Mark G. Bolyard

Expression of a modified Dutch elm disease toxin in Escherichia coli.

The fungal toxin associated with Dutch elm disease, cerato-ulmin, has been produced in the bacterium Escherichia coli by the assembly of oligonucleotides according to the unpublished amino acid sequence of the toxin. This toxin was produced at approximately 80 micrograms/L of cell culture as a fusion to glutathione S-transferase. We synthesized the toxin as a fusion protein to improve purification and stability. Recombinant cerato-ulmin was analyzed by immunoblot analysis and then separated from its fusion partner by thrombin. We incorporated this molecule into an appropriate medium to test the activity of the toxin on the growth of American elm callus cultures.

Transient gus expression in zygotic embryos ofTaxus Brevifolia

SummaryGerminated zygotic embryos of Pacific yew (Taxus brevifolia) were transiently transformed with the expression vector pBl121, as demonstrated by expression of the β-glucuronidase (GUS) gene. Embryos exhibited sectors of blue color following treatment with DNA and dimethyl sulfoxide (optimal concentration of 2.25%). However, transient expression depended on the presence of ions in the buffer, as no GUS activity was observed when deionized water was used in place of other buffers. GUS gene expression was dependent on the developmental stage of the embryo, since the frequency of GUS expression was elevated in embryos incubated with the expression vector during a period from 4 to 8 wk after culture of DCR medium. In addition, the utility of using zygotic embryos from mature seeds for tissue culture and transient expression experiments was also examined.

Genetic Transformation in Ulmus Species (Elms)

American elm (Ulmus americana) is believed to be the most aesthetically pleasing tree in the North American landscape. Its height, symmetrical crown, and arching vase shape made it a favorite of arboriculturists and nurserymen, and it was planted in great numbers. Prior to its devastation by Dutch elm disease (DED), the American elm was also prized by American and Canadian timber industries. Since the arrival of DED to North America, millions of American elms have been lost.

Refinement of physiological roles for cerato-ulmin by analogy with other hydrophobins

Cerato-ulmin, a hydrophobic protein produced by the Dutch elm disease fungus Ophiostoma ulmi, is toxic to susceptible elms. The physiological role of this protein may be related to its location on hyphal surfaces. Cerato-ulmin is a member of the hydrophobins, proteins used in the formation of structures involved in asexual reproduction (sporulation and conidiation).

Strategies for the Production of Disease-Resistant Elms

This work was supported by the National Park Service of the Department of the Interior (MBS; CA-3040-9-8003).

“Lonesome Ginkgo” Lab

Developing plant tissue culture experiences for high school students can be challenging, particularly when the instructor wants to incorporate aspects of plant anatomy into the lab. This lab describes a straightforward way to perform zygotic (seed derived) embryo culture. It involves removing developing embryos from seeds and observing their development into trees in culture (Camper et al. 1997). Students may wonder why one would want to use this method, rather than just plant the seeds in soil. There are two major advantages to this method. First, germination of the embryo is easy to observe in culture. Second, it is not unusual for seeds from certain trees, including ginkgo, to require specific environmental cues to germinate. Embryo culture can be used to remove the requirement for these cues. The plant selected for this lab is the ginkgo, Ginkgo biloba. It was selected in part because of its large, easily observable zygotic embryo. Ginkgo is also interesting for its medicinal properties. Students have probably seen advertisements for ginkgo supplements on TV, as these formulations are possibly useful for preventing memory loss (there have been some serious scientific studies looking at ginkgo metabolites as treatments for Alzheimer’s disease, for example Maurer et al. 1997). Finally, plant cell culture provides an introduction to the sterile technique required for even more sophisticated projects, such as those working with eukaryotic cells in culture. This beginning lab can be expanded to ask a variety of experi-