Micropropagation of blackberry and blueberry: assessing the effects of subculture duration and explant density through the integration of traditional measurements and smartphone 3D imaging

The cultivation and consumption of blackberry and blueberry are expanding, with micropropagation emerging as the dominant propagation method. In the present study the influence of explant density and subculture duration on blackberry (Rubus spp. cv. ′Thornfree′) and blueberry (Vaccinium corymbosum L. cv. ′Brigitta′) was investigated. Four initial densities (6.64, 4.43, 3.32, and 2.21 cm² per explant, considering a vessel base area of 66.48 cm²) and two subculture durations (30 and 45 days for blackberry; 45 and 60 days for blueberry) were tested. Hereafter, the four densities will be referred to as 10, 15, 20, and 30 explants per vessel. Growth performance was assessed through shoot viability, number and length, callus formation, fresh and dry biomass, chlorophyll content, and canopy area, the latter quantified via high-resolution 3D modeling using an iPhone 15 Pro Max, viDoc RTK Rover, and Pix4Dcatch. In blackberry, subculture duration was the primary determinant of growth. Rooting occurred only at 45 days, while mean shoot length was lower at 45 days and this was due to increased shoot number and total shoot length. Explant density affected the dry weight that was higher with 10 explants due to a higher callus development. The chlorophyll content decreased at the higher explant density tested. The covered area per explant increased with explant density and subculture duration, reaching maximum values at 30 explants and at 45 days; shoot density followed the same trend. In blueberry, subculture duration significantly affected shoot length, callus fresh weight, and biomass accumulation. Longer subcultures (60 days) enhanced shoot length, callus weight, and dry weight, although shoot fresh weight remained unchanged. Chlorophyll content was not affected by either the number of explants or the duration of subculture. The highest values of the covered area per explant were observed with 10 explants and the lowest with 30 explants. Covered area also increased at 60 days of subculture. Shoot density decreased with increasing explant number but increased with subculture duration. These findings identify subculture duration as the key driver of micropropagation efficiency and demonstrate, for the first time, the potentiality of integrating digital 3D phenotyping to optimize protocols for blackberry and blueberry micropropagation. These findings identify subculture duration as the key driver of micropropagation efficiency and demonstrate, for the first time, the potentiality of integrating digital 3D phenotyping to optimize protocols for blackberry and blueberry micropropagation. Digital imaging further revealed species-specific responses: in blackberry, shoot-covered area and shoot density increased with both explant number and subculture duration; in blueberry, instead, shoot-covered area and density decreased with increasing explant number but increased with subculture duration.