[Arabidopsis] Optimal germination of older Arabidopsis seeds

Charles S. Gasser via arab-gen%40net.bio.net (by csgasser from ucdavis.edu)
Fri May 15 16:46:30 EST 2009

Thanks very much to all who replied to my request 
for information on ways to aid older Arabidopsis 
seeds in germination.  I asked if anyone had 
suggestions for helping us as some samples of 
seed were failing to germinate on either soil or 
plates. Some replied to both me and the group, 
but I summarize them all here.

June Medford pointed out that the plates (media) 
was the better of the two methods we were using 
since "you can at least see if the seed swells 
and the radicle protrudes but I don't know how 
well this works (e.g., no quantitative data)."

Jim Tokuhisa provided a useful reference:

>Here is a relevant citation.  I have not looked at the article nor have
>I tried the protocol but the abstract covers the important bits.
>Usmanov,P.D. (1999). Aging of Arabidopsis thaliana seeds and its
>overcoming. Russian Journal of Plant Physiology 46, 423-425.
>Ref ID: 18276
>Keywords: nail/Arabidopsis/seed/viability/cytokinin/Lazarus
>Reprint: Not in File
>Abstract: The viability of seeds of Arabidopsis thaliana (L.) Heynh
>seeds (race Enkhein) declined in the course of their storage at room
>temperature. In the eighth year of storage, the germination percentage
>of Arabidopsis thaliana seeds collected from plants grown in soil was
>50%, and for plants grown on agar medium in tubes, 22%. In both cases,
>seeds fully lost their capacity for germination by the ninth or tenth
>year of storage. Fertile plants were obtained after the treatment of
>such seeds with 1 mg/l. kinetin or after the addition of 1% yeast
>extract to the agar medium. A. thaliana seeds stored for 11 years or
>longer did not regain their capacity for germination even after
>treatment with kinetin, yeast extract, a mixture of amino acids,
>vitamins, and purine and pyrimidine bases. Thus, during long-term
>storage, irreversible loss of seed viability takes place.
>Jim Tokuhisa, Ph.D.
>Assistant Professor
>Horticulture Department
>Virginia Tech  (0390)

Jyoti Shah and Eva Huala suggested physical assistance:

>In the past I have had moderate success with 
>using flat tweezers to gently break open the 
>seed after it has been sitting on MS plates for 
>3-4 days.
>Dr. Jyoti Shah
>Associate Professor
>University of North Texas
>Department of Biological Sciences
>In a former life when I was working with GA 
>mutant seed and I wanted to germinate them 
>without GA treatment I would sterilize them, 
>imbibe them and then carefully remove the seed 
>coat with tweezers and leave them on an agar 
>plate where they would begin to grow.  I don't 
>know if this would work for old seeds though, 
>maybe it would be too stressful.

A number of people suggested various means for 
use of GA to promote germination:

Jerry D. Cohen
>I am not sure about the scratching working with 
>older seeds, but here my colleagues use GA4 to 
>induce germination with Arabidopsis in total 
>darkness (works better than GA3).  From our work 
>years ago with long dormant seeds, I would guess 
>that would be your best bet. Regards, Jerry

>Yes, of course, I should have said 'my 
>colleagues who wish to germinate seeds in 
>darkness' use GA4.  Light + GA is better.  I 
>will add that GA4 is also preferred over the 4+7 
>mixture, but either is choice is better than GA3.
>Thanks for the chance for clarification, Jerry

>Typically 100 uM is what they are using here. 
>That is the level that gets good germination of 
>new seeds even without light.  For older seeds I 
>would use that amount with light treatment as 
>well.  Hope this works, Jerry

Peetambar Dahal
>This would be quite an achievement if someone could make seed(s) that
>might have lost viability. Since germination phenomenon of normal
>distribution has been described in mathematical terms, it is possible
>some of the seeds in the population may still be viable and might
>respond to 1-100 uM GA(4+7) treatment after cold stratification. If
>there are enough seeds, treatment with ACC, careful incision at the
>micropylar end might help. I have not tried this in Arabidopsis but hope
>that our experience with tomato and lettuce might to Arabidopsis as
>Light should be better than total darkness to 
>enable germination of the viable fraction of 
>seeds in the presence of GA4+7.

Luis Lopez-Molina provided the most detailed 
response suggesting GA, and possibly the addition 
of sucrose and norflurazon as well.

>Dear Dr. Gasser,
>Everything you say is fine.
>Avoid pots if you have a delicate seed batch.
>I would recommend that you don't drown the seeds 
>in a GA solution. Rather, have the GA in the 
>plate (10µM is plenty). 1% sucrose helps.
>GA should obliterate the need of scratching the 
>seed coat. However, if they still don't 
>germinate it makes sense to try to mechanically 
>remove the seed coat:  dissect them yourself 
>with fine forceps (instead of the carborundum 
>powder). We perform dissection experiments 
>routinely, if you have some seeds we can try to 
>help you with pleasure.
>A last thing you may try is to add norflurazon 
>to the medium (100µM). If you dissect the seed 
>and have it in a medium with GA and 
>norflurazon....then your embryo is most likely 
>I can't think of anything else. We store the 
>seeds at 4C and they last years.....some people 
>store them at -20C (I have never tried).
>Best wishes
>Luis Lopez-Molina

We will likely try the GA4 in plates first I and 
pick at the seeds if they appear to swell but not 
split the seed coat.

Best wishes,
Chuck Gasser

Charles S. Gasser
Section of Molecular and Cellular Biology
University of California
1 Shields Ave.
Davis, CA  95616

csgasser from ucdavis.edu
Tel. 530 752-1013
FAX 530 752-3085

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