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.for gene expression analysis.
During the petal sampling of on-tree flowers, in-vase treatment of P. suffruticosa flowers was conducted. Flowers at soft bud stage (S1) were trimmed to 25 cm in stem length with all leaves removed and placed with their stem end in 80 mL distilled water in glass vials (one flower per vial) for in-vase treatment. Vase conditions were 21–23◦C and 50–60% RH, and
a 16 h photoperiod with an illumination of ∼40 mol m−2
s−1 The solution was replaced every 24 h. Sampling for anthocyanin accumulation and gene expression analysis in the in-vase flowers at later developmental stages (S2–S5) during vase life was carried out in the same way as mentioned above for on-tree flowers.