## 4 Plant Tissue and Organ Cultures in Bioreactors
### Temporary Immersion System
There are essentially three strategies for producing recombinant proteins in plant systems: first, the use of cell culture-based systems that are equivalent to mammalian, microbial, and insect cell models; second, the transient expression of foreign genes in plant tissues, which are transformed by agro-injection or viral infection; and thirdly, the development of transgenic plants carrying stably integrated transgenes. The presented systems enable for high production efficiency through simple manipulation, thus enabling quick validation of the expression constructs and production of large amounts of recombinant protein in a short time [155; 156]. The general strategy of obtaining transgenic plants, cells, or tissue cultures expressing recombinant proteins in bioreactors is shown in Figure 4.  
Most research involving the production of recombinant proteins in plants has focused on early-stage goals, such as expression verification, optimization of production, and purification to some extent, and completion of initial functional tests without any cultivating trials on an industrial scale [152; 157; 158; 159]. For example, [PERSON] et al. examined the expression and glycosylation pattern of a recombinant therapeutic protein--GA733-FcK in transgenic _N. tabucum_ seedlings [160]. [PERSON] et al. showed high expression levels of a prostate cancer antigen with fusion of the Fc fragment of human IgG1 to the glycoprotein GA733 (PAP-IgA Fc and PAP-IgA FcK) and a high level of dimerized proteins in _N. tabucum_ leaves [161]. In turn, [PERSON] et al. revealed the expression of the colorectal cancer vaccine candidate GA733 and the antigen-antibody complex protein GA733-Fc in tobacco plant expression systems. Additionally, fusion of the Fc fragment of human IgG to the C-terminus of GA733 and the ER retention KDEL in GA733-FcK-generating oligomannose glycosylated proteins can be an ideal strategy to easily purify the recombinant GA733 vaccine candidate proteins and to enhance accumulation of the recombinant proteins with oligomannose for comparable immunogenicity of the non-KDEL-tagged mammalian-derived proteins in a plant expression system [162]. [PERSON] et al. noted that there were expression levels up to 85 mg recombinant human erythropoietin (rhEPO)/kg in fresh leaves of _Nicoliana benthamiana_ (_N. benthamiana_) [163]. In another example, [PERSON] and [PERSON] showed that the transient expression of human epidermal growth factor (hEGF) in _N. benthamiana_ is capable of producing large amounts of recombinant protein in combination with a P19 silencing inhibitor and codon optimized constructs [164]. Finally, [PERSON] et al. reported a higher activity of human interferon alpha-2b in young leaves of _Daucus carrota_ plants (up to 50.7 \(\times\) 10\({}^{3}\) IU/g FW) compared to mature leaves, probably due to the susceptibility of this protein to degradation. Additionally, they reported that the taproot expression system could also provide sufficient protein (up to 16.5 \(\times\) 10\({}^{3}\) IU/g FW) and can optionally be used to produce interferon alpha-2b protein for the prevention and treatment of infectious diseases [165]. Additionally, recombinant protein production may be modulated via the CRISPR/Cas9 system. Two proteins named \(\beta\)(1,2)-xylose and \(\alpha\)(1,3)-fucose were reduced in _N. tabucum_ BY-2 suspension cells, knocking-out \(\beta\)(1,2)-xylosyltransferase and \(\alpha\)(1,3)-fucosyltransferase, two genes that are responsible for the addition of plant-specific glycans and result in glycoproteins without plant-specific glycans [166]. The _N. benthamiana_ dicer-like protein 2 and 4  
Figure 4: General strategy for the use of in vitro plant cultures in bioreactors for the production of recombinant proteins on an industrial scale. double-knockout genes might produce higher amounts of human fibroblast growth factor 1 [167].