Agrobacterium is a natural genetic material delivery system that humans use to produce genetically modified plants (GMO). In nature, GMOs also occur with the participation of agrobacteria. In 2019, the list of known natural GMOs was expanded by an order of magnitude, and facts were found in favor of the expression of agrobacterial genes in natural GMOs. The frequency of this phenomenon for dicotyledon plants has been estimated at 7 percent. Opine synthase genes turned out to be the predominant ones of agrobacterial origin in natural GMOs. They probably perform important functions in natural GMOs. In 2021, an article was published with an updated list of natural GMOs, but the list of genes expressed in natural GMOs has not been updated since 2019.The aim of this work is to update the list of opine synthase genes expressed in natural GMOs. The research methods included bioinformatic search using queries based on the sequences of opine synthase proteins from Agrobacterium rhizogenes, A. tumefaciens and A. vitis, their homologues from Ipomoea and Nicotiana plants, in the TSA database of the National Center for Biotechnology Information (NCBI) using the TBLASTN algorithm with default settings.The study resulted in the addition of another 18 species to the list of natural GMOs with expressed opine synthase genes, 12 of which belong to genera where natural GMOs were not previously described (Albizia, Cenostigma, Averrhoa, Gynostemma, Eurycoma, Gypsophila, Myosoton, Camptotheca, Gustavia, Eschweilera, Cestrum, Jasminum, and Paulownia).An analysis of the diversity of the detected sequences showed that homologues of cucumopine and mikimopine synthase predominate among them. The end products of these genes are optical isomers. In the future, it makes sense to start studying the functions of opine synthases in plants from these genes.

Naturally transgenic plants represent the result of Agrobacterium-mediated gene transfer. T-DNA of soil bacteria Agrobacterium integrated into plants genome is called cellular T-DNA (cT-DNA) [1]. Today, more than 50 species of naturally transgenic plants, or natural GMO (nGMO) are known [2, 3]. The function of cT-DNA in plants remains unknown. It is assumed that the fixation of transgenes could give plants different selective advantages depending on which genes had been integrated into the plant [4]. In order to clarify this issue, it is necessary to study more naturally transgenic plants. Until recently, the list of nGM plants contained less than 2 dozen species, but a search through genomic and transriptomic sequencing data made it possible to more than double this list [2]. In this work, we used the same approach, looking for cT-DNA genes in whole genome sequencing data that have appeared in the NCBI WGS since 2021. We found 14 new species of naturally transgenic plants, among which the most extended cT-DNAs were found in Triadica sebifera, Lonicera japonica, and Lonicera maackii. The cT-DNAs in these species are organized as imperfect inverted repeats. In the genomes of the species Paulownia fortunei, Apocynum venetum, Elaeagnus angustifolia, Erythroxylum havanense, E. densum, E. daphnites, E. cataractarum, Ceriops decandra, Camellia oleifera, Silene uniflora, short cT-DNAs containing only opine synthesis genes were found. We also estimated the approximate age of the cT-DNAs. The first described examples date back to the Late Paleogene, and the process continues to the present. Thus, we can conclude that natural GMOs are a widespread phenomenon, many aspects of which remain unclear, requiring additional research on the topic. The article was made with support of the Ministry of Science and Higher Education of the Russian Federation in accordance with agreement No. 075-15-2022-322 dated 22.04.2022 on providing a grant in the form of subsidies from the federal budget of the Russian Federation. The grant was provided for state support for the creation and development of a world-class scientific center, Agrotechnologies for the Future.