Vol.:(0123456789) 1 3 Plant Cell, Tissue and Organ Culture (PCTOC) (2019) 139:621–634 https://doi.org/10.1007/s11240-019-01703-6 ORIGINAL ARTICLE Production of marker‑free tomato plants expressing the supersweet protein thaumatin II gene under the control of predominantly fruit‑specifc promoters Vadim Timerbaev 1,2,3  · Alexander Pushin 1,3  · Sergey Dolgov 1,2,3 Received: 30 May 2019 / Accepted: 17 September 2019 / Published online: 27 September 2019 © Springer Nature B.V. 2019 Abstract Despite the lack of evidence of the danger of genetically modifed organisms the presence of marker and antibiotic-resistant genes in transgenic plants causes concern to consumers. Genetically modifed plants with viral and bacterial genes are adopted by consumers, but with concerns; in addition, constitutive promoters have a number of disadvantages in industrial-scale cultivation of plants. In our study, we used the pMF vector system (Wageningen Plant Research, Wageningen, Netherlands), which combines inducible site-specifc recombinase and a bifunctional selectable gene to obtain marker-free tomato plants. The gene of interest was the supersweet thaumatin II protein from the tropical plant Thaumatococcus daniellii under the control of tomato predominantly fruit-specifc early-light inducible protein (ELIP) or E8 promoters and tomato Rubisco terminator. The use of this gene in our laboratory allowed enhancing sweetness, as well as improving the taste character- istics of fruit such as apple, strawberries, carrots, tomatoes, and pears. By using diferent strategies of early and delayed selection we developed a protocol for obtaining fully marker-free tomato plants, which was checked by polymerase chain reaction and Southern blotting. The thaumatin II gene expression was confrmed by reverse transcription-PCR and western blotting analyses. The fruit of transgenic and marker-free tomato plants displayed a sweet taste. A quantitative comparative assessment of the level of expression of the thaumatin protein under the control of two promoters was carried out using enzyme-linked immunosorbent assay. Multiple and/or incomplete T-DNA inserts that often occur during transformation of Solanaceae greatly reduced the efciency of the system used. Key message The strong tomato ELIP promoter provides a high level of expression of the supersweet thaumatin II protein gene in the fruit of marker-free tomato plants. Keywords Solanum lycopersicum · E8 · Early-light inducible protein (ELIP) · Cytosine deaminase · R/RS recombination system · High expression level Abbreviations 5-FC 5-Fluorocytosine ANOVA One-way analysis of variance BCIP 5-Bromo-4-chloro-3-indolyl phosphate CaMV35S Caulifower mosaic virus 35S promoter CRISPR/Cas9 Clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 Dex Dexamethasone ELIP Early-light inducible protein ELISA Enzyme-linked immunosorbent assay HSP Heat shock protein IAA Indole-3-acetic acid Communicated by Goetz Hensel. * Vadim Timerbaev timerbaev@gmail.com 1 Branch of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Pushchino, Russia 142290 2 Nikita Botanical Gardens – National Scientifc Center, Russian Academy of Sciences, Yalta, Russia 298648 3 All-Russia Research Institute of Agricultural Biotechnology, Russian Academy of Sciences, Moscow, Russia 127550