Share The Seeds
Gardening Area => Advanced Cultivation Techniques => Topic started by: ONandONandON on January 05, 2019, 10:57:52 PM
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Greetings STS, nothing to post about yet, hopefully eventually, still in the gathering supplies and researching stage.
So i thought i'd invite everyone to join in! Feel free to post your questions, videos, experiments, helpful information..
Also trying to raise any interest in a group buy?! of "Murashige and Skoog medium" that's MS medium for short..
That's the most commonly used growing medium in micropropigation suitable for a wide range of plants.example:
(https://proxy.mind-media.com/proxy.php?url=http%3A%2F%2Fwww.lapshin.org%2Fcultivar%2FN34%2Fin-vitro%2F24.jpg)
https://en.wikipedia.org/wiki/Murashige_and_Skoog_medium
There are 22 chemicals needed, in mg amounts, each can be purchased in large amounts cheap..
i was thinking if people are interested, we could each purchase some of the chemicals, then trade.
Even if only two people, it would make it half-price, and the more people the cheaper it would be.
So post here if interested in the "Murashige and Skoog medium" group buy ;D
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Dude, OMG I am so down for this!!
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Count me in.
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An interesting project, but unfortunately my financial situation is not that good at the moment.
Nevertheless I would be very happy if you could keep us updated on your progress :)
I can offer 2 PDFs which might be interesting for doing experiments with tissue culture, but I´m not sure if it is allowed to post the links here:
Hirenkumar Sherathiya - Practical manual for Plant Tissue Culture: Basic Techniques of Plant Tissue Culture and Molecular Biology
Roberta H. Smith - Plant Tissue Culture: Techniques and Experiments
Edit:
Why don´t you just buy the MS Medium as dehydrated powder for making a solution, wouldn´t that be easier?
Edit 2:
I looked a bit closer, powder for 10L solution is not that expensive, depends on how much solution is needed?
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Yea Im down to just buy my own mixture and experiment with plants I have here right now. Id love to share results, and try experiments with other people. Can any of this be applied to fungi possibly?
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On second look at prices, it would be MUCH cheaper and easier to buy it premixed :P
i found some from this guy in Germany on ebay: 100L for 55$ 50L for 36 10L for 20
https://www.ebay.com/itm/Murashige-Skoog-Medium-mod-with-Gamborg-Vitamins-powder-premix-for-50-L/113205340598?hash=item1a5b9069b6:g:7wIAAOSwel5bjO-V:rk:5:pf:0
i guess i was only looking at the USA sellers, but thanks for the suggestion Neebu!
Awesome to see others interested in micropropigation! i'll be getting the 50L or 100L soon as i can..
so the group buy of individual chemicals is off.. but i guess we could have one for the premixed 100L ..
i only got 10$ paypal ATM, but i have ebay store selling plants, so not to long and i'll have more funds.
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Can any of this be applied to fungi possibly?
Of course, it's very similar techniques and equipment needed for both.
-sterilizer / pressure cooker
-still air glovebox or hepa flowhood
-growing containers
-chemicals for cleaning
that's most of the basic equipment.
There is a ton of how-to videos on youtube..
Just search micropropigation or tissue culture.
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I found the same product by dephyte, but on another site (it´s not cheaper there).
If you don´t mind, I would like to participate with 15$ for a bit of powder, this seems affordable.
I don´t have any other Equipment yet, but hope to start something some day ;D
I attached the product datasheet.
Thanks for posting that thread ONandONandON.
Wish you good luck and all the best for your experiments!
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Awesome! ;D
that's two people in for 15$, so we're at 10L for 20$ at least (but surely someone else will get in on this)
Let's try for 100L (it's the best deal) one more person makes 18.66$ each..
Two more (total of four people) is 14$even, each.. that's 25L worth each..
So two more people in would be perfect, and easy to divide into 25% each.
56$ https://www.ebay.com/itm/Murashige-Skoog-MS-Medium-for-plant-micropropagation-powder-for-100-L/123309579751?hash=item1cb5d2dde7:g:G90AAOSwt0dcEAcy:rk:11:pf:0
Edit: Or if shamichael and darkpines is in for 14$, that makes four and this groupbuy is closed, going onto next stage.
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It seems like, besides sugar and agar, that also at least an auxin is needed for the preparation of the MS medium.
In the books NAA is suggested for callus and root growth induction and it seems to be the cheapest choice (not sure if it is really necessary to add a cytokinine as well?).
I looked on the internet a bit and found a company which offers 25g of NAA for around 29 USD, shipping included.
I´m not sure if they will sell it to individuals, but buying from ebay might be an alternative.
I could take care of this (but only next month), so you don´t have to bear all the risk.
Also not sure if some kind of antifungal/antibacterial additive is needed?
Afair many antifungal/antibacterial substances are toxic to the explant.
Although Sherathiya writes that he uses an antibiotic called Cefotaxime, idk where to get something like this, which can be used for tissue culture and is not toxic for the explant.
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Thanks for posting that product data sheet BTW! very useful.. anyways...
i think it's optional to add hormones and/or antibiotics, but might be needed for the best results.
The MS-medium already has some growth hormones (for example Indole Acetic Acid 1-30 mg/l and Kinetin 0.04-10 mg/l)
(i believe cytokins and auxins can be used alone or together in different concentrations to get different effects)
i recently bought some hormones to experiment with from ebay.. and might can trade/spare some of:
6-BA 6-Benzylaminopurine 99% 5g CYTOKININ type
6-KT Kinetin Furfurylaminopurine 99% 10g natural CYTOKININ type
IBA Indole-3-butyric acid Indole butyric acid 98% 10g Water soluable
Gibberellic Acid GA3 90% 5g
NAA Naphthaleneacetic acid Naphthalene acetic acid 98% 10g
DA-6 Diethyl Aminoethyl Hexanoate 98% 5g
Triacontanol 99% 5g
Also for cheap experiment trying out Antibiotic Neomycin Sulfate Ointment 1 Oz Tubes
(neomycin is similar in action to the common geneticin commonly used in tissue culture)
(will try water extracting the neomicin sulfate, also inactive-ingredients don't look harmful.)
Several antibiotics can be bought for farm-animal use, or for fish antibiotics petstore ebay etc.)
There are many types that can be tried and just need some researching, here is some good information:
https://www.sigmaaldrich.com/life-science/core-bioreagents/learning-center/antibiotic-selection.html
https://www.sigmaaldrich.com/technical-documents/protocols/biology/antibiotics.html
https://www.sigmaaldrich.com/technical-documents/articles/biology/antibiotic-kill-curve.html
Following reagents are not included and need to be purchased separately:
Distilled water
Gelling agent, e.g. Agar or Gellan (if a solid media is required)
Sucrose (20 - 30 g/l, depending on the culture requirements)
Plant Growth Regulators (if necessary)
@lso Darkpines and Shamichael please confirm if you are in or out, thanks :)
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ONandONandON, you seem to be really well prepared in regard of plant growth hormones ;D
I still need to do much more research on this topic.
Many thanks for the links and additional information!
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I can throw down about 20$ for this! My only problem is how to pay for it? If we are using ebay, I can go buy a gift card and just load up the code when needed. Let me know! Ill go buy one in the next few days.
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Ok cool, i currently have 24$papal, and two sales 'awaiting payment' so i think we got enough!
it's better if you have paypal and can just send it to me that way, and i will make the purchase.
(One of you hopefully has a paypal to send, or this will take longer)
IF not, the best way i know of is send me cash in a card in the mail,
and i should have enough paypal by then to cover online purchase.
Or, to use an ebay card, i can post a special listing.. loses $ to fees.
(56$100L)14$each=25% =25L each, i'll also send some hormones..
i estimate shipping about 3-5$each? though it's hard to say exactly,
it doesn't say how many grams we will be getting, just 100L worth.
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Ok cool, i currently have 24$papal, and two sales 'awaiting payment' so i think we got enough!
it's better if you have paypal and can just send it to me that way, and i will make the purchase.
(One of you hopefully has a paypal to send, or this will take longer)
IF not, the best way i know of is send me cash in a card in the mail,
and i should have enough paypal by then to cover online purchase.
Or, to use an ebay card, i can post a special listing.. loses $ to fees.
