In vitro production of high-value compounds using cell suspension cultures from Coffea arabica (L.)

Nowadays, a large part of the world’s population relies on plant-origin drugs for primary health care needs, mostly supplied by plant harvesting and chemical synthesis with detrimental impacts on the natural environment. The development of green production strategies is of outstanding importance to support the growing demand of bioactive compounds from medicinal plants. Amongst plant tissue culture techniques, cell suspension cultures reveal as a promising biotechnological tool for achieving such purpose. Indeed, plant stem cells cultivated in in vitro conditions preserve the ability of synthesize secondary metabolites belonging to the parental plant. Despite the great economic value of Coffea spp., its significance goes beyond the popular beverage, as it is a major source of health promoting phytochemicals, among which caffeine, chlorogenic acids and diterpenes. Tissue cultures of coffee has been mainly focused on plant regeneration, whereas the establishment of cell suspension cultures has been exclusively considered for basic research purposes or beverage production attempts. In order to obtain an in vitro cell culture, as a first step, we developed a protocol aimed at the establishment of C. arabica cell suspension cultures starting from the callus induction. The protocol was focused on the determination of the best type and concentration of plant growth regulators, in particular auxins and cytokinins, as they control specific cell division checkpoints. The combination of 1 mg L-1 of both kinetin and 2,4,5-trichlorophenoxyacetic acid successfully induced the proliferation of a non-embryogenic and friable callus mass from young leaves explants and was selected to develop a fine and homogenous cell suspension in the liquid medium. The productive potential of valuable phytochemicals of such in vitro system has been evaluated through elicitation strategies aimed at triggering a cell stress-response. In particular, this was evaluated as the biosynthesis of two diterpenes, exclusively identified in the coffee plant, namely cafestol and kahweol, which exhibit anticancer and analgesic activities. The elicitors tested (i.e., low molecular weight chitosan and salicylic acid), at their maximum dose, significantly increased the concentration of cafestol. However, low molecular weight chitosan strongly inhibited kahweol accumulation. Furthermore, the concentration of both diterpenes was significantly affected in cells grown under photoperiod conditions, compared to complete darkness. No synergistic effect has been found in the presence of photoperiod and elicitors. Finally, C. arabica cell suspension cultures have been evaluated also as a source of extracellular vesicles (EVs), lipid bound particles with heterogeneous cargoes such as secondary metabolites, lipids, proteins and nucleic acids. EVs were isolated from the conditioned culture medium by differential centrifugation and characterized on the basis of their size distribution, morphology, protein content and proteomic profiles. The isolated vesicles had an average diameter of 120 nm and exhibited protein markers indicating their origin via unconventional mechanisms that bypasses the Golgi apparatus, depending on multivesicular bodies (MVBs) secretion, as well as from plasmalemma budding. These findings demonstrate the validity of coffee cell suspension cultures as biotechnological platforms to obtaining high-value compounds, including secondary metabolites and extracellular vesicles, with promising applications in the therapeutic field.