We have developed a protocol to produce axenic plants (plants without associated microbes) from field-gathered seed of Sporobolus alterniflorus . We can then propagate the plants under sterile conditions, producing many clones of each individual, for experiments re-combining plants with selected, known microbial partners.
The protocol requires sterile technique but is quite easy and produces axenic seedlings with high efficiency.
Protocol DOI for producing axenic Sporobolus seedlings:
Protocol DOI for confirming that seedlings are axenic:
Briefly, S. alterniflorus seeds are germinated, the glume, endosperm, and primary root removed, then they are soaked in 10% bleach. Seedlings are then grown in sterile jars in agar with MS media and rooting hormones until they develop a vigorous root system.
This protocol avoids tissue culture from undifferentiated "callus" (i.e. growing masses of undifferentiated and unorganized cells), which tends to produce many spontaneous mutations. The new propagation method more closely mimics the way plants naturally propagate themselves in the marsh.
A previous (less successful!) strategy:
Another approach was to take advantage of the natural ability of S. alterniflorus to make new shoots and roots rapidly from robust rhizomes (belowground stems). We easily grow field-gathered plants hydroponically in the lab, producing far more clean new plant material. Standard approaches such as rinsing with soap and 10% bleach solutions can then be used to reduce microbes in and on the tissues, but we found they could not produce completely axenic plants in vitro.