(56$100L)14$each=25% =25L each, i'll also send some hormones..
i estimate shipping about 3-5$each? though it's hard to say exactly,
it doesn't say how many grams we will be getting, just 100L worth.
Yea thats my dilemma lol. I dont have paypal, and all my information is in an account I cant access because of forgotten security questions/answers. I might be able to access it if I call them maybe or email or something. Ill have to check into it in the future. I guess I can send cash, I have some trusty envelopes for it. Shipping would only be about 4$ or so. Not a big deal for this experiment. I lack all knowledge of this area and want to learn more by just kind of diving straight in to it lol.
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I lack all knowledge of this area and want to learn more by just kind of diving straight in to it lol.
That also applies to me^^
I don´t have much experience in mushroom cultivation and sterile work is not my strength, but working with petri dishes went actually fine.
There is also another useful book on micropropagation linked by ONandONandON in another tread:
https://hackteria.org/wiki/images/6/64/Plants_From_Test_Tubes_Complete.pdf
http://www.scielo.br/pdf/sa/v72n6/0103-9016-sa-72-6-0540.pdf
Also I gathered some other interesting threads on this topic on this forum:
Antibacterial/antifungal substance for the medium:
http://sharetheseeds.me/forum/index.php?topic=2296.msg17906#msg17906
More Documents on Tissue Culture:
http://sharetheseeds.me/forum/index.php?topic=1374.msg9399#msg9399
Micropropagation resources:
http://sharetheseeds.me/forum/index.php?topic=408.msg1276#msg1276
Plants from Test Tubes:
http://sharetheseeds.me/forum/index.php?topic=1777.msg38439#msg38439
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I also found a YT video, it´s not intended to advertise this product, but one can get an idea of the working routine, seems a bit similar to mushroom cultivation.
But I´m not sure if it´s the best example for working sterile properly, it should start at 21m54s.
Edit: Linking the time-position didn´t work, you can go to 21minuetes 54seconds, you will just miss some advertisement.
https://www.youtube.com/watch?v=nRXU_AWmRU4#t=21m54s
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Pretty good video after advertising, but they definitely use antibiotics, and not Very sterile technique..
Which apparently they get away with... probably have to change out the growing medium more often.
here is a couple good ones..
https://www.youtube.com/watch?v=qDOGrEhUe8A
(Tissue Culture Propagation: Class 10m)
https://www.youtube.com/watch?v=Cw4oNRVxwSY
(boring but informative 3 part-series 1h)
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This supplier has several types available. for example:
orchid medium
banana medium
WPM | Lloyd & McCown's Woody Plant Basic Medium
Murashige & Skoog Basic Medium
Murashige & Skoog Medium mod. with Gamborg Vitamins
Murashige & Skoog (1962). Modified to contain vitamins as described by Gamborg et al. (1968).
This combination is often used when plant species have different requirements on the vitamins included in the media, especially concerning a higher thiamine concentration.
Murashige & Skoog Shoot Multiplication Medium C
This popular modification additionally contains sodium phosphate monobasic (147.5 mg/l), adenine hemisulfate monohydrate (80 mg/l), kinetin (1 mg/l), NAA (0.1 mg/l).
not sure which would be the best.. i have some kinetin and NAA, i don't have the adanine or phosphate.
6BAP is benzyl adanine.. i wonder if that's similar? i'll just stick with basic MS medium.. then again... idk :P
some more random info:
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/General_Information/culture-media.pdf
http://cdn.intechopen.com/pdfs/40181/InTech-Plant_tissue_culture_media.pdf
http://himedialabs.com/TD/PT040.pdf LINSMAIER & SKOOG (1965)
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So we cant just use a basic formula for a most plants? I have a ton of plants and wanted to try a lot of them out lol. Maybe I should focus on one? Maybe if we are working on this together, we could each choose a specific project to work on. For example, one person can do a certain plant, another can do a cactus, another can do a plant different from the rest, and so on. Idk just some ideas. I also dont have much space for many things to be going on at once. At least until spring comes around.
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i think the antibiotics we use most in the lab are benomyl, refampicin, kanamycin, & streptomycin. i'll have to check some notes, but pretty sure these can all be used in plant tissue culturing. but we almost always use two or more antibiotics, i think it helps limit the amount of microbial resistance that may develop over time. we dont want to breed antibiotic resistant bacteria and such...
if you are working with healthy plants and clean the exterior surfaces well, you might can get away without any antibiotics
as to which media to use, i would google scholar search some primary lit and try to find folks who cloned related plants to what you want to work with, read through the materials & methods sections, and see what was included in the media used. that stuff gets a bit dense sometimes, but it will probably tell you why the particular media was used as well, and then maybe you can adjust according to your needs. of course this wont be of much help for really obscure plants, but worth checking out, could save a lot of time and resources and headache!
i think the hormone levels are really important, and different groups of plants are likely to prefer different ratios.
rooting hormone is a form of auxin- if used too generously on easy-to-root plants such as succulents or vines, it tends to be counteractive, inhibiting root development. these plants root easy because their endogenous auxin levels are "perfect", so to say. whereas, hard to root plants really benefit from small amounts of rooting hormone, and some can be almost impossible to root without hormonal assistance.
the same is going to happen in your media- some plants will appreciate a generous amount of auxin, others will die at these concentrations.
same goes for cytokinins
and then these groups of hormones play off each other, so relative ratios to each other are also important, and preferred ratios likely differ between species.
i think growing two different species or even genera of for example Solanaceae family will probably both work well in similar media,
but something like Diplopterys from the Rubiaceae family in the Asterids, might require a totally different media than Acacia from the Fabaceae family in the Rosids... i have no idea really, but i would not be surprised.
really, if you have the time, space, and patience, itd be awesome if you set this up like an experiment, and created multiple sets of cloning media, with different ratios of antibiotics and hormones, and record how the clones of a single plant type perform. then repeat with more plants. we all could learn a lot from that work.
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Many thanks XDX for your help, it´s really appreciated!
It´s a good idea to try different media for different plants and to record different ratios of antibiotics and hormones.
Since I don´t have much experience I thought it would be good, to start with something boring to develop proper technique and not to waste plant material.
I thought of something like broccoli florets, which were used in an experiment with MS Medium in the book by Roberta H. Smith.
I think the McCown Woody Plant Medium in the pdf posted by ONandONandON could be interesting.
There are tissue culture experiments conducted with Mitragyna speciosa and McCowns Woody Plant Medium.
Could be worth a try as well.
But, anyway, don´t have the rest of the equipment yet, this will take a while^^
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broccoli is probably a great place to start, there's tons of info on brassicas!
heres some papers i dug up, looks like MS media is also good place to start!
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annnnd one on Mitragyna in WPM....
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Plenty to read, thank you :)
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I havnt much edible veggies to work with, but I have many others. Id like to try a single media, across many plants experiment!! I can record all of the data. Since my payment options are limited, im down to purchase my own mixtures if possible, so Im not dragging this out longer than it should be. Once summer comes , I will have more room to start working on this as well. My entire garden is in my room next to me right now LOL I am out of space atm.
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Sorry I haven't responded, I am definitely in and can pay through PayPal or Venmo.
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Bought my own MS premix in the meantime, because shipping from the US is too expensive, but great that you are still onboard :)
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i've ordered the 50L of BASIC-MS-MEDIUM for 36$ Shamichael; i could send you 20L+some hormones for 15$ ,when it arrives.
The BASIC medium i ordered doesn't have the added hormones, but there are others with premixed hormones also available.
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I actually got access to my paypal again, so if im not able to afford my own bottle, I wouldnt mind paying a little from someone else? I am ready to start doing some experiments. Do I need some equipment, like petri dishes or anything maybe?? I planned on buying some soon for agar plates anyway.
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I actually got access to my paypal again, so if im not able to afford my own bottle, I wouldnt mind paying a little from someone else? I am ready to start doing some experiments. Do I need some equipment, like petri dishes or anything maybe?? I planned on buying some soon for agar plates anyway.
I just bought enough premix powder for 10L, because it was the least expensive.
If you are satisfied with a little powder, I can send you a bit of mine.
I did not study the literature on this topic further because I had too little time, but I think that test tubes or small Erlenmeyer flasks would be useful.
Small glass jars, like the ones baby food is sold in, could work either.
