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Nepenthes in vitro . . .

  • #61
So, you like to snuggle under the blankets too.......
 
  • #63
I've seen this with Venus Fly Traps and sarrs, but not with neps, although I guess it's pretty much the same idea.... very interesting. Best of luck with the seedlings!
 
  • #64
BigBella,

An off-topic TC question... what would cause Nepenthes to turn brown and die part-way through germination in vitro? Assuming a standard media of 1/3 MS with vitamins and 25g/L sucrose with no PGRs.

And while I'm asking, have you used activated charcoal in any of your cultures? I think I remember seeing in some literature that it helps in Nepenthes TC and it certainly has its place in orchid TC... BE also has a video that shows some of the Nepenthes TC stock and some cultures have the charcoal while others do not.

Thanks!

-Jimmy
 
  • #65
BigBella,

An off-topic TC question... what would cause Nepenthes to turn brown and die part-way through germination in vitro? Assuming a standard media of 1/3 MS with vitamins and 25g/L sucrose with no PGRs.

And while I'm asking, have you used activated charcoal in any of your cultures? I think I remember seeing in some literature that it helps in Nepenthes TC and it certainly has its place in orchid TC... BE also has a video that shows some of the Nepenthes TC stock and some cultures have the charcoal while others do not.

Thanks!

-Jimmy

Jimmy, the death of seedlings in vitro can occur for a few reasons -- chiefly among them, sustained higher temperatures (I rarely keep anything in culture above 20˚ C); though some seedlings simply spontaneously "abort" upon germination. That unfortunately does happen.

In terms of the use of activated charcoal (AC), I have used it for a number of purposes. It removes plant exudates (such as phenols and excess PGRs) from media; to promote root formation (partially due to the darkness provided); and it diminishes unwanted callus growth. I used AC on a handful of occasions with Nepenthes seed (having read some of that same literature); though I was unimpressed by its supposed benefit to germination media. I had far better results, by nearly a third, by eliminating it. Its chief drawback is that it all-too efficiently removes organic nutrients and PGRs from media, especially auxins for some odd reason (one paper I had read suggested adding anywhere from 10-100x the normal amount if "high" levels of activated charcoal were present to offset that effect). AC also affects the given pH of media, by increasing it by about .75.

If you insist upon using it, get some laboratory grade AC from a laboratory supply (Caisson Labs, for example); and don't bother to purchase some pecker-wood charcoal supplements from health food stores. You never know what you're getting; and the AC for tissue culture use has to be precisely prepared . . .
 
  • #66
Thanks for the detailed reply, BigBella! Temps may definitely have been an issue, as they were kept in about 80-degree-Fahrenheit temps after I set up the cultures... There were other factors too, but I wanted to ask if there are any key things to watch out for and you certainly helped!

That's an interesting remark on AC... I'll keep it off my list and out of my cultures for a while until I have more room for experimentation. I'm still relatively new to TC and I'm more interested in the science of it and the degree of control it allows for plant growth, but I'd rather be TC'ing Nepenthes than anything just because they're my favorite. I may try orchids one of these days...
 
  • #67
That's an interesting remark on AC... I'll keep it off my list and out of my cultures for a while until I have more room for experimentation. I'm still relatively new to TC and I'm more interested in the science of it and the degree of control it allows for plant growth, but I'd rather be TC'ing Nepenthes than anything just because they're my favorite. I may try orchids one of these days...

I have no intention of denigrating activated charcoal's use in tissue culture; it's just that there is a mistaken, though popular notion among hobbyists, that it is simply an inert additive. Some AC even contains some trace amounts of PGRs, even after processing; and I have used it to encourage rooting in genera that are occasionally difficult -- some highland Nepenthes and Heliamphora, for example.

Some Nepenthes species -- N. rajah, in particular, though for no apparent reason -- are long-lastingly affected by some PGRs (especially BAP); and activated charcoal plays a beneficial role in diminishing it from the media -- and even withdrawing some of it from the explants themselves . . .
 
  • #68
Here is a recent update on a second batch of 2012 seed, plated in December. All are on 1:3 MS with 3% sucrose and 1-2 mg/L 6-BAP . . .

Nepenthes attenboroughii 5 February

ATTEN2013_zpsa0534e51.jpg


Nepenthes villosa (Tambuyukon)
VILLOSA2013_zps1ca55e05.jpg
 
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  • #69
In light of the forum's NASCAR fantasy league, "Git her done."
 
  • #70
Lots of beauties coming along David! Can't wait to see how the progress.
 
  • #71
Hi BigBella, my name is Ryan and I am newish to this forum and have been involved in in vitro propagation for a while now. Seeing this thread has sparked some real interest primarily with the N. villosa Tambuyukon variety. I am currently growing many seedlings in vitro of the N. villosa (Mt. kinabalu of course) variety, at this point I have counted maybe thirty or forty that have germinated as of today (2/9/2013). Anyhow, I was wondering if you would be interested in either trading some flasks or seedlings, I love villosa and I in particular would be very interested in the Tambuyukon variety. Well either way, it would be an honor to chat micropropagation with you some time, I have many questions and have enjoyed many of your threads and learned a lot! Thanks for your time.
 
  • #72
Here are a couple of shots of a particularly stubborn Nepenthes species -- finally beginning to multiply in vitro, after a succession of both media and plant growth regulators -- including 6-BAP and TDZ:

Nepenthes edwardsiana (Tambuyukon) 6 March

EDDIE2_zpsb7e766af.jpg


EDDIE-2_zps85d5436e.jpg
 
  • #73
Stop teasing me........ :evil:
 
  • #74
Here is yet another exercise in patience, especially for pokie22; and why those vials at the back of the shelf should be spared for another thirty days . . .

As I was rummaging through my jars with a magnifying loupe, and determining what needed to be transferred or even tossed, I decided to check upon some "hopeless" vials from last November. To my pleasant surprise, I saw several sprouts (only one of which is visible in this crappy photo), after only one hundred and sixteen days, especially after the lion's share germinated after four weeks . . .

Nepenthes rajah 15 March
RAJAH_zpsef436b54.jpg
 
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  • #75
Here is yet another exercise in patience, especially for pokie22; and why those vials at the back of the shelf should be spared for another thirty days . . .

Next thing, you will be telling me I need beta blockers, yoga classes, and a day off from lab. And I have patience, really I do. I am not as cruel as you portray me BigBella! Every culture is given their Jen process and after that, they simply await their demise. My motto in life: next .

Of course, if they are cute, furry, and cuddley, there might be some leniency in their sentencing. :grin:
 
  • #76
Next thing, you will be telling me I need beta blockers, yoga classes, and a day off from lab.

Beta-blockers couldn't hurt -- that, and a little sunlight. . .
 
  • #77
Those wretched UV rays will give me wrinkles. Although, in the summer time I am usually 4-5 shades darker. Where are you big fishies.....

Flame boy to the culture= Jen beta-blocker
 
  • #78
i do like this.... i like it a lot.
 
  • #79
My planned dalliance with patience is quiet costly - insofar as recapitulating infantilism.....

8562320607_60392cba04_c.jpg


And in response to the bit about necessitating sunshine, I heard this song this morning and directly thought of your sagacious words BigBella. Especially those lyrics about "speedos" and "tanned cheeks"....

http://www.youtube.com/watch?v=wyx6JDQCslE
 
  • #80
More strained beets for pokie22!

Damn it, woman -- the dedication!
 
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