Hi Tomáš,

A couple of things you might want to try:

- The reddish color comes from anthocyans produced by the seedlings in response to stress; it is commonly observed if you have high amounts of sugar in your medium (which doesn't seem to be the case here, as you don't mention it) or from light stress; check the conditions of your growth chamber for light intensity and light/dark cycle (what timing do you use?).

- Another cause for stress can be the pH. Do you adjust the pH of your MS medium, if yes what pH do you adjust to and which buffer do you use (MES etc)?

- You can synchronize and enhance germination of your seeds by stratification; just after sowing in medium, keep the plates in the fridge for two to three days before transfer to growth chamber;

- After sterilization with EtOH, you shouldn't keep the seeds for long before using. Can you be more specific in respect to "elongated EtOH wash"?

- You can complement the medium with 0.25% sucrose to boost their growth.

Some more questions: Do you grow you plants in solid (with Agar/Agarose/Phytagel?) or liquid medium? How many is a "small fraction"?

Also have a look at here:

http://tinyurl.com/lpavv39

for more tips.

Hope that helps and good luck with your experiments,

C j

In this case, stratification seems to be problem. Try this as suggested by Jens Maintz above.

In no particular order;

Avoid excessive contact with ethanol; it beaks dormancy and kills the seed. We use bleach to surface sterilise seeds - see previouse posts on seed sterilisation or try Wilco Ligeterinks' dry sterilisation method.

Try 1/2 MS this is sufficient. We also adjust the pH to 6.0 before autoclaving it the drops to 5.8.

Avoid sucrose unless using at low concnetration as suggested above as you will introduce stress and will get increased bacterial growth.

Try stratifying plates in the dark at 5C for two days before transfering to growth conditions that also helpd to redmove residual dormancy and synchronise germination.

As a substrate agar is best avoided, agarose (0.7%) or phytogel are better.

Hi Tomáš,

we have a similar problem and with bleach-water sterilisation the germination is better. If seeds are old that can also be a problem =)

Hi Tomáš,

If you do go down the bleach-water sterilisation route be careful about how long the seeds are in the bleach for, I would recommend no more than 5 minutes, and make sure that the bleach has been completely removed before you sow your seeds. We check this using the blue paper towel which just happens to turn pink in the presence of bleach. Otherwise pH paper can be used to confirm.

For short term storage (a couple of years), room temperature is perfectly fine. (Longer storage I would recommend 4 oC).

I normally wait a week after collecting before sowing my seeds, and then I would always do a 2 day stratification step at 4 oC on the plates, before moving to the growth room.

Hi,

In my lab, Arabidopsis seeds are surface-sterilized in 70% ethanol for 1 min followed by 1% NaOCl for 10 min. Then, seeds were washed four times in sterile distilled water, mixed in molten 0.1% water agar (Biowest), and plated on top of solid

Murashige and Skoog medium with 1% sucrose. The Petri dishes were sealed with Parafilm, incubated at 4 °C in darkness for 2–3 d, and then moved to a growth room and incubated at 22 °C under 12 h of light and 8 h of darkness per day.

The seeds will be fine for years if a drying box is used for strorage at room tepmprature (25 °C).

Hi Thomas,

You are seeing anthocyanin production = stress response. I could be the medium or the growth conditions.

Seed sterilization: work in sterile conditions. Sterilize the seeds by vortexing 100-200 seeds in 1mL of 10% bleach for 2 min in a 1.5 mL epp tube . Spin down and repeat twice. Spin down and wash with 1 mL sterile water three times. To coordinate germination, you can leave the sterile seeds in water at 4C for 1-2 days.

MS Medium: add 0.5 grams of MES per Liter of MS medium plus vitamins. Adding 5 grams of sucrose per Liter (0.5% sucrose) of MS helps the seedlings to start growing. At this point the pH should be close to 4. Add KOH to bring the pH to 5.7. After that, add 7 grams of Phyto-agar or agarose per liter of MS. Do not use common bacteriological agar!!!

Incubate the plates at temp between 18-22C in a light cycle of 8 to 16h (do not use constant light). The light intensity should be somewhere between 50-100 100 μE.m-2.s-1

Perhaps the below article may further help.

Ann Bot. 2011 Oct;108(5):933-44. doi: 10.1093/aob/mcr198. Epub 2011 Aug 5.

Seeds of Brassicaceae weeds have an inherent or inducible response to the germination stimulant karrikinolide.

Long RL1, Stevens JC, Griffiths EM, Adamek M, Gorecki MJ, Powles SB, Merritt DJ.

Hi, thanks for all the answers and suggestions.