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bottles/jars:
i ordered a few 25mm( the size of a quarter) test tubes just on a whim to try them out, but they are pricey; 9$ for 10..
ive been saving clear bottles, mainly beer bottles, which are perfect shape i think, and only 9$ for 12, also full of beer!
The tops could be stuffed with polyfil and wrap with tinfoil, or maybe wrap tops with tyvek and tinfoil with rubberband.
glove box:
Set up glove-box/ still-air-box in closet with UVlight and ozone-generator which are turned on a few hours before use.
idk if this is necessary, when using ozone, i wait an hour or so for ozone to clear, so it doesn't 'absorb' into any liquids.
measurments:
Gram scale for larger measurements, and a milligram scale for smaller amounts of hormones, vitamins, antibiotics, etc..
Glass ml pipette with suction-bubble, or i guess a ml syringe would work also for measuring/mixing.
sanitizing and pre-treatment:
H2O2, alc.100%, bleach5%, lysol spray, good butain lighter/or alc. lamp, pressure sterilizer
surfactant:
dish soap, or tween-20, or other
antibiotics:
neomycin, amoxycilin (only ones i have right now, other kinds can be used)
lighting/incubation:
i recently ordered some LED light strips from walmart, and built some shelves for plants to grow on!!!;D
and of course the MS-medium, i have 50ml willing to trade up to half.
Shamichael and DarkPines; please pm me soon if interested, thanks.
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ONandON, I will let Darkpines decide if he wants to split, if not then I will. I already have some basic hormones.
If you're using ETOH for sterilization of surfaces and instruments I believe it's best to use 70%, anything above 91% is less effective. Not sure if the 100% is used as part of a solution or as part of a soak.
This is very interesting look forward to seeing your results. I want to play around with it, and have a flow hood and PC but not much time at the moment and a list of other tasks to do first like build a spectometer, diy led lights, make custom soil, and start several trays of seeds.
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I am currently broke Af. I will need some more time. But I will definitly get on this eventually! I promise!
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Are the efforts being made here specifically being towards cacti microprop or just plants in general?
I’m currently wanting to get serious about cacti microprop to begin. Not to exclude other plants but I imagine cacti will require a bit of a diff media than plants or perhaps diff cytokines and auxins??
Funds are currently limited however I am fully willing to throw down on this soon. I can manage $10 or so atm but give me a month or so and I can manage $50 easy
What do we need to do this, I’m serious as can be. What are we lacking here that I can provide by buying it? I have a flow hood as well seems like we can do this together and get it going quicker with our combined experiences
Does a guy need pipette to dispense the cytokines or auxins? Man I just need to buy a book I think lol
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Hey guys!
I just wanted to say I am down for joining this :D .. haven't been active here, but i hail from Bluelight, Shroomery and Discord, experienced with chemistry, fungi and plants. I already have a set of hormones, some random inorganic salts, an antibiotic (tho i have heard about a superior antibiotic product for this application) and a lot of other compounds, but I have been looking for MS salts to get efficiently together with other people (and love to trade and share experience).
I am expecting tween 80 and want to use it for various things but also try to dissolve / emulsify non-polar plant hormones. I wonder if that would also potentially work for gel media.
What kind of options are there for getting a quantity of MS salts from or with you guys that really won't run out soon? Is there just one type of MS salt mix you have or more types?
What are your ideas about affordable containers for growing cactus seedlings? Maybe polypropylene containers? I have glass reaction tubes for plants with significant roots but it seems potentially very impractical for growing something like Strombocactus. And idk about small glass jars if they don't have transparent lids...
So yeah cacti will be my main focus too although not exclusively.
Since I live in the Netherlands, if you only have liquid MS salt solution it will surely not be worth the postage though, is there no crystal/powder available from any one of you?
Will definitely not try to sneak in for darkpines' share of course, but it's a bit unclear to me if onandonandon still has some left to spare.
Am willing to put in some money and/or trade, I have not looked into it deeply enough but there may be some nutrients that would be nice to have extra of to be able to tweak the most commonly tweaked ones besides the hormones of course.
Let me list some materials I have:
Plant hormones: rooting powder i can extract IBA from, pure: 6-BAP, triacontanol, brassinolide, GA3
Inorganic salts: Calcium sulfate, calcium chloride, sodium bicarbonate, sodium carbonate, sodium phosphate, aluminum sulfate, calcium sulfate. May get some nitrate salt or could perhaps just make some ammonium chloride for N. Should have a sulfite and sodium hydrogen sulfate.
Perhaps relevant organic pure chems: Tartaric acid, citric acid, HPBCD. Working on extracting aspirin and making salicylic acid from it.
Gelling agent: agar powder, expecting methocel
Other nutrients: yeast nutrient (nutrivit), peptone expected later, yeast extract based nutrient mix powder for pets
Also expecting black food-grade pigment that is perhaps good to stop light from getting to roots?
Not sure if there is a point to carbs other than regular sugar but i have some
a little bit of azomite and comparable substances.
gentamicin but not enough to spare.
Have pH strips to spare cause I have a digital meter now.
Flowhood here too. In the future may be open to suggestions regarding chemistry i could perform for the benefit of others here doing TC. Perhaps it couldn't hurt to have a pH buffer but not sure if that is already in MS.
I'm also quite interested in mutants [teratopia], so things like colchicin seem cool (though quite tricky for various reasons) - advanced and definitely not for this stage though. But witch's broom on a cactus, i would be super interested in a sample of this.
Afaik you should never use regular dish washing soap with plants, which is a detergent other than soap.. but instead soft soap.
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im lacking funding here also, not much to update, group-buy was a bust..
i think we decided it would be best for everyone to order their own bottle.
ive been collecting glass juice bottles, they seem like perfect growing containers,
(http://www.hitokiri.com/blog/archives/misc/martinellis-glass-thumb.jpg)
instead of using the questionable antibiotic ointment, i found cheap fish antibiotic of all types,
including gentamycin, and penacillin.. Still waiting for them, and also ordered some tween 20.
i will try the cacti, but also any rare plants.. i don't think there is much difference in technique..
One difference might be using less than full strength ms-grow medium, like 1/2 or 1/4 or 1/8th..
it's common to use 1/2 strength medium in any case, bcause it still works fine, and saves costs.
i read about in rooting stage, it's good to use less, so roots are encouraged to search for food.
Good to see more people interested, i know we can get this to work, it's like following a recipe.
There are lots of informative research papers just search google.. filetype:pdf micropropagation
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Not sure what kind of plastics absorb light energy and if they would be bad, but like I said I don't like the idea of losing light to a lid at the "apex". Mind you I grow indoors so will often have lighting right overhead of the plants.
Hence my consideration of PP containers with clear lids fitted with polyfill filters.
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Edit: never mind.. I just checked out more of the details and pieced it together with this thread. Probably not worth mixing it myself and that German eBay source just seems quite reasonable honestly. I'm planning on getting at least the 10L worth bottle of powder, but will hold off a little for some like cactiqueen. Again, I do not live in the US, but if cactiqueen or any of you want to get part of a larger amount of MS medium, please let me know this week. But honestly, the point would rather be to also share hormones or other add-ons to tweak the medium as needed.
For just the MS salts maybe you might as well buy your own bottle because of the free shipping.
You will need to look into that more yourself but I guess you basically need:
- Basic MS salt medium
- Growth regulating hormones in a certain proportion depending on the stage of growth
- Possibly some added macronutrients basically like a custom NPK fert
- Antibiotics
is that right?
https://www.plantcelltechnology.com/plant-preservative-mixture-ppm-30-ml/ This is supposedly very good, but yes it's more expensive than just some gentamicin I guess, but those others aren't really so broad spectrum against molds etc i think.
My hormones are pure powders I weight with a sub-milligram scale but yes you could also do volumetric measurement: you would weigh out more and make a stock solution, depending on the concentration you would need a micropipette or just a measured syringe if you don't make it quite so concentrated. As I said before plenty of these hormones don't dissolve well in water normally and I would rather not do it by pH or solvents, however I have tween 80 coming in to facilitate the dissolution.
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Also still collecting equipment for micropropagation, maybe I am ready to start a try in 2 months or so :P
The apple juice bottle looks really nice, but the neck seems a bit tight…
And besides that the juice tastes probably better than caper.
I was also looking for some test tubes alternatives and found a nice caper jar for 99 cents, which could work.
