Gene tagging and transgenic approaches for the study of legume nodule and root development

Experiments were carried out in the model legume L. japonicus to determine the efficiency of promoter trapping and insertional mutagenesis using Agrobacterium tumefaciens (hypocotyl) and A. rhizogenes (hairy root) transformation systems. Fourteen trapped lines showed root and nodule tissue-specific GUS expression. The trapped lines were divided into two classes based on the dependence of GUS expression upon rhizobium inoculation. Based on the results it was concluded that lateral root and nodule formation processes share at least some genetic control.; Transgenic plants of the model legume Lotus japonicus were regenerated by hypocotyl transformation using a bar gene as a selectable marker. The bar gene encodes Phosphinothricin acetyl transferase that detoxifies phosphinothricin (PPT), the active ingredient of herbicides such as Ignite (AgrEvo) and Basta (Hoechst). Typically, 15–20% of transformed L. japonicus plants are lost to tissue culture-induced sterility when antibiotic selectable markers are used. All regenerated plants were fertile using PPT selection and void of visible abnormalities. The lack of tissue culture-induced variation, ease of PPT application and low cost of PPT make this protocol an attractive alternative for the regeneration of transgenic L. japonicus.; Lotus japonicus plants expressing the dominant, negative etr1-1 allele from Arabidopsis thaliana were constructed and found to be ethylene insensitive by their lack of the triple response. The numbers of infection foci were enumerated using a Mesorhizobium loti strain constitutively expressing the lacZ gene. There was a direct correlation between the degree of nodulation and the level of ethylene insensitivity. The position of infections was examined relative to root anatomy. Approximately 30% of the nodules or nodule primordia in transgenic plants were found between xylem poles, compared to only 5% in wild-type plants. The study indicated a role for ethylene in nodule initiation by influencing root cell infection by rhizobia and position of nodule initiation in relation to protoxylem pole. The hypernodulation of ethylene insensitive plants was nitrate sensitive. It is proposed that the hypernodulation due to ethylene insensitivity is due to defective‘local autoregulation’ of nodulation.