|Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2003: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2002: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2001: ¥1,500,000 (Direct Cost: ¥1,500,000)
We have improved our previously published technique for plant regenerationfrom seed-derived embryogenic callus of bahiagrass by keeping the callus size small through regular partition. This modifiedculture remains regenerable for at least 18 months and results in a decrease of albino plants among regenerants. Also, in transformation experiments, an increased surface area is available for gene delivery, and a greater number of cells can potentially regenerate. Using a simple self-built particle inflow gun with improved technical features, we have optimized delivery conditions and bombarded the small calli with pDB1, a construct carrying both the GUS reporter gene and a bialaphos resistance gene (bar). Numerous calli have been selected on bialaphos-supplemented medium and subjected to regeneration conditions. These calli remained resistant and continue to display GUS activity. Shoots as well as embryo-like structures formed from resistant calli under regeneration conditions and green pla
nts were obtained.
Super roots of Lotus corniculatus are a fast growing legume root culture that allows continuous root cloning, direct somatic embryogenesis and mass regeneration of plants under entirely growth regulator-free culture conditions. These features are unique to root cultures and are now stable expressed since the, culture has been isolated almost 5 years ago. In several years of experimentation, all our attempts to directly transform super roots with Agrobacterium failed for unknown reasons. Only recently, by using super root-derived callus as acceptor tissue, we were able to demonstrate transient expression of a reporter gene (GUS) in a tissuederived from super roots. GUS expression was also, visible in roots and shoots regenerated from transformed calli. The super root character is stable maintained through the callus phase. Roots derived from these calli can serve as explants for new super root cultures. This stability of the callus phase makes it unlikely that transformation was made possible through the loss of protective genetic information, such as a spontaneous A. rhizogenes infection, through callus initiation. Less