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Thread: N. villosa/ N. macrophylla TC help

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    SimonTheSkink fiercedeity's Avatar
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    N. villosa/ N. macrophylla TC help

    I am looking here to see if anyone has experience with working with villosa and macrophylla tissue samples and being able to get a callus to form for multiplication. Each Nepenthes species seems to have its own preference for how much BAP and MS it likes, so by getting a good idea for both these plants I can be able to find a common ground media that may work well for both. My theory behind this is the genetic similarities to N. villosa and N. macrophylla, since they are so close on a family level that means their genetic structure is similar and therefore their metabolic functions would be similar as well. Using this data will help me in my studies to help clone the tissue of living plants instead of relying solely on seeds in TC.

    I know many people say it's near impossible to grow Nepenthes from meristem samples but I believe it can be done and made more easy! I am starting some experiments in my home and my plan is to first properly sterilize the meristem tissue and to then cut the tissue length wise down through the center of the plant to expose the meristem to the media. This i believe has been widely overlooked as a possible cause of why so little samples ever got the chance to grow, the leaves surrounding the meristem acts as a shield which prevents the media from reaching the core of the sample. By exposing this tissue I think it will have a much higher chance of growing and forming a callus.

    Ok so lets get some responses in here, media mixes and other advice is greatly appreciated.

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    i dont do pots. amphirion's Avatar
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    With regards to meristems... I wonder if recently activated nodes could be sterilized and utilized for callus propagation.
    " You keep using that word. I do not think it means what you think it means." -Inigo Montoya
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    SimonTheSkink fiercedeity's Avatar
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    Technically couldn't an auxin be applied to the nodes and help encourage the tissue to become active in growth? I believe they can do this with orchids.

    Also congrats on your 3,000 post

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    i dont do pots. amphirion's Avatar
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    hahaha. and i will break my 3000th post with this one.
    yes i believe that would be an easier way to go about it. the reason i believe these nodes would be better than taking traditional method of meristem is because the meristem tissue from these nodes are already actively growing, as opposed to forcing the meristem to activate in the TC. also, their small, smooth size would make it much easier to clean and sterilize.
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    BigBella's Avatar
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    Quote Originally Posted by fiercedeity View Post
    I am starting some experiments in my home and my plan is to first properly sterilize the meristem tissue and to then cut the tissue length wise down through the center of the plant to expose the meristem to the media. This i believe has been widely overlooked as a possible cause of why so little samples ever got the chance to grow, the leaves surrounding the meristem acts as a shield which prevents the media from reaching the core of the sample. By exposing this tissue I think it will have a much higher chance of growing and forming a callus.
    Actually, dissection has been the standard process for the sterilization of Nepenthes explants, along with a host of fungicides and antibiotics. It's simply that the process is generally too costly and prone to contamination than are seeds. That being said, the procedures you're looking into often fall under the aegis of proprietary knowledge. I would suggest pursuing the published literature available online and adapt it, if necessary, for use with those highland species . . .

    Quote Originally Posted by fiercedeity View Post
    Technically couldn't an auxin be applied to the nodes and help encourage the tissue to become active in growth? I believe they can do this with orchids.
    You're thinking of keiki pastes, which are cytokinin-based, often containing 6-BAP, among others. Auxins are generally used to facilitate rooting, though both classes of growth regulators can "overlap" a bit, in terms of their function; and Nepenthes are not as easily receptive to the pastes . . .
    “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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    SimonTheSkink fiercedeity's Avatar
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    Quote Originally Posted by BigBella View Post
    Actually, dissection has been the standard process for the sterilization of Nepenthes explants, along with a host of fungicides and antibiotics. It's simply that the process is generally too costly and prone to contamination than are seeds. That being said, the procedures you're looking into often fall under the aegis of proprietary knowledge. I would suggest pursuing the published literature available online and adapt it, if necessary, for use with those highland species . . .



    You're thinking of keiki pastes, which are cytokinin-based, often containing 6-BAP, among others. Auxins are generally used to facilitate rooting, though both classes of growth regulators can "overlap" a bit, in terms of their function; and Nepenthes are not as easily receptive to the pastes . . .

