I have 4 lines of Arabidopsis which I am trying to compare under salt stress conditions. I sterilize the seeds using 70% ethanol and 50% bleach and then washing with water 7-9 times. Incubation is at 25°C day and 22°C night 16:8 cycle.
The first time I tried this, all plates showed nice growth.
I have tried replicating the same again about 6-7 times, but it has not been successful. Just one of the plates shows some growth whereas others don't show any growth even after prolonged incubation.
Please someone guide me.
This can be due to several reasons. May be the carbon source in your media was charred due to excess autoclaving or you had over-exposed the seeds to bleach while sterilization.
Hi Zahid Jawed ,
50% bleach is a very high concentration. I usually use 3-4%, if I use bleach. I prefer to use 70% EtOH for 5-7min though, followed by a wash with 100%EtOH.
If you kill the seeds with the bleach, there should be no germination at all. So I would rather think that it either has something to do with your salt concentration (being to high or something), or with your carbon source, as
Aditya Banerjee mentioned. Another thing connected to this might be the pH of your media. If your sugar caramellized, the pH of the media can drastically change and then you would have two types of stress on top of each other.
Teresa Wulf Thank you for the wonderful explanation. It seems the bleach is also one of the factors i should remove from the equation.
Hi Zahid Jawed!
As it has been said the bleach concentration you used seems to high. It also depends on the source/type of the bleach you use. Different manufacturers have different products. In our lab we use 3% for sterilizing tomato seeds from a 12% or 6% stock solution by manufacturer X, while up to 30% works from a stock manufacturer Y. The Y produce is commercial bleach we use at home. I use 70% EtOH for 1-2 min followed by the bleach treatment, but there are seeds very sensitive to EtOH. I suggest that: 1. You 1st check what optimum concentration and duration of sterilization is enough not to kill your seeds. This has to be done on plates without salt (control plates) 2. I think you need to use 1/2 MS, to reduce stress from the MS itself. In the following link I don't see EtOH. Article Standardized Method for High-throughput Sterilization of Ara...
Hi Zahid Jawed!
In the M & M of this article you'll get a better a pieace of information, how they sterilize and grew Arabidopsis seed. Article A Simple Plant Gene Delivery System Using Mesoporous Silica ...
Good Luck
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