Overexpression of black rice OsC1 confers tissue-specific anthocyanin accumulation in indica rice cv. Kasalath and its potential use as a visible marker in rice transformation

Anthocyanin biosynthesis, a process regulated by distinct MYB and MYC transcription factors, plays a crucial role in determining pigmentation in various tissue of plants. This study aimed to investigate the impact of overexpressing the OsC1 gene from black rice, encoding a MYB transcription factor, on anthocyanin pigmentation in red indica rice cv. Kasalath. Anthocyanin pigmentation was readily observed as purple spots in calli and as purple shoot tips and purple leaf sheath in transgenic seedlings. We confirmed the presence of the transgene using GUS assay and PCR analysis, and the pigmentation segregated following a 3:1 Mendelian ratio. T₀ and T₁ transgenic plants exhibits anthocyanin accumulation in various tissues including leaf sheaths, auricles, nodes, stigma, apiculus and awns, excluding the pericarp. Notably, the pigmentation in node tissues has not been previously reported for the OsC1 gene, and this gene does not involve in pericarp pigmentation. RT-PCR analysis of transgenic seedlings demonstrated that the overexpression of the OsC1 gene upregulated anthocyanin structural genes, particularly OsDFR, leading to anthocyanin accumulation. Intriguingly, the absence of OsB2 expression, encoding a MYC transcription factor, in transgenic seedlings suggests the involvement of alternative MYC factors in purple leaf sheaths. This study not only expands our understanding of OsC1's role in tissue specific anthocyanin pigmentation but also proposes OsC1 as a potential visible marker in rice transformation. Utilizing OsC1 as a marker provides an alternative approach to address concerns related to antibiotic-resistant genes while providing visually striking pigmentation.