2.2. General processing
Petals of outer (the 1st to 3rd layer), middle (the 4th to 6th layer),and inner (the left layer) layers (Fig. 1B), and blotch and the non blotch parts of petals of middle layers (Fig. 1C) from on-tree flowers of five developmental stages, were separated, weighed and sampled for anthocyanin measurement. As well, petals of middle layers from on-tree flowers of five developmental stages and various tissues including carpels, stamens, middle petals, sepals, stems and leaves detached from on-tree flowers at the full opening stage, as
well as roots, were sampled, immediately frozen in liquid nitrogen and stored at −80◦
C for gene expression analysis.