Sharon A. Bates

Thidiazuron stimulates shoot organogenesis and somatic embryogenesis in white ash (Fraxinus americana L.)

Immature and mature nonstratified seeds of white ash (Fraxinus americana L.) were dissected transversely and 2/3 of each seed was placed onto agar-solidified Murashige and Skoog medium. Adventitious buds, shoots, and somatic embryos formed on callus, cotyledons, and hypocotyls of the resulting seedlings. Shoot organogenesis was induced on explants cultured on medium with 10 μM thidiazuron but not on explants on media with benzyladenine (BA) or isopentenyladenine. Not all seed sources were equally capable of shoot organogenesis and embryogenesis. Atypical of adventitious regeneration of other woody plants, mature seed explants of white ash were more organogenic with shoots that elongated better than explants from immature seeds. Somatic embryogenesis was observed in cultures where mature seeds were first cultured for 4 weeks on a medium containing 10 μM adenine 2,4-dichlorophenoxyacetic acid in combination with 0.1 and 1.0 μM thidiazuron, followed by transfer to a medium containing 0.05 μM 6-benzyladenine and 0.5 μM naphthaleneacetic acid. Adventitious shoots and epicotyls from both seedlings and germinated somatic embryos were rooted under intermittent mist and acclimatized to the greenhouse.

Somatic embryogenesis in white ash ( Fraxinus americana L.)

White ash (Fraxinus americana L.), a member of the Oleaceae, is an important hardwood tree species and the most common native ash found in North America. The fast growing dioecious trees can attain heights of more than 30 meters and trunk diameters of up to 1.3 meters. Younger trees have a pyramidal or ovoid shape and mature trees have rounded tops. In landscaping, white ash is well suited for large yards and open areas such as parks and along roadsides. The wood is excellent for tool handles and sporting equipment because it is hard, strong, stiff, shock resistant, and lightweight.

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.