins that can be in MS salts? Does it depend on how long you plan on growing a plant in a medium?
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inski
Cortinariologist



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Re: Micropropagation [Re: Solipsis] 1
#26006576 - 05/22/19 12:33 AM (4 years, 8 months ago) |
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Quote:
Solipsis said: Respect!
Could you shine a light on how an explant is best sterilized?
I would be interested in growing Strombocactus on agar and have a flowhood now and should be getting MS salts, but the seeds are so incredibly tiny (and explants can be as well).
(Would also like to clone my Salvia as it is doing badly due to desiccating conditions of my apartment during winter - i believe - and there isn't quite enough to just make cuttings)
For the seeds would it be smart to rinse in a bleach solution and then with distilled water a number of times, using a coffee filter which would just as the rest would have to be autoclaved first? Then finally you place them on the medium by hand?
Also most people are not too eager to share medium recipes but are there good points of departure just based on very rough classification of cactus / other succulents / woody plants / leafy plants ?
P.S. a propagule would be an explant that has meristematic tissue correct?
Hi Solipsis Sorry for the late reply, so far I have successfully surface sterilised and germinated Lophophora williamsii and Ariocarpus kotschoubeyanus seed on half strength MS with vitamins, first I stirred the seeds in distilled water with one drop of organic plant based dishwashing liquid for one hour in a beaker covered with foil on my magnetic stirrer then moved the beaker to the flow hood. Next I immersed and stirred the seed in 70% isopropyl alcohol for 30sec then rinsed in sterile distilled water, next I immersed and stirred the seed in 30% bleach with two drops of Tween20 for 30min then rinsed three times in sterile distilled water before inoculating onto half strength MS with vitamins.
I have yet to attempt to multiply these plants but will soon, I think one useful modification to the MS media for Cacti could be slightly higher levels of calcium chloride.
A propagule can be any part of a plant that can give rise to the growth of a new plant.
Quote:
Solipsis said: How important are the vitamins that can be in MS salts? Does it depend on how long you plan on growing a plant in a medium?
The main vitamins included in culture media are from the B complex and are essential for metabolism and growth, in the lab they are prepared as stock solutions and added to MS media at a rate which has been found to be best for the particular plant being cultured.
At present I use a pre made MS media that has vitamins included so I just weigh the media in powder form, dissolve in distilled water on the hotplate magnetic stirrer, add hormones which are stored in the refrigerator as stock solutions if required then adjust the pH with 1M HCl or 1M NaOH accordingly before adding the agar and heating, once the agar has dissolved I pour into culture tubes and autoclave. I hope this is useful
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Mateo
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Re: Micropropagation [Re: inski] 2
#26007398 - 05/22/19 01:12 PM (4 years, 8 months ago) |
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How nice you do Micropropagation and tissue culture.  I hope it´s going well for you, it looks promising in the pics. I hope more people find this intresting and start experimenting. After all, if one can grow mushrooms it´s not so different with TC & Micropropagation.
I have had plans for trying tissue culture with coca leafs for a long time but never started it. I have collected needed items as media, hormones, plastic tubes, PH meter and stuff. I hope i finally get around to start it. Coca likes Anderssons Rhododendron media mix, i collected some PDFs where they tried different media with coca. It would be fun to try some cactus also, and plants that are difficult to get growing from seed.
I will surley follow this thread and hope you post your progress.
-------------------- A wise rat has many holes
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Solipsis
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Re: Micropropagation [Re: Mateo]
#26009748 - 05/23/19 04:20 PM (4 years, 8 months ago) |
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Ow!
Thanks for the answers guys!
I don't have any B vitamins or any for plants really... but I did order the MS salts without vitamins already. But with invoice so I can cancel that... should I get the ones with vitamins then?
Some of the less common PGRs seem to decay in solution, but I have no idea what that means for degradation in gel media.. Either GA3 or brassinolide at least I think. But I guess they are not essential anyway, but are interesting for germination.
Are stock solutions of the others that are normally not really water-soluble more easily / better made with NaOH/HCl or something like tween 80 like I have (no 20)?
