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Author Topic: micropropigation preparation workspace  (Read 1786 times)

Neebu

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Re: micropropigation preparation workspace
« Reply #30 on: January 22, 2019, 03:53:28 pm »

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.
« Last Edit: January 22, 2019, 03:54:35 pm by Neebu »
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ONandONandON

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Re: micropropigation preparation workspace
« Reply #31 on: January 24, 2019, 08:50:35 pm »

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.
« Last Edit: January 25, 2019, 07:00:36 pm by ONandONandON »
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Shamichael

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Re: micropropigation preparation workspace
« Reply #32 on: January 25, 2019, 05:25:05 pm »

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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DarkPines

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Re: micropropigation preparation workspace
« Reply #33 on: January 29, 2019, 08:17:40 pm »

I am currently broke Af. I will need some more time. But I will definitly get on this eventually! I promise!

Cactiqueen

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Re: micropropigation preparation workspace
« Reply #34 on: March 31, 2019, 07:48:35 pm »

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
« Last Edit: March 31, 2019, 08:01:33 pm by Cactiqueen »
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Solipsis

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Re: micropropigation preparation workspace
« Reply #35 on: April 01, 2019, 02:09:08 am »

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.
« Last Edit: April 01, 2019, 03:14:02 am by Solipsis »
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ONandONandON

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Re: micropropigation preparation workspace
« Reply #36 on: April 01, 2019, 04:50:49 am »

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,


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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Solipsis

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Re: micropropigation preparation workspace
« Reply #37 on: April 01, 2019, 02:45:43 pm »

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.

====

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/
« Last Edit: April 01, 2019, 04:18:40 pm by Solipsis »
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Neebu

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Re: micropropigation preparation workspace
« Reply #38 on: May 11, 2019, 05:29:48 pm »

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.



« Last Edit: May 11, 2019, 05:41:34 pm by Neebu »
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ONandONandON

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Re: micropropigation preparation workspace
« Reply #39 on: May 16, 2019, 01:45:00 am »

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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Neebu

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Re: micropropigation preparation workspace
« Reply #40 on: May 16, 2019, 09:36:05 am »

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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ONandONandON

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Re: micropropigation preparation workspace
« Reply #41 on: May 18, 2019, 01:32:55 am »

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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CJS

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Re: micropropigation preparation workspace
« Reply #42 on: May 19, 2019, 10:17:39 pm »

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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Cactiqueen

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Re: micropropigation preparation workspace
« Reply #43 on: June 13, 2019, 09:36:30 pm »

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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Solipsis

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Re: micropropigation preparation workspace
« Reply #44 on: July 03, 2019, 12:34:43 pm »

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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