Marzocchi, Adua (2025) Vertical farms and plant stem cells as biofactories for enhanced, safe, and sustainable phytocomplexes. [Tesi di dottorato]

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Abstract

Plant biofactories are production systems that use plant cells, tissues, or whole seedlings to synthesize health-relevant phytochemicals. These systems provide year-round supply, improved standardization, and reduced land and water requirements compared to conventional field crops. Environmental parameters such as light, nutrients, or elicitors can be adjusted to enhance the accumulation of target metabolites. This thesis develops two complementary plant biofactory systems: radish microgreens (Raphanus sativus L.) grown under controlled conditions, and plant callus cultures (PCCs) derived from peel and pulp of the Italian apple cultivar Annurca (Malus pumila Mill. cv ‘Annurca’). Microgreens are edible, young seedlings typically harvested one to three weeks after germination, characterized by well-formed cotyledons and elevated concentrations of nutrients and phytochemicals. These seedlings exhibit rapid growth in limited space and demonstrate predictable responses to controlled environmental conditions. Plant callus cultures (PCCs) consist of dedifferentiated plant cells cultivated in vitro on media containing salts, sugars, vitamins, and phytohormones. PCCs function as biochemical production platforms that can be modulated using elicitors. Chapter 3 focuses on optimizing the production of bioactive compounds of nutraceutical interest from radish microgreens. The chapter outlines a systematic approach involving cultivar screening and setting up growth parameters to enhance the yield of these compounds. Radish cultivars were screened under two LED light spectra (red and blue) and at three harvest times (3, 6, and 9 days) to identify the optimal conditions for maximizing glucosinolate (GSL) levels and co-occurring bioactives. Moreover, a comprehensive extraction and HPLC–HESI–MS/MS method was designed and validated for the quantification of intact GSLs. The Saxa cultivar demonstrated the highest GSL content, with total GSLs reaching 126.5 ± 8.5 mg/g dry weight under red lighting at an intermediate harvest (6 days). Glucoraphasatin was identified as the predominant GSL. These findings demonstrate the advantage of selecting an appropriate cultivar and harvest time under specific light conditions, in order to obtain microgreens with high nutritional and health-promoting value. Chapter 4 investigates the anti-hypertensive potential of Tango radish microgreens. Based on the screening results, a rapid targeted HPLC-qQq-MS/MS method for GSLs was developed and applied to the Tango cultivar. Additionally, the antioxidant capacity was confirmed using FRAP, ABTS, and DPPH assays. The optimized microgreen extract (OME) behaved as an H₂S donor. Hydrogen sulfide (H₂S) release was quantified using the fluorescent probe SF7-AM. In cell-free assays at concentrations ranging from 1 to 1000 µg/mL, Tango extract induced a concentration-dependent increase in H₂S, reaching approximately 2.5 µM at 1000 µg/mL after 15 minutes. In bovine aortic endothelial cells (BAEC), live-cell imaging with SF7-AM demonstrated sustained intracellular H₂S for up to 120 minutes at concentrations between 10 and 1000 µg/mL. These results indicate that Tango microgreens are standardized, GSL-rich, H₂S-donor candidates with potential relevance to endothelial function. Chapters 6 and 7 address Annurca apple plant callus cultures (PCCs) derived from pulp and peel tissues. PCCs were established from both tissue types, and yeast extract (YE) at concentrations of 300 to 500 mg/L was applied as a biotic elicitor. In pulp-derived calli, YE treatment increased both biomass and polyphenol content. Specifically, HPLC-DAD-FD analysis revealed that chlorogenic acid increased from 0.95 to 2.06 mg/g dry weight with YE 500, and catechin increased from 0.018 to 0.216 mg/g dry weight with YE 300. The elicited extract exhibited approximately 20% higher antioxidant activity, protected HaCaT cells from UVA-induced reactive oxygen species (ROS) and glutathione (GSH) imbalance, and accelerated re-epithelialization as measured by the scratch assay (4-fold wound-area reduction at 24 h). In peel-derived calli, YE mainly increased dihydrochalcones and triterpenoids. Phloridzin increased with YE, and ursolic acid rose by 132% (YE 300) along with the induction of MdOSC1 and MdCYP716A175. The peel extract was biocompatible, completely blocked UVA-induced ROS, and demonstrated strong antibacterial activity. For example, the inhibition zones for B. cereus were 35 to 37 mm with YE 500, compared to 9 mm with an antibiotic mix. Overall, this thesis establishes radish microgreens and Annurca callus cultures as sustainable plant biofactories. Microgreens as quick, inducible, and standardized sources for GSLs, with measurable H₂S-donor activity, and Annurca PCCs as soil-free, elicitor-responsive sources for polyphenols and triterpenoids that have antimicrobial and dermoprotecting activities. This highlights the potential of plant biofactories for developing nutraceuticals and cosmetics.

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