In Vitro Studies with White Ash (Fraxinus Americana) Nodules
Calli were obtained from cotyledonary tissue of cut zygotic seeds that had been cultured in vitro on agarsolidified MS medium with 5 μM 2,4-D and 5 μM BA for four weeks. Seedlings were subcultured onto MS with 0.5 μM NAA and 0.5 μM BA. After two to four months, callus tissue was excised and placed into liquid MS with 1 μM NAA and 1 μM BA on a shaker at 100 rpm. Within four weeks, spherical clusters (nodules) formed that regularly sloughed-off and produced additional clusters. To speed multiplication, nodule clusters could be physically broken-up with forceps and the pieces subcultured. Nodules had a distinct morphology compared to callus clumps. Scanning electron microscopy (SEM) studies of nodule cross sections revealed a cortical region composed of parenchyma cells surrounded by a layer of closely packed cells (possibly epidermis). Areas of vascularization containing xylem and a cambium-like tissue were evident within the cortical region. Nodules exposed to moderately high levels of thidiazuron produced large amounts of callus. Nodule clusters could be removed aseptically from liquid medium and dehydrated for up to 14 days. After desiccation for various times, the nodules were placed on agar-solidified medium where they rehydrated and grew as callus. Thidiazuron in the rehydration medium stimulated callus growth. Nodules showed sensitivity to glyphosate herbicide on agar-solidified and liquid media. On solidified medium, glyphosate at 0.1 and 1 mM was not lethal, and nodules remained green; 10 mM glyphosate resulted in nodules with areas of green and brown, and 0.1 and 1 M glyphosate were lethal. In liquid medium, nodules exposed to 1 mM herbicide became brown and died, at lower concentrations, both green and brown sectors were evident.
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