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Author Topic: micropropigation preparation workspace  (Read 660 times)

Neebu

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Re: micropropigation preparation workspace
« Reply #15 on: January 10, 2019, 12:06:36 pm »

Quote from: DarkPines
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
« Last Edit: January 10, 2019, 12:08:01 pm by Neebu »
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Neebu

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Re: micropropigation preparation workspace
« Reply #16 on: January 10, 2019, 12:08:41 pm »

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.

#t=21m54s
« Last Edit: January 10, 2019, 12:11:08 pm by Neebu »
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ONandONandON

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Re: micropropigation preparation workspace
« Reply #17 on: January 10, 2019, 08:37:45 pm »

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..


(Tissue Culture Propagation: Class 10m)


(boring but informative 3 part-series 1h)
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ONandONandON

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Re: micropropigation preparation workspace
« Reply #18 on: January 10, 2019, 10:27:11 pm »

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
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DarkPines

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Re: micropropigation preparation workspace
« Reply #19 on: January 10, 2019, 10:57:59 pm »

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.

XDX

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Re: micropropigation preparation workspace
« Reply #20 on: January 11, 2019, 12:08:31 am »

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.
« Last Edit: January 11, 2019, 12:44:06 am by XDX »
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Neebu

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Re: micropropigation preparation workspace
« Reply #21 on: January 11, 2019, 04:32:49 pm »

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^^
« Last Edit: January 11, 2019, 04:36:28 pm by Neebu »
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XDX

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Re: micropropigation preparation workspace
« Reply #22 on: January 11, 2019, 07:29:40 pm »

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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XDX

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Re: micropropigation preparation workspace
« Reply #23 on: January 11, 2019, 07:47:07 pm »

annnnd one on Mitragyna in WPM....
« Last Edit: January 11, 2019, 07:47:49 pm by XDX »
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Neebu

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Re: micropropigation preparation workspace
« Reply #24 on: January 11, 2019, 08:17:47 pm »

Plenty to read, thank you :)
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DarkPines

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Re: micropropigation preparation workspace
« Reply #25 on: January 12, 2019, 02:13:45 pm »

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.

Shamichael

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Re: micropropigation preparation workspace
« Reply #26 on: January 21, 2019, 04:05:11 am »

Sorry I haven't responded, I am definitely in and can pay through PayPal or Venmo.
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Neebu

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Re: micropropigation preparation workspace
« Reply #27 on: January 21, 2019, 11:43:01 am »

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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ONandONandON

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Re: micropropigation preparation workspace
« Reply #28 on: January 21, 2019, 08:15:28 pm »

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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DarkPines

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Re: micropropigation preparation workspace
« Reply #29 on: January 21, 2019, 08:58:52 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.
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