Plant Tissue Culture (Cloning)

Discussion in 'Cannabis Community' started by Vibe, Sep 16, 2016.

  1. Vibe

    Vibe New Member

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    I would like to have your opinion about this technique. Does it work? Is it worth the hassle?
    http://cals.arizona.edu/pubs/garden/mg/propagation/grafting.html#Top

    Plant Tissue Culture for the Home
    [​IMG]

    Although technical procedures for aseptic culture of plant cells, tissues, and organs are as diverse as the plant material on which they are practiced, a simplified general procedure can be followed in the home. All that is needed are a few basic supplies which can easily be obtained. The procedures outlined in this section can be used in the home to propagate various species of plants, both easy (African violets, coleus, chrysanthemums) and difficult (orchids, ferns, weeping figs) to propagate.

    Medium Preparation
    For 2 pints of tissue culture medium, mix the following ingredients in a 1-quart home canning jar:

    • 1/8 cup sugar
      1 teaspoon all-purpose, soluble fertilizer mixture. Check the label to make sure it has all of the major and minor elements, especially ammonium nitrate. If the latter is lacking, add 1/3 tsp. of a 35-0-0 soluble fertilizer
    • 1 tablet (100 mg) of inositol (myo-inositol) which can be obtained at most health food stores 1/4 of a pulverized vitamin tablet which has 1 to 2 mg of thiamine
    • 4 Tablespoons coconut milk (cytokinin source) drained from a fresh coconut. The remainder can be frozen and used later.
    • 3 to 4 grains (1/400 teaspoon) of a commercial rooting compound which has 0.1 active ingredient IBA


    Fill the jar with distilled or deionized water. If purified water is not available, water that has been boiled for several minutes can be substituted. Shake the mixture and make sure all materials have dissolved.

    Baby food jars with lids, or other heat-resistant glass receptacles with lids can be used as individual culture jars. They should be half filled with cotton or paper to support the plant material. The medium should be poured into each culture bottle to the point where the support material is just above the solution.

    When all bottles contain the medium and have the lids loosely screwed on, they are ready to be sterilized. This can be done by placing them in a pressure cooker and sterilizing them under pressure for 30 minutes or placing them in an oven at 320oF for 4 hours. After removing them from the sterilizer, place them in a clean area and allow the medium to cool. If the bottles will not be used for several days, wrap groups of culture bottles in foil before sterilizing and then sterilize the whole package. Then the bottles can be removed and cooled without removing the foil cover. Sterilized water, tweezers, and razor blades, which will be needed later, can be prepared in the same manner.

    Plant Disinfection and Culture
    Once the growing medium is sterilized and cooled, plant material can be prepared for culture. Because plants usually harbor bacterial and fungal spores, they must be cleaned (disinfected) before placement on the sterile medium. Otherwise, bacteria and fungi may grow faster than the plants and dominate the culture.
    [​IMG]


    Various plant parts can be cultured, but small, actively growing portions usually result in the most vigorous plantlets. For example, ferns are most readily propagated by using only 1/2 inch of the tip of a rhizome. For other species, 1/2 to 1 inch of the shoot tip is sufficient. Remove leaves attached to the tip and discard. Place the plant part into a solution of 1 part commercial bleach to 9 parts water for 8 to 10 minutes. Submerge all plant tissue in the bleach solution. After this time period, rinse off excess bleach by dropping the plant part into sterile water. Remember, once the plant material has been in the bleach, it has been disinfected and should only be touched with sterile tweezers.

    After the plant material has been rinsed, remove any bleach-damaged tissue with a sterile razor blade. Then remove the cap of a culture bottle containing sterile medium, place the plant part onto the support material in the bottle making sure that it is not completely submerged in the medium, and recap quickly.

    Transferring should be done as quickly as possible in a clean environment. Therefore, scrub hands and counter tops with soap and water just before beginning to disinfest plant material. Rubbing alcohol or a dilute bleach solution can be used to wipe down the working surface.

    After all plants have been cultured, place them in a warm, well-lit (no direct sunlight) environment to encourage growth. If contamination of the medium has occurred, it should be obvious within 3 to 4 days. Remove and wash contaminated culture bottles as quickly as possible to prevent the spread to uncontaminated cultures.

    When plantlets have grown to sufficient size, transplant them into soil. Handle as gently as possible because the plants are leaving a warm, humid environment for a cool, dry one. After transplanting, water the plants thoroughly and place them in a clear plastic bag for several days. Gradually remove the bag to acclimate the plants to their new environment; start with one hour per day and gradually increase time out of the bag over a two-week period until the plants are strong enough to dispense with the bag altogether.
     
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  2. FallenOak

    FallenOak Mushroom Cultivator Mushroom Doctor

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  3. Vibe

    Vibe New Member

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    Wow, very interesting. This thread fits perfectly into this forum with agar and pressure cooking and stuff.

    I hope to see more discussion here. Lets see.
     
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  4. FewTrueSeed

    FewTrueSeed Many meiosis Supporter

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    I have tried numerous times with some sucsess. Much harder than growing mycelium. I never tried an antibacterial additive. Could be usefull. I would like to try again as well. I was able to get calus tissue wih coleus.
     
  5. Essence

    Essence Well-Known Member

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    Excellent topic. I've been playing around with this a little myself. I'm finding the recipe needs to be a little different and more specific for cannabis. You can actually buy the medium on a wholesale level, but of course I'm stubborn and have to do it the hard way lol. Good luck finding anyone's recipe that works specifically for cannabis. Top secret affair that is and too much money involved at the corporate levels lol. I, however, don't really give a shit so long as one of us gets it done and out there for everyone to see and do. :grouphug:

    My current recipe in process

    Recipe #1

    250ml / 1 cup well water warm
    5 grams Agar Agar (Agar flakes)
    2 teaspoons common processed cane sugar
    1cc Canna Start
    .5cc Superthrive
    .5cc Canna Rhizotonic
    50mg Thiamin
    200mg Folic Acid
    15mg Ba 6 Benzylaminopurine (Plant Hormone Cytokine)
    15mg Ga3 Gibberellic Acid (Plant Hormone)
    15mg IBA 3 Auxin / Indole Butyric Acid (Plant Root Hormone)

    My last attempt came very close and I believe would have worked (actually seeing cell division), but a late Fusarium outbreak ended it. I'll be sure to extend the sterilization/clean up of the tissue next time. The failure instigated a lot of research into symbiotic relationships between plants, mold/fungi and yeasts. Fascinating stuff!

    At any rate, does it work? Yes. Is it worth it? Depends on your goals. Ultimately I would like to accomplish synth seeds >>> https://skunkpharmresearch.com/tissue-culture/ and http://growblox.com/white-papers/cannabis-tissue.html
     
    Last edited: Sep 16, 2016
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  6. Titus

    Titus Creative PITA

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    Great thread!
     
  7. Pistilwhipped

    Pistilwhipped Grower

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    Me too! Was considering this as a side endeavor. "I can save your sick heirloom plants" and whatnot. Got a bunch of hormones and supplies, just looking for the time to play.
     
  8. frosty.dog

    frosty.dog New Member

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    ***BUMP***

    Did this recipe end up working out???

    Anyone else doing tissue culturing work for cannabis with some knowledge or stories?