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Author Topic: micropropigation preparation workspace  (Read 2037 times)

Solipsis

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Re: micropropigation preparation workspace
« Reply #60 on: July 25, 2019, 06:09:40 pm »

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.
« Last Edit: July 25, 2019, 06:14:03 pm by Solipsis »
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ONandONandON

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Re: micropropigation preparation workspace
« Reply #61 on: July 26, 2019, 07:08:43 am »

..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.
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dissolves both polar and nonpolar compounds and is miscible in a wide range of organic solvents as well as water

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solipsis; Black tea and green tea inhibit different types of bacteria (Gram positive vs negative but probably not that simple)
...
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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 $$$.$$
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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
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Cactiqueen

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Re: micropropigation preparation workspace
« Reply #62 on: July 26, 2019, 04:32:54 pm »

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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Solipsis

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Re: micropropigation preparation workspace
« Reply #63 on: July 27, 2019, 02:56:44 pm »

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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ONandONandON

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Re: micropropigation preparation workspace
« Reply #64 on: July 28, 2019, 03:50:52 am »

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...
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Solipsis

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Re: micropropigation preparation workspace
« Reply #65 on: July 28, 2019, 02:31:28 pm »

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.
« Last Edit: July 28, 2019, 02:36:01 pm by Solipsis »
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ONandONandON

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Re: micropropigation preparation workspace
« Reply #66 on: July 29, 2019, 12:49:24 am »

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.
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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?
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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)
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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?
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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?
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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.
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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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CJS

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Re: micropropigation preparation workspace
« Reply #67 on: July 29, 2019, 02:49:44 am »

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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Solipsis

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Re: micropropigation preparation workspace
« Reply #68 on: July 29, 2019, 01:01:21 pm »

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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ONandONandON

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Re: micropropigation preparation workspace
« Reply #69 on: July 31, 2019, 05:25:25 am »

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?

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Solipsis

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Re: micropropigation preparation workspace
« Reply #70 on: August 04, 2019, 03:44:22 pm »

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.
« Last Edit: August 04, 2019, 03:50:01 pm by Solipsis »
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Solipsis

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Re: micropropigation preparation workspace
« Reply #71 on: September 04, 2019, 09:19:50 pm »

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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ONandONandON

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Re: micropropigation preparation workspace
« Reply #72 on: September 06, 2019, 10:24:22 pm »

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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Solipsis

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Re: micropropigation preparation workspace
« Reply #73 on: September 08, 2019, 02:33:37 pm »

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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