The alternative approaches to anthocyanin production by callus culture of Vaccinium arctostaphylos L. and the ultrastructure of anthocyanin-producing callus

Abstract

In this study, the aim was to try to increase the anthocyanin-producing in callus and to investigate the ultrastructure of the developing callus cells of leaf, apical meristem, and node explants of Vaccinium arctostaphylos L. To generate callus, sterilized explants were seeded in Woody Plant Medium (WPM) containing different concentrations of indoleacetic acid or zeatin. The most calluses containing anthocyanin were obtained from the apical meristem explant cultured on medium containing 2.0 mg L⁻¹ indoleacetic acid. The medium containing 2.0 mg L⁻¹ indoleacetic acid was modified with different sucrose concentrations and were prepared to increase anthocyanin production and callus biomass. The best callus production was obtained with apical meristem explants cultured on medium containing 50.0 g L⁻¹ sucrose and was observed as a pink color callus when examined under a fluorescence microscope with 525-nm green and 610-nm red wavelengths. The callus had autofluorescent. The cell ultrastructures of the pink- and yellow-colored callus growing in medium containing 50.0 g L⁻¹ sucrose were examined using a transmission electron microscope (TEM) after the preparation process. Cells that developed from yellow callus and were called type 1 had an elliptical or long, angular shape. Type 1 cells had a large vacuole. There were electron-dense vesicles around the vacuoles attached to the inner surface of the tonoplast, and anthocyanic vacuolar inclusion–like inclusions were seen in the vacuole. The cytoplasm and organelles were trapped between the vacuole and the cell wall. It was observed that cells developed from pink color callus (called type 2) often had an elliptical shape. Similar to type 1 cells, it has a large vacuole; its cytoplasm is trapped between the cell wall and the vacuole with very few intercellular spaces. Some of the vesicles had an anthocyanic vacuolar inclusion appearance within the vacuole. A macroautophagy-like fusion was placed between the cytoplasmic vesicles and the vacuole. These observations showed that the anthocyanin-producing cells of the V. arctostaphylos L. transported anthocyanins to the vacuole using the vesicular transport method. The current research is the first study to fill the gap in the literature, so it is extremely important as a source for future studies.