I want to learn more about this thanks for the great thread.
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Learning together on this one 
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MS formulation adapted for a couple of different volumes, for convenience:
200 mL:
500 mL:
1 Liter:
2 Liter:
An alternative to the Murashige & Skoog nutrient base is the Gamborg B5 formulation:
Macroelements
113.23ppm CaCl
2500.00ppm KNO3
121.56ppm MgSO4
134.00ppm (NH4)2SO4
130.44ppm NaH2PO4
Microelements
.025ppm CoCl2.6H2O
.025ppm CuSO4.5H2O0.025
36.70ppm FeNa EDTA
33.00ppm H3BO
0.75ppm KI
10.00ppm MnSO4.H2O
0.25ppm NaMoO4.2H2O
2.00ppm ZnSO4.7H2O
Vitamins
100.00ppm myo-Inositol
1.00ppm nicotinic acid
1.00ppm Pyridoxine HCl
10.00ppm Thiamine HCl
Carbon / energy source
30000.00ppm Sucrose
An alternative to the Murashige & Skoog nutrient base is the Modified Gamborg B5 formulation:
Macroelements
113.23ppm CaCl
2500.00ppm KNO3
121.56ppm MgSO4
134.00ppm (NH4)2SO4
130.44ppm NaH2PO4
Microelements
.025ppm CoCl2.6H2O
.025ppm CuSO4.5H2O0.025
36.70ppm FeNa EDTA
33.00ppm H3BO
0.75ppm KI
10.00ppm MnSO4.H2O
0.25ppm NaMoO4.2H2O
2.00ppm ZnSO4.7H2O
Vitamins
50.00ppm myo-Inositol
1.00ppm nicotinic acid
1.00ppm Pyridoxine HCl
10.00ppm Thiamine HCl
Amino Acids as Protein
1000ppm casein hydrolysate
Carbon / energy source
30000.00ppm Sucrose
Additional references:
Lata H., Chandra S., Khan I., ElSohly M.A. (2009) Thidiazuron- induced high-frequency direct shoot organogenesis of Cannabis sativa L. In Vitro Cell. Dev. Biol. 45:12-19.
Wang R., He L. S., Xia B., Tong J. F., Li N., Peng F. (2009). A micropropagation system for cloning of hemp (Cannabis sativa L.) by shoot tip culture. Pak. J. Bot., 41: 603-608.
West, DP (1998) Hemp & Marijuana: Myths and Realities. North American Industrial Hemp Council Inc. http://www.naihc.org/hemp_information/content/hemp.mj.html
Sayed Hussein Farag Hussein (2014) Cannabinoids production in Cannabis sativa L. Thesis.
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A quick update, being busy with things and am looking at different ways to handle an aseptic work area, at the moment. Dithering at the moment on how much effort to exert into a DIY box. May go cheap with a still air box. Or not. Havenât quite decided.
Will continue updating the original posts shortly once I determine a reasonable enough path forwardâŚ
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Good day Loki. Can you tell me the chlorine to water ratio you are using to introduce cuttings to sterile environment?
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For disinfection of plant material, the suggested concentration appears to be 10% of (fresh) household bleach along with 0.1% of Tween20 or other surfactant.
So, for one liter youâd add 100 ml bleach + 1 ml Tween20 to ~899ml sterile water.
Verbeck Lab Protocol for Disinfection of Cell Culture 052212 GV.pdf (94.9 KB)
p.s. sorry, slow on the updates. Itâs been something of a whirlwind around here lately with work and guests from out of townâŚ
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Thank you! Also, unacceptable with lack of updates! Sort your priorities 
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Did you have success with this?
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OK, cool. Donât trip, work is always first amigo. I saw a plant being sold today online locally but in agar. It appears to be a tissue culture, so I got to thinking, if someone down here can do it for commercial plants it canât be That hard. 
When searching I came across this thread.
Quick question, doesnât tissue culture reset the plant? If it has a disease or any other issue will doing a tissue culture eliminate it? Specifically p. M
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There are a bunch of different techniques under what we are calling tissue culture. I believe that if you are doing cell dedifferentiation, callus generation, and then differentiation (e.g. producing roots and stems) by hormonal manipulation, then yes. The clock is reset. Kinda of like stem cells. I could be wrong, though.
A âcallusâ or somatic embryogenesis is a bunch of unorganized cells that donât know what to do yet. It looks weird. E.g. photo of a callus that is starting to produce shoots:
Image sourced from Wikipedia.
