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Thread: Nepenthes in vitro . . .

  1. #65
    BigBella's Avatar
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    Quote Originally Posted by Jimmy View Post
    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 . . .
    “Sì perché l'autorità dell'opinione di mille nelle scienze non val per una scintilla di ragione di un solo . . ."

    -- Galileo "Biff" Galilei

  2. #66
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    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...
    "I'm just a scientist without the proper documentation."

    My Growlist: http://www.terraforums.com/forums/sh...d.php?t=113724

  3. #67
    BigBella's Avatar
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    Quote Originally Posted by Jimmy View Post
    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 . . .
    “Sì perché l'autorità dell'opinione di mille nelle scienze non val per una scintilla di ragione di un solo . . ."

    -- Galileo "Biff" Galilei

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    BigBella's Avatar
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    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



    Nepenthes villosa (Tambuyukon)
    Last edited by BigBella; 02-06-2013 at 12:06 AM.
    “Sì perché l'autorità dell'opinione di mille nelle scienze non val per una scintilla di ragione di un solo . . ."

    -- Galileo "Biff" Galilei

  5. #69
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    In light of the forum's NASCAR fantasy league, "Git her done."

  6. #70
    Hear the Call of Nepenthes carnivoure12's Avatar
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    Lots of beauties coming along David! Can't wait to see how the progress.
    -Carnivoure12
    →Growlist

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    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.

  8. #72
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    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



    “Sì perché l'autorità dell'opinione di mille nelle scienze non val per una scintilla di ragione di un solo . . ."

    -- Galileo "Biff" Galilei

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