A domestic company offering rather cool tubes awkwardly offered me non-sterile ones for free at first, more than 50k pcs even, but another employee took over communication and explained that they couldn't offer any [even decent samples for me] at only the cost of shipping whatsoever.
So I will be kinda thrown back for now to the 50 mL culture tubes I have (but virtually all in use right now by like expired fungal slants.. but they are not flat bottom.
Par for the course, of course... but this is how it goes
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Mateo
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Re: Micropropagation [Re: Solipsis]
#26012161 - 05/25/19 05:17 AM (4 years, 8 months ago) |
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I look for any fitting plasic tubes/jars on ebay. The tubes/jars made especially for TC are usually very expensive but probably very good also. The only requirement is that they can withstand sterilization (pressure cooking). Many uses baby food jars and buy lids for them.
-------------------- A wise rat has many holes
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Solipsis
m̶a̶d̶ disappointed scientist



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Re: Micropropagation [Re: Mateo]
#26013913 - 05/26/19 08:22 AM (4 years, 7 months ago) |
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I have UV-C now for "sterilization" although i still need to build it into a hood cabinet, so autoclavability should not be required.
Some parts that are PS couldnt be autoclaved anyway..
I guess I will keep looking for containers to improvise with.
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inski
Cortinariologist



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Re: Micropropagation [Re: Solipsis] 1
#26108969 - 07/15/19 01:58 AM (4 years, 6 months ago) |
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Here are two of my current projects, both were propagated from surface sterilised seed germinated on 1/2 MS media.
Lophophora williamsii Ariocarpus kotschoubeyanus v. macdowellii
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Solipsis
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Re: Micropropagation [Re: inski]
#26158660 - 08/29/19 08:53 AM (4 years, 4 months ago) |
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Very nice, inski!
What is the medium you used for continuing to grow after germination?
How about: - 0.5 - 0.75 MS - 5 mg/L BAP - 1.1 mg/L NAA - sugars?
For a balanced growth of shoots and roots? Would you normally not want to put an emphasis on either root development or shoot development?
I am currently just trying to germinate some seeds on 0.5 MS, at least I am preparing the medium right now. I think also Lophs or something for now, Strombocactus has such small seeds, I don't wanna lead with them. [Aw damn, just realized I forgot to pH correct my media, I tend to do that :S ]
Are you going for a callus culture next, inski? I myself will want to just start with germinating seeds and then developing a plantlet/seedling, before looking at things like callus.
Not sure if rooting a cutting of certain species in vitro would be an economically viable way for easily sending a small plant in the mail. I guess it is not warranted.
What are the biggest challenges? The procedures are just a matter of learning routine and the bigger challenges are in finetuning those recipes? Is basic info of even like cultivating Loph or Trich and making callus culture so valuable that nobody (few) wants to open source it?
Edited by Solipsis (08/29/19 08:54 AM)
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denger
Mycelium keeper



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Re: Micropropagation [Re: Solipsis]
#26159593 - 08/29/19 07:24 PM (4 years, 4 months ago) |
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So, per your suggestion Solipsis, I am moving our discussion here. To answer your question, I really am not lacking any materials at all: I got 6-BAP, kinetin, a boatload of 2,4-D, NAA, enough MS, coconut water, dishes and jars and basically everything I think I need (besides time and clear recipe for callus induction and differentiation).
So far I got a few dishes with this:
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Solipsis
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Re: Micropropagation [Re: denger]
#26162334 - 08/31/19 12:24 PM (4 years, 4 months ago) |
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That's nice! Does kinetin have some rather unique application or is it non-essential? I also thought coconut water is non-essential and can be substituted fine.
"A boatload of 2,4-D" - haha copy that mofo.
Did you have a lot of losses getting dishes like that? I hope you don't quickly run out of space for the buttons. Although i have been wondering about cubic ones like Japanese watermelons.
For callus I guess we'll get there when we get there and hopefully with some tips about how to adjust PGR concentrations for cacti.
Just gotta get on it.
