Agrobacterium tumefaciens-mediated transformation of Artemisia annua L. and regeneration of transgenic plants.

A transformation system was developed for Artemisia annua L. plants. Leaf explants from in vitro grown plants developed callus and shoots on medium with 0.05 mg/L naphthaleneacetic acid and 0.5 mg/L N(6)-benzyladenine after transformation with the C58C1 Rif(R) (pGV2260) (pTJK136) Agrobacterium tumefaciens strain. A concentration of 20 mg/L kanamycin was added in order to select transformed tissue. Kanamycin resistant shoots were rooted on naphthaleneacetic acid 0.1 mg/L. Polymerase chain reactions and DNA sequencing of the amplification products revealed that 75% of the regenerants contained the foreign genes. 94% of the transgenic plants showed a ß-glucuronidase-positive response.

A new bioassay for auxins and cytokinins.

The authors have developed a sensitive bioassay that can be used to detect auxins as well as cytokinins. The bioassay is based on the expression in transformed tobacco (Nicotiana tabacum) mesophyll protoplasts of a chimeric gene, consisting of the upstream sequences of the Agrobacterium tumefaciens gene 5, coupled to the coding sequence of the beta-glucuronidase. The expression of this gene is induced by the presence of both auxin and cytokinin in the culture medium. Using this assay, indole-3-acetic acid was detected at 5 x 10(-8) molar, whereas trans-zeatin could be detected at 5 x 10(-11) molar. The assay can be performed in microtiter plates, allowing numerous samples to be analyzed simultaneously. Only 2.5 x 10(5) protoplasts are required for one individual assay in 250 microliters of culture medium and for qualitative results, the reaction is readily visualized by ultraviolet light.

Differential regulation of superoxide dismutases in plants exposed to environmental stress.

Superoxide dismutases (SODs) are metalloproteins that catalyze the dismutation of superoxide radicals to hydrogen peroxide and oxygen. The enzyme is ubiquitous in aerobic organisms where it plays a major role in defense against oxygen radical-mediated toxicity. In plants, environmental adversity often leads to the increased generation of reduced oxygen species and, consequently, SOD has been proposed to be important in plant stress tolerance. Here we describe the isolation of a cDNA clone encoding a cytosolic copper/zinc SOD from Nicotiana plumbaginifolia. Using this, together with previously isolated cDNAs encoding the mitochondrial manganese SOD and the chloroplastic iron SOD as probes in RNA gel blot analyses, we have studied SOD transcript abundance during different stress conditions: in response to light, during photoinhibitory conditions (light combined with high or low temperatures), and in response to a xenobiotic stress imposed by the herbicide paraquat. Evidence is presented that iron SOD mRNA abundance increases whenever there is a chloroplast-localized oxidative stress, similar to the previous finding that manganese SOD responds to mitochondria-localized events. The diverse effects of the different stress conditions on SOD mRNA abundance thus might provide an insight into the way that each treatment affects the different subcellular compartments.

The extensin signal peptide allows secretion of a heterologous protein from protoplasts.

Extensins are hydroxyproline-rich glycoproteins which are amongst the most abundant proteins present in the cell wall of higher plants. Here, we describe the structural analysis of an extensin-encoding gene from Nicotiana plumbaginifolia. The encoded protein (46 kDa) has a highly repetitive structure and contains 37% proline, 18.1% tyrosine, 13.4% lysine, 8.1% serine and 7.1% histidine. The extensin-encoding sequence contains a typical signal peptide for translocation of the protein to the endoplasmic reticulum. By using chimeric genes consisting of different 5' parts of the extensin-encoding gene and the neomycin phosphotransferase II-encoding gene (nptII) as reporter gene, we show that the N-terminal part of extensin can mediate the secretion of NPTII from electroporated N. tabacum protoplasts.

Primary structure of a hormonally regulated beta-glucanase of Nicotiana plumbaginifolia.

A cDNA clone for a hormonally regulated beta-glucanase from Nicotiana plumbaginifolia has been isolated by using an oligodeoxynucleotide probe, synthesized to match the previously determined N-terminal amino acid sequence. The cDNA has the complete sequence of the mature protein and contains at least part of a hydrophobic signal peptide. At the amino acid level, the beta-glucanase of N. plumbaginifolia is 73% homologous to a beta(1,3)-glucanase from tobacco and 52% homologous to a beta(1,3;1,4)-glucanase from barley. Southern-blot analysis clearly demonstrated that N. plumbaginifolia contains at least two related genes encoding beta-glucanase. The extent of the complete signal peptide of the cloned beta-glucanase was determined by sequencing part of the corresponding gene. Northern analysis showed that the expression of the beta-glucanase gene is influenced by auxins and cytokinins.

Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity.

A Ti plasmid mutant was constructed in which all the on-cogenic functions of the T-DNA have been deleted and replaced by pBR322. This Ti plasmid, pGV3850, still mediates efficient transfer and stabilization of its truncated T-DNA into infected plant cells. Moreover, integration and expression of this minimal T-DNA in plant cells does not interfere with normal plant cell differentiation. A DNA fragment cloned in a pBR vector can be inserted in the pGV3850 T-region upon a single recombination event through the pBR322 region of pGV3850 producing a co-integrate useful for the transformation of plant cells. Based upon these properties, pGV3850 is proposed as an extremely versatile vector for the introduction of any DNA of interest into plant cells.

Interactions and DNA transfer between Agrobacterium tumefaciens, the Ti-plasmid and the plant host.

Agrobacterium tumefaciens is a gram-negative bacterium with the unique capacity to induce neoplasmic transformations in dicotyledonous plants. Recently, both the mechanism and the biological significance of this transformation have been elucidated. Agrobacterium tumefaciens strains contain a large extrachromosomal DNA plasmid (the Ti-plasmid). This Ti-plasmid is responsible for the oncogenic properties of Agrobacterium strains. A particular segment of the Ti-plasmid, containing information determining the tumorous growth pattern and the synthesis of so-called 'opines', e.g. octopine (N-alpha-(D-1-carboxyethyl)-L-arginine) and nopaline (N-alpha-(1,3-dicarboxypropyl)-L-argine), is transferred and stably maintained and expressed in the transformed plant cells. This phenomenon can be understood as a 'genetic colonization' of the plant cells by bacterial plasmid DNA so that the transformed plant cells will produce and secrete into the medium amino acid derivatives (the opines) that Ti-plasmid carrying agrobacteria can selectively use as carbon and nitrogen sources.

In vivo transfer of the ti-plasmid of Agrobacterium tumefaciens to Escherichia coli.

The Ti-plasmids are naturally self-transmissible from their normal host Agrobacterium to E. coli. They are however unable to stably establish themselves as a replicon in E. coli. It is nevertheless possible to study the Ti-plasmids in E. coli with the help of Ti::RP4 cointegrate plasmids that transfer and maintain themselves very efficiently in E. coli. An E. coli harbouring such a Ti::RP4 plasmid is unable to catabolize octopine and unable to induce crown-gall tumours on plants.