### Tocopherol Synthesis
#### 2.1.3 Genetic Approaches for Boosting Isoprenoid Production in Plants
[136], who found that a polymorphism of one nucleotide in the \(\epsilon\)LCY gene (\(H523L\)) led to an increase in the accumulation of carotenoids in rice calli. This experiment demonstrates the possibility of the replacement of the genes by \"elite alleles\" within one generation, which makes an invaluable contribution to the development of new varieties of agricultural plants. _Nicotiana tabacum_ K326 plants, which are the knockouts of the homologous genes encoding \(\epsilon\)LCY, _Nte-LCY1_ and _Nte-LCY2_, were obtained using CRISPR/Cas9 by [PERSON] et al. [137]. The authors studied the phenotypes of the mutants, the expression pattern of carotenoid biosynthesis pathway genes, and the response to light stress. This made it possible to identify functional differences in the expression of homologues. Mutations in the _Nte-LCY_ gene regions led to an increase in growth rate of leaves, an accumulation of carotenoids and chlorophyll, and an increase in stress resistance to strong light of tobacco plants. All these effects were most pronounced in _Nte-LCY2_ mutant plants [137]. The use of the Target activation induced cytidine deaminase (Target-AID) technology results in obtaining the alleles of the _DNA damage UV binding protein 1 (SIDDBI)/de-etiolated1 (SIDETI)_ genes, and the genes encoding \(\beta\)LCY (SICYC-B) that affects the accumulation of carotenoids in tomatoes. The content of carotenoids, lycopene and \(\beta\)-carotene in the edited lines was higher compared to the wild-type tomato plants [138; 139]. This study showed the possibility of simultaneous replacement of nucleotides in several target genes in one plant within one generation, that has potential advantages in breeding work. Another promising approach to increase the content of carotenoids in plants is the expansion of storage space for carotenoids together with intensification of their biosynthesis. The previously described _Or_ gene is not only a chaperone capable of binding to PSY, but is also involved in chromoplast formation, a storage site for carotenoids [63]. This gene encodes the cysteine-rich protein DnaJ and regulates the accumulation of carotenoids in chromoplasts [140; 141]. The use of the CRISPR/Cas9 system makes it possible to introduce targeted mutations into various parts of the _Or_ gene and to identify the parts of the gene where the changes would lead to an increase in carotenoid accumulation. A model cell line of orange rice callus was obtained by targeted mutagenesis of the _OsOr_ gene. The authors showed that it was the single guide RNA targeting region between the third exon and the third intron which produced the orange calli phenotype. The transformed cell line accumulated more lutein and \(\beta\)-carotene compared to the wild-type callus line and was characterized by an increase in the level of transcripts of the genes encoding the enzymes of the carotenoid metabolic pathway: PSY2, PSY3, PD, ZDS, and \(\beta\)LCY. Orange callus plants also showed increased tolerance to salt stress [142]. An interesting and promising approach involves simultaneously overexpressing genes in the isoprenoid biosynthesis pathway that are located upstream of the target product and reducing the activity of downstream enzymes. For example, the knockdown of the activity of \(\beta\)-OHase in wheat endosperm led to an approximately 10-fold increase in \(\beta\)-carotene content [143]. Overexpression of the _PSY_ gene resulted in an approximately 14-fold increase in \(\beta\)-carotene content. As a result, the simultaneous use of both of these strategies led to an approximately 30-fold increase in \(\beta\)-carotene content in wheat endosperm [143]. There are examples of successful editing of genes encoding the proteins of isoprenoid catabolism resulting in an increase in isoprenoid content. One such example is the editing of _Carotenoid cleavage dioxygenes_ (_CCDs_), which belong to a small family of genes that play an important role in carotenoid degradation.