Procedure

A novel system of applying low levels of the herbicide glufosinate (commercially known as BASTA/Challenge/Rely) was used to inhibit GS in the root tissues of Lotus japonicus. Nitrogenase activity was assayed using a closed system acetylene reduction assay (Parsons, Baker 1996). Transgenic plants were obtained through the development of a transformation protocol in which seedling cotyledon attachment sites were wounded and infected with Agrobacterium (Chang el al. 1994). Regenerant shoots were selected and screened for transgene insertion by kanamycin resistance and PCR. Plant tissues were profiled for changes in N metabolism by MTBSTFA derivatization and analysis by capillary GC-MS.

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