First update on my seeds - they look better, they are not so reddish, at least those which manage to grow, but the germination rate for WT is stil 50%, the rest of plants either does not start to grow at all or the plant stays very tiny

So my complete protocol:

MS (4.3 g/l of Basal Salt Mixture NH4NO3 free)

1% sucrose (I just realised we add it before autoclaving, is that OK?)

0.6% phytagel (we used agarose for plants previously)

MES 0.5 g/l

pH 5.7

after autoclaving, antibiotikum is added (in this case 25 mg/l hygromycin) and poured

###

so the sucrose seems to be quite high, in comparison to your suggestions, but as I do not have the reddish plants in my last batch, that doesn't seem to be the problem

###

the seeds are sterilized as follows:

5 min 70% EtOH

7-8 min 5% bleach + 0.5% Triton

washed with water 5-6×

taken up in 96% EtOH and spread over filter paper and left to dry

###

this is what I meant with the prolonged exposure to EtOH, either with the sterilisation with 70% EtOH or then the spreading with 96% EtOH as it takes some time until it dries

###

afterwards immediately put on the plates with toothpick, the plates are sealed with some permeable tape, left in fridge for 2 nights and put into fytotron to grow

###

the fytotron is used by other people and they do not seem to have the problems, so I do not expect that to be the cause

However, I was thinking about two/one thing. As I put the seeds on the agar (OK, phytagel), sometimes I bury them a little below the surface (like 1 or 2 mm or something like that) so if they start growing and they are immediately on light (as the agar is transparent), they start growing leaves and everything already inside the agar.

What about to leave the plates covered for one or two days?

Hi,

Firstly your growth media, although not what I would use (1/2 MS, no sucrose and KOH to get the pH to 5.9) your plants should still be growing well, I would suggest increasing the pH a little as it drops again after autoclaving. There is no harm adding the sucrose before autoclaving.

The hygromycin concentration for Arabidopsis is fine, but you will notice that your plants will grow a lot slower and have a lower germination on hygromycin than just MS and it will take 7-14 days before any selection is actually noticeable. I would suggest testing your protocol without hygromycin to see the germination rate and growth to make sure everything else is good.

I do my sterilisation the other way round with 20% bleach for max 3 mins (0.75ml), add 0.75ml 0.05% triton H2O mix for max 3 mins, then pour away excess liquid. Wash 5-6 times with 0.05% triton H20 until all the bleach has been removed. 70% EtOH wash for approx 3 min, then 70% EtOH again and pour immediately onto filter paper to dry. The contact with EtOH that you are using should be fine for seed viability, and by only using 5% bleach and then 5-6 washes it is unlikely that you have bleach contamination. The Triton concentration may be a little high and there there is no harm reducing this.

Burying the seeds a little bit below the surface should not be a problem as there will still be a gap (where you placed the seed) for the leaves and shoot to grow through. Where they are noticeably buried you could use the tooth pick to make a slightly bigger hole above them as you do this? Being buried and surrounded by hygromycin may be exaggerating the problem of their growth on hygromycin anyway. I personally always place the seed on the top of the agar... we either scatter the seeds from the filter paper using a scalpel to gently remove them from where they have dried (if they do not need to be lined up for root analysis), or we do the sterilisation with just bleach and water, and then using a leaky pipette, pipette the seeds onto the agar using a P200 (there is a knack to this) so that the seeds are in the surface on a drop of water.

Good luck :)

Yeh, I mentioned the hygromycin, just to have it all in one place, but the 50% of WT is meant on MS without antibiotics. On hygromycin it doesn't grow of course. And the mutants have germination rates between 10 and 60% (for three lines I've tried last time) on plates without antibiotics.

That's the problem, that I need to count how many plants germinated to determine which plants are homozygous.

Hmm yes that doesnt help at all to determine homozygous lines. However if you grow them both on MS and on Hyg plates then you could compare the two to determine this. Also they should still be germinating on Hyg plants whether they are resistant or not, so counting the seedlings at a young age, approx 5 days to see how many are growing and then again after the selection kicks in (normally 7-14 days), you should get an idea if they are segregating or not. If you are just looking for segregation, then you can scatter the seeds directly onto the surface and see if that helps germination as well.

It does not seem like you are doing anything that should case the low germination, increasing the pH and lowering the triton content may help. Sometimes it can be the seed condition itself that is affecting the seed germination, if the parent plants were stressed (heat, amount of water) when setting seed you can have a reduction in germination.

What should be the difference between the 5 and 7-14 days?

