ORIGINAL ARTICLE The epigenetic phenotypes in transgenic Nicotiana benthamiana for CaMV 35S-GFP are mediated by spontaneous transgene silencing Seong-Han Sohn • Min Sue Choi • Kook-Hyung Kim • George Lomonossoff Received: 17 April 2011 / Accepted: 3 May 2011 / Published online: 24 May 2011 Ó Korean Society for Plant Biotechnology and Springer 2011 Abstract Diverse epigenetic phenotypes are frequently found during research on transgenic plants. To understand the factors underlying such diversity, hundreds of indepen- dent 35S-GFP transgenic N. benthamiana plants were ana- lyzed. The diverse GFP-expression phenotypes of the transgenic plants were classified into three major types based on the GFP expression patterns and their response to 35S- GFP agroinfiltration: steady-green, silenced and non-uni- form phenotype. The non-uniform phenotype was further sub-divided into five minor phenotypes: variegated, red- dropped, on-silencing, partitioned and misty, according to the distribution of GFP expression on the leaves. Many of transgenic plants continuously generated diverse phenotypes over several generations despite the transgene identity. Such epigenetic GFP phenotyping was found to be the result of spontaneous transgene silencing mediated by either or both of post-transcriptional gene silencing (PTGS) and tran- scriptional gene silencing (TGS). This finding was verified by the detection of 21- and 24-nt small interfering RNA (siRNA) molecules, and DNA methylation in the transgenic plants that showed repeated epigenetic variation. Agroinfil- tration demonstrated that irregular distribution of GFP on a leaf was the result of erratic transgene silencing, and the technique also proved to be a rapid and effective method for selecting fully silenced plants within 3 days. Furthermore, two novel phenotypes described are potential materials for in-depth investigations into the genes and mechanisms responsible for spontaneous transgene silencing. Keywords Spontaneous transgene silencing Á Agroinfiltration Á GFP-transgenic Á Nicotiana benthamiana Á CaMV 35S promoter Á CMV 2b Abbreviations GFP Green fluorescent protein CaMV Cauliflower mosaic virus CMV Cucumber mosaic virus TGS Transcriptional gene silencing PTGS Post-transcriptional gene silencing siRNA Small interfering RNA Introduction Agrobacterium-mediated plant transformation has been extensively applied for the introduction of useful genes with the aim of creating novel traits. However, transgenes are frequently incorporated into the genome in more than one copy, and transgene silencing is often facilitated by Electronic supplementary material The online version of this article (doi:10.1007/s11816-011-0182-3) contains supplementary material, which is available to authorized users. S.-H. Sohn (&) National Academy of Agricultural Science (NAAS), Rural Development Administration (RDA), Suwon 441-707, Korea e-mail: sohnseonghan@korea.kr M. S. Choi 161 Sandspit Rd, Cockle Bay, Auckland 2014, New Zealand K.-H. Kim College of Agricultural and Life Sciences (CALS), Seoul National University, Seoul 151-741, Korea G. Lomonossoff Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, Norfolk, UK 123 Plant Biotechnol Rep (2011) 5:273–281 DOI 10.1007/s11816-011-0182-3