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The ability of legumes to recognize and respond to ␤-glucan elicitors by synthesizing phytoalexins is consistent with the existence of a membrane-bound ␤-glucan-binding site. Related proteins of approximately 75 kDa and the corresponding mRNAs were detected in various species of legumes which respond to ␤-glucans.

We have constructed a novel micro-projectile accelerating system for efficient gene transfer into cells in situ that avoids binding DNA to micro-projectiles and keeps the DNA in solution. Further, instead of a macro-projectile (or the equivalent), it accelerates the particles in a Bernoulli air stream. The micro-targeting approach directs highly dispersed particles to sites with diameters as little as 0.15 mm, allowing precise aiming to restricted tissues. The system is physically flexible and should therefore be adaptable to different tissues and species. Transient expression of the Escherichia coli beta-glucuronidase gene in immature wheat embryo scutella was obtained at a frequency of up to 3% of the treated cells in the surface layer. In tobacco SR1, we achieved many transgenic plants, and the efficiency of stable transformation with the neomycin phosphotransferase (NPTII) gene was approximately 10(-3) per exposed cell.

A new culture method for the injection of tobacco mesophyll protoplasts has been established. The protoplasts are embedded in a thin layer of alginate and are nourished from the medium in the underlying basis layer. In the alginate layer the protoplasts regenerate to calli at a frequency of up to 80%. Embedded protoplasts can be selected either with 50 mg l-1 kanamycin or 5 mg l-1 paromomycin. Single resistant cells can be recovered from about 10,000 sensitive cells in one alginate layer. Injection of the neo gene (coding for neomycin phosphotransferase II) into protoplast derived single cells in the alginate layer results in kanamycin resistant colonies that can be regenerated to mature plants. These plants express the neomycin phosphotransferase as shown by enzyme activity assay. The integration of the transgene into the plant genome could be proved by Southern hybridization to high molecular weight DNA. With this culture method 100 cells can be injected per hour. Transformation frequencies range from 2 to 20%. In crossing experiments, it was shown that the foreign gene is transmitted to the next generation in a Mendelian fashion.

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