Date: Wed, 13 Aug 1997 08:21:59 +0200 (MET DST) From: Guy Van Der Kinderen <Guy.VanDerKinderen@rug.ac.be> To: cp@opus.hpl.hp.com Message-Id: <aabcdefg3086$foo@default> Subject: more coco's and cyto's
Dear Loyd, John, Kent,...
First I have to clear out some things about the coconut milk. I
suggested the liquid for experimenting with difficult seeds in vitro (not
as a pretreatment), and only as an alternative if other methods fail.
Although it is mostly used to promote cell division and (axillary) bud
formation, it also proved to enhance germination in temperate terrestrial
orchid seeds. I have no idea what the effect will be with CP seeds! I
do not have to tell you that cocnut milk, when used without sterilization,
will spoil very soon. So, if you want an alternative to leaching, then you
better try out the hypochlorite affection method. And do not forget GA's
as they did help in some CP's (Byblis), or scoring as in Drosophyllum, or
cold stratification as in certain Pings. As far as I know, pure
cytokinins, such as zeatin or its riboside, are never used as a
pretreatment to germination.
Regarding thermostability of plant hormones, it is now generally
agreed, as John states, that most of them are stabile indeed, and hence
may be autoclaved. When I was experimenting with coconut endosperm,
cytokinins and orchids (some fifteen years ago), much less data were
available on this matter, and I did not want to take the risk of loosing
all activity upon heating. Nevertheless, it is known that GA's may loose
90% of activity after autoclaving. At the end, filtersterilization did not
appear that elaborous to me, Kent.
BTW Kent,
>While there is probably some degradation of the hormones when autoclaved;
>as a practical matter it is easier to add the hormones before autoclaving
>aand adjust the concentration to what works after sterilization.
I was wondering,
1) How do you know the losses, since you suggest to adjust 'to what works'
after sterilization?
2) and how do you add the 'adjusting aliquot of hormone' to your already
sterilized medium?
The technique of adding an overdose of compound, in order to
compensate for losses after sterilization, has also been used for
vitamins. Finally, certain hormones can (need to) be dissolved in pure
alcohol. One may add a small amount of a concentrated stock solution with
a sterile pipet to the autoclaved medium, without filtersterilization,
since the alcoholic solution is sterile. The small and diluted amount of
alcohol won't disturb culturing.
Loyd, if you still want to experiment with pure cytokinins, I
would suggest not to start with the expensive zeatins, but try kinetin or
benzylaminopurin (BAP), which I think are cheaper. In addition, they won't
give you the problem of choosing between cis or trans, a problem which I
did not manage to solve yet.
It's very hot here today in Belgium, I could use some ice cream, Loyd.
Kind regards,
Guy~
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