    This leaves me with more questions then answers. First off why would people withhold this type of information? Shouldn't the knowledge be freely spread to help encourage the growth of these plants and help prevent removal from their natural environment? This could make the plants more widespread and help save the few left in the wild from poaching, or at least reduce it to the point that that it doesn't go the same way that N. clipeata did.

    In any case I suppose my method on lateral dissection of meristem tissue is not original, still if you have a very rare plant in cultivation such as N. edwardsiana there is almost no chance of growing it and being able to get a flower and fertile seeds. So rarer plant don't give us much of an option and make us take a step off the edge to try something harder, yet significantly more possible then raising seed from an already rare specimen. Nevertheless you did mention literature and I would be very happy if you could point me in the right direction as to what i can read. I have read your posts on cloning on other threads here in the past and you have provided a lot of insight for me, in fact I was kind of hoping you would reply to this thread.

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    i dont do pots. amphirion's Avatar
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    easy answer: money, and controlling supply and demand.

    value of many nep species are dependent upon their accessibility and rarity. to make these plants common place would drastically lower the monetary value.

    N. campanulata is a great example of this. after discovered, the entire species was thought to be lost due to fire destroying the only known location. however, another location was found, and the plant entered TC. demand for this plant was extremely high, so production went full steam ahead. however, production overshot demand, and within a short period of time, a $150 plant became a $100, then a $50, then $25, and even $12, IIRC.

    The CP hobby is still small and extremely specialized. Many vendors and nurseries have a hard time breaking even or making profit---could you imagine if an N. hamata ended up being for sale for the value of $5.95? N. platychilla for $4.99? would be great for us, but a nightmare for vendors. not to mention all the bitter cp growers who spent at least ten times that amount to obtain them. Then there's another factor of limiting the number of clones in the market, but that's another story for another time...
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    BigBella's Avatar
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    Quote Originally Posted by fiercedeity View Post
    This leaves me with more questions then answers. First off why would people withhold this type of information? Shouldn't the knowledge be freely spread to help encourage the growth of these plants and help prevent removal from their natural environment? This could make the plants more widespread and help save the few left in the wild from poaching, or at least reduce it to the point that that it doesn't go the same way that N. clipeata did.
    Consider for a moment the expense of setting up a tissue culture laboratory, above that of, say, the average hobbyist with a cut-up Rubbermaid® tub; a collection of baby food bottles; a spray-bottle of bleach; and a Sacred Heart of Jesus votive candle. Consider then the expense involved in primary research and the myriad and often costly failures; and the very-western concept of intellectual property, which is seen as somehow legitimate in every other realm of human endeavor.

    As an aside, Nepenthes has been tissue cultured for decades; yet poaching is, arguably, as rampant as it always has been; and black markets still exist throughout Asia.

    Consider, also, how small the market is for carnivorous plants, which is, admittedly, far greater than when I began; but is still insignificant, when compared to orchid cultivation. Growers would naturally wish to maximize their profit in so small a venue.

    That said, I had moderate success with isolating the growing tip of Nepenthes hamata a few years back, for a Japanese client; and that involved the preparation of a carefully-cleaned 3 cm or so section of plant. At the time, I had ready access to fungicides no longer legal within the US, and to perhaps a dozen or more antibiotics and antimycotics at my disposal. It took serial re-plating to obtain a "clean culture;" but it can be done. The standard media was 1:3 MS with 8.8 µM 6-BAP; but meristematic tissue is a real coin toss, since you cannot be sure which growth phase the tissue is in when you make the attempt to establish it. Nepenthes also has a reputation for being "woody;" and there are some other medias, namely WPM, where growers have had some success.

    There are many papers involving in vitro culture of Nepenthes online. A twenty minute search should find a few. Still others are on costly academic databases; but abstracts are generally available. Check also the ICPS database for TC cultivation and the www.flytrapcare.com tissue culture forum for sources. There have been some threads within the last year or two . . .
    Last edited by BigBella; 02-26-2013 at 05:56 PM.
    “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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