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[ONandONandON]

the few jars that survived contams have been sitting 5-6 months...... a couple days ago i noticed a new growing pup from an areole!
it's still small so ill add pictures later, also a couple others have something happening at the areole, one small callus forming at edge.

 

1 Like chronic likes this.

[ONandONandON]

..about six months later.. growth from a node..

i must get some agar to start more experiments.

[DarkPines]

Thats amazing! I actually have some extra money this year and might be able to afford geting into this lol.

I was in a bit of legal trouble last year so thats why I wasnt able to join sooner with the group buy.

Id like to get into it this year though.

[ONandONandON]

go for it! i just got some agar to do some more experiments soon..
it's pretty cheap to get started, and mostly basic sterile technique.
ms-medium 30$
hormones 10$
agar 5$
sterile supplies:
alc. 1$
h2o2 1$
gloves 1$
bleach 1$
soap 1$
glovebox homemade-free
containers, i use glass apple juice bottles..
pressure cooker, ~40$
so if you have a pressure cooker already, about 50$ total starting investment.

[DarkPines]

Oh true! All I need is the ms and hormones lol. I have everything else from growing mushrooms.

Ill be inquiring later this year about it some more. For now Im going to just follow this and study how you guys are doing it. Thanks man <3

[ONandONandON]

interesting development, ive been watching these tiny white specs growing bigger,
ever since the x-plant got knocked out of place, i had to tap it back into the middle.
(they are cell clusters, that grew from where the x-plant slid around on the agar)
..the x-plant was knocked out of place again, and two clusters ended up on top of the x-plant..



in this one an air-root has formed.


and this one has some strange tan growth.. maybe callus

[woolmer]

I'd like to join in on this thread 

Below are some entheos that I put in vitro on 23/5. In order: P. Alba seeds, B. Caapi node, and T. Pachanoi explants.

Recipe followed was 1/2 strength MS, 5.4 uM (1mg/L) NAA, and 22.5 uM (5mg/L) BAP. I kept this the same for all explants because this is my first go at it... Though this is a recipe suggested in a paper for T. Spachianus.

1 Like ONandONandON likes this.

[ONandONandON]

Thaat's awesome!  it's inspiring, i hope to be doing some more soon..
it took about 6 months before mine had any growth worth photographing,
but it's fun watching and growth seems to speed up once it's 'established'.
Keep us updated on progress! i'm especially interested in the alba seeds.

[woolmer]

Yea, it's really exciting to try something like this as a hobby 

How are your cacti looking that you put in vitro a while ago? What ph were you growing at? I have read ph is quite important in TC, but I did not have a probe for my cultures so I have no idea what my readings were  . I have ordered one now for my next cultures. 

Some have had great success producing growth in a matter of weeks with trichs and lophs:
https://www.shroomery.org/forums/showflat.php/Number/10835098#10835098
https://www.shroomery.org/forums/showflat.php/Number/1932583/fpart/all/vc/1 (check the date of each update)

The following quote is from the 2nd link.

Loph Media/Pedro pupping [my original formula, causes a little vitrification]:
1/2 MS w/ vit./L
0.7mg/L PPM [trying 1.0 mg/L]
5.0mg/L BAP
0.5mg/L NAA
25g/L sucrose [trying 20g sucrose + 5g dextrose]
~5.80 pH adjusted w/ H2SO4

Loph Media [my newest recipe, no vitrification and stronger growth]:
1/2 MS w/ vit./L
1.0mg/L PPM
5.0mg/L BAP
0.5mg/L NAA
20g/L sucrose
5g/L dextrose
~5.80 pH adjusted w/ H2SO4

So far, this is the best recipe I've been able to find for actual shoot elongation in columnar cacti [still far from ideal, growth is very slow]:

1/4 MS w/ vit./L
0.8mg/L PPM
2.0mg/L BAP
0.1mg/L IBA [this seems to be key, swapping IBA for NAA pretty much stops all growth, if anyone is experimenting with this you might want to pay special attention here]
25g/L sucrose
~5.80 pH adjusted w/ H2SO4

And here is an interesting post on FB of mutations done with 2.5mg/L of BAP.

[ONandONandON]

i didn't have any ph meter either, and ph paper can be unreliable,
had just got some premixed ms medium (just add agar and sugar)
..so did it without measuring the ph.. but it would be a good idea.

..the cacti look about the same as last pictures but a little bigger..

That post by UNA was one of the first that got me interested in tissue culture..
but it's very disappointing that he wouldn't share his 'hard earned recipes' 
Looking back at the thread, it's hard to tell the timeline,
According to those pictures, unless im missing something..
huge quarter size double pups can grow in about a month.

[woolmer]

Some really interesting posts I found on the Cactus Lab FB group.

https://www.facebook.com/groups/429525097582007/permalink/865872710613908/

I made then crest !!!! Let’s see if pattern keep rocking

using 3 mg per L BAP +++ 0,3 ANA

https://www.facebook.com/groups/429525097582007/permalink/829755950892251/

Scopulicola showing pups after new formula for the media - only 14 days after replicated

it’s about using 2,0 Bap per L on media

https://www.facebook.com/groups/429525097582007/permalink/844763266058186/

I am stoked the small pieces of the Trichocereus pachanoi monstrose crested variegated is pupping in vitro

this case only Bap 2,5 but I also use 4x1 Bap x iba or Ana

And then some more where 2.5mg/L is often used.

So it seems around 2-3mg/L BAP is ideal along with a ratio higher than 4:1 and up to 10:1 of BAP:NAA (or IBA perhaps). Higher concentrations of BAP also seem to induce mutations.

Received my ph meter and will be making some more mediums today. I have read in various places ph should be around 5.7-5.8.

Any ideas perhaps what ph to put caapi and psychotria in? I understand it to be slightly acidic... Think I will stick to the same ph as the cacti.

[ONandONandON]

that's cool but i don;t have a FB so unable to view it but thanks for the quotes"
..id stick with that 5.7-5.8 if that's what it sais, makes sense, on the acidic side..

so it looks like if you drink lots of coffee, then your pee should be about perfect Ph!

[woolmer]

I'm struggling a lot to sterilize explants. Either they seem to die from over sterilization or they get contaminated.

Any tips?

[ONandONandON]

i had a lot die from the sterilizing also, i guess you just have to find a balance.
also some that just looked dead, but later turned out to be alive.
i regret throwing out the failed ones, because they might have just looked dead,
and it's possible to remove the dead piece and re-sterilize jar for another chance.

alc.75%-90% only few seconds 3-5
h2o2 long as you want,
bleach<only few seconds, make sure all bleach is washed off, or just skip the bleach.
colloidal silver, long as you like
distilled water, between each wash, and fresh distilled water wash for last step.

[woolmer]

Thanks for the tips

Glad you told me you had some dead-looking ones that turned out to be alive. Was thinking of throwing out some but may keep them around to see if something happens.

I realized I might have been using explants with too few areoles. I've read suggestions of needing at least 3-4 areoles.

What was your contamination rate On&On?