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A reproducible protocol has been established for the transformation of Ginkgo biloba by Agrobacterium tumefaciens. Embryos were co-cultivated with Agrobacterium tumefaciens GV3101 (pGV2260) carrying the binary vector pTHW136, which contained the gus reporter gene and the nptII selectable gene, encoding the enzymes β-glucuronidase (GUS) and neomycin phophotransferase II, respectively. Transient GUS activity has been used to screen the effects of different factors on the transfer of DNA into embryos (age of embryos, infection method, composition of co-cultivation medium). Then, experimental conditions have been defined to obtain transgenic kanamycin-resistant G. biloba calluses expressing GUS activity. The highest rate of transformation (45%) was reached using 1.5-month-old embryos co-cultivated on a medium lacking mineral elements. The integration of gus and nptII genes in calluses was confirmed by polymerase chain reaction analysis and Southern blot analysis.

Scopolin-producing cells of Nicotiana tabacum were immobilized within Ca-alginate gel beads. Free cell suspensions accumulated scopolin within cytoplasmic compartments and cell disruption was necessary to recover scopolin. On the contrary, immobilized plant cells excreted considerable amounts of scopolin. Scopolin diffused throughout the gel matrix and reached the culture media. A large fraction of produced scopolin could then be recovered from the culture medium without disrupting cells. Immobilized N. tabacum cells produced more scopolin than free cell suspensions did (3.8 mg/g fresh weight biomass [into the culture media] versus 0.2 mg/g fresh weight biomass [intracellular]). Variation of the immobilization conditions revealed a marked influence on the behavior of N. tabacum plant cells: production of scopolin and enhanced excretion, cell growth, and morphological aspect of plant cell colonies. This excretion phenomenon could be used advantageously at an industrial production level.

Endogenous scopolin and its aglycon scopoletin were tested in various plant cell suspensions by HPLC. The results indicate that the levels of these compounds varied greatly from one plant cell suspension to another (10 −3 –5 mg/g dry mass). Both scopoletin and scopolin were detected in the Nicotiana tabacum L. cell suspensions while only scopolin was detected in the Duboisia myoporoides R.Br., Solanum aviculare Forst. and Papaver somniferum L. cells. The identity of this scopolin was confirmed by NMR and MS data. Neither of these compounds was accumulated in the Nicotiana plumbaginifolia Viv. cell suspensions. Moreover, all the cell suspensions, except N. tabacum could bioconvert exogenous scopoletin to scopolin. The yield of the bioconversion varied from one cell suspension to another (21–92%). No correlation could be established between the capacity of the cells to bioconvert scopoletin to scopolin and the endogenous scopolin level.

We have developed a procedure for opium poppy (Papaver somniferum) transformation using Agrobacterium tumefaciens-mediated gene delivery. Hypocotyl-derived cell suspension cultures of P. somniferum were cocultivated with A. tumefaciens strain GV3101 (pMP90) harbouring either the binary vector pTHW136 or the binary vector pO35SSAM. The former contained the uidA reporter gene and the nptII selectable gene, encoding the enzymes β-glucuronidase and neomycin phosphotransferase II, respectively. The latter contained the saml gene encoding the enzyme S-adenosyl-L-methionine (SAM) synthetase and the nptll gene. Putatively transformed cell lines were selected on media supplemented with paromomycin. Integration of the foreign genes was confirmed by Southern blot analysis with and without PCR amplification prior to hybridization. SAM synthetase activity was measured in extracts of 5 transformed cell lines. One of them expressed a significant overactivity while two others had a lower activity than the control cell line, leading us to question the possible partial cosuppression of both the resident and the foreign sam genes. To our knowledge, this is the first report of Agrobacterium tumefaciens-mediated transformation of Papaver somniferum cell suspension cultures.

From a solanaceous cell suspension an acetophenone glycoside, androsin, has been isolated along with one polyphenol glycoside, scopolin. The androsin presence is described for the first time in solanaceous cell suspension.

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.

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.

A competitive enzyme immunoassay (EIA), using anti-nornicotine antibodies is described. The specificity of these antibodies was checked by a high pressure liquid chromotographic (HPLC) procedure. Following purification, the alkaloid extracts were simultaneously analysed by EIA and HPLC and similar results were obtained. The high sensitivity of EIA and its good specificity toward the major tobacco alkaloids enabled us to perform analysis on crude extracts prepared from very small samples (ca. 1 mg dry weight) of plant material. The method can therefore be applied to the localization of alkaloids in Nicotiana plants and cell suspensions.

