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Thread: Nepenthes in vitro . . .

  1. #101
    BigBella's Avatar
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    Here is a recent update of some 2014 Nepenthes cultures -- all of which have been subdivided any number of times; and which have been on multiple media, with and without plant growth regulators, since last year. The vial on the right has given rise to three separate clone lines since then.

    This was always my favorite species; and I had thought it just a pipe dream that I would ever gave access to it, much less have an opportunity to micropropagate it . . .


    Nepenthes villosa (Tambuyukon) 18 June 2014


    28 April


    Last edited by BigBella; 04-28-2015 at 02:04 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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    Carnivorous plant enthusiast vraev's Avatar
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    Looking amazing D. Man! I would love to be able to do that and have an "almost" endless supply of edwardsiana...much less variants from different mountains.

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    n bicalcarata's Avatar
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    Tissue culturing any hamata red hairy's

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    BigBella, could you possibly give me a bit of details if you don't mind?

    I looked at the link from message #13. In the link, you used PPM in the flask media in addition to the sterilization. Do you do the same for Nepenthes?

    What concentration of sucrose are you using to 1/4 MS? I looked at this paper:
    Mekong et al 2000. Vegetative Propagation of selected Nepenthes species Borneo Science 7:1-9
    They found 1/2 MS +6% sucrose had the highest germination rate (66%) with N. mirabilis, N. ampullaria. With 3% sucrose, the germination rate became less than 1/2. So I'm curious what concentration you are using (I went through the thread, but I might have missed it).

    With sterilization, what is the rationale to use MS + PPM instead of water + PPM?
    Mekong paper used 30% Clorox (2.5% NaClO) for 3 min. They had 10-20% contamination rate. You mentioned that Nepenthes seems to be sensitive to bleach. So I haven't decided the method. What kind of contamination rate and germination rate (ball-park) are you getting with PPM?

    Final question, are you using vented flasks?

    I have flasked orchids, but I have never done it with Nepenthes, so this thread was very helpful. Thanks!

  5. #105
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    Quote Originally Posted by naoki View Post

    In the link, you used PPM in the flask media in addition to the sterilization. Do you do the same for Nepenthes?

    Yes, PPM is used, both for sterilization and as a bio-static preservative of sorts, for the media . . .

    What concentration of sucrose are you using to 1/4 MS? I looked at this paper:
    Mekong et al 2000. Vegetative Propagation of selected Nepenthes species Borneo Science 7:1-9 They found 1/2 MS +6% sucrose had the highest germination rate (66%) with N. mirabilis, N. ampullaria. With 3% sucrose, the germination rate became less than 1/2. So I'm curious what concentration you are using (I went through the thread, but I might have missed it).

    I typically use 2.5- 3% of sucrose; and have never had successful experiences at those high concentrations that you had mentioned. Also, explants grown with such high carbohydrate loads, take longer to eventually get established, ex vitro. Further, Nepenthes ampullaria and N. mirabilis are real outliers in terms of that high sucrose tolerance; and few highlanders would ever respond well to that concentration. It is also a direct invitation for algal or bacterial contamination; and it comes as little surprise to me that Mekong et al faced such issues with their cultures. They may as well have been making bread . . .


    With sterilization, what is the rationale to use MS + PPM instead of water + PPM?
    Mekong paper used 30% Clorox (2.5% NaClO) for 3 min. They had 10-20% contamination rate. You mentioned that Nepenthes seems to be sensitive to bleach. So I haven't decided the method. What kind of contamination rate and germination rate (ball-park) are you getting with PPM?

    The Plant Preservative Media (PPM) producers have established a protocol, for their product, which involved the use of full MS salts, without pH adjustment, along with the PPM. Presumably, that combination -- along with the high solute (hypertonic) concentrations -- lowers the pH of the already acidic solution that much more; and, thereby furthers its effectiveness, as an antibacterial agent. I do recall the paper that you had mentioned; and all that I can say, is that Mekong, et al must have been using their unwashed feet, along with serious sinus infections to achieve that stellar level of contamination. Even, while at university, our contamination rates, in our first rookie experiences with micropropagation, were well below five percent. With the use of PPM and improved sterile technique, my contamination rate hovers around .87 percent; and the overall germination rate -- highly dependent upon the condition and freshness of the seed (lowland Nepenthes have seed viability which may be measured in weeks) -- is over sixty-seven percent. Forget the bleach with Nepenthes seed; they are far too sensitive, rare and fragile . . .


    Final question, are you using vented flasks?

    I no longer use vented flasks, since cultures tend to quickly become desiccated; the lids don't tolerate much autoclaving; and the venting is yet another invitation for contamination. Simply, swap out the cultures more often; and the gas exchange issues become moot . . .

    I have flasked orchids, but I have never done it with Nepenthes, so this thread was very helpful. Thanks!
    I hope that that answered your questions . . .
    Last edited by BigBella; 02-26-2016 at 06:46 PM.
    “Sì perché l'autorità dell'opinione di mille nelle scienze non val per una scintilla di ragione di un solo . . ."

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    Thank you very much for detailed information, BigBella!

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    Here is a recent update, having just re-plated what seemed like dozens of vials. Time for wine. That wad of Nepenthes goodness, on experimental media, is about the size of a Titleist, if not a bit larger . . .

    Nepenthes villosa 16 July
    “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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    nimbulan's Avatar
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    Yikes that is quite the ball of pitchers. What are you going to do with it all?

  9. #109
    BigBella's Avatar
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    Quote Originally Posted by nimbulan View Post
    Yikes that is quite the ball of pitchers. What are you going to do with it all?
    Well, it will eventually be divided -- sooner rather than later, considering its size. Some will be planted ex vitro, while others will grow, in vials, into new Titleists. Others will eventually be shipped overseas.

    Also, I had been wanting to do a more formal study on the potential effects of coffee on Nepenthes for some time, but had previously lacked a sufficient number of cloned plants upon which to experiment. That is currently not an issue. Here are a few more examples . . .

    Nepenthes villosa 17 July


    Nepenthes attenboroughii
    Last edited by BigBella; 07-17-2016 at 01:33 PM. Reason: forgot the jello . . .
    “Sì perché l'autorità dell'opinione di mille nelle scienze non val per una scintilla di ragione di un solo . . ."

    -- Galileo "Biff" Galilei

  10. #110
    BigBella's Avatar
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    I happened upon an article in one of the more stultified journals, Protoplasma; one if those publications with all the joy and tolerance of a Calvinist sermon -- an article which confirmed what many if us have already observed, having micropropagted Nepenthes species; that variability is often visible within individual cultures, particularly upon re-plating; that genetic fidelity is often bandied about; but is quite illusory.

    Occasionally, odd variegation may initially be observed; stunted growth is common among some highland species (especially, N. rajah cultures which were originally attributed to Kew "askew" Gardens, decades ago, which refuses to grow, for most, beyond 5-6 centimeters).

    The study did happen to choose a wonky species, in my estimation: N. khasiana, of which, I have found, over the years, to vary a great deal from one TC generation to the next. What the authors managed to do was to actually quantify changes from one generation to the next. They determined that, genetically, the first regeneration after an initial plating, showed a chromosomal variance in excess of 23%; the second, in excess of 33%; and a third, over 40%.

    True, without getting into the weeds, we're dealing with cytogenetic "offtype" differences such as "heterochromatin distribution;" but it does put into some perspective, the somewhat dubious value of micropropagated soecies -- especially when the potential reintroduction of endangered species, from TC sources, has been considered . . .
    Last edited by BigBella; 02-10-2017 at 06:50 PM. Reason: Typing with boxing gloves . . .
    “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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