Factors Affecting Agrobacterium Mediated Transformation of Broccoli(Brassica oleracea var.botrytis)

An Agrobacterium mediated transformation system was used to evaluate some factors influencing transformation with the strain LBA4404 in broccoli varity Lv Xiu(using transient and stable transformation assays).These included the precultivation time,the bacterial density,the inoculation duration time with Agrobacterium tume faciens,the concentration of acetosyringone,the delay selection and the concentration of kanamycin in selection.Using cotyledons with 1mm petioles as explants,the best transformation parameters were:OD600 0.3 of bacterial density,five minute infection with Agrobacterium tume faciens,100μmol/L acetosyringone in the cocultivation medium.Stable integration of the transgenes in the plant genome was demonstrated by PCR and Southern hybrization analysis.Expression of the transgenes was confirmed by GUS staining.