GA mutants - summary

[Carol Auer]

Hello 

As requested, I am posting  a summary of the excellent responses I 
received regarding the use of GA mutants for plant physiology labs. 
Thanks to those with who responded.  There is enough information here 
to make several interesting labs.  Several people have noted that, 
although the mutant lines are available from the ABRC, they only  
send a small amount of seeds.  Therefore, more seeds must be produced 
to use in a large lab class.

Carol Auer
Dept. of Plant Science
Univ. of Connecticut
cauer at canr1.cag.uconn.edu

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Dear Carol Auer

I've read with great interest your mail on the use of BA mutants for 
teaching purposes.  I've been thinking of doing the same and I msut 
say I just did a trial this term.  I have been working as a post-doc 
in Nick Harberd's group at the John Innes Centre for Plant Science in 
Norwich (Great Britian).  The work done was mostly molecular genetics 
on the GA deficient and GA insensitive mutants of arabidopsis.  My 
plan for the use of GA related mutants would be as follows. 1) The 
use of a severe mutant like ga1-3 to show that GA deficient plants 
cannot germinate without GA.  Add GA at a nano to mico molar 
concentration to see the dose dependent effect of GA on the 
percentage of germination.

2) Remove seed coat from the ga1-3 mutant seeds after imbibition.  
This shows that mutant seeds can germinate without GA providing the 
seed coat is removed.  There must be a GA antagonist in the seed coat 
(probably ABA)?

3) Grow on soil ga1-3 plants that have been germinated on GA.  Show 
that the plant phenotype is severe dwarfism and thre is an altered 
flowering response as well.  Water with millimolar concentration of 
GA within few days there is a spectacular reversion of the phenotype 
that goes back to almost wild type.  The same can be done on less 
severe mutant like ga3, ga4 or ga5.

4) I have been using also GA mutant for genetic purposes.  Showing 
that GA deficient are recessive mutation.  Also I am using gai, that 
is impaired in GA response.  The mutation is semi-dominant so its a 
very interesting model to study.  Since I've been working on this 
particular mutant during my post-doc I msut say  that's my pet 
project.

Pierre Carol
Bio. Mol. Veg.
CERMO
Universite Joseph Fourier - BP 53X
Grenoble 38000
France
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Hi, I have done some work with ga-deficient mutants in Arabidopsis.  
ga1 is probably the best mutant.  I use 25 micromolar GA4+7 to rescue 
for germination.  GA3 is available from Sigma, and should work well.  
Bump up the concentration to 50-100 micromolar.  GA dissolves in 
dimethyl formamide.  I also have sprayed the non-elongating bolts and 
have rescued them in that way.  I would try a similar concentration 
of GA in a spray bottle.  Again, first dissolve GA in DMF, and then 
dilute in H2O. Good luck, 

Judy Brusslan
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Dear Dr. Auer,

I have some experience working with Arabidopsis GA mutants.  To 
answer your questions on the net:
1. ga1, ga2 and ga3 require exogenous GAs for germination and require 
GA treatment to set seed.  ga4 and ga5 do not.

2. On plant height, ga1, ga2 and ga3 are severe dwarf. ga4 and ga5 
are only semi-dwarf.  They all respond to GAs with shoot elongation.

3. the GA concentration that I use is 0.01 mM GA3.  Spray plants 2-3 
times each week since seed germinates.

Hui-Hwa Chiang
Dept. of Molecular Biology
Massachusetts General Hospital
Boston, MA 02114
chiang at frodo.mgh.harvard.edu
---------------------------------

Carol,

I use the ga1, ga4 and ga5 mutants of Arabidopsis in my research.  
ga1 lacks ent-kaurene synthase A, probably a single copy gene, and so 
is an exteme dwarf, requiring added GA for germination, bolting and 
flower development.  ga and ga5 lack 3b-hydroxylase and 20-oxidase, 
respectively, and are semi dwarf, completing their life cycles 
without needing extra GA.  The 20-oxidase has at least 3 genes 
expressed in different tissues, and it's assumed that the 3b-
hydroxylase must also have more than one gene, although only one has 
been identified.

You can germinate ga1 in the soil by application of 10 uM GA3 
(dissolved in EtOH then dilute in water), but this sometimes carries 
over into the rosette, promoting flowering and bolting.  
Alternatetively, you can germinate the seeds on filter paper soaded 
in the GA solution and transplant, or imbibe the seeds in water with 
GA for 1-2 days.  I use a spray of 10uM GA3 for inducing flowering 
etc on all the mutants.  They show a measurable response within 4 
days, best observed after a week.

Paclobutrazol is applied as a pot drench as its taken up through the 
roots.  You need to apply for some time to see an effect - I can get 
full details if you need them.

Andy Phillips
University of Bristol
IACR-Long Ashton Research Station
Long Ashton
Bristol, BS18 9AF, UK