Abstract
An important optimization step in plant-based recombinant protein production systems is the selection of an appropriate cultivar after a potential host has been determined. Previously, we have shown that transgenic tomatoes of the variety ‘Micro-Tom’ accumulate incredibly high levels of miraculin (MIR) due to the introduction of MIR gene controlled by a CaMV35S promoter and a heat-shock protein terminator. However, ‘Micro-Tom’ is unsuitable for commercial production of MIR as it is a dwarf cultivar characterized by small-sized fruit and poor yield. Here, we used the crossbreeding approach to transfer the high MIR accumulation trait of transgenic ‘Micro-Tom’ tomatoes to ‘Natsunokoma’ and ‘Aichi First’, two commercial cultivars producing medium and large fruit sizes, respectively. Fruits of the resultant crossbred lines were larger (~ 95 times), but their miraculin accumulation levels (~ 1,062 μg/g fresh mass) were comparable to the donor cultivar, indicating that the high miraculin accumulation trait was preserved regardless of fruit size or cultivar. Further, the transferred trait resulted in a 3–4 fold increase in overall miraculin production than that of the previously reported line 5B. These findings demonstrate the effectiveness of crossbreeding in improving MIR production in tomatoes and could pave the way for a more efficient production of recombinant proteins in other plants.
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Acknowledgements
We thank the T-PIRC Gene Research Center of the University of Tsukuba for allowing us to use their netted greenhouses. We also thank Dr. Oscar W. Mitalo for editing our manuscript.
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This study was supported by a joint research project with Inplanta Innovations, Inc.
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OS and HTK performed most of the experimental work. NK supported cultivation and ELISA analysis. AO produced the crossbred lines. TS investigated the ration of fruit tissue mass to fruit mass. HTK and HE designed the experiments and wrote the manuscript. All authors read and approved the final manuscript.
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Hiwasa-Tanase, K., Ohmura, S., Kitazawa, N. et al. Improvement of recombinant miraculin production in transgenic tomato by crossbreeding-based genetic background modification. Transgenic Res 31, 567–578 (2022). https://doi.org/10.1007/s11248-022-00320-y
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DOI: https://doi.org/10.1007/s11248-022-00320-y