Dear Keli,

There is a chance that the agent needs to act on the plant at a certain time after germination. So it is useful to do it on time. As long as you have enough seedlings, you can replicate the experiment. If you have a lot of seedlings, you can do it on time and then do the same experiment after the seedlings have sufficient length roots.

It would have been easier to answer if you elaborated on what the agent you are going to use and if the short length of seedlings is due to a mutation, transformation or due to a physiological condition imposed by your growing conditions on wild-type seedlings.

Good luck!

Dear Ranjith，

Thanks for your answer first.

There is an agent can inhibit the length of primary root, and I want to know whether DPI can recover this phenotype.However,my wild-type seedlings grow slower than normal condition. It has been a long time.However,this time is too sever.

As to reasons，I think  it is due to my medium or growing conditions. So I bought a can of MS salt from Duchefa rencently. Wish it works!

Sincerely.

I agree with Ranjith. Also, if the growth rate is 'severe' slow as you stated, the medium may have problem. Use new Wt seeds if possible and find out the standard receipt for Arabidopsis medium. Commercial companies sell different forms of MS powder, some only has MS macro-nutrients, some with both macro- and micro-nutrients, some with B5-vitamin added, some with vitamins originated from Murashige and Skoog medium formula. So, Check it out before use.

Dear Kali,

As suggested by Yuan-Yu, please check the MS catalogue number you got from Duchefa. Full MS is too strong, use half strength to grow the seedlings. Many plants do not like full-strength MS, so is Arabidopsis. You need to supplement iron anyway.

Their Cat No M0221 has micro and macro elements only, so you need to supplement  vitamins. If you bought M0222, then it has vitamins included. They also supply macro separately in M035  and Micro in M0301. To grow plants in tissue culture we use Lindsmaier & Skoog vitamins (L0406) or Gamborg B5 vitamins (G0415), not MS vitamins. If the seeds are grown under cool white fluorescent light with a 14 to 16 h photoperiod at 24 °C plants should grow normally.

Regards

You may also need to check light intensity, day length and temperature. Usually we use 120-150 µmol/m2sec light intensity, long-day 21°C for Arabidopsis on 1/2MS agar plates. To get longer primary root, you may try a longer day length.Were your wt seeds also germinating slower? If the seeds are too old, extend the stratification time.

Cheers.

Dear Yuan-Yeu,

Thanks for your suggestion,  

I bought M0222 which has micro and macro elements both and vitamins included. It has been used for a long time in other labs in NKU.

Dear Ranjith,

Your suggestions are resonable. I bought M0222. And I just use half of MS salt but without MES. I don't know why should we use MES.And I don't understand why should we add iron either. Could you tell me?

Regards.

Dear Xi,

Your idea is heuristic! Though medium is the main cause,light intensity can be another important reason. My mutant's development is connect with light intensity, and we just adjust our photoperiod and light intensity for another experiment. Maybe this interfered my result. Thanks.

Regards.

Hi Keli,

You are welcome.

Sounds good, and set up half strength (x 1/2) of the medium, right pH, and right photoperiods plus temp as Ranjith and Xi suggested. I think you are good to go.. Update us later to let us know how does it go. By the way, what ecotype of Arabidopsis are you testing? A common one right?

Hi Yuan-Yeu,

Experiments are underway. Of course I will tell you the result. Wish it goes well this time!  As to your question, I am working on Col-0 and Ler now.

Regards

Hi Kia,

Thanks for your recommendation. It's a good literature to learn. I will read it.

Regards

I am sorry for to late to tell you the recent condition. It's almost become normal while using Duchefa MS salt (M0222). However, things are different on different position of incubator.  So, I can only use a part of the incubator.

Hi Keli,

You meant, the growth behaviors were different depending on the location inside an incubator (or growth chamber)? That's odd. Is it because of the different light intensity?

Hi Yuan-Yeu,

Things are just like you said. It's really a mystery. We have two incubators, the middle layer of one incubator is abnormal, but root can grow vertically. The growth behaviors were different depending on the location inside the other incubator，while roots  grew towards the light. So light can be one reason but not all.

Did you stand the plates, so the roots can grow towards the ground?