Tobacco shoot regeneration from calli in temporary immersion culture for biosynthesis of heterologous biopharmaceuticals

‘Molecular farming’, the use of transgenic plants to produce biopharmaceutical proteins is emerging as a new biotechnological paradigm. Transgenic plants offer several advantages over conventional microbial and mammalian cell host technologies. In particular, transplastomic plants, with transformed plastid genomes, are capable of massive expression of foreign proteins and represent a promising platform for biopharmaceutical synthesis. The main theme of this PhD thesis is the investigation of in vitro regeneration of tobacco (Nicotiana tabacum) shoots from callus tissue in temporary immersion (TI) culture for heterologous biopharmaceutical synthesis. There is special emphasis on subunit vaccine expression in transplastomic tobacco, in which foreign protein accumulation is correlated with chloroplast number and development during the organogenesis process. Studies using transplastomic N. tabacum expressing TetC (tetanus toxin fragment C) investigated the influence of several culture parameters on biomass regeneration and recombinant protein expression. The parameters investigated include medium nitrogen source ratio, sucrose concentration and hydrodynamics. These studies highlight the sensitivity of transplastomic protein yields to the culture microenvironment, and provide a starting point for further optimisation. Further studies demonstrated the feasibility of TI culture for biosynthesis of proteolytically-unstable transplastomic subunit vaccines, p24 (HIV antigen) and VP6 (rotavirus antigen). TI culture is also demonstrated as a means for nuclear expression of functional Guy’s 13 monoclonal antibody. Finally, the use of TI culture as the basis of novel technological innovations is investigated. This includes the demonstration of transplastomic protein expression in a prototype large-scale mechanical temporary immersion bioreactor. Encapsulation of callus aggregates in an alginate matrix for long-term germplasm preservation was trialled, prior to temporary immersion regeneration. Overall, this work presents a novel in vitro propagation method for the contained large-scale biosynthesis of biopharmaceutical proteins, as a potential alternative to conventional plant propagation platforms based on agricultural cultivation or cell suspension culture.Open Acces