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denger
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Re: Micropropagation [Re: Solipsis]
#26162958 - 08/31/19 08:07 PM (4 years, 4 months ago) |
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Quote:
Solipsis said: That's nice! Does kinetin have some rather unique application or is it non-essential? I also thought coconut water is non-essential and can be substituted fine.
Well, I certainly have some redundancies there, but did not know which is going to be useful as far as hormones go, so I picked up a set of whatever was mentioned in cactus papers I read. Coconut water IS essential because it prevents death by phenolic compounds which tend to accumulate once you cut the buttons.
Quote:
Solipsis said: Did you have a lot of losses getting dishes like that? I hope you don't quickly run out of space for the buttons. Although i have been wondering about cubic ones like Japanese watermelons.
It seems like I had about 25% germination rate once I figured out sterilization procedure. First round was a complete bust because I used too much bleach. Low germ rate I attribute also to high concentration of salts in the media, since I used full strength MS. Will go 1/2 next time. And perhaps dial down bleach again because I got 0 contaminations, so must have overdone it at 10% bleach for 10 min (with some tween-80 for surface action). Space wise, they all still have at least 3mm to go up before they hit the lid, and they are going very slow, perhaps 1/2 speed or worse then out in the wet sand. I am intentionally not putting them under strong light until I figure out next steps. If I manage to induce callus and then induce plantlet formation from it, I will not let them grow big or worry about roots. They will go straight to pereskiopsis from the dishes, at least that's my plan. If I wanted to go slow I wouldn't bother with tissue culture. Seeds are cheap enough, and plentiful.
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Solipsis
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Re: Micropropagation [Re: denger] 1
#26165282 - 09/02/19 12:37 PM (4 years, 4 months ago) |
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Thanks thats good to know about the coconut water. Maybe there are other (antioxidants?) one could use for the phenols.
I thought that you are supposed to be able to achieve full potential of the viability of your seeds with TC? Idk if that is really possibly near 100% with e.g. Lophs. If you don't get much above 70% I would say there is no point in sowing seeds for TC. It could be interesting to add certain PGRs like iirc GA3? to the medium, at least some of them seem to help germinate even dormant seeds.
When I can grow seeds reliably for me it is valuable though when I have only a few quite valuable seeds of a rare type. And callus culture would be awesome eventually for me when I have only one small plant or tissue specimen of some rare genetics.
Apart form it being very interesting, what value are you hoping to get doing callus culture etc? 
Maybe dumb question but: is it possible to graft a callus culture directly?
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denger
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Re: Micropropagation [Re: Solipsis]
#26166502 - 09/03/19 08:36 AM (4 years, 4 months ago) |
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I would not expect to get better germination rates in sterile conditions vs regular soil, just because sterilization procedure is so harsh by necessity. Then there is always risk of contamination. Now, after germination survival rates should be close to 100% barring secondary contaminations. It only makes sense to me to do for massive propagation of a desirable plant. Lophs pup readily when grafted on the Pereskiopsis, and pupping can be induced easily with hormones without the effort of going through tissue culture, so only truly massive multiplication is practically useful. I am talking a 100 plants from one seed or better. Because a dozen or three can be had easily by regrafting pups. To answer your question about grafting callus - I do not believe it to be possible since there is no vascular tissue to connect in the callus tissue, and it is quite fragile and susceptible to infections due to lack of any type of protective layer on the outside. It will also dry out very rapidly. However, it should be straight forward to differentiate it quickly into plantlets that can be grafted easily and readily.
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Solipsis
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Re: Micropropagation [Re: denger]
#26166591 - 09/03/19 09:44 AM (4 years, 4 months ago) |
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About the sterilization being so harsh by necessity: I don't buy it. 
Whether chemical oxidation or irradiation, it makes a lot of difference whether you need to penetrate just one cell thickness of like a contaminant, vs penetrating a seed's tissue layers and damaging it.
Not that I have got this tackled, but it should be possible to find a middle ground where you do kill all unicellular contaminants but don't get through to your seeds' embryos. So I guess I am saying: if we are not yet able to do it, that doesn't mean it can't very well be done just fine.
What do you use for pupping a grafted Loph? The regular BAP in lanolin?