I also read some explant sterilization protocols and think I will try it that way:
1. Wash explant with tap water to remove soil and dust particles
2. Transfer washed explant into glass beaker containing tap water, add a few drops of liquid detergent or Tween 20
3. After that, wash it thrice with distilled water, each washing should be 3-4 minutes
4. Transfer explant to glove box
5. Treat it with JIK bleach (containing 3,5% Sodium hypochlorite) for 20-30 minutes
6. After treating it with JIK, wash it with sterile distilled water for thrice, each washing should be 3-4 minutes
7. Wash it with 70% alcohol for 30 seconds to remove water from the surface of the explant
8. Cut explant into small pieces with a sterile blade
9. Now the explant is ready for inoculation
I attached a document on surface sterilization which might be helpful.
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ive been ready to start for two months, i have everything except the antibiotics..
this is a scam alert: DO NOT BUY ANTIBIOTICS FROM EBAYSELLER: angellover520
they had a large selection and the cheapest prices, but items never arrive..
the seller added a shipping number, but it was never updated to 'in transit.'
Then two months later, the date when i can file for a refund, seller marked items as cancelled..
which makes difficult to apply for refund, so still working on getting money back, and annoyed.
>:( :-[ :'(
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As I don´t bought antibiotics yet, I can´t give you any of mine :(
But I plan to order some plant preservation mixture at kitchenculturekit.com next month, as someone already ordered there and they seem reliable.
If someone in Europe still needs MS premix powder, I bought premix powder for 10 L, so I can give a bit of mine.
I hope that you get your money back soon, OnandOnandOn :(
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Thanks Neebu! seller message today to send refund, only after i open case with ebay and paypal, and found a way to leave bad feedback ;D
just frustrating having everything setup sitting around collecting dust, but it's all good.. shouldn't be much longer to get experiments started.
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FWIW, I have ~20 years of tissue culture experience.
Recommended containers: baby food jars and Mason jars.
Antibiotics: Skip them. You either get clean cultures, or you don't. Anticontaminants like PPM are hazardous for use in the home environment.
Any specific questions, I can help with. Developing the formulation is 90% of the work. Disinfection of explants is where most of the problems are. Workspace cleanliness is kind of an intrinsic factor, you either have a good HEPA-filtered workstation or some sort of clean transfer box, or you don't.
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You said it all, the recipe is all the work and here we have an amazing member not trying to be secretive how awesome!!
I’m prepared to start experimenting with ozone water sterilization of explants
If you could help with auxin and cytokines to use and concentrations I would be ecstatic
I’m looking to propagate trichocereus cacti initially and others as well soon after.
I would be willing to buy the cytokines and auxins enough to share with other members at cost
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Thanks for the tips..
I really wanted to get into conical (coffin-shaped) tubes for micropropagation but i haven't been able to source them yet except mega-bulk..
Another container that seems interesting to me is (small-ish) filterboxes used a lot in mushroom growing. Transparent lids for top-lighting, autoclavable and with 0.2 micron filters.
But my first trials will probably just be with babyfood-like jars.
cactiqueen, i still have plenty of hormones and considering how potent they are i would rather do a friendly trade than more being bought pointlessly but thats up to you.
What i listed earlier, but NAA Na+ added.
I'm also very interested in where to start with the specific recipe for cacti, especially trichocereus or loph / other geophytes. Strombocactus is one i would like to grow on agar...
I kinda get why something like PPM is not recommended, but with a flowhood isn't it nice to prevent some absolutely minimal contamination from escalating? And if PPM is hazardous, aren't compounds like 6-BAP just as much?
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...here is a quick google search... "filetype:pdf cacti micropropagation"
https://bioone.org/journalArticle/Download?fullDOI=10.2985%2F026.019.0107
https://journals.ashs.org/jashs/view/journals/jashs/115/2/article-p337.pdf
https://www.researchgate.net/profile/Mutasim_Khalafalla/publication/238659095_Micropropagation_of_cactus_Opuntia_ficus-indica_as_strategic_tool_to_combat_desertification_in_arid_and_semi_arid_regions/links/54b7770f0cf24eb34f6ea83d/Micropropagation-of-cactus-Opuntia-ficus-indica-as-strategic-tool-to-combat-desertification-in-arid-and-semi-arid-regions.pdf
http://www.scielo.br/pdf/sa/v72n6/0103-9016-sa-72-6-0540.pdf
http://ggfjournals.com/assets/uploads/1-8.pdf
https://core.ac.uk/download/pdf/35347196.pdf
http://www.iscientific.org/wp-content/uploads/2018/02/2-IJCBS-12-1-06.pdf
http://jpacd.org/downloads/Vol11/Vol11_3.pdf
https://academicjournals.org/journal/AJB/article-full-text-pdf/BDA6FB363601
http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.877.4954&rep=rep1&type=pdf
http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.652.9367&rep=rep1&type=pdf
https://www.arpapress.com/Volumes/Vol37Issue1/IJRRAS_37_1_01.pdf
https://umexpert.um.edu.my/file/publication/00013217_117097.pdf
http://www.hortorumcultus.actapol.net/pub/11_4_77.pdf
https://d-nb.info/981533078/34
http://www.cazri.res.in/annals/1984/1984J-1.pdf
https://www.ajol.info/index.php/ajb/article/viewFile/101990/92036
http://www.indianbotsoc.org/admin/uploaded/13%20ANUPAM%20KUMARI%20AND%20MD%20NASEEM.pdf
i've read half strength MS medium is used, because cacti need less nutrients... 1/2 or less strength MS is also used for rooting.
ready to start this experimenting soon! as soon as i can clean the house, and if i can ever get some spare time.. ok GTG TTYL :)
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15 jars
5mg 6BA
3.7g MS medium
7g agar
20g sugar
trial 1; disaster, because i mixed all dry ingredients then added hot water, everything wouldn't dissolve, even with heating,
resulting after cooling, extra water in the jars that had to be poured off, but used jars anyway, cut up cacti into small pieces..
bleach might be to strong for sterilizing cactus.. soaked 5-10 min in common bleach 3-5% (it bleached out the chlorophyll)
...washed few minutes in H2O2 and then 70%alc. then few seconds in 3 separate jars of sterile water, then into bottles.
Hard to get a good pic of just white cacti wedges, but at least they are still white and not brown, we'll see what happen...
(https://files.shroomery.org/files/19-27/244361130-cactissu.jpg)
Both times jars were pre-poured using a funnel, and then sterilized, then moved to glove-box, easier than pouring inside glovebox.
(note jar lids should be very loose when cooking so they don't explode)
15 jars
7mg 6BA
3g MS medium
8g agar
20g sugar
Trial 2; better, i used more agar (8g/L instead of 7) dissolve ingredients separately, and there was no excess water after cooling.
leaves were not badly effected by bleach, new-growth plant leaves used; ficus religiosa, star anise, nova, surinam cherry, juniper.
ficus religiosa...
(https://files.shroomery.org/files/19-27/244361751-ficusreli.jpg)
first measured out 1L of H2O,
..in about 2/3rds (of 1L) NEAR-boiling dissolve in SMALL-DASHES of agar until all dissolved.
with some of remaining water dissolved the sugar in separate cup (usually about 20-30g/L)
but i mixed MS together with the 5mg of rooting hormone 6-BA, which i think the 6BA didn't completely dissolve..
might try DMSO ive read can dissolve most hormones, and be added to the medium without harm to the plant,
anyway definitely next time dissolve everything separately, they could also dissolve in basic solution or alcohol.
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I kinda get why something like PPM is not recommended, but with a flowhood isn't it nice to prevent some absolutely minimal contamination from escalating? And if PPM is hazardous, aren't compounds like 6-BAP just as much?
PPM is comprised of isothiazolone biocides, which work by breaking disulfide bonds, such as those that hold your proteins together, and have been implicated as neurotoxins. Unfortunately, they're used in shampoos and cosmetics. But they're never put into an autoclave or microwave in the home where they can vaporize and be inhaled. Plant growth regulators (PGRs) have been tested for carcinogenicity etc., and for the most part they seem pretty safe- but are heavy enough (possessing a low vapor pressure) that they're not going to be emitted in the steam while autoclaved. Then again, none of this stuff is intended for home use in this fashion.
PPM just isn't very effective. It's an expensive crutch. It's like spraying all your plants with fungicide, all the time, and then wondering why your cacti rot because you overwater them anyway. Chemical prophylaxis doesn't make up for good methods and techniques.
