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Thread: Micropropagation/Tissue Culture - seed germination

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    Moderator Joseph Clemens's Avatar
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    Micropropagation/Tissue Culture - seed germination

    When I was still in college, back in the late 1980's, I had some older, abused seed that I surface sterilized and sowed in vitro. Most of the different species I tried this with had fairly good germination after a month, and I eventually transplanted them into typical media and grew them in more traditional ways. An exception was Drosera aliciae. The Drosera aliciae seed did not germinate in a typical way, in vitro the Drosera aliciae seed began to produce small callus-like growths that appeared to expand from the testa in the vicinity where the embryo would have been. Eventually some of the callus differentiated into roots and shoots, usually more than one per seed.

    I was wondering if anyone else working with tissue culture had similar experiences from any CP species.
    Joseph Clemens
    Tucson, Arizona, U S A

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    WRC Fan adamtekium's Avatar
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    I know you're familiar with tissue culture, but I'm just trying to understand how much callus grew before you saw differentiation. Is it possible that you used equally high levels of cytokinin and auxin to begin with, and when regulator levels in the media were finally in a state of imbalance, you saw the differentiation?

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    Moderator Joseph Clemens's Avatar
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    Back when I was busy being a college student I was allowed access to a small lab with a flow hood and a small shelf space where I mounted a fluorescent light and kept my TC.

    I had a few vials of Knudsen orchid germination media, each vial was pre-measured to make up one liter of media. For each batch of media I added enough purified water, sucrose, and gelrite to make up two liters at half strength - I didn't worry about the pH.

    I used a variety of protocols to pre-treat the seed, soaking with tween-20 in water, sodium hypochlorite solution, 95% ETOH, 3% hydrogen peroxide, and finally rinsing with sterilized/purified water.

    The preliminary callus-like growth I observed took at least a month to escape the confines of the seed testa and make itself visible. I had only kept this culture around that long because I was a preoccupied college student. When I first noticed it, it was only pink and white, and I first thought it might have been contamination. I thought it still might be contamination almost until buds formed and plantlets developed. Chlorophyll only formed once buds had formed leaf primordia. One reason I suspected it might not be contamination but rather callus, was its very slow progression, and that the callus rarely exceeded one or two millimeters from the seed in any direction. And it did look like callus rather than bacterial colonies or fungal hyphae.

    Part of my normal job in this lab, was to assist graduate students to culture plant pathogens and produce quantified disease innoculants to use for testing plants for disease resistance. Organisms we routinely cultured were, Verticillium dahliae, Rhizoctonia solani, and Phytophthora capsici.
    Joseph Clemens
    Tucson, Arizona, U S A

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