Marker-free transgenic rice lines with a defensin gene are potentially active against phytopathogenic fungus Sarocladium oryzae

In this work it was developed marker-free transgenic indica rice plants (cv J-104) by biolistic co-transformation and segregation approach. We attempted to express the NmDef02 antifungal defensin. Primary transformants were regenerated from embryogenic callus on culture medium with 50 mg/L hygromycin. Screening of hpt-marker-free transgenic lines was made by PCR in T1 progeny lines, germinated on semisolid medium without hygromycin. Relative expression of NmDef02 mRNA was examined by quantitative RT-PCR in marker-free T1 plants. In vitro antifungal test was performed by disk diffusion assay against Sarocladium oryzae. PCR assay verified that 15.12% of T1 plants were marker-free (NmDef02+/hpt−). RT-PCR analysis indicated that NmDef02 gene was successfully transcribed and the transgenic lines displayed different expression levels of the NmDef02 cDNA. Protein extracts of marker-free lines with high relative expression of NmDef02 inhibited fungus mycelial growth around disks. In contrast, it was confirmed fun...