Date: Fri, 20 Feb 1998 14:01:12 +0000 From: Peter Cole <carnivor@flytrap.demon.co.uk> To: cp@opus.hpl.hp.com Message-Id: <aabcdefg677$foo@default> Subject: Re: tissue culture
"Kamikaze" <011114@hillstrath.on.ca> writes:
> I have tried tissue culturing many varities of CP...especially VFt
> with some white bulb material attached. When culturing, I sterilized
> the plant material in 15% bleach solution for 10 minutes. I then
That sounds fine, but VFT tissue is difficult to sterilise (compared
to most Drosera, Ping, Utric.) I would recommend washing the
explant prior to sterilisation for 30 mins to 1 hr (amazing how much
contamination is reduced just by washing with plain water!) The
easiest way is to suspend it in a piece of nylon mesh (bridal veil
or similar,) under a running tap. You may also wish to soak for
5-10 mins in Hydrogen peroxide as well before bleaching, and adding
a few drops of detergent to the bleach to ensure good wetting.
It's much easier to work from seeds with this sp., as they're so
nice and shiny, but of course you can't do red/dentate/other
interesting cultivars and forms from seed...
> carefully removed the cuttings into an aquarium that had been filled
> with rags soaked in isopropyl alcohol and left inverted on the rags
Leaving the cutting overnight is perhaps not ideal - all sorts
of spores etc. can waft in with small air currents and settle.
The alcohol will almost certainly evaporate from the glass
surfaces in a short time, even if the rags are still saturated (one
reason I prefer bleach - nearly burning the house down last year is
another!) Better to sterilise the aquarium immediately prior to
culturing. I prefer to use diluted bleach (same as for plant
tissue or stronger,) for sterilising - mist or swab all interior
surfaces and leave for 20 mins. Then just blot lightly to absorb
excess (it will do no harm to leave the surfaces damp with bleach,
but you don't want it dripping into the flasks!)
> overnight...in short the aquarium was quite sterile. The media I used
> was also well sterilized and did not have any fungal or bacterial
You can narrow down the point at which the problem is occuring
by trying some "dry runs" - opening, prodding around in and closing
flasks in the (hopefully) sterile aquarium. If the aquarium,
tweezers, etc. are sterile then they should remain
contamination-free. But indeed the problem is more likely with the
tissue than the aquarium.
> contamination. I then carefully transplanted the sterile (so I
> thought) VFT into the TC media inside the aquarium. The VFT culture
> after 4 days was contaminated with fungus. I repeated this experiment
> several time with varying strengths of bleach and alcohol. In each
> case, I either failed to sterilize properly or over sterilized (thus
> killing the plant material) the plants. I am at wits end.
>
> Please let me know any experiences that people have had with
> effective sterilization agents. I know that the media and aquarium
> were quite sterile.
Hope this helps,
Happy cloning,
Peter
+++ Peter Cole, 17 Wimmerfield Cr.,Killay,SWANSEA SA27BU,WALES,UK +++
mailto:carnivor@flytrap.demon.co.uk - http://www.flytrap.demon.co.uk/
++++ Carnivorous Plants, seeds and tissue culture kits for sale ++++
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