I am trying to isolate plant growth promoting endophytes from legume seeds, however, there is often very little growth if any even in the 10^0 plate. I surface sterilise the seeds (2 washes with sterile water, 1min 70% ethanol, 2 washes, 30s NaOCl, 5 washes in excess sterile water) then macerate after ~5days. I then use synthetic malate wash buffer or PBS to grind the seedlings/ shoots before serially diluting in the respective buffer and plating onto modified synthetic malate medium or TSA plates. The plates are incubated at room temperature for 3-5days before being moved to the incubator at 30 degrees Celsius for another 48-72hrs. In most cases there is no growth and when there is growth they are a few colonies (1-5 total).
May you please advise the best way to isolate seed endophytes or how I can improve my isolation procedure?
Your sterilization methods are questionable, the alcohol and NaOCl performed the same function. I suggest you surface sterilised seeds using 1% NaOCl for one minute. Let us also know seed endophytes you are interested in.
I wish you well.
@Kouadri Mohamed El Amine thank you.
Osabutey Samuel thank you. The 2 sterilizers are used to ensure that only endophytes are isolated, but I admit they may be too harsh. I am interested in plant growth promoting endophytes.
Please check it.
Article Isolation and characterization of culturable seed-associated...
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