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Thread: JDW's Tissue Culture

  1. #17
    BigBella's Avatar
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    Quote Originally Posted by JDW View Post
    With my 5% for 15 minutes I could see the "wings" of the seed were much lighter in color while the embryo area stayed dark. BigBella, do you know if the wings are projection on the testa, or the entire coat itself? I wonder if the the embryo area has a second layer of protection, where I could bleach the seeds longer so the outside gets sterilized yet not damage the embryo. Also, with the last seeds I did, I put way too many per jar and couldn't spread them out well. With the contamination in the PPM cultures it appears that the molds started growing on a large clump of seed where not all of them were touching the media.

    I also wonder if there's some way to remove the wings, they are annoying....
    Your best bet is to leave the seed intact; you don't ever want bleach solutions, etc to come into contact with the embryo; just sow fewer in number per vial. I admire your courage, since you've also managed to choose some of more difficult to sterilize seed of the genera -- and without the benefit of a laminar flow hood. The lowland species are notoriously difficult to clean and may take an excess of twelve hours in a 4% PPM-MS solution; or else, serial bleach-alcohol treatments may eventually work.

    Another method is to actually encourage the mold and fungal spores to grow, by "hardening" -- soaking the seed in ten percent solutions of sucrose for twelve hours or more; then, successive washes with alcohol, sterile water, H202; and then air-drying in a sterile environment.

    Given the time involved and the effort, PPM is cheaper and simpler in the long-run . . .
    “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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    mcmcnair's Avatar
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    I just had seeds of the same ones sprout actually, same source for the seeds too . Best of luck to you
    NCSU's Carnivore Nut
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    JDW's Avatar
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    Quote Originally Posted by BigBella View Post
    Your best bet is to leave the seed intact; you don't ever want bleach solutions, etc to come into contact with the embryo; just sow fewer in number per vial. I admire your courage, since you've also managed to choose some of more difficult to sterilize seed of the genera -- and without the benefit of a laminar flow hood. The lowland species are notoriously difficult to clean and may take an excess of twelve hours in a 4% PPM-MS solution; or else, serial bleach-alcohol treatments may eventually work.

    Another method is to actually encourage the mold and fungal spores to grow, by "hardening" -- soaking the seed in ten percent solutions of sucrose for twelve hours or more; then, successive washes with alcohol, sterile water, H202; and then air-drying in a sterile environment.

    Given the time involved and the effort, PPM is cheaper and simpler in the long-run . . .
    The issue I have about the wings is that the soaked seeds stick together and it is hard to spread them out evenly on the media. Using a forceps to pull the seeds out of the soaking solution I get 10-25 at a time, and trying to spread them out with a spatula or glass rod they just roll in a mass and don't separate. What I had to do is stir them so they aren't all floating at the top and grab one or two to place on the media. This took way too long for sowing 500 seeds!

    Honestly I didn't know at the time that lowland seed is more difficult to sterilize. I just bought what I like and was available. But for me there hasn't been a difference. Only the first 10 milk bottles were done in a clean box. Everything after that I did inside a flow hood.


    Just to note, my 10% bleach soak for 10 minutes didn't kill the seeds. I rescued some bleached ampullaria seed from contaminated flasks and placed them on soil. They are now sprouting just fine.

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    BigBella's Avatar
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    Quote Originally Posted by JDW View Post
    The issue I have about the wings is that the soaked seeds stick together and it is hard to spread them out evenly on the media. Using a forceps to pull the seeds out of the soaking solution I get 10-25 at a time, and trying to spread them out with a spatula or glass rod they just roll in a mass and don't separate. What I had to do is stir them so they aren't all floating at the top and grab one or two to place on the media. This took way too long for sowing 500 seeds!
    First of all, the prospect of even sterilizing five hundred Nepenthes seeds at one time is a tall order, even under most commercial settings (I usually deal with a maximum of one hundred at any given time) -- the chances for contamination being far higher than doing serial smaller batches; and the method I have found most helpful for manipulating that "annoying" seed, is to soak them in the sterilization solutions within filter paper packets affixed with plastic paper clips (which can be color-coded for different species, as seen below). The seed is far easier to handle individually, while still attached to the damp paper.

    With that method, my contamination rate remains below two percent . . .

    Last edited by BigBella; 12-06-2012 at 04:23 PM.
    “Sì perché l'autorità dell'opinione di mille nelle scienze non val per una scintilla di ragione di un solo . . ."

    -- Galileo "Biff" Galilei

  5. #21
    JDW's Avatar
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    Oh I didn't do 500 at a time of one variety. I sowed 17 different varieties with only 10-50 seed per batch, in three different weeks. Each batch was disinfected separately in vials with 5mL of ppm or bleach.

    How you do it with the filter paper is a great idea, I'll have to try that next time.

  6. #22
    BigBella's Avatar
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    The filter paper method is a pleasure to deal with, especially after years of having to ensure that loose seed always remained in solution and not adherent to the glass wall. The Grade-1, 7 cm Medium Flow Rate (11 µM) is a good choice and allows for easy flow-through.

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

    -- Galileo "Biff" Galilei

  7. #23
    JDW's Avatar
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    I've got my first Nepenthes seed germinating in vitro!!


    This is albomarginata red form, so far there are 4 seedlings. None of the other varieties are germinating yet... I aslo planted out my first plant! A Drosophyllum from seed.


    And two days ago I flasked some Sarracenia and Darlingtonia seed in my newly finished flow hood. I had to buy a different fan that was already wired with a 3-prong cord :/

  8. #24
    sarracenia lover dionae's Avatar
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    Congrats on your success.

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