To begin, wash certified Pinus pinaster seeds in running tap water to remove larger debris. Then wash the finer debris using a common detergent solution with vigorous agitation. In a flow hood, add a commercial bleach solution until the seeds are covered and mix vigorously for 15 minutes.
After discarding the bleach solution, rinse the seeds three times with sterilized tap water. Now immerse the seeds in 80%ethanol for 15 minutes with vigorous agitation. After disposing of ethanol, wash the seeds three times with sterilized ultrapure water.
For pine germination, break the sterilized seed coats with a sterile mechanical lathe. Isolate the pine nuts and set them in a closed container with sterilized wet filter paper to hydrate overnight. The next day, stratify hydrated pine nuts at four degrees Celsius for seven days to break dormancy and synchronize germination.
Then maintain the pine nuts in the dark at 25 degrees Celsius until germination. Transfer the aseptic germinated pines to controlled environmental conditions for two weeks, or until the main stem begins developing. After two weeks, cut the aerial portion of the seedling above the root with a scalpel and transfer it to a sterilized Schenk Hildebrandt culture medium.
Maintain the culture in controlled environmental conditions as described previously. Subculture periodically by cutting the callous tissue at the base of the shoot with a sterile scalpel. Transfer the shoot cluster using sterile tweezers to a fresh multiplication culture medium.
Next, inoculate the microshoots into the Schenk Hildebrandt culture medium containing three grams per liter of activated charcoal for micro stem elongation. Now place the elongated shoots in the Schenk Hildebrandt culture medium without phytohormones or activated charcoal. Add 100 to 150 mixed life stage sterilized pinewood nematodes at the bottom of the microshoot.
Maintain the culture in controlled environmental conditions as described previously. The establishment and infection of in-vitro pine shoots showed the appearance of chlorotic needles within a month of co-culture with pine wood nematodes.