BTW, "Lacon" snap-lid containers made of polypropylene will survive the autoclave, and are decent at keeping the contents sterile for weeks or months.
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update about a week after xplant inoculation.. only three jars contamd so far, which all originated from explants.
star anise leaves turned brown. Not much to report, except this one juniper jar that grew two things overnight..
(o just remembered i left lights on all night, where i usually turn them off at night, may have encouraged growth)
(https://files.shroomery.org/files/19-28/289308930-juni.jpg)
sorry for poor quality pic, (there are three xplants in one jar)
First explant stood up, because stem got stuck into the agar,
i think formed 'calus' it's bumpy, looks like tiny batch of eggs,
quickly overgrowing the explant, what i guess are plant cells?
Second Xplant, in back of picture, overgrowing some kind of mycelium.
Third explant to the right in picture nothing happening.
..considering making bottles tonight, to save the one forming a calus.
Cacti not much change, surprisingly no contams yet, no antibiotic added.
i have noticed the slightest bit of green chlorophyll return to white pieces.
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Nice work your doing on this, it's a learning process :) And yeah my Salvia turned brown the only time i half-assed tried, but i washed it in bleach for way too long..
Good to know about the PPM, and am pleased to not have to pay that money. I never meant that it could be a substitute for clean/sterile work and methods, but that working sterile is not such a completely black and white situation and more about orders of magnitude of cells present, so if a single rogue bacterium can be prevented from multiplying it could end up making a difference. Another example is how HEPA filters can be 99.999% efficient which illustrates my point: there is a certain tolerance at a logarithmic scale I think, is that not true?
And yea half strength MS sounds good, I think it's especially nitrogen that should not be present too much for cacti.
Upgrade to flowhood FFU is pretty much done so i'm ready to go after vacation and well needed rest.
Still looking for proper containers. And sure PP containers should work fine, there are filterboxes used for both mushroom growing and TC which are made of PP. There are those sold by SacO2, a Mycelia.be partner.. But i also really dig the "De Wit" culture tubes which are conical.
They are sold in bulk unfortunately... I would buy some but only if I could split it with at least 2 other people or so, and the international shipping might not exactly be pretty and most of us don't sound very rich..
I guess the filterboxes are sold for nearly 1€ a piece, I guess I could do that. :-\
I hope this hasn't been answered somewhere in between, but any suggestions for when absolutely tiny seeds have to be sterilized/disinfected and handled and placed on agar? (I kind of understand the point of elongated culture tubes, but it can be already enough of a nightmare without the tube being cramped and narrow)..
You would use autoclaved coffee filters to filter the tiny seeds after they have been in bleach or distilled water subsequently, and then placed on the agar with a lab spatula?
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Anyone ever considered using a black tea agar for micro propagation? Or black tea as an addition to knock back molds and bacteria??
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Thanks, congrats on flow=hood, we look forward to seeing your experiments!
i had two saly jars, one turned brown, the other grew fungus from explant.
they were tiny, unhealthy, dying plants already, last chance to save them.
i dislike PPM (PlantPreservativeMix) because brand-naming,
the over-pricing, and not-revealing their real-ingredients.
will eventually try antibiotic experiments, if it's needed.
i do like these apple juice jars! so far they seem to have a good seal..
if ever needed i might try using tyvec added to the lid for air exchange.
also apple juice (also coconut water) contain cytokin hormones, eventually want to try
experiments using apple-juice as sugar and water content, or maybe 1/2 diluted juice.
TRIAL3
8g agar
20g sugar
~0.4mg NAA
~20mg 6GA
3.3g MS
trichocereus 'lumberjac'
(https://files.shroomery.org/files/19-28/307495822-lumberjac_trial_three.jpg)
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solipsis says:
I hope this hasn't been answered somewhere in between, but any suggestions for when absolutely tiny seeds have to be sterilized/disinfected and handled and placed on agar? (I kind of understand the point of elongated culture tubes, but it can be already enough of a nightmare without the tube being cramped and narrow)..
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NOt sure maybe someone else knows, but i have read seeds are a good starting material, because they can be sterilized for longer time.
Also seeds have better germination rates on agar, it said like 90%, there is a pdf somewhere about it,
Really though what is the point of elonged culture tubes?
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solipsis says:
You would use autoclaved coffee filters to filter the tiny seeds after they have been in bleach or distilled water subsequently, and then placed on the agar with a lab spatula?
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sounds like a good plan! maybe if seeds sink, you could pour out most of the water before filtering,
or maybe even pour out all water and use spatuala to remove seeds from cup, and drop onto agar.
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Cactiqueen says:
Anyone ever considered using a black tea agar for micro propagation? Or black tea as an addition to knock back molds and bacteria??
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i have heard coffee is good supplement for mushrooms, it would be interesting experiment,
i think tea would be more supplemental than antibacterial,
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I hope this hasn't been answered somewhere in between, but any suggestions for when absolutely tiny seeds have to be sterilized/disinfected and handled and placed on agar? (I kind of understand the point of elongated culture tubes, but it can be already enough of a nightmare without the tube being cramped and narrow)..
Disinfect in small vials. The vials should come new/sterile or be autoclaved prior to use, to further reduce the risk of contamination. Add seeds, add disinfectant + wetting agent, soak for however long. If at this point the seeds have sunk, it's a simple matter to wash with sterile DI, and retrieve with a sterile tool such as a microbiology loupe, a sterile wire, or sterile microspatula of stainless steel. If they float, it's easier to skim off the surface, put into a new vial with sterile water, shake, and then retrieve from that and sow onto media.
If you want, it may be easier to wash with sterile DI, decant the wash water, then add one more aliquot of sterile water, shake, and then pour the seeds + water onto sterile media. The extra water on the medium will not matter. If you wish, add a small amount of 3% hydrogen peroxide to either your wash water or the final aliquot to further reduce contamination. Seeds and plant tissue are quite resistant to hydrogen peroxide.
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Solipsis let’s try black tea agar
Do you see anything to prevent you from doing so?
I know you will see the possible uses others here are not familiar with the concept
What are your thoughts solipsis?
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Solipsis let’s try black tea agar
Do you see anything to prevent you from doing so?
I know you will see the possible uses others here are not familiar with the concept
What are your thoughts solipsis?
well why not explain this concept so we can all learn, instead of random suggestion of tea with no explanation? rude ::)
i was just thinking, leave a cup of tea and a cup of water sitting on the table for a month, the tea will be full of bacteria growth..
after googling it appears black and green tea do have "some inhibitory effects on Gram-positive but not on Gram-negative bacteria."
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3249787/
since i have antibiotics, i won't be trying this anytime soon, but the best of luck to you.
Update trail 3 lumberjac masacree,= :'(
all contamed, not from explant, it was something in the air or glovebox, or mixing process or not sterilized enough. but all trashed.
the juniper i tried to remove myceleum from bottle, and it became contamed, trashed.
Some ficus religiosa jars are growing callus on sides of leaf.
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Rude?
The concept is used in growing fungi. Fungi spores will not germinate on black tea agar. With fungi you must germinate spores on regular agar then place mycelium onto black tea agar to set back bacteria and gain the upper hand if you will
The info can be found at the shroomery for those who are truly interested common knowledge on that site among mushroom growers. I can provide link if it’s not a no no
Not sure if mold spores will germinate on the black tea agar or not but w what I do know seems possibly useful and possibly a way to not need antibiotics. Possibly a bad idea tho as well I have done no research the thought just dawned on me less than a week ago
Cleaning dirty cultures with tea agar, is the thread title.
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Something I have learned recently, perhaps this is already known here I’m not sure
If wanting to maintain a cultivars, “stick with auxiliary shoot multiplication”. Callus culture will yeild genetic variance not a good idea for maintaining cultivars
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interesting thanks, i haven't heard of that tek before, worth an experiment.
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So aux shoot as well as seeds would be good to start with anyway? If talking about cacti that would mean somehow getting your cactus to pup then, right? Well I'm experimenting with pupping cream anyway but it is (grafted) Lophs that seem more tricky to induce it in.
About the T-gel agar: I don't know.. it's worth a shot but not sure if the plants will like all those polyphenols around in the medium like tannins from the tea and I would suggest to correct the acidic pH you would expect from using it.