A high performance liquid chromatographic (HPLC) procedure for the analysis of tobacco alkaloids in plant material is described. It allows the detection of up to 10 ng of nicotine, nornicotine, anabasine and nicotinic acid. It is more efficient with regard to the sensitivity, simplicity and suitability for phytochemical analysis than the gas chromatographic (GC) procedure previously used by the authors. Alkaloid extraction and purification prior to HPLC analysis can be carried out more quickly and easily. The method has been applied to the study of the bioconversion of nicotine into nornicotine by Nicotiana plumbaginifolia cell suspensions. The yield of biotransformation which was estimated at 53.2% by GC analysis is now found to be nearly 100% by HPLC analysis. Moreover, it has been shown that the method is reliable for the evaluation of high or low quantities of endogenous alkaloids produced by plant organs or tissue cultures.

The alkaloid content and profile of roots and aerial parts of diploid, haploid and hypohaploid plants of Nicotiana plumbaginifolia regenerated in vitro from leaf explants was determined by enzyme immunoassay and gas chromatography. Roots and buds separately neoformed in vitro were examined by the same methods. An interesting trait was found: buds, even without roots, contained alkaloids. Each of the tested hypohaploids exhibited a peculiar alkaloid content and profile compared to diploid and haploid genotypes, confirming the novel character of these hypohaploids. No correlation was observed between the degree of ploidy and the alkaloid content or profile.

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...

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 .

Three anther-derived haploid and three haploid-derived dihaploid tissue lines of tobacco were transformed with three strains of Agrobacterium tumefaciens: B6S3, pGV2215 and pGV2255. Unorganized, shooty and rooty tumours were induced, respectively. Tumour morphology and the growth rate of transformed tissues depended mainly on the bacterial strain. The presence of T-DNA genes in transformed tissues was confirmed by polymerase chain reaction (PCR). By means of high performance liquid chromatography (HPLC) nicotine was found only in the control plants and in the roots transformed with Agrobacterium tumefaciens pGV2255. Its content did not depend on the ploidy level of the tissue. Transformed roots could not be subcultured because of callogenesis. The callus which overgrew transformed roots did not produce nicotine.

Different matrices, obtained by varying calcium (0.1 to 1.5M) and alginate (1 to 1.5%) concentrations, were used to study the influence of immobilisation parameters on the behaviour ofS. aviculare. A significant modulation of cell growth, cell release, and scopolin production and excretion has been observed. Physiological and morphological characteristics ofSolanum aviculare cells immobilised within Ca-alginate beads were notably different from those of suspended cells. ImmobilisedS. aviculare have accumulated scopolin (up to 120 μg·g-1 FWB) within beads and excreted it into the culture medium (up to 8 μg·g-1 FWB). Contrary to suspended cells which have accumulated only traces of this metabolite within intracellular compartments (1 μg·g-1 FWB), no scopolin has been found into the culture medium.

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.

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.

Direct densitometric determination by absorption of flavonoids of the yellow-weld (Reseda luteola L.) separated by thin-layer chromatography This paper describes a method for the determination of the flavonoids of the yellow-weld (Reseda luteola L.) by analysis of ethanolic extracts of the different parts of the plant on cellulose thin layers, followed by measurement of the intensity of ultra-violet absorption. Standard curves have been established for each of the following flavonoids: luteolin, luteolin 7-O0flucoside, and luteolin diglucoside. The optimum ranges for measurements are: 0.17-0.85 μg for luteolin, 0.17-1.50 μg for luteolin 7-O-glucoside, and 0.20-1.0 μg for luteolin diglucoside. The observed variation factors are less than

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.

Antibodies were obtained from the immunization of rabbits with DL-tropic acid conjugated to bovine serum albumin. Their wide specificity enabled us to analyse simultaneously every main tropane alkaloid of Solanaceous plants: hyoscyamine, atropine ( DL-hyoscyamine), and scopolamine. The competitive ELISA assay used for this analysis was found to be a very rapid, sensitive method with good accuracy for the dosage of alkaloids in plant material: purified extracts, crude extracts, and dry powdered material. A tropine could be detected with a sensitivity of 0.1 ng. A racemization step was necessary to detect hyoscyamine and scopolamine with the same sensitivity.