Early on you should see everything germinating (or 10-60% in your case), these should grow normally albeit slowly and should start green. After 5 days everything is often still green depending on the seed density ( a lower seed density will start to die earlier than a high seed density). From 7 days onwards you will start to see white seedlings as the sensitive seedlings are dying, and it should be obvious which seedlings will survive and which will die (resistant seedlings will have longer roots at this point), by 14 days everything that is sensitive should have died (white or yellow and very small) while the resistant plants will be growing (slower than they would on MS though) with a good root system and a healthy green colour

One more thing Alison. You wrote, that you spread the seeds in 70% EtOH on filter paper. Does it evaporate quickly enough? I have spread it in 96% EtOH to speed up the evaporation and limit the contact with EtOH.

The ethanol evapourates off very quickly in either case, the 30% water in my case normally takes a little longer, I normally leave them to dry for an hour of two (depending on seed density, the more seeds the longer it needs to dry) before plating, and I never have any problems. Another trick which you probably already do is that I put blue paper towel down and then put the filter paper on top of these. The blue paper towel soaks up the excess liquid letting the seeds and filter paper dry quicker.

we used Arabidopsis plantlets to find out if they are capable of starting mycorrhiza.

Thus we used the medium M for the growth of the fungus, and sew the Arabidopsis on those plates. Medium M is low in the nitrates and has 1% sucrose and o.2% of phytagel, and pH 5.5. The normal seeds grew very well on this.

Alison's sterilisation process is somewhat similar to ours, thus I don't need to specify.

Adress your quest to Kim Gamburg please, he is most experienced in this area. his email is [email protected]

So I've tried a new experiment. I took the same WT seeds as last time, three media plates and tried three protocols for sterilization:

the media plates:

MS - w 1% sucrose, the plates which I used the first time. As it turned out, the MS I used was NH4NO3 free and because of that it was basically 2x MS (I used 4g/l as for normal MS, while it should be only 2g/l)

1/2MS - plates I prepared in accordance to your advices - 0.5% sucrose, pH 5.9, however, again it was NH4NO3 free, so it was basically 1x MS

MS plates fro a friend

the sterilization procedures:

long - 5 min w 70% EtOH, 10 min with bleach, 5x H2O

short - 3 min w EtOH, 8 min w bleach, 5x H2O

reversed - 3 min w bleach, 5x H2O, 3 min w EtOH (as somebody here wrote)

after one week, I see basically no effect of sterilization (maybe the reversed one has little lower germination rate), but the medium has great effect. The MS from friend had germination rate almost 100% and all the plants are nice and big. The 1/2 MS has a little lower germination rate, but once it grows, the plants are more less OK. On the other hand, the old MS medium had the lowest germination but most importantly about half of the plants was small and reddish. So I assume the nitrogen is important for the germination, but the sucrose is the stressor.

The first time I did it, I counted only the good-looking plants as germinated. Now I'm aware that I should count everything that grows at least a bit into germinated plants.

@Allison - you wrote you keep the seeds to dry for some hours. I always keep them just few minutes, untill they seem dry. Could that be problem for the germination as well?

(the last time I left them for longer, so maybe that's why they germinated so much)

A longer wait for the EtOH to dry would help. I used to sterilize seeds by adding 70% EtOH for 15 min (in 1.5 ml tubes on a rotator), then 95% EtOH for 5-10 min (again, rotating). I would let the seeds dry completely by pipeting them (EtOH and all) onto filter paper in a sterile hood. The EtOH will sterilize the paper. I would store the seeds in petri dishes (still on the paper) and use a pipet tip to transfer them to growth plates.

Also, if the plants are germinating but are reddish in color that is normally a sign of plant stress. What kind of growth conditions are you using? Too high of a temperature with a high light intensity can lead to reddish/purple seedlings. Arabidopsis germinates well at 25 degrees C.

I am glad that you have got to the bottom of the issue with the MS medium :).

The longer wait would have ensured that all the EtOH would have evaporated off and therefore would not have a negative effect on the germination. Depending on the density of your seeds a 20-30 minute wait to dry them to up to 2 hours is required. As I often scrape my seeds off the filter paper by lightly touching them with a sterilised blade I can tell by eye if they have dried properly. If they fall off the paper with barely a touch they are completely dry and ready for plating, if the seeds are clumping or sticking to the filter paper or blade then they require more time. You will hopefully be able to tell this when you use your toothpick as well so you can work out the optimium time required in your flow hood.

Hopefully everything will go smoothly from now on :)

Wow, they really got off the paper much easier when I left them for longer time to dry. I thought they just stick to the paper somewhat irreversibly due to the EtOH. And it's also faster when I don't have to fight with every single seed to release it off the paper :)

I have had good success germinating arabidopsis using the following procedure:

Plates: 1/2 MS with no sucrose, 1% agar, and pH to 5.7

Surface sterilization of seeds: 2 baths of 70% EtOH followed by three rinses using ddH2O.

