Tissue culturing.

Discussion in 'Plant Help' started by mr. luke, 4 Mar 2010.

  1. mr. luke

    mr. luke Member

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    Has anyone ever thought about a bit of home tissue culturing?
    There are some basic diy tutorials online that people use for house plants with low germination rates and i thought it might be a cool idea to give it a try on some aquatic plants? 8)
    One reason im keen to try it is I often find nice mosses when im out and about and im very hesitant to add them to my aquariums incase of pesticides and whatnot.
    I know you cant be 100% sterile at home (equipment not me :rolleyes:) but I think there is a chance of it working.
    Anyone else up for it?
    Thought we could share our experiences on amounts of minerals vs. growth rates etc.

    The basic principle is a mineral and nutrient rich agar in a sterilised jar and a sterilised sample of 'chosen plant' then leave and relax.
     
  2. a1Matt

    a1Matt Member

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    Interesting post Luke :thumbup:
    I always assumed tissue culturing needed some serious chemistry knowledge. You make it sound quite straightforward :) Do you have any links to tutorials or pages with background info?
     
  3. Lisa_Perry75

    Lisa_Perry75 Member

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    This sounds interesting. But how would you sterilise a sample of plant without damaging it? As I work in a molecular biology lab and do tissue culture (but with animal cell lines) I could probably do something myself...
     
  4. mr. luke

    mr. luke Member

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    http://www.world-of-carnivores.com/mediadirections.html
    This website has soe great information and makes it sound fairly home do-able.
    Ill be using this mainly for mosses and crypts which aparantly are very easy to grow like this.

    Many other websites say you can use staralised tinfoil as a lid, which is what ill be using.
    Im also putting everythin into the pressure cooker and skipping the alcohol part (unless anyone knows where to get some cheapish)
    Ill be using bleach to staralise the plant sample, watered down with de ionised water.
    Ill also be taking on the gelatin aproach as aposed to agar.
    Ill be trying this with some aquarium macro's and micros mixed with the gelatin.


    Be aware the site in the link is aimed at carnivourus plants, but the same principles can be applied to all plants.
    The only thing that will difer is the ph required, mosses for example could handle almost any ph (another reason why im trying it out on mosses first :thumbup: )
     
  5. Lisa_Perry75

    Lisa_Perry75 Member

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    I believe you can buy agar by the way, its a vegetarian alternative to gelatine.
     
  6. mr. luke

    mr. luke Member

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    Thanks for the heads up :)
    Ill have a see how much it is, but if it costs more than id like to spend ill try gelatin.
     
  7. Lisa_Perry75

    Lisa_Perry75 Member

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    Ah my second post has disappeared. Basically you might be able to buy alcohol from a pharmacy (used for window cleaning), dilute to 70% is fine for killing bugs. Please don't spray your hands with this stuff. Buy some gloves, wear a jumper or shirt which is very clean and tuck into the gloves.
     
  8. dw1305

    dw1305 Expert

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    Hi all,
    First thing is that mosses are extremely unlikely to harbour anything that would be damaging in the aquarium, they don't have any roots or any vascular tissue, this why they aren't effect by simazine etc, so if you soak them in a bucket of R.O water before use they are basically sterilized anyway.

    The link is OK, but have a look at this one <http://www-saps.plantsci.cam.ac.uk/docs/tissue.pdf>,I've done this and I have the complete "recipe" for what you need, if anybody does want to try it?

    As Lisa has said it is almost impossible to maintain sterile conditions away from a microbiology lab, but you can flame and alcohol sterilize your scalpel etc. and use a pressure cooker as an autoclave to sterilise the jars (Kilner jars were the best, but they are difficult to get hold of now) and agar media etc as you are doing. You can't tissue culture Bryophytes in the same way you would with Orchids or Carnivorous plants, as in those cases you use an "apical meristem" as your "explant" and then subculture the resulting plant (you can vary the ratio of the plant hormones - auxins and cytokinins to increase shoot or root initiation), the advantage of this is the meristem is fairly sterile to start off with, and doesn't contain viruses (old cultivars of plant are often virused due to many years of vegetative propagation). If you wanted to go into mass production of some of "Tropica's" PBR registered coloured Echinodorus cultivars this would be an easy way to bulk up hundreds of plants.

    I've just grown a load of Pinguicula "moranensis" (Mexican Butterwort) plants from detached leaf fragments, swirled in 2% sodium hypochlorite bleach and placed on micro-waved damp vermiculite (will swap for other insectivorous plants, ferns etc. if anyone is interested.)

    Bryophytes don't have any tissue which is differentiated into meristems etc. (they are much simpler structures without vascular tissue, true leaves etc. (the green things are microphylls), but they do have the ability to regenerate from very small fragments (both bryophytes and the higher plants can potentially regenerate the whole organism from a single cell, this is called "totipotency", so are very different from mammals where you need "stem cells" or "neural crest cells" - step forward the Zebra Danio a cell biology legend.

    For mosses you don't need the agar or hormones, just a sterile container. Remove a very small section from the tip of each moss frond, place in the container on a rockwool, peat or sand medium (sterilized in a microwave), place in the sealed container (a jar is excellent) and spray the stem fragments with an extremely dilute liquid feed.

    You can also do moss or fern spores very easily using the same method.

    cheers Darrel
     
  9. Mortis

    Mortis Member

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    Shouldn't be too hard but it obviously depends on the species, conditions you provide to it and the starter culture used.

    Random : Hey Lisa I'm a molecular biologist too !
     
  10. Lisa_Perry75

    Lisa_Perry75 Member

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    Cool! I'm just doing my PhD now, what stage are you at/where?
     
  11. Mortis

    Mortis Member

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    Just finished my Msc from Nottingham a year and a half ago. Im working right now and plan on doing so for another couple of years atleast before doing my PhD.
     
  12. dw1305

    dw1305 Expert

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    Hi all,
    I re-found this post earlier today when I was looking for a micropropagation thread, and I thought I'd post a picture of the same Pinguicula mentioned, I have grown a few more, but this is the original leaf cutting from 2010.

    It was pretty dried out while I was away from work in the summer (you can see the dead moss), but it recovered after watering and it's dice with death has obviously stimulated it to flower.

    Pinguicula.JPG

    You can also see a sporeling fern at the front of the bowl, I rogue these out on a regular basis, but there are always new ones growing.

    cheers Darrel
     
  13. Edvet

    Edvet Forum Moderator Staff Member

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    Plants: try to kill them and they will flower:cool:
     
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  14. Aquahorti

    Aquahorti Member

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    Ahh that is what my wife is doing to the orchids when I'm away with work, she is not trying to kill them, she is just trying to make them flower...
     
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  15. Edvet

    Edvet Forum Moderator Staff Member

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    I loved my greenhouse. It will be restored when i grow up. Full Cattleya collection.
     
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