Les premières cultures cellulaires végétales in vitro ont été développées à partir de carotte, grâce à Gautheret en 1939. Il a obtenu ce résultat par la découverte au préalable du pouvoir de totipotence des cellules végétales (Haberland 1902). Afin d’obtenir les cellules indifférenciées Gautheret a utilisé des milieux de culture contenant des macroéléments (K, N et P), des microélements (Mg, B,…), des vitamines, du sucre et des phytohormones. Dans la littérature, plusieurs compositions sont souvent utilisées comme White (1934), Murashige and Skoog (1962)) ou Gamborg, Miller et Ojima (1970). Les nuances entre ces milieux se basent sur des concentrations modifiées en phosphates, nitrates ou en phytohormones (auxine/cytokinine). Chaque espèce a besoin d’un milieu particulier pour induire la callogénèse. En effet, selon l’origine (géographique) et le type d’explant (feuilles, racines, tiges,…) traité les conditions d’induction de la callogénèse varieront. De nombreuses personnes portent un intérêt aux cultures cellulaires pour leur utilisation en cosmétique ou en pharmacie. Actuellement, deux entreprises produisent ces cellules à l’échelle industrielle. Ainsi, Phyton Biotech (entreprise allemande) purifie du taxol à partir d’If (Taxus baccata) et Mitsui petrochemical produit de la skinonine à partir de grémils (Lithospernum erythrorizon). / The first plant cell culture has been developed by Gautheret in 1939 based on carrot plant cell tissus. He obtained these results through the discovery of the plant totipotency power (Haberland, 1902). He used for cal production a culture medium composed by macroelements (K, N, P…) and microelements (Mg, B …), vitamin, sugar. Later on, several mediums were used like and described in literature i.e. Murashige and Skoog (1962), White (1934) or Gamborg, Miller and Ojima (1970). The differences between such medium consisted mainly concentrations especially of phosphates, nitrates or auxin/cytokinine balance. However, each species needs specific medium for growth. Any people works of this subject and the interest for pharmaceutical and cosmetical industry grow up. Plant cell culture is a difficult technology an industrial use. Recently, two companies performed industrial production of taxol (Taxus baccata) and shikonin (Lithospernum erythrorizon) respectively PhytonBitotech and Mitsui petrochemical. My thesis work was to develop plant cell cultures fom unusual plants which can be used for the industrial production of cosmetics.

The supply of precursors from primary metabolism is often overlooked when engineering secondary metabolism for increased product yields. This is because precursor supply may be assumed to be non-limiting, and it is considered difficult to engineer primary metabolism, because control of carbon flow (flux) is generally distributed among most enzymes of the pathway. The aim of this thesis was to increase the production of sterols, part of the isoprenoid class of secondary metabolites, in tobacco (Nicotiana tabacum) Bright Yellow 2 (BY-2) cell cultures. This was achieved by genetically engineering increased activity of mitochondrial citrate synthase, an enzyme of the tricarboxylic acid (TCA) cycle that is involved in the provision of cytosolic acetyl coenzyme A, the primary metabolite precursor to sterols. Metabolic flux analysis revealed that citrate synthase exerts significant control over cyclic TCA cycle flux in BY-2 cells and suggested that increasing the activity of downstream enzymes within secondary metabolism could lead to a further redirection of TCA-cycle-derived precursors into sterol biosynthesis. Attempts were made to achieve this by genetically engineering increased activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), a key enzyme of secondary metabolism involved in sterol biosynthesis. Consistent with previous research, transgenic lines had increased sterol levels. However, the high sterol phenotype was unstable, and attempts to co-express HMGR and citrate synthase genes were unsuccessful. The thesis demonstrates that increasing the provision of precursors to secondary metabolites can result in increased yields of those secondary metabolites but suggests that in most cases the activity of enzymes within secondary metabolism has a greater effect on those yields. It also reveals that single enzymes can exert significant control of flux within primary metabolism, although the control exerted by specific enzymes probably changes with the demands placed on metabolism.