Oh i just checked and found this:
"Tannins interfere with seed germination, radicle elongation, root activity, growth of the plant, hormonal action, membrane permeability [...]"
Need I go on? :D
https://www.insa.nic.in/writereaddata/UpLoadedFiles/PINSA/Vol51B_1985_2_Art15.pdf
Of course this is for the plant species tested but I don't think it bodes well for T.
My BAP wouldn't dissolve in 1M NaOH :( I'm gonna try citric acid I guess, or alcohol or glycerol idk.
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Bummer lol that’s what I was looking for your the man solipsis
This makes me wonder, what exactly is it that gives the tgel it’s antibacterial properties
I’m gonna make put some Shungite powder into agar and see what happens. It purifies water so maybe it’ll help keep the dishes clean longer I’m curious
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This makes me wonder, what exactly is it that gives the tgel it’s antibacterial properties
I’m gonna make put some Shungite powder into agar and see what happens. It purifies water so maybe it’ll help keep the dishes clean longer I’m curious
Yeah too bad. :) What makes tea antibacterial seems to be non-polymeric phenolic compounds. Black tea and green tea inhibit different types of bacteria (Gram positive vs negative but probably not that simple). If it's really about the antibacterial effect one might mix green and black tea, for fungi.. worth a try. But I think quite a few commonly found materials and foods etc can have antibacterial properties. The question is which ones are nicely broad spectrum and efficacious.
Shungite idk.. for something to lend properties to the agar I would say you want it to be dissolved in the agar so that it is everywhere, or if not, to make up the surface of the medium or something. A well mixed suspension. If you have it powdered extremely fine then yeah possibly. Interesting! Would the roots of various plants also appreciate being able to hang on to high surface area materials? Such as perlite dust otherwise? Or is this just not a factor with agar medium?
My 6-BAP didnt want to dissolve in 1M NaOH, what the hell.. It can't be that it's the salt form and the vendor failed to mention or label this - that would also just dissolve fine in 1M NaOH right? (Before diluting with water). Otherwise I'm gonna try glycerol and other options to make my stock solution haha.
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..i had the same problem with hormones, got them mostly dissolved using 95%alc. but not completely, then hot water to evaporate alcohol.
ive read DMSO can dissolve hormones and be added without harm to xplants. it's a natural solvent found in wood.. and farm stores.
dissolves both polar and nonpolar compounds and is miscible in a wide range of organic solvents as well as water
solipsis; Black tea and green tea inhibit different types of bacteria (Gram positive vs negative but probably not that simple)
...
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3249787/
Antibacterial properties of hot water extracts of green, black, and herbal teas showed inhibitory effects on Gram-positive, but not on Gram-negative bacteria.
most info on about shungite seems like hippy dippy crystal flower power for just $$$.$$
wiki
Shungite is a black, lustrous, non-crystalline mineraloid consisting of more than 98 weight percent of carbon. 98% carbon
.. but activated carbon also has antimicrobial and has water cleansing effect...
The Antibacterial Activity of Activated Carbon, Silver, Silver Impregnated Activated Carbon and Silica Sand Nanoparticles against Pathogenic E. coli BL21 (https://pdfs.semanticscholar.org/1409/b7f113ecef362c3d2a788151900722e3d872.pdf)
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Either you believe in energy or you don’t ;)
The hippy dippy effect of Shungite happen to be able to be measured scientifically and quantified. Showing physical proof that the hippy crap is real. But that’s topic for a diff thread.
If anything cool happens in relations to plants w agar and shungite I’ll report back.
The Shungite will be in a powder form and it’s the energetic field that would be acting not the physical granulated/powder Shungite particulate matter. However the powder will be evenly mixed in also for the most part.
I have a special Shungite magnet that can keep an avacado green for days doesn’t turn brown. Avacado salad last for a week doesn’t go brown. The magnet simply is placed on the outside of the fridge. Pretty sweet science but I bet science couldn’t explain why it works haha
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I bet there are compounds like ethylene which play a role in ripening of fruit and which is a gas plants themselves can exude, and fruits too... and which adsorb (sic, NOT absorb) onto the activated carbon like structure. So then you would get less ripening.
So far what we see is that we get progressive insight from science, although it is a fallible and provisional method and thus does not pretend to be the truth. However scientific methods do try to minimize the fallibility of humans and their limitations, by taking them out of the picture as much as possible.
Anyway, I find it best to stick to attachment of scientific articles / literature when making a claim. I don't know about what you are saying about electromagnetic radiation, but would like to see those studies that prove something, not to be an asshole about this but I am genuinely curious.
Everyone is entitled to their beliefs but they are just not a part of the scientific method and interfere with it. It's perfectly fine that beliefs exist, they only shouldn't be confused with scientific claims, but you can have a hunch that something works and experiment with it of course. Other than that, throwing claims and references around can be a nice dialectic sort of way to get to some provisional truth together and not be considered a fight.
I worry a little with something like shungite that if too much of it is present like powdered in agar, it might adsorb way too many compounds including good ones, the nutrients. Not sure if it works so well for the roots to.. "retrieve" the nutrients from it, haha.
At least dissolved NAA Na+ easily in water just now, at least that's something. ;D
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trial4 19jars
20g sugar
8g agar
4g MS
(eyeball estimate~)
~1mg 6BA
~0.2mg GA3
XPLANTS:t.bridge, guava, d.illinois, edulis
got hormones completely dissolved but it took about 40 minutes.
dissolved in 1tsp. 90%alc. 'heated and swirled in shot glass about 10 min.. until mostly dissolved,
then 1tsp. h20 added, and shot glass heated until below the 1tsp. line, to be sure all alc evaped.
ficus religiousa growing some tiny leaflets...
(https://serving.photos.photobox.com/2161617643b7a54c50e4d7dc017e23476864b7cae2fdca3cca3c70f8154307b2d4cf1e14.jpg)
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Technicality: I think you cannot really boil off all the alcohol out of a mixture with water. The reason is that water and alcohol form an azeotrope. This is a non-ideal mixture in which the two kind of stick to each other and form a new boiling point, they are kind of integrated and behave more as one.
That might be a good thing because the little bit of alcohol left may serve to facilitate dissolution. If you got rid of it and were only left with water, your hormones would just drop out solution again i think?
Shouldn't there be an auxin in your medium for root development? (probably dumb noob question)
Are recipes for rooting a cutting explant or germinating seeds basically the same?
Is there a medium fine for both rooting and shoot development at the same time, and is it instead something like a callus culture which would be tedious / laborious
What is the role of GA3 in your case and would you only want it in *some* media / growth stages, but not all?
By the way i was able to dissolve 6-BAP using citric acid and HCl. Especially when diluting with water i needed magnetic stirring to help keep the BAP in solution.. i just gave it extra time and it all dissolved eventually.
i guess i will need to adjust / neutralize the pH of the medium anyway.
I was personally considering trying glycerol if other methods didn't work, instead of alcohol.
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Technicality: I think you cannot really boil off all the alcohol out of a mixture with water. The reason is that water and alcohol form an azeotrope. This is a non-ideal mixture in which the two kind of stick to each other and form a new boiling point, they are kind of integrated and behave more as one.
Good point i googled it ... seems it would all boil off .. or maybe a small amount left idk.
Each azeotrope has a characteristic boiling point. ... A well-known example of a positive azeotrope is 95.63% ethanol and 4.37% water (by mass) boils at 78.2 °C. Ethanol boils at 78.4 °C, water boils at 100 °C, but the azeotrope boils at 78.2 °C, which is lower than either of its constituents.
That might be a good thing because the little bit of alcohol left may serve to facilitate dissolution. If you got rid of it and were only left with water, your hormones would just drop out solution again i think?
Yes possible.. i might have observed this, as i was watching it closely, i noticed some crystals droping out, and immediately mixed into the hot agar/ms solution.
Shouldn't there be an auxin in your medium for root development? (probably dumb noob question)
hey good qwestion, iam dumble noob also, but you are correct sir, there is a second stage for rooting..
i was following a recipe from paper about growing guavas invitro,
irronically the guava xplants turned brown almost immediately... they didn't survive sterilization steps.
Generally ive read it's
high cytokinin low auxin for shoots,
high auxin low cytokinin for rooting
Are recipes for rooting a cutting explant or germinating seeds basically the same?
iam thinking seeds are sprouted on something like 1/2ms medium with no hormones.
