ORIGINAL PAPER A direct method for genetically transforming rice seeds modelled with FHVB2, a suppressor of RNAi Sudhanshu Sekhar Das • Neeti Sanan-Mishra Received: 15 May 2014 / Accepted: 21 August 2014 / Published online: 4 September 2014 Ó Springer Science+Business Media Dordrecht 2014 Abstract RNA interference (RNAi) is a novel method of gene regulation and one of the potent host defense mech- anisms against viruses. It however acts as a deterrent in transgene-technology by constraining the expression of the introduced gene. The virus-encoded suppressors have the ability to restrict host RNAi to promote pathogenicity. They thus have tremendous potential to ameliorate low transgene expression and have important applications in the biofarming sector. Unfortunately the suppressors sev- erally reduce plant regeneration potentials in the standard procedures. In this study, we report a simple, fast and efficient method for in planta transformation of rice seeds that can be used for over-expressing FHVB2, a well- characterized suppressor of RNAi. The protocol involves agro-inoculation of embryos without vacuum infiltration or injury followed by their growth ex vitro. Following trans- formation the transgene integration, expression and stable inheritance was confirmed. We observed that the FHVB2 transgenic survival in this methodology was 15-fold higher compared to that in available callus-based methods. The protocol has the potential to be extended for transforming rice with any gene as exemplified by the use of control constructs. Keywords Rice seeds Á Transformation Á In planta Á RNAi suppressors Á FHVB2 Abbreviation FHVB2 B2 protein of Flock House Virus Introduction The transformation of plants has emerged as a key research tool in the scientific understanding of plant biology and for improving existing varieties. Agrobacterium mediated transformation remains a powerful and preferred method for genetic modification of plants. This is due to its high efficiency of transformation, integration of small numbers of copies of T-DNA into chromosomes, minimal rear- rangement of transgene and transfer of relatively large segments of DNA upon transformation (Birch 1997; Han- sen and Wright 1999). Rice, the staple food of more than half of the world’s population, has emerged as an ideal monocot model system because of its commercial value, relatively small genome size and close relationship with other important cereal crops. In mid 1990s, rice transfor- mation via soil bacterium, Agrobacterium tumefaciens, emerged as a powerful tool to introduce foreign gene(s) into rice (Hiei et al. 1994, 1997). Since then several protocols and their modified versions are available for efficient rice transformation (Dong et al. 1996; Rashid et al. 1996; Toki 1997; Cho et al. 1998; Yara et al. 2001; Kart- hikeyan et al. 2012). However, the potential of the trans- genic plants are limited by unpredictable levels of transgene expression between independent transformants and in passage to subsequent generations. This is mainly Electronic supplementary material The online version of this article (doi:10.1007/s11240-014-0604-3) contains supplementary material, which is available to authorized users. S. S. Das Á N. Sanan-Mishra (&) Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India e-mail: neeti@icgeb.res.in S. S. Das e-mail: ssdas@icgeb.res.in 123 Plant Cell Tiss Organ Cult (2015) 120:277–289 DOI 10.1007/s11240-014-0604-3