Micropropagated plantlets of sugar cane were inoculated with the N2-fixing bacterium Acetobacter diazotrophicus.
Various modifications on the basic plant culture medium MS were made for the plant/bacteria association. The
protocol required the inoculation of the bacteria at the end of the rooting period in a medium without hormones
or vitamins, and with the concentration of sugar and mineral nutrients reduced by a factor of 10. Individual plants
were inoculated with A. diazotrophicus and maintained under the appropriate light and temperature condition used
for micropropagation up to 7 days. The system favored the infection and the establishment of the bacteria within the
plant tissue. Bacteria colonized the plant tissue and accumulated in inter-cellular cavities and the region of lateral
root emergence and also colonizes the xylem vessels. The inoculated plantlets were subsequently transferred to
the acclimatization phase and after 30 days it was possible to isolate the bacteria from plant tissue. This protocol
permitted studies of infection and comparison among strains.