Is there a medium fine for both rooting and shoot development at the same time, and is it instead something like a callus culture which would be tedious / laborious
What is the role of GA3 in your case and would you only want it in *some* media / growth stages, but not all?
GA3 was used in the guava recipe i used.. i think it encourages sprouts, ive seen it used in 'cacti pupping cream'
By the way i was able to dissolve 6-BAP using citric acid and HCl. Especially when diluting with water i needed magnetic stirring to help keep the BAP in solution.. i just gave it extra time and it all dissolved eventually.
i guess i will need to adjust / neutralize the pH of the medium anyway.
excellent.. i thought they only dissolve in base?
i havn't been checking/adjusting ph, but ms medium is suppose to be slightly basic,
will try the DMSO as soon as i can save up extra 9 dollars.
I was personally considering trying glycerol if other methods didn't work, instead of alcohol.
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That might be a good thing because the little bit of alcohol left may serve to facilitate dissolution. If you got rid of it and were only left with water, your hormones would just drop out solution again i think?
Not necessarily. That which dissolves in alkaline solution (KOH or NaOH to dissolve) or in solvents may be perfectly soluble at 1/1000th the concentration in water, without any respect to the trace of alcohols used to dissolve them in the first place.
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Oh yes of course.. Serious dilution. :)
Im going through "plant tissue culture" by R. Smith and thought i would post my notes, questions and maybe suggestions based on my background. Forgive and correct me if i make wrong suggestions.
- About microscopes: can i use my compound microscope as a dissecting microscope? Esp if i add a lower magnification objective?
- Apparently 2,4-D is used for callus induction, is this important / very useful to have on hand when i get into that?
- GA3 (i think i saw it in you guys' recipes) can inhibit callus growth and auxin-induced adventitious root formation. It is also not thermostable so would need to be filter sterilized and added after autoclaving the medium. So maybe not a good idea to just add to media?
- Sucrose or glucose are suggested as carbs but starch can work (i wonder whether maltodextrin could also be utilized). Is there an advantage to adding a little complex carb as more of a time release C source? I wonder this for fungi as well.
Maillard / caramelization is also mentioned so my suggestion would be to use glucose and not sucrose or fructose (havent checked maltodextrin), because if it contains a sugar like fructose it degrades at autoclave temp of 121C while glucose should not. I dont mean interaction with ammonium etc which shouldnt be a problem or could be avoided by autoclaving some media components separately.
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GA3 is good for shoot formation and budding i think, and according to data sheet from supplier, all the hormones are somewhat heat sensitive.
Also the data sheet suggested to dissolve in 95%alc. then dilute in water, which is what i did last time, seems to be working so far..
2-4-D (i think i read somewhere) expresses widest range of DNA mutations, useful for creating new varieties of plants.
ive seen corn starch used as an agar substitute, maybe one could have two-in-one use from corn starch.
well i think i found source of contam problem in trial 3 and 4; i didn't have bottle water at the time, so i used tap water from fridge dispenser,
which contained white something that survives pressure sterilizer, and grows ontopof and inside agar of 3 and 4..
and it is not present in trails 1, 2, and 5 which i used bottled distilled water.
Better picture of ficus religiousa; some new growth white cells forming at the bottom, maybe roots?
(https://i.postimg.cc/nhf27gGL/firi.jpg)
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Ooh nice pic
Interesting.. 2,4-D does seem to be a mutagen of sorts but not sure if it is relevant for the purpose: i think at the concentrations where it would be practical to apply as mutagen it would cause aberrant growth which screws up the plants (hence the herbicide use)?
I ordered way more 2,4-D than i would ever need, so feel free to contact me for a generous trade. It's quite toxic so only for those who are skilled to handle it.
Hmm if it turned brown that sounds like you did it for too long (even if another plant could handle it, perhaps by a thicker cuticle?) The bleaching
Tap water here is at least as good as bottled mineral water so as with fungi i dont think i will use distilled.
Do you know perhaps what makes that agar opaque? Do MS salts always do this?
Afaik on the one hand roots shouldnt get any light at all but otoh it would be nice to be able to see roots better.
If you have a more balanced ratio of auxins to cytokinins, do you get both kinds of growth not much of either? I guess the latter..
Still im interested whether you can make the medium less one-sided or whether its just best practice to do one mode of growth at a time.
I think i may have added GA3 to my pupping cream too yes.. But then its not autoclaved.
Was nothing mentioned about the GA3 for the guave being added through filter sterilization?
Also with something like De Wit conical microprop tubes (im expecting 75 pcs), im curious whether it can breathe well enough.
Is one layer of parafilm recommended, for example over a loose cap of a culture tube? Oh yes pretty sure ive seen it
And no, BAP can dissolve in both acid and base.
Do you know what % of alcohols can be tolerated in agar medium?
Also with the azeotrope: it makes a mixture become like a new thing like... Brangelina
Haha
Bad analogy but the point is you cant really see them as separate anymore and it makes it so that separating them is limited.
Anyway nvm it should be too little left to matter, stock solutions of alcohol are used but i guess you want an as saturated solution as possible to limit the volume you need to add.
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I'm sterilizing some seeds as we speak, to put in culture tubes..
What are some practical ways to rinse seeds will all those liquids and filter them out again, in a clean way?
Also, when moving to explants from plants, what are the smallest recommended sizes or just generally the recommended sizes for the explant?
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Hello Solipsis! im on a break from this for a while, at least until i can restock supplies..
Guess whatever size fits your jars.. it's been said they can grow from just a few cells,
but i used about 1 centimeter to 1 inch size pieces, that contained meri-stem sections.
i tried to grow some premature seeds, used tweezers for transferring, but didn't grow,
a spoon may be good for moving seeds, let us know if you discover a good procedure.
Update on my jars nothing much, ficus looks the same, cacti not dead but not growing.
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You can grow from a few cells but you shouldn't try to haha :) Bigger explants can grow out at a more reasonable timescale.
If you have meristem in your explant then you don't need callus culture but can just root and bud? Seems much easier if so.
yeah i used large forceps for the seeds i planted too this time but i don't like it. Supposedly large swabs can be used, I guess they can be kinda sticky for picking up seeds? Idk.
Too bad about your cacti.. they ran out of a certain nutrient the medium was deficient in in the first place or the medium is depleted by now?
Hopefully we can find PGR concentrations that are right for callus induction in cacti, and hopefully having PGRs meant specifically for it on hand means it should be doable.
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I have my first germinated Loph seed on MS medium!
Too bad it's quiet here.. :(
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the few jars that survived contams have been sitting 5-6 months...... a couple days ago i noticed a new growing pup from an areole!
it's still small so ill add pictures later, also a couple others have something happening at the areole, one small callus forming at edge.
:D
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..about six months later.. growth from a node..
(https://files.shroomery.org/files/20-005/042161163-IMG_20200126_190239.jpg)
i must get some agar to start more experiments.
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Thats amazing! I actually have some extra money this year and might be able to afford geting into this lol.
I was in a bit of legal trouble last year so thats why I wasnt able to join sooner with the group buy.
Id like to get into it this year though.
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go for it! i just got some agar to do some more experiments soon..
it's pretty cheap to get started, and mostly basic sterile technique.
ms-medium 30$
hormones 10$
agar 5$
sterile supplies:
alc. 1$
h2o2 1$
gloves 1$
bleach 1$
soap 1$
glovebox homemade-free
containers, i use glass apple juice bottles..
pressure cooker, ~40$
so if you have a pressure cooker already, about 50$ total starting investment.
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Oh true! All I need is the ms and hormones lol. I have everything else from growing mushrooms.
Ill be inquiring later this year about it some more. For now Im going to just follow this and study how you guys are doing it. Thanks man <3
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interesting development, ive been watching these tiny white specs growing bigger,
ever since the x-plant got knocked out of place, i had to tap it back into the middle.
(they are cell clusters, that grew from where the x-plant slid around on the agar)
..the x-plant was knocked out of place again, and two clusters ended up on top of the x-plant..
(https://files.shroomery.org/files/20-48/660152163-100_3995.jpg)
in this one an air-root has formed.
