Development of transgenic plants with non-plant antibacterial protein genes for resistance to bacterial pathogens

Recent advances in genetic engineering have made it possible to transform non-plant genes into crop plants. Some progresses have been made in the development of transgenic plants with non-plants with non-plant antibacterial protein genes for resistance to bacterial pathogens. The mammalian gene encoding human lactoferrin (HLF), an iron-binding glycoprotein, has not been used previously in developing transgenic plants for bacterial disease resistance. In an attempt to develop transgenic plants using antibacterial protein genes to enhance bacterial disease resistance, we first developed transgenic calli using a tobacco suspension cell culture to express HLF. Transgenic calli produced truncated HLF which were much smaller (about 48 kDa) than full length HLF (80 kDa). Total protein extracts made from transgenic calli containing the truncated HLF exhibited much higher antibacterial effects than calli containing the full length HLF on four representative phytopathogenic bacteria. Consequently, transgenic tobacco (Nicotiana tabacum) plants expressing the human lactoferrin cDNA were further developed using Agrobacterium tumefaciens strain A2760. All T$\sb0$ and most of T$\sb1$ lactoferrin transgenic plants inoculated with Pseudomonas solanacearum (E. F. Smith) showed a significant delay of bacterial wilt. Western blots showed differences in gene expression levels among different transgenic plants. Quantification of HLF by ELISA revealed a positive correlation between HLF and resistance levels. Additionally, synthetic bovine lactoferricin and shiva-1 were compared in vitro for antibacterial activity against three phytopathogenic bacteria. Bovine lactoferricin was more bactericidal than shiva-1 against all three pathogens. In order to introduce a lactoferrin gene into Phaseolus vulgaris, factors influencing both Agrobacterium-, or biolistic-mediated transformation of this plant were studied using a GUS intron containing construct. Factors significantly affecting Agrobacterium transformation included preculture and cocultivation conditions, explant age and maturity, host-range, and selection pressure. Using optimized transformation conditions, about 4% of total shoots or buds were putatively transformed. Parameters influencing biolistic transformation of P. vulgaris were also optimized, based on embryonated cotyledons as target tissues