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

Abstract

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 fungus proliferation on disks impregnated with protein extracts of non-transgenic plants. The results of the present work demonstrated that the expression of the NmDef02 defensin in transgenic rice plants is effective against the phytopathogenic fungus Sarocladium oryzae under in vitro conditions. Thus, NmDef02 defensin could be a useful tool for J-104 rice improvement.