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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.

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.

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.