For an experiment I want to germinate lettuce seeds. For logistical reasons it would be convenient to imbibe the seeds by placing them in the cold for 2 days, afterwards I would sterilize the seeds using dilluted bleach. I mostly see people apply stratification or imbibition after sterilization, does the order matter? Would there be a difference for cold treatment (stratification) or wet treatment (imbibition)?
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Personal anecdote: Once I planned to sterilize seeds of Silene latifolia for tissue culture but after the first step (washing the seeds in water for an hour) I had to do other things and the seeds did spend the night wet in room temperature. The next day some of the seeds were showing first signs of germination - a little crack in seed coat. I decided to sterilize them anyway, since there was nothing to lose. The result: Some seeds did germinate after sterilization and were fine. Some seeds died. Interestingly, in some seeds the bleach destroyed only the exposed root meristem resulting in rootless seedlings.
I have never worked with lettuce seeds but I would consider questions:
- How fast the seeds germinate and what conditions they need for germinating? You cannot sterilize seed that have already germinated.
- Could the inbibition change the properties of the seed coat and make it easier for the bleach to pass through and kill the embryo? I suspect not, but cannot be sure.
- Wet environment is good for bacteria and fungi growth. "Dirtier" seeds are sometimes harder to sterilize - they need longer exposure to bleach or stronger bleach concentration to completely remove contamination. Stronger bleach or longer treatment can sometimes kill some (especially small) seeds. It is hard to say weather two days of wetness can make any difference. The cold should slow down the bacterial and fungal growth.
At the end of the day I reccomend you to try with few seeds and see the result. Maybe include negative control with usual order of steps. If the survival is the same for both groops you are good to go.
