## 3 Flavonoids
### Genetic Approaches for Boosting Flavonoid Production in Plants
The growth of larvae on the transgenic tobacco plants was inhibited by 10-40% compared to wild-type plants, likely due to the reduced ability of the larvae to feed on tobacco with increased flavon-3-ol content. Similar results were obtained in the same study for transgenic tobacco plants overexpressing ANR, an enzyme involved in the synthesis of proanthocyanidins from anthocyanins, for which leucoanthocyanidins are the precursors (Figure 4). Overexpression of DFR from _P. hybrida_ in potatoes resulted in an increase in anthocyanin content. Pelargonidin content increased four-fold and petunidin content increased three-fold [240]. Furthermore, extracts from the tubers of these transgenic potato plants exhibited enhanced antioxidant activity compared to that of wild-type plants [240]. Since anthocyanins are also the precursors of anthocyanins (Figure 4), the _DFR_ gene is often used as a target for genome editing in various plant species. The genome of _Ipmoaa nil_ contains three tandemly arranged copies (_DFR-A_, _DFR-B_, and _DFR-C_). All these copies are expressed, but _DFR-B_ is the dominant one responsible for stem and flower pigmentation. Targeted knockout of the _InDFR-B_ gene of _I. nil_ resulted in a change in stem color to green and to the appearance of white flowers without anthocyanins (Table 2) [154]. Editing of the _OsDFR_ gene in rice resulted in reduced anthocyanin accumulation and a change of the rice grain color from black to ocher (Table 2) [149]. The tomato _DFR_ gene was used for two-stage editing in order to develop the technology of inserting a transgene into a given region of the genome. Initially, the authors used CRISPR/Cas9 to obtain tomato plants with a 1013 bp deletion of the DFR gene. In the second stage, this deletion was corrected by restoring the original gene sequence through the use of donor DNA. Knockout of the _DFR_ gene led to green color of hypocotyls and calli of seedlings homozygous for the deletion, which were able to regenerate in vitro. When the integrity of the gene was restored by knocking, the purple color of these plants was also restored due to the accumulation of anthocyanins (Table 2) [152]. FNS catalyzes the final step in the formation of one more flavonoid class, flavones (Figure 4, Table 3). Overexpression of FNS from the Antarctic moss _Pohlia nutans_ in Arabidopsis resulted in an increased flavone content in the transgenic plants [246]. These transgenic plants demonstrated greater resistance to drought compared to wild-type plants. For instance, seed germination on MS medium with mannitol (osmotic stress) was approximately 45% higher in the transgenic lines compared to the wild-type plants [246]. Transgenic tobacco plants overexpressing FNS from _Morus notabilis_ also accumulated more flavones and exhibited increased resistance to UV-B radiation [210]. Genome editing of gentian plants, which produce blue flowers due to the accumulation of the polyacylated anthocyanin gentiodelphin, helped to identify the functions of three genes of the FGT family: _anthocyanin 5-O-glycostransferase (Gt5 GT)_, _anthocyanin 3'-O-glycostransferase (Gt3'GT)_, and _anthocyanin 5/3'-aromatic acyltransferase (Gt5/3'AT)_. In mutant gentian lines, the effect of gene knockouts on pigment accumulation was distinct for each gene. When the _Gt5 GT_ gene was knocked out, deplhinidin 3G became the primary accumulated pigment. For lines with a _Gt3'GT_ knockout, the dominant floral pigment was delphinidin 3G-5 CafG. Conversely, plants with a _Gt5/3'AT_ gene knockout accumulated two types of pigments: delphinidin 3G-5G-3'G as the primary pigment and delphinidin 3G-5G as the secondary pigment. Therefore, there are two possibilities for modification of delphinidin 3G-5G in gentian flowers: one involves glycosylation by the 3'GT enzyme, and the other involves acylation by 5/3'AT. The flowers of the knockout plants were pale red-violet, dull pink, and lavender, in contrast to the bright blue flowers of wild-type plants [156].