[Arabidopsis] brands of agar

Hi Tanya

For the last 15 years or so we have been routinely studying Arabidopsis
root growth and branching on vertical 'agar' plates, for up to two weeks
after germination. We have used 0.8% agar-agar in the past with
reasonable results, but found that batches were variable and sometimes
root growth would be inhibited. We now choose to use PhytagelTM, which
is purer and gives a clearer gel, and we have had no problems with
different batches (note that you have to make sure that the divalent
cation concentration in the medium is at least 2 mM, otherwise it won't
set!).

As an important aside, I would strongly recommend that you *don't* use
MS (or even 1/2 MS) for anything other than tissue culture or antibiotic
selection. It is an extremely rich medium with non-physiological N
concentrations that far exceed anything that plants are ever likely to
have experienced in nature (40 mM nitrate + 20 mM ammonium)and root
growth in this medium is also very slow. We use a dilute form of B5
medium that allows much faster root growth. We have done direct
comparisons with Ler and Col-0 growing in MS, 1/2 MS and in our modified
B5 medium at different rates of N supply and found that all growth
parameters (lateral roots, primary roots, shoots) are faster on the
modified B5 medium. The modified B5 medium also allows you to vary
individual components of the nutrient supply without creating severe
nutrient imbalances that cause plant stress. The composition of our
medium was originally published in Zhang & Forde (1998) Science 279,
407-409, but I can give you more details if you wish to email me.

Good luck!

Brian


=====================================
Brian G. Forde
Prof. of Environmental Plant Biotechnology
Department of Biological Sciences
Lancaster Environment Centre
Lancaster University
Bailrigg
Lancaster LA1 4YQ
tel. +44 (0)1524 510207 (direct line)
email [Only registered users see links. ]
[Only registered users see links. ]

Editor-in-Chief Plant Methods
email [Only registered users see links. ]
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=====================================



-----Original Message-----
From: [Only registered users see links. ]
[mailto:[Only registered users see links. ].indiana.edu] On Behalf Of Falbel, Tanya
G.
Sent: 09 February 2008 21:11
To: [Only registered users see links. ]
Subject: [Arabidopsis] brands of agar


Colleagues:

Over the years, I've used several brands of agar for growing Arabidopsis
seedlings on MS medium, from Gibco, Sigma, and others. I've heard that
some groups use Noble agar, others, bacto-agar, others in the past have
washed their own agar, used phytagel or other
gelling agents. I have two questions:

1) What brands of agar are most commonly in use now by groups? I've
found something similar to Gibco's Phytagar that is now available
through Caisson labs. Other groups seem to like Sigma's A1296. But
besides those, what do most groups use? I'm especially interested in
the opinion of groups that measure root length or root branching -
growing roots vertically on plates for more than just a couple of days.
(as opposed to just antibiotic selection)

2) Does anyone know what inhibitors are washed away? Are micronutrient
ions or other toxic compounds bound to the crude agar? Has anyone
looked into this? This may have been a question that came up among
researchers 10 years ago, but I couldn't find any record in the
archives.

Here's why I'm asking. I made up a batch of medium with a brand of
'purified agar for microbial use', and got a very strong inhibition of
root meristems. Seed germination was good, but roots failed to elongate
any further after about 4 days of growth. The root meristems became a
swollen mess, reminiscent of what happens in weak gnom alleles. I'm
trying to decide if this is something worth looking into.

I know that for regeneration of plants in tissue culture, folks are very
fussy about particular brands of agar.

Please let me know what brands you have used with success or failure.
I'd be especially interested in other similar experiences - where a
brand wreaked havoc on root meristems.

Thanks

Tanya Falbel
Department of Biology
105 Garfield Avenue
University of Wisconsin-Eau Claire
Eau Claire, WI 54702-4004, USA

Tel: 715-836-5087
Fax: 715-836-5089


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