Hmm yeah I wanna believe your suggestion/answer about grafting callus, but since it is undifferentiated I would still like to know whether something like vascular tissue can be differentiated out of necessity, using the same sensing mechanisms that would normally notice where such infrastructure is needed.
Good point about having to harden in vitro samples though: it remains unclear to me how sensitive plants are that have been grown sterilely and in vitro for their entire lives. Some reports make me think that it's quite hard and requires professional measures to adjust to the outside world while other sources make it seem more reasonable.
Differentiating into plantlets may be more practical - it is also much easier to understand this path or approach. Still doesn't mean callus culture doesn't hold promise - just saying that because I think we might be surprised by the potential of undifferentiated tissue. I think if it falls short it will be more due to the complexities of being able to guide the differentiation as we would like to.
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denger
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Re: Micropropagation [Re: Solipsis]
#26166717 - 09/03/19 11:08 AM (4 years, 4 months ago) |
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I want to agree with you on sterilization. I definitely want to experiment more, as I have alluded before. Ultimately, want to tweak the procedure till I start getting some contamination. Also, if we can harvest whole fruit and sterilize the intact fruit, it will protect seeds inside - that might be an easy way out of damaging seeds.
For pupping a grafted loph I use sun and fertilizer :-) They do it all by themselves when overfed. Basically treat it as a pereskiopsis plant and it behaves like a pereskiopsis plant with no top growth. But yes, BAP in lanolin (or simply "orchid keiki paste" from amazon or ebay) if you need to force it. I personally never had to, have more stuff to graft then space for it. Need to build that greenhouse.
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Solipsis
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Re: Micropropagation [Re: denger]
#26197691 - 09/19/19 03:28 PM (4 years, 4 months ago) |
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My Loph seeds are not germinating on 0.5 MS for some reason.. i thought I had sterilized too long at 8% bleach for 8 minutes but now i see 30% bleach at 30 minutes???
I have condensation problems from taking the tubes out of the PC erroneously i guess, but i drained some and keep the tubes at an angle. No signs of contamination on any tube.
Very strange.
(Did quick 70% rinse then bleach then 3x sterile water.) its been about 2 weeks
I don't have lanolin but use cocoa fat for the BAP i believe.. i have not seen clear results from it honestly - not like pupping from the treated areole.
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inski
Cortinariologist



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Re: Micropropagation [Re: Solipsis]
#26198511 - 09/19/19 11:43 PM (4 years, 4 months ago) |
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I stirred 10 seeds in distilled water for an hour on my magnetic stirrer then moved to the flowhood, under aseptic conditions and using sterile technique I soaked the seed in 70% isopropyl alcohol for 15 seconds then rinsed in sterile distilled water, 15% bleach for 20 minutes then three consecutive rinses in sterile distilled water.
I inoculated one seed per test tube containing 1/2 MS media as follows.
MS------------------2.2g/L Agar----------------8.8g/L Sucrose------------20g/L Distilled Water---1liter
pH was adjusted to 5.9 with 1M NaOH or 1M HCl before autoclaving and before the addition of agar, it's believed that pH drops slightly after autoclaving so my final pH should be about 5.7, I haven't tested that yet, the plants are growing and looking alright so I'm not too worried.
My surface sterilisation was inadequate because all but one of the seeds became contaminated with Trichoderma after a couple of days, the only seed that wasn't contaminated germinated after a few more days and is the plant I posted earlier in this thread.
Since then I have used that plant as a source of explants, I removed the plant under aseptic conditions from the original germination media and removed the roots, the plant was cut vertically down the middle and each half with roughly 5 areoles each was inoculated onto a multiplication media containing auxin and cytokinin, for some reason one half has undergone areole activation and the other half hasn't. The half that is multiplying has at least 7 pups, if these pups are grown on until each has 10 or so areoles then individually inoculated onto the same multiplication media it's possible I'll have up to 70 pups all from only half of that original seedling, so to me even if only 10% germination success is achieved it is worth it if areole activation is successful.
Solipsis, at what temperature and light cycle are you growing your cultures under?
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