Hi, Ana, how old are your seeds? How they look like?And what do you mean as "normal embryo"? If these  10% seeds what germinate have a normal embryos (normal cotyledon and normal roots structure), and your line is homozygotic, the rest embryos should be normal as well. I would suggest to generate new seeds ( it take less than 2 months) and use new generation. I hope this will help.

Hi,

Has that line naturally such a low germination rate? Why do you think that it could be due to embryo morphology, and not to a physiological limitation (apart from GA)?

If you are not sure about the age or quality of the seeds, I would try to grow the plants you get to obtain another generation of seeds.

Cheers,

Ruben

Dear Lopez,

did you try to geminate the sesds on the artificial nutrient medium e.g. 1/2 MS?

Thank you everybody for your answers,

My seeds are 3 months old so not too old. They are different genetic lines generated by different treatments. I´m doing analysis in different generations so I cannot go to the next generations because I´m trying to characterize this generation (next one is genetically different). I have my control lines that were treated in the same way (same growth cabinet, same time and flowering conditions, same time of seed collection and storage....) and the germination is always 100%. Because of that I know the inhibition of the germination is a phenotype related with my treatment (actually I have different treatments differentially affected in germination).

I have tried to germinated the seeds in different mediums including MS and for example the GA and long stratification assays were done in 1/2 MS. I didn´t increase the germination with any treatment. Because of that I started thinking that maybe the seeds that don´t germinate have aborted embryos or any other defect in the seed (I have never worked in seed development). The seeds look the same as the control lines for me (looking at them using a stereo-microscope I can not tell them), same color, same size...

Can anybody imagine a reason for that low germination rate? Does anybody recommend any staining or other method to check the embryo?

Thanks again for all your attention and help,

Ana   

Dear Lopaz,

You can perform this Test for embryos germination.

Tetrazolium test:

Soak seeds overnight in deionized water. A solution of 2, 3, 5 triphyenyl tetrazolium chloride add to water to prepare 1% tetrazolium salt solution.

Incubate the seeds in this solution at 37°C for 30 min.

After incubation, remove the seed coat and red embryo will be red observed.

Red color means seed (embryo) is still alive

Ana,

Could you send about 100 non-germinated seeds to me, and a photo of a normal seedling? I´ll send all of them back to you.

[email protected]

Thank you very much! I´m looking forward doing it!

I also recommend TTC test for testing if your seeds are viable or not. We use 0.6% 2,3,5-triphenyl tetrazolium chloride for testing seed viability or dehydrogenase activity (you may refer to our paper that is likely to appear tomorrow i.e. 3rd September, 2014 in PLoS ONE for details of this test). All living cells possess dehydrogenases and hence embryos of all viable seeds turn red. In case embryos of all or majority of your seed turn red, then it is likely that embryos in seeds of lines you are testing might not be fully mature/developed or have some developmental defect. GA helps in overcoming dormancy and generally we do not experience much dormancy in Arabidopsis.

Dear Ana, If your non germinating seeds are alive (tetrazolium test will give the answer) they could have a strong dormancy. In this case there is other ways than GA treatments which have been described to stimulate the germination of Arabidopsis dormant seeds . You will find them easily in detail in literature, for instance longer stratification, or scarification or afterripening, or NO3, or fluridone treatments.

Regards

Hi, Ana, do you compare different lines (mutants?) after treatment? Which treatment have you used? At which stage? It looks like your treatment have some effect on embryo-genesis or post-embryonic development. You can do an easy test: embryo isolation after 7-12 days after pollination. This test will show you viability of the embryos and it morphology. If during formation embryos are OK, it just mean that the problems are really maturation or realization from the dormancy.   

You may try low levels of IAA or NAA (1 to 2 uM) as suggested by Liu and co-workers. You may consult attached PNAS paper for details.

If you want to see the embryo inside the seeds, you can use the technique of clearing and staining method given by Stelly et.al., 1984, Stain technology Vol. 59: 155-161. or  by Young et.al., 1979, Can. J. Bot. vol. 57: 1668-1672. Worked with tomato seeds.

What ecotype are you using?

And on what medium are you sowing ?