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The antimicrobial peptide MSI-99, an analog of magainin 2, was expressed via the chloroplast genome to obtain high levels of expression in transgenic tobacco (Nicotiana tabacum var. Petit Havana) plants. Polymerase chain reaction products and Southern blots confirmed integration of MSI-99 into the chloroplast genome and achievement of homoplasmy, whereas northern blots confirmed transcription. Contrary to previous predictions, accumulation of MSI-99 in transgenic chloroplasts did not affect normal growth and development of the transgenic plants. This may be due to differences in the lipid composition of plastid membranes compared with the membranes of susceptible target microbes. In vitro assays with protein extracts from T 1 and T 2 plants confirmed that MSI-99 was expressed at high levels to provide 88% (T 1 ) and 96% (T 2 ) inhibition of growth against Pseudomonas syringae pv tabaci, a major plant pathogen. When germinated in the absence of spectinomycin selection, leaf extracts from T 2 generation plants showed 96% inhibition of growth against P. syringae pv tabaci. In addition, leaf extracts from transgenic plants (T 1 ) inhibited the growth of pregerminated spores of three fungal species, Aspergillus flavus, Fusarium moniliforme, and Verticillium dahliae, by more than 95% compared with non-transformed control plant extracts. In planta assays with the bacterial pathogen P. syringae pv tabaci resulted in areas of necrosis around the point of inoculation in control leaves, whereas transformed leaves showed no signs of necrosis, demonstrating high-dose release of the peptide at the site of infection by chloroplast lysis. In planta assays with the fungal pathogen, Colletotrichum destructivum, showed necrotic anthracnose lesions in non-transformed control leaves, whereas transformed leaves showed no lesions. Genetically engineering crop plants for disease resistance via the chloroplast genome instead of the nuclear genome is desirable to achieve high levels of expression and to prevent pollen-mediated escape of transgenes.Antimicrobial peptides (AMPs) with ␣-helical structures are ubiquitous and found in many organisms. They are a common component of innate defense mechanisms in the animal kingdom and help to control normal microbial flora and combat pathogens (Tossi et al., 2000). AMPs have been isolated from frogs, insects, and mammalian phagocytic vacuoles (Biggins and Sansom, 1999; Tossi et al., 2000). The AMP used in this study (MSI-99) is an analog of magainin 2, a defense peptide secreted from the skin of the African clawed frog (Xenopus laevis), first discovered by Zasloff (1987).Magainins and their analogs have been studied as a broad-spectrum topical agent, a systemic antibiotic, a wound-healing stimulant, and an anticancer agent (Jacob and Zasloff, 1994). However, the possible agricultural use of magainin-type AMPs has received limited attention until recently. Li et al. (2001) have reported disease resistance, to both a fungal and a bacterial pathogen, conferred by expression of a magainin analog, M...

Exploiting the biolistic process we have generated stable transgenic bean (Phaseolus vulgaris L.) plants with unlinked and linked foreign genes. Co-transformation was conducted using plasmid constructions containing a fusion of the gus and neo genes, which were co-introduced with the methionine-rich 2S albumin gene isolated from the Brazil nut and the antisense sequence of AC1, AC2, AC3 and BC1 genes from the bean golden mosaic geminivirus. The results revealed a co-transformation frequency ranging from 40% to 50% when using unlinked genes and 100% for linked genes. The introduced foreign genes were inherited in a Mendelian fashion in most of the transgenic bean lines. PCR and Southern blot hybridization confirmed the integration of the foreign genes in the plant genome.

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