Annie Jacquin-Dubreuil

Tropane alkaloid production by hairy roots of Atropa belladonna obtained after transformation with Agrobacterium rhizogenes 15834 and Agrobacterium tumefaciens containing rol A, B, C genes only.

Atropa belladonna leaf disks were infected by a wild strain Agrobacterium rhizogenes 15834 harboring the Ri-TL-DNA and by a disarmed Agrobacterium tumefaciens strain harboring a construction with only rol ABC and npt II genes. Thirteen root lines were established and examined for their growth rate and alkaloid productivity to evaluate the possible role of rol genes in morphological differentiation and in tropane alkaloid formation. A great diversity has been observed in the growth rate of these 13 root lines. The root biomass increased up to 75 times. The total alkaloid contents were similar in the root lines obtained by infection with A. rhizogenes 15834 and A. tumefaciens rol ABC. The last ones accumulated between 4 (1.1 mg g(-1) DW) and 27 (8 mg g(-1) DW) times more alkaloids than the intact roots (0.3 mg g(-1) DW). This work has shown that the rol ABC genes were sufficient to increase tropane alkaloid production in A. belladonna hairy root cultures.

Simultaneous analysis of l-hyoscyamine, l-scopolamine and dl-tropic acid in plant material by reversed-phase high-performance liquid chromatography

Abstract A sensitive reversed-phase high-performance liquid chromatographic (HPLC) procedure for the analysis of the main parasympatholytic tropane alkaloids in plant material is described. It uses an acidic aqueous acetonitrile mobile phase and UV detection at 204 nm. It allows a good simultaneous separation of l-hyoscyamine, l-scopolamine and tropic acid, their acidic precursor. The detection limits are 20 ng for the alkaloids and 5 ng for tropic acid. A simple and rapid method, very convenient for HPLC analysis, is also described for the preparation of purified alkaloid extracts. The procedure was applied to the evaluation of the alkaloid content of Datura leaves. The results are in good correlation with those obtained with a tropic acid derivatives- specific enzyme immunoassay.

Glucosylation of butyric acid by cell suspension culture of Nicotiana plumbaginifolia

Abstract Suspension cells of Nicotiana plumbaginifolia rapidly absorbed exogenously applied butyric acid and converted it into a new product within one day. The conversion product was found to be 6- O -butyryl- d -glucose. This molecule could be of great interest because of its capacity to transport and release butyric acid in vivo .

Ability of a Nicotiana plumbaginifolia cell suspension to demethylate nicotine into nornicotine

Abstract A cell suspension of Nicotiana plumbaginifolia which does not accumulate tobacco alkaloids was found to keep the ability to demethylate nicotine into nornicotine. The highest bioconversion yield was 53.2%. The influence of some environmental factors upon the reaction has been studied. In particular, it appears that light enhances the catalytic activity of the cells which leads to the hypothesis that this metabolic step of tobacco alkaloids is bound to photodependent systems.

Biotransformation of a synthetic compound, 1,5-diphenylsulphinyl-3-methyl-3-nitropentane, by cell suspensions of Catharanthus roseus

Abstract Biotransformation of a synthetic substrate, 1,5-diphenylsulphinyl-3-methyl-3-nitropentane, by cell suspension cultures of Catharanthus roseus was investigated. It was found that this substrate was (i) incorporated into cells and (ii) converted within three days into a new product, 1-phenylsulphonyl-5-phenylsulphinyl-3-methyl-3-nitropentane through a regioselective oxidizing process not previously described. This original organic substrate used for bioconversion is entirely synthetic and the functions (nitro, sulphoxide) are rarely involved in biotransformation studies. Such a biotechnological process could be of great interest for the production of new chemical compounds.

Biotransformation of ethyl 2-acetylamino-2-carbethoxy- 4-(phenylsulphinyl)-butanoate by cell suspension of Catharanthus roseus and Thevetia nefiifolia

Abstract Biotransformation of the synthetic substrate, ethyl 2-acetylamino-2-carbethoxy-4-(phenylsuphinyl)- butanoate, by cell suspension cultures of Catharanthus roseus and Thevetia neriifolia was investigated. It was found that only three of six cell lines of T. neriifolia tested biotransformed the test substrate into a new product, ethyl 2-acetylamino-2-carbethoxy-4-(phenylsulphonyl)-butanoate, through a selective oxidizing process not previously described. Such a biotechnological process could be of great interest for the production of new chemical compounds.

Cardenolide Formation in Cell Suspension Cultures of Thevetia peruviana (Pers.) K. Schum. (synonym Thevetia neriifolia Juss.)

Abstract Six cell suspension cultures established from different organs of Thevetia peruviana were initiated under various growth conditions. Even after two years of subculture, cardenolides proved to be present in all the strains. The cardenolide content varied from one strain to another according to the nature of the original explant and was always lower in comparison with that of the seeds of the original plant. Kinetic studies showed that the cardenolide content varied over a complete cycle of culture.

Selection and improvement of Datura innoxia Mill.-Morphological variability in in vitro propagated plants

SummaryTo select high-alkaloid-producing plants, we have developed an experimental model producing the largest possible number of plants differing from the source plant. On the basis of the commonly accepted idea that the formation of an intermediate callus would increase chances of recovering altered plants, we propagated Datura innoxia Mill, through axillary buds (micropropagation) and through regeneration from explants giving a callus at various degrees of development before bud shoots are differenciated. Seventy-eight plants were obtained; their morphological characteristics were observed at the first floral bud stage. Numerous plants, including those propagated through axillary buds, were smaller and had less leaves than seed-grown plants. Very peculiar features (flowers, leaves, ramifications) were observed in five plants (“abnormal plants”): two were produced from thin cell layers without intermediate callus, three from internodes after formation of a well-developed intermediate callus. No “abnorma...