Transformation of tobacco and tomato with a fungal endochitinase gene and assays for resistance to nematodes and fungi

Four different constructs containing the endochitinase gene from the biocontrol fungus Trichoderma harzianum were used to transform tobacco and tomato. Plants transformed with pStudI (double 35S CaMV promoter, AMV enhancer, cDNA, fungal signal peptide) displayed up to 220X control endochitinase activity and had the expected 41 kDa band in Western blots. The highest chitinase activity was in roots and older leaves of transgenic plants. Enzyme activity of progeny and primary transformants was correlated.; T₁ tobacco seedlings were inoculated with Meloidogyne hapla eggs in two experiments. Significant differences in number of hatched nematodes and egg counts were found between some transgenic lines and controls but were not consistent for lines tested in both experiments.; Disease development caused by the fungus Rhizoctonia solani on T₁ tobacco in vitro was delayed up to six days; transgenic plants had somewhat lower disease severity scores. No increased resistance was found in transgenic tobacco grown in R. solani-infested soil. T₁ tomato also showed no improved resistance to R. solani in vitro or to Fusarium oxysporum f. sp. lycopersici and to Alternaria solani. It was concluded that the increased levels of endochitinase in the transgenic plants are not sufficient to enhance resistance to nematodes and fungi.; Calli obtained from tobacco transformants expressed high levels of endochitinase activity. Endochitinase was detected in the medium surrounding calli and in the medium from transgenic cell suspensions, indicating that the enzyme is secreted from cells. Medium in which the protein concentration was increased by ammonium sulfate precipitation inhibited germination of Penicillium digitatum spores. Some inhibition was also observed in concentrated control suspension, probably due to secretion of endogenous chitinases and glucanases. Endochitinase levels in concentrated transgenic medium were at least 20 times higher than in transgenic tomato and tobacco plants; however, even the concentrated medium did not completely inhibit germination of P. digitatum spores.