(https://files.shroomery.org/files/20-48/660152284-100_3999.jpg)
and this one has some strange tan growth.. maybe callus
(https://files.shroomery.org/files/20-48/660152428-100_4000.jpg)
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I'd like to join in on this thread :)
Below are some entheos that I put in vitro on 23/5. In order: P. Alba seeds, B. Caapi node, and T. Pachanoi explants.
Recipe followed was 1/2 strength MS, 5.4 uM (1mg/L) NAA, and 22.5 uM (5mg/L) BAP. I kept this the same for all explants because this is my first go at it... Though this is a recipe suggested in a paper for T. Spachianus.
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Thaat's awesome! ;D it's inspiring, i hope to be doing some more soon..
it took about 6 months before mine had any growth worth photographing,
but it's fun watching and growth seems to speed up once it's 'established'.
Keep us updated on progress! i'm especially interested in the alba seeds.
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Yea, it's really exciting to try something like this as a hobby :)
How are your cacti looking that you put in vitro a while ago? What ph were you growing at? I have read ph is quite important in TC, but I did not have a probe for my cultures so I have no idea what my readings were :-X. I have ordered one now for my next cultures.
Some have had great success producing growth in a matter of weeks with trichs and lophs:
https://www.shroomery.org/forums/showflat.php/Number/10835098#10835098
https://www.shroomery.org/forums/showflat.php/Number/1932583/fpart/all/vc/1 (check the date of each update)
The following quote is from the 2nd link.
Loph Media/Pedro pupping [my original formula, causes a little vitrification]:
1/2 MS w/ vit./L
0.7mg/L PPM [trying 1.0 mg/L]
5.0mg/L BAP
0.5mg/L NAA
25g/L sucrose [trying 20g sucrose + 5g dextrose]
~5.80 pH adjusted w/ H2SO4
Loph Media [my newest recipe, no vitrification and stronger growth]:
1/2 MS w/ vit./L
1.0mg/L PPM
5.0mg/L BAP
0.5mg/L NAA
20g/L sucrose
5g/L dextrose
~5.80 pH adjusted w/ H2SO4
So far, this is the best recipe I've been able to find for actual shoot elongation in columnar cacti [still far from ideal, growth is very slow]:
1/4 MS w/ vit./L
0.8mg/L PPM
2.0mg/L BAP
0.1mg/L IBA [this seems to be key, swapping IBA for NAA pretty much stops all growth, if anyone is experimenting with this you might want to pay special attention here]
25g/L sucrose
~5.80 pH adjusted w/ H2SO4
And here (https://www.shroomery.org/forums/showflat.php/Number/1932583/fpart/all/vc/1) is an interesting post on FB of mutations done with 2.5mg/L of BAP.
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i didn't have any ph meter either, and ph paper can be unreliable,
had just got some premixed ms medium (just add agar and sugar)
..so did it without measuring the ph.. but it would be a good idea.
..the cacti look about the same as last pictures but a little bigger..
That post by UNA was one of the first that got me interested in tissue culture..
but it's very disappointing that he wouldn't share his 'hard earned recipes' ::)
Looking back at the thread, it's hard to tell the timeline,
According to those pictures, unless im missing something..
huge quarter size double pups can grow in about a month.
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Some really interesting posts I found on the Cactus Lab FB group.
https://www.facebook.com/groups/429525097582007/permalink/865872710613908/
I made then crest !!!! Let’s see if pattern keep rocking
using 3 mg per L BAP +++ 0,3 ANA
https://www.facebook.com/groups/429525097582007/permalink/829755950892251/
Scopulicola showing pups after new formula for the media - only 14 days after replicated
it’s about using 2,0 Bap per L on media
https://www.facebook.com/groups/429525097582007/permalink/844763266058186/
I am stoked the small pieces of the Trichocereus pachanoi monstrose crested variegated is pupping in vitro
this case only Bap 2,5 but I also use 4x1 Bap x iba or Ana
And then some more where 2.5mg/L is often used.
So it seems around 2-3mg/L BAP is ideal along with a ratio higher than 4:1 and up to 10:1 of BAP:NAA (or IBA perhaps). Higher concentrations of BAP also seem to induce mutations.
Received my ph meter and will be making some more mediums today. I have read in various places ph should be around 5.7-5.8.
Any ideas perhaps what ph to put caapi and psychotria in? I understand it to be slightly acidic... Think I will stick to the same ph as the cacti.
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that's cool but i don;t have a FB so unable to view it but thanks for the quotes"
..id stick with that 5.7-5.8 if that's what it sais, makes sense, on the acidic side..
(https://cdn.kastatic.org/ka-perseus-images/7ca9aecccf7e9d5caaf1ea10d2835c81f4036708.png)
so it looks like if you drink lots of coffee, then your pee should be about perfect Ph!
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I'm struggling a lot to sterilize explants. Either they seem to die from over sterilization or they get contaminated.
Any tips?
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i had a lot die from the sterilizing also, i guess you just have to find a balance.
also some that just looked dead, but later turned out to be alive.
i regret throwing out the failed ones, because they might have just looked dead,
and it's possible to remove the dead piece and re-sterilize jar for another chance.
alc.75%-90% only few seconds 3-5
h2o2 long as you want,
bleach<only few seconds, make sure all bleach is washed off, or just skip the bleach.
colloidal silver, long as you like
distilled water, between each wash, and fresh distilled water wash for last step.
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Thanks for the tips ;D
Glad you told me you had some dead-looking ones that turned out to be alive. Was thinking of throwing out some but may keep them around to see if something happens.
I realized I might have been using explants with too few areoles. I've read suggestions of needing at least 3-4 areoles.
What was your contamination rate On&On?
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just had one areole on each of mine.
but a bigger piece would grow faster.
i can't remember exactly, but it was pretty bad..
it was something like 40-50% became contamed,
and 30-40% no contams, but dead from sterilizing.
then i threw away some that might have survived.
so i'm left with three jars that are growing good.
they have root and pups, so i'm planning to
transfer to sterile sandy soil sometime soon.
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Yea mine is terrible. I've got 70% contam so far from like 10 jars.
What was the condition of the plants before you took cuts?
I cut some pieces from the base of a T. Pachanoi that has been in the process of rooting for 3 months. This is likely my issue because there is already some very slight rot going on in the base. It has etiolated terribly so I might just cut the tip and give it a go as it's fresh and hopefully somewhat cleaner growth. I haven't got much stock to experiment with so I'm just going with this for now.
The guy on FB with lots of Cacti TC experience is almost always using seed. Seems the better route to go as sterilization is simpler/easier.
Please post some pics once you get those in the soil! :)
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it's been about 2 years since the last updated, and the experiment started about 4 years ago..
Oddly, one cacti pup was growing strait down into the agar, and living happily in a pool of liquid..
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ive been waiting to build a clean room first, but i decided enough waiting,
i removed the plant from the jar, (in unsanitized open-air)
with some slight pressure it popped into three pieces...
one piece has two heads and roots,
the other piece was the one growing down in the agar
the third piece is the original xplant, it's turned grey, i assume it's mostly dead.
i put the original piece back in the bottle, even though it was surley contamed..
..and a few days later, it has some slime mold, pennacillin, and a fungi growing.
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Finally, the two pieces were planted in sandy soil cups in open air, they are looking good so far.
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and some other pictures..
first two pics: ficus religiosa 4 year old callus from leaf tip
second two: white tissue growing from microscopic cells
last two pics: cactus eye xplant, that grew a root and a small pup,
note: pup was green, but left in the dark for couple of weeks, it turned white.
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Nice update… I never got really far with my experiments since stillair box was too much work for me.
Do you let them sit in natural light or have shop lights? Do you know why your callous is black?
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thanks, it's under LED shoplights for now, they'll move outside later.. the ficus callus is actually brown, i don't know,
i think brown is the typical callus color, occasionally it grows a layer of translucent cells, which eventually turn brown.
yeah creating clean environment and sterile workspace seems to be the most difficult and the most important part.
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… I never got really far with my experiments since stillair box was too much work for me.
I am planning on building an enclosed cabinet for a 3d printer with a built in HEPA filter and I came across this and thought you may be interested. It's about the easiest and cheapest flow hood I've seen.
https://youtu.be/lInfdAVvBts?si=xybXLrb_gXCWQBne
I also came across these:
https://sciplus.com/squirrel-cage-blower-motor-assemblies-120vac/
The trick would be how to funnel the air correctly using them.