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Author Topic: micropropigation preparation workspace  (Read 1769 times)

ONandONandON

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Re: micropropigation preparation workspace
« Reply #45 on: July 04, 2019, 12:58:32 am »

...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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ONandONandON

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Re: micropropigation preparation workspace
« Reply #46 on: July 06, 2019, 10:35:33 pm »

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...


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...

 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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CJS

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Re: micropropigation preparation workspace
« Reply #47 on: July 07, 2019, 08:42:26 pm »

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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ONandONandON

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Re: micropropigation preparation workspace
« Reply #48 on: July 12, 2019, 03:24:31 am »

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)


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.
« Last Edit: July 12, 2019, 03:30:48 am by ONandONandON »
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Solipsis

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Re: micropropigation preparation workspace
« Reply #49 on: July 12, 2019, 11:03:25 pm »

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?
« Last Edit: July 12, 2019, 11:09:18 pm by Solipsis »
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Cactiqueen

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Re: micropropigation preparation workspace
« Reply #50 on: July 13, 2019, 11:46:34 pm »

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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ONandONandON

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Re: micropropigation preparation workspace
« Reply #51 on: July 14, 2019, 05:49:14 am »

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'


...........................................
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)..
...........................................
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?
...........................................
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?
...........................................
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.
............................................
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??
....................................................
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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CJS

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Re: micropropigation preparation workspace
« Reply #52 on: July 14, 2019, 06:05:55 am »

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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Cactiqueen

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Re: micropropigation preparation workspace
« Reply #53 on: July 14, 2019, 03:31:58 pm »

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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ONandONandON

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Re: micropropigation preparation workspace
« Reply #54 on: July 17, 2019, 04:52:12 am »

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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Cactiqueen

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Re: micropropigation preparation workspace
« Reply #55 on: July 17, 2019, 04:32:42 pm »

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.
« Last Edit: July 17, 2019, 06:49:44 pm by Cactiqueen »
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Cactiqueen

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Re: micropropigation preparation workspace
« Reply #56 on: July 17, 2019, 04:46:31 pm »

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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ONandONandON

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Re: micropropigation preparation workspace
« Reply #57 on: July 18, 2019, 05:45:54 am »

interesting thanks, i haven't heard of that tek before, worth an experiment.
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Solipsis

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Re: micropropigation preparation workspace
« Reply #58 on: July 24, 2019, 12:07:26 pm »

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
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Cactiqueen

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Re: micropropigation preparation workspace
« Reply #59 on: July 24, 2019, 04:00:20 pm »

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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