The efficiency of surface sterilization procedure was checked for each sterilized plant segment following the imprint method.
The most known and standard isolation procedure is the surface sterilization followed by plating of small sterilized piece of plant material onto nutrient agar [28, 29].
Seeds were treated with 4% NaClO solution, 75% ethanol and sterilized water in turn for surface sterilization, then sowed on MS culture medium (1×MS salts, 1×MS vitamins, 3% sucrose, 1% agar, pH 5.7).
Between the control egg masses and the sterilized egg masses, no significant differences were found in time to hatching (control, 4.1 ± 0.5 days, n = 182; sterilized, 4.1 ± 0.4 days, n = 180) and hatching rate (control, 95.4 ± 6.1%, n = 14; surface sterilized, 97.3 ± 4.0%, n = 14), indicating that surface sterilization of eggs did not affect the embryonic development of the insect.
Twelve pods were allowed to dehisce naturally and seeds were removed for surface sterilization by gently tapping the dehisced pod in a sterile Petri dish.
To verify proper surface sterilization three 0.5-cm root pieces of each individual plant were excised and surface sterilized as described above and incubated at room temperature on potato dextrose agar plates.
To evaluate the efficacy of the surface sterilization method the water used to rinse the tissues after surface sterilization was plated on selective medium and was incubated, too.
Regarding the surface sterilization technique, the collected plant must be processed immediately after collection.
After surface sterilization the seeds were treated with cultures, F1, G2 and FC, GC respectively.
This indicated that the five-step surface sterilization protocol was effective at killing the epiphytic microorganisms.
Surface sterilization steps are normally performed to ensure the elimination of surface microorganisms.
