New achievements in tissue culture of the vegetable and medicinal brassica Diplotaxis tenuifolia (L.) DC: Axillary shoot proliferation, somatic embryogenesis and histological analysis, and polyphenolic compounds profile of in vitro and acclimatized plants

Abstract

Wild rocket (Diplotaxis tenuifolia (L.) DC) is widely cultivated as a vegetable crop known for its nutritional and medicinal benefits, but also for its value as an oil, pasture and beekeeping crop. Biotechnological approaches are essential to respond to the new challenges in breeding, as conventional breeding methods have proven insufficient for further improvements. The aim of this study was to develop a shoot regeneration and propagation protocol for D. tenuifolia and to investigate its metabolic profile. Optimal shoot multiplication was achieved using Murashige and Skoog medium (MS) supplemented with 0.2 mg L^-1 6-BA, yielding 3.04 shoots per explant. IBA 0.5 mg L^-1 promoted shoot rooting and acclimatization (80 %) in the greenhouse. Somatic embryogenesis was developed for the first time using immature zygotic embryos (IZE) cultured on MS medium without plant growth regulators (PGR-free) with an embryo-forming capacity of 3.3. Histological observation revealed that the somatic embryos (SEs) have unicellular or multicellular origin and passed through the globular, heart, torpedo and cotyledon stages. PGR-free and MS media supplemented with low doses of cytokinins 6-BA or KIN were satisfactory for both SE germination into plants and secondary SE regeneration. SE-derived plants survived efficiently when transplanted into the soil. The entire SE process from IZE isolation to flowering of the acclimatized plants took a total of 24 weeks. LC/MS analysis revealed that in vitro cultured shoots contained a broader spectrum and higher amount of bioactive polyphenols, especially the selected clone SR1 obtained from the super-regenerative IZE. These results can shorten the breeding process through targeted selection, large-scale propagation and maintenance of desirable clones, but also form the basis for breeding through advanced biotechnological strategies.