**2.4 Callus and plant cell suspension cultures**

Plant cell suspension cultures grow as individual cells or small aggregates and are usually derived from *callus* tissue by the disaggregation of friable callus pieces in shake bottles and are later scaled up for bioreactor-based production. Recombinant pharmaceutical protein production is achieved using transgenic explants to derive the cultures or by transforming the cells after disaggregation, usually by co-cultivation with *A. tumefaciens*. The co-cultivation of plant cell suspensions and recombinant *A. tumefaciens* has also been used for the transient expression of proteins [85]. Since these plant cell suspension cultures are grown in sterile contained environments, they provide a cGMP-compatible production environment that is more acceptable to the established pharmaceutical industry and regulatory authorities [3, 86]. These systems have added benefits of complex protein processing compared to bacteria and yeasts and increased safety compared to mammalian cell systems, which can harbor human pathogens. Another advantage of plant suspension cultures is the very low maintenance cost in comparison to other fermenter-based eukaryotic systems such as mammalian or insect cells. Moreover, the possible secretion of the target protein into the culture medium simplifies downstream processing and purification procedures [87, 88]. Nevertheless, plant cell cultures also have some limitations such as poor growth rates, somaclonal variation (particularly due to chromosomal rearrangements, common in plant cell cultures generated by *calli*), and gene silencing, together with the inhibition of product formation at high cell densities, formation of aggregates, cell wall growth, as well as shear-sensitivity for some species [89]. However, high levels of functional recombinant protein in plant cell suspension cultures were already obtained [87]. Besides, the previously mentioned first licensed recombinant pharmaceutical protein, Elelyso™, was produced in plant cell suspension cultures (reviewed in [88]). Tobacco has been the most popular source of suspension cells for recombinant protein production. Tobacco plants proliferate rapidly and are easy to transform, but other plant species have also been used to generate suspension cells, including rice and *Arabidopsis thaliana,* alfalfa, soybean, tomato, *Medicago truncatula*, and carrot [85, 88, 90]. Carrot suspension cells have been used by the aforementioned Protalix Biotherapeutics to produce a recombinant glucocerebrosidase. This case of commercial success shows that suspension cell cultures have potential as a viable system for large-scale protein production. Recently, carrot callus cultures, expressing epitopes from the cholesteryl ester transfer protein, were accessed for the potential of becoming an atherosclerosis oral vaccine [91].
