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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 T 1 progeny lines, germinated on semisolid medium without hygromycin. Relative expression of NmDef02 mRNA was examined by quantitative RT-PCR in marker-free T 1 plants. In vitro antifungal test was performed by disk diffusion assay against Sarocladium oryzae. PCR assay verified that 15.12% of T 1 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.

This paper describes a subculture system of rice calli (cultivar J-104) that maintains the potential for long-term regeneration. We examined the effects of 2.4-D (1.0, 2.0, 3.0, 4.0 and 5.0 mg/L) and time of subculture on the morphogenesis and regeneration potential. Calli were subcultured for two years on semisolid medium, in the dark. During this time, calli with similar weight were monthly transferred to the regeneration medium, under photoperiod regime. Plant regeneration took place through two morphogenic pathways: indirect somatic embryogenesis and indirect organogenesis. The concentration of 2.4-D used in the callogenesis determined the prevalence of one path or another. In the first ten months of subculture, embryogenesis prevailed over organogenesis. However, from this point to the final stages, the shoots were organogenic. Callus lines retained their regeneration potential by organogenesis until the end of the second year, without presenting a significant number of abnormal plants. These results indicate that it is possible to maintain for two years J-104 rice callus lines potentially morphoge-nic, which can be applied to a plastid transformation protocol, specifically to the design of an efficient and long-stage selection, for reaching of homoplasmy during the callus period, before the regeneration of the plants.

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