By manipulating the horomes added to the agar, you can start different phases of differentiation. E.g. callus->roots. Then,stems. At each stage, you transfer a portion of the prior culture into the next stage until you have produced an entirely new plant.
But there are also several other techniques that fall under tissue culture. Stem propagation and Micro-propagation, for instance, which I do not believe will have the same effect. Essentially, miniature clones. At least, I think thatâs the case but this could be wrong (callus vs propagation). I really need to study up more on this.
In the order of complexity / difficulty:
Stem propagation, e.g. for cloning
Micro propagation, e.g. for removal of microbiological contamination / infections using shoot-tips
Meristem Culture, e.g. for removal of viruses using shoot-tip dissection (meristem)
Protoplast Culture, e.g. undifferentiated cell culture for many engineering / research techniques
For viruses, yes. From what Iâve research so far, it is possible and common to create a virus free clone from an infected mother. Iâm not certain on PM. Also,needs some research this.
The key difficulty for this stuff, which is tricky but isnât too big of a hurdle, is to ensure that the environment and plant material is sterile. All kinds of unwanted stuff will grow vigorously in the Agar. A bit of plant preservative mixture may help in the short term.
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Even if the unwanted virus,bacteria etc was able to transfer and reproduce in the agar it could be separated and made sterile threw repeat separations and examination, would be time consuming and powder mildew can be cured alot easier than that, but yes itâs common to use agar plates to seperate good from bad. One prime example is how a wild mushroom spore sample is isolated and made sterile.
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Bro, a week or so I stumbled across a paper on using oryzalin treatments to induce polyploid in cannabis, interesting enough in itâs own right, though maybe more interesting to you is that the process uses auxillary bud tissue culturing to establish plantlets after treatment. Included in the paper is the method and protocols used for this culturing process. It makes for an interesting read and it may contain some useful information for your experiments.
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Good stuff, never heard of Oryzalin before this.
According to Phytotech: https://phytotechlab.com/oryzalin.html
Oryzalin is an antimitotic that has been used for doubling chromosomes, 4-(dipropylamino)-3,5-dinitrobenzenesulfonamide
Apparently, itâs also used as a pre-emergent broad leaf herbicide and can also be found under the tradenames Oryzalin 4 and Surflan.
From their study:
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Anything new to report on this experiment bro? Just asking out of curiosity having just been reading about somaclonal variation with tissue culture reproduction⌠i.e genetic variation/mutation that is speculated may be partly caused by various types of growth regulators medium which may act as mutagens when used at elevated levels in the medium. . I am curious as to whether the weed plantlets will exhibit this when cultured and if they do what kind and frequency of variation that turns up⌠It could be a useful and happy side occurrence should somaclonal variation be an outcome of your experiments.
Some reading on the topic.
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Unfortunately, not at the moment.
My timing was a bit off relative to some other things that I supposed to get accomplished. Spring time, plating season, etc. It is still on my radar to complete and document this. My apologizes for the delay.
Iâm not well versed enough in this to have a good understanding of some of advancements in knowledge and techniques in cloning. I have seen some similar papers and the really interesting results. Some of the techniques are getting down to cell level manipulation where they end-up merging cells in what amounts to genetic manipulation. Itâs pretty cool.
What weâll have here, Iâm hopeful, is something in the middle. I would like to generate a callus and go from there. If we can learn the overall technique and be successful at it (at home), we can probably start getting in to some of the deeper and promising possibilities. Playing with the hormone ratios and such.
So keep an eye out. Iâll get this going again. Once we get past some of the first runs, we can start to talk about possible things to try, pay closer attention to, and experiment with.
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Hey mate, I found a paper you may be interested in on reverse breeding using anther tissue culturing to create haploid plantlents, these are then doubled with cochicine or similar to produce double haploid plants to create homozygous pure lines in a single step. It might be a bit out of reach at this stage, but itâs a good read anywayâŚ
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Oh very cool, quickly scanned over this. Iâll need to read through this more thoroughly.
It seems that some of the more interesting things being researched is fairly advanced with the extraction of the meristem / protoplast. In this case, it appears that theyâre utilizing an âembryoâ from seed, which may actually be an easier method to obtain undifferentiated cells early-on than through shoot tip dissection. At least one thing Iâd noticed at first glance. Interesting.
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Definitely reads as a more approachable technique that many I have looked at thatâs for sure. There is a wealth of additional information in the references too, including some of the well established protocols used in creating DH plants in various grain crops etc.
The other method discussed, the gene elimination technique is cool also⌠hops maybe?
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