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Thread: Nepenthes, Cephalotus, Drosera, Heliamphora ...etc, in vitro

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    pokie22's Avatar
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    Nepenthes, Cephalotus, Drosera, Heliamphora ...etc, in vitro

    All purpose thread for all the cultures I have in the works. Most of my cultures are Nepenthes, but I should have a couple of everything...In the last two months, I have amassed nearly 100 cultures. I have listed the media in cases where it is not clear. Here are a few of the cute sprouts:


    Nepenthes Attenboroughii (Jeremiah) 2 month old seedling on 1/4MS+2.5%sucrose+1mg/L BAP, ph5.6


    Nepenthes Attenboroughii (Jeremiah)2 month old seedling; compost control


    Nepenthes Glabrata putative


    Nepenthes Glabrata; compost control putative



    Drosera Regia in vitro


    Drosera Regia; compost control


    Pinguicula Lusitanica on MS + 1x Vits ph 5.75


    Dionaea Fused Fuzzy on ⅓ MS + 1X Vits+ 3% sucrose ph 5.6


    Dionaea Sawtooth II on ⅓ MS + 1X Vits+ 3% sucrose ph 5.6


    Sarracenia Alabamensis; 24hr h2O presoak + 42hr 500ppm ga3 soak onto 1/3MS+1xvits+3%sucrose ph5.8



    Sarracenia Jonesii -- red/HV;24hr h2O presoak + 42hr 500ppm ga3 soak onto 1/3MS+1xvits+3%sucrose ph5.8


    Sarracenia Oreophila;24hr h2O presoak + 42hr 500ppm ga3 soak onto 1/3MS+1xvits+3%sucrose ph5.8


    Darlingtonia Californica, Siskiyou Co; 42hr 500ppm ga3 soak onto 2/3KC+3%sucrose+0.5mg/L NAA ph5.8


    Darlingtonia Californica, Del Norte Co; 42hr 500ppm ga3 soak onto 1/3MS+3%sucrose+1mg/L BAP ph5.6


    Darlingtonia Californica, Del Norte Co; 42hr 500ppm ga3 soak onto 2/3KC+3%sucrose+1mg/L NAA ph5.6

    In my hands, Darlingtonia California necessitates a ga3 treatment (or stratification) to break dormancy. My controls without ga3 +/- NAA or BAP, are not showing any signs of germination. Also, no cute sprouts on any media with NAA. Instead, they look like those white Chinese steamed buns (callus perhaps..) during the first rise.

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    sarracenia lover dionae's Avatar
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    Very good looking cultures pokie.

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    Very nice! It looks like everything is coming along great. Thanks for sharing your formula for success.

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    zesty. BioZest's Avatar
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    Nice seedlings! I hope they do well for you

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    Most of the sprouts will be for experiments. Extras will be given away. As far as the Nepenthes sprouts are concerned, I will be trying some protoplast fusion with the more abundant ones, and if a suitable SOP can be devised, then some ultra highlanders. I have made many yeast protoplast fusions for senescence experiments in the past, as well as dual mammalian species hybrids - those require sendai virus..etc. In most of those cases, one phenotype is dominant and silences the other spp by epigenetic means. Any requests Don?

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    Getting There...
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    Pokie, have you been doing this long? Any advice for someone looking to get started in it?

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    DonH's Avatar
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    Quote Originally Posted by pokie22 View Post
    Most of the sprouts will be for experiments. Extras will be given away. As far as the Nepenthes sprouts are concerned, I will be trying some protoplast fusion with the more abundant ones, and if a suitable SOP can be devised, then some ultra highlanders. I have made many yeast protoplast fusions for senescence experiments in the past, as well as dual mammalian species hybrids - those require sendai virus..etc. In most of those cases, one phenotype is dominant and silences the other spp by epigenetic means. Any requests Don?
    As soon as I decipher what you just wrote, I'll come up with a request.

    Quote Originally Posted by Vbkid View Post
    Pokie, have you been doing this long? Any advice for someone looking to get started in it?
    Pokie's a lab geek. Lol!

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    pokie22's Avatar
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    Quote Originally Posted by Vbkid View Post
    Pokie, have you been doing this long? Any advice for someone looking to get started in it?
    You should read my previous thread: http://www.terraforums.com/forums/sh...Tissue-Culture

    My first question is: How earnest are you in getting started?

    For me, this is a past time when I am waiting on an experiment. And even so, there was an initial investment needed to acquire TC consumables that I did not have.

    I started my first TC of CPs on Thanksgiving of 2012, so ~ 2 months. I have a few plants in compost, but watering is not for me. I started CP TC with Jeremiah's attenboroughii seed in late November. However, I am a scientist. It would be unfair to use me as a measure of how easily one can perform TC, as my normal work entails: cloning, mammalian tissue culture for antibody production purposes or small synthetic proteins, hESC, mESC, iPS cells, differentiation of primary human cells, phage display, algae for biodisel, anaerobic thermophillic fungi, lentivirus production....etc.etc. Therefore, sterile technique comes almost as a reflex to me, and almost no tissue culture will be as difficult as the coddling necessary to grow hESC on feeder layers. However, that is only part of the equation. The other part is to perform one's due diligence and educate yourself on what media or PGRs are necessary for the CP in question. Everything I have learned has either come from this forum, **********, WOC, ICPS boards or scientific journals. If you are unsure,there are many people with more experience that are usually happy to lend a helping hand. I know I have asked BigBella more than a few. Although, expecting someone else to just tell you some exact protocol when it is already freely available, is not the best choice. In the grand scheme of things, CP TC is rudimentary, and fairly easy. First, it is an organism which inherently has some self-defenses against potential nasties that would like to eat the jello. Second, one does not need to enumerate, count, or passage (very often). I am actually very surprised that most would wait to know the sex or spp of their Neps- it has been a great while since the dawn of molecular biology.

    Tissue culture is not that difficult. One needs to be cogniscent of where your hands are at all times and everything that touches something sterile, needs to be sterile. If you lived close to me, I would invite you over. Seeing and doing, is different from reading and hearing. As I find with most of my students, learning by immersion is the best way. Of course, some media will be contaminated and there will be initial losses, but that is expected with the learning curve. It is only the idiot who does not learn from this - I have had a few of these too. After some trial and error, you will have a methodology that works for you. Have you visited the HTCG? They do sell starter kits. I do my TC where all other TC is performed - laminar flow hood. There are many DIY plans out there for. Albeit, it is not necessary, but does inhibit any contamination arising airborne spores.

    I like to try new things all the time, and for the most part I am successful. Unlike most people, I like to do it right the first time. Just as I don't try to live, I never try to do anything - I just do. If you met me, you would understand.
    Last edited by pokie22; 02-03-2013 at 11:01 PM.

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