Sweet Cherry Rootstock Micropropagation Using SETIS™ Bioreactor: Evaluation, Challenges, and Biochemical Characterization of Regenerated Shoots

In vitro propagation of Prunus sp. using semi-solid media allows clonal multiplication but requires frequent subcultures and higher labor and material costs. In contrast, temporary immersion bioreactors (TIBs) promoted faster shoot elongation, higher biomass accumulation, and improved overall shoot quality, while reducing handling time and the cost per regenerated plant. This work aimed to establish in vitro selected rootstocks of Prunus avium and Gisela 6 ( Prunus cerasus x Prunus canescens ) and compare the multiplication and rooting processes of shoots obtained in different culture conditions, in semi-solid media and SETIS ^TM bioreactors a system that alternates brief liquid immersion with aeration to enhance growth and shoot quality. Process efficiency and shoot growth parameters were evaluated, including in vitro establishment percentages, shoot length, number of shoots, and Relative Growth Rate (RGR). Physiological and biochemical analysis were also conducted, such as photosynthetic pigments content, total flavonoids, malondialdehyde (MDA), and proline levels. The in vitro percentage establishment was 56% for P. avium and 62% for Gisela 6, consistent with seasonal conditions favoring lower contamination. While no statistical differences were observed in the number of shoots between systems or genotypes, shoot elongation was significantly higher in TIB, particularly for Gisela 6. The RGR index confirmed the superior performance of TIB in promoting shoot proliferation, and rooting capacity was also improved, with 57% rooting in Gisela 6 and 44% in P. avium from TIB compared to 16% and 10% in semi-solid medium, respectively. Biochemical analyses revealed that P. avium propagated in TIB showed reduced pigment levels and signs of stress likely related to hyperhydricity, whereas Gisela 6 exhibited greater stability across both systems. Overall, the results demonstrate that TIB is a more efficient alternative to semi-solid media for the micropropagation of sweet cherry rootstocks, enhancing shoot quality, growth, and ex vitro rooting, although genotype-specific responses highlight the need for further optimization.