Abstract
Diverse epigenetic phenotypes are frequently found during research on transgenic plants. To understand the factors underlying such diversity, hundreds of independent 35S-GFP transgenic N. benthamiana plants were analyzed. 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-uniform 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 transcriptional 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. Agroinfiltration 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.
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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
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Acknowledgments
This study was carried out cooperatively with the John Innes Centre (JIC), and was funded by the National Academy of Agricultural Science (NAAS, PJ006437) and additionally supported by the Biogreen21 program (BG21) of Rural Development Administration (RDA), the Republic of Korea. JIC is grant-aided by the Biotechnology and Biological Sciences Research Council (BBSRC), UK.
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11816_2011_182_MOESM1_ESM.pdf
Fig. S1. Production of epigenetic phenotype variations from specific phenotypes and their transgene numbers. The different phenotypes selected at T3 (red-lined squares) and their progenitors were confirmed of transgene numbers. The numbers on the down and upper-side of photos indicate the serial pedigree number of each transgenic plant. The G, S, O, P, M and V indicate the steady-green, silenced, on-silencing, partitioned, misty and variegated phenotype, respectively, and black arrowheads at the right-side of Southern blot indicate the locations of the incorporated GFP transgenes. (PDF 88 kb)
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Sohn, SH., Choi, M.S., Kim, KH. et al. The epigenetic phenotypes in transgenic Nicotiana benthamiana for CaMV 35S-GFP are mediated by spontaneous transgene silencing. Plant Biotechnol Rep 5, 273–281 (2011). https://doi.org/10.1007/s11816-011-0182-3
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DOI: https://doi.org/10.1007/s11816-011-0182-3