After I am done plating the seeds I stratify the seeds on the plates in the dark at 4 degrees Celsius for three days prior to putting them in the growth chamber. This procedure typically produces 100% germination after 7-10 days in growth chamber.

When I store my seeds I make sure to poke a hole through the lid of the tube they are in to allow moisture to escape. I also keep them in the dark. 

I use the following protocol for my experiments: 

Surface Sterilization: 

1. 1 ml 70% EtOH and vortex gently for two minutes. Discard the supernatant. 

2. 1 ml sterilization solution (20%NaClO+1%SDS) and shake it horizontally at 200 rpm on a tabletop shaker. 

2. Wash the seeds 5-6 times with DW or DI water. 

4. Transfer them to Hoagland agar. 5. two days in dark at 4

5. two days in dark at 4 degrees and transfer them to the growth chamber (23degree,) 

Hi there,

I think nutrient addition is important. Vitamins are essential for the Arabidopsis seed germination, because they nearly do not have bran in seeds. The bran contains vitamins and minerals and more. It is also a source of phytic acid (myo-inositol mainly) and antinutrients. Due to the poor presence of bran content, during seed respiration, the germination capacity is quickly lost (depending on the storing conditions mainly). Germination is also a high energy consuming process. So vitamin and salt supplement is important for long term seed operations. So, try to use MS medium with suitable salts and vitamins (Gamborg's B-5).

And I found a poster that will summarize most of the porbs in germination probability. Hope it'll help smhow. (http://arabidopsis.info/information/kill.pdf).

good luck

Hello!

we have a better germination rate using bleach sterilization (1/4 v/v bleach in water and 0.1% tween) for 5-8 min mixing time to time. Wash the bleach using sterile water for 4-8 times.

In the media we usually add gelrite and only in rare case agar, but usually the germination rate is not affected.

If all the plants are reddish I will also double check the temperature in the growth chamber.

I hope it helps

Tomáš Hluska Hi!

If you still have problems growing Arabidopsis on MS plz notice me. I am growing a lot of them (WT, mutants) on MS and long time ago I went through many problems growing nice plants. But now i have good experience, enough to help you with your problems (growing green and nice up to 3 weeks in Petri dishes). Because there are a lot of answers to your question so i am not sure you will see my answer.

Hello everyone,

 can please someone tell me the  clear portocol of (MS) for Arabidopsis ?

hi,i am trying to grow transgenic arabidopsis on half MS plates...but its not giving fruitful results..germination is little better but seedlings are very much fragile and these higly susceptible to contamination...as water drops become in petries...and their growth is very slow

my protocol is...

2.216g/l MS

30g/l sucrose

2g/l phytagel

ph 5.8

seed sterlization protocol

seeds are washed with 1ml 70% ethanol by vortexing for 1min and removing Etoh.

then washing with autoclaved distilled water three times and then drying on filter paper and plating seeds by toothpicks..

stratification by placing petries in 4c for 2 days.

and then placing in growth chamber...

kindly suggest what should i do?

Hi Dureschehwar, is there any contamination on the plants themselves (just seeing if sterilisation method and making plates is fine?).

There shouldn't be water drop within the petri dishes till quite late in seedling development, so maybe you are not leaving your plates or seeds long enough to dry properly? To help the seeds dry I often do a final 70% EtOH as the last step and quickly pour them onto the filter paper. When you are stratificatying the plates do you leave them in the fridge agar up? (this will also prevent water droplets from forming).

I would also avoid vortexing the seeds, instead use a smaller amount of seeds within the eppendorf (approx 1-2mm high - this will be hundreds of seeds). Then invert the tube at least 8 times to clean them. When looking at transgenic lines (T1) I often do a number of these tubes per line. I assume you sterilise the toothpicks? Personally I sterilise a scapel blade and use that to lightly sprinkle the seeds from the filter paper onto the plates - if they are completely dry then they will just fall off the paper as you touch them?

Good luck :)

As per your storage is concerned, It's better to put it in Eppendorf tube and then keep at freezing temperature preferably at -20 C.

hi Alison....thanks for your suggestions...i use autoclaved toothpics ....and stratify plates in 4c fridge.....and then vapors form on petricover and then i place them inside growth chamber at22 they become little water droplets...is this due to high humidity?and in growth chamber there is 55 humidity.....

Dureshehwar Saleem, what kind of problems do you still have with growing plants?