Bioscience Methods 2024, Vol.15, No.4, 184-195 http://bioscipublisher.com/index.php/bm 1 89 \ Figure 2 Knockout and overexpression of OsMYB3 in the black rice cultivar Zixiangnuo1 (Adapted from Zheng et al., 2021) Image caption: (A) Sequencing for the CRISPR/Cas9-targeted sites close to the 5′end of OsMYB3 knockout plantlet lines. (B) Grain color of the wild type Zixiangnuo1 and three knockout lines of OsMYB3. (C) Anthocyanin content in grains of Zixiangnuo1 and OsMYB3 knockout transgenic lines. KO-5, KO-8, and KO-10 were three independent knockout lines of OsMYB3. (D) Grain color of the wild type Zixiangnuo1 and three OsMYB3-overexpressed transgenic lines. (E) Anthocyanin content in grains of Zixiangnuo1 and OsMYB3-overexpressed transgenic lines. The asterisk (*) and double asterisk (**) indicate significant differences as compared to Zixiangnuo1 at P < 0.05 and P< 0.01, respectively. OE-Z3, OE-Z4, and OE-Z5 were three independent overexpression lines of OsMYB3. Error bars represent the standard deviation in (C) and (E) (Adapted from Zheng et al., 2021) 6 Overexpression Studies of R2R3-MYB Genes 6.1 Methodology for gene overexpression The overexpression of R2R3-MYB genes in plants is achieved through the insertion of the target gene into the plant genome, typically via Agrobacterium-mediated transformation. This method has been widely employed to elucidate the functional roles of these TFs in a range of species. For example, the overexpression of the OsC1 gene in white rice plants was achieved through Agrobacterium-mediated transformation, resulting in increased anthocyanin production and enhanced oxidative stress tolerance (Upadhyaya et al., 2021). Similarly, the OjMYB1 gene from Oenanthe javanica was overexpressed in Arabidopsis thaliana, resulting in elevated anthocyanin content and the up-regulation of anthocyanin biosynthesis genes (Feng et al., 2018). 6.2 Development of transgenic black rice lines To develop transgenic black or purple rice lines, the R2R3-MYB genes are cloned into a suitable expression vector under the control of a strong promoter, such as the CaMV 35S promoter. Subsequently, the vector is introduced into rice calli via Agrobacterium tumefaciens. The transformed calli are selected on a medium containing an appropriate antibiotic or herbicide, and the regenerated plants are screened for the presence of the transgene using
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