Stable expression of promoterless bar gene in transgenic rape plants

Phosphinothricin resistant plants of two rapeseed (Brassica napus L. var. oleifera DC.) spring industrial cultivars were obtained by Agrobacterium tumefaciens leaf disk transformation. Vector constructions contained the promoterless coding sequence of phosphinothricin acetyltransferase (bar) gene located between two inverted lox-sites (elements of Cre/lox recombination system of P1 phage) and selective neomycinphosphotransferase II gene (nptII). Integration of the alien genes was confirmed by the PCR analyses. Stable and linked inheritance of foreign genes in T1 and T2 progeny was shown.     

Anther-specific expression of ipt gene in transgenic tobacco and its effect on plant development

Isopentenyl transferase (ipt) gene from Agrobacterium tumefaciens T-DNA was placed under the control of a TA29 promoter which expresses specifically in anther. The chimeric TA29-ipt gene was transferred to tobacco plants. During flowering, mRNA of the ipt gene in the anthers of the transgenic plants accumulated and the level of iPA + iPs increased 3–4-fold in the leaves, petals, pistils, and stamens compared with those in the wild type plants. This cytokinin increase affected various aspects in development indicating that the alterations of endogenous cytokinin level by using anther-specific expression of the TA29-ipt gene affected morphology, floral organ systems and reproductivity of the transgenic plants.     

Analysis of insecticidal activity in transgenic plants carrying the ipt plant growth hormone gene

The expression of a bacterial cytokinin biosynthesis gene (PI-II-ipt) in Nicotiana plumbaginifolia Viviani plants has been correlated with enhanced resistance to Manduca sexta and Myzus persicae. We expressed the PI-II-ipt gene in N. tabacum and Lycopersicon esculentum and observed similar antifeedent effects with the transgenic tobacco but not tomato. A 30 to 50 % reduction in larval weight gain was observed with some of the tomato plants but these results could not be repeated consistently. Leaf surface extracts from transgenic N. plumbaginifolia leaves killed 100 % of M. sexta second instars at concentrations of 0.05 % (w/v) whereas the N. tabacum extracts were at least 20 times less active. Extract suspensions were stable for up to 2 days at ambient temperatures below 42 °C and for at least 3 months at 4 °C when stored in the dark. HPLC analysis of the N. plumbaginifolia extracts yielded an active fraction that reduced hatching of M. sexta eggs by 30 % and killed first, second and third instars within 24, 48 and 72 hours of exposure, respectively. The activity appears to be associated with oxygen-containing aliphatic compounds, possibly diterpenes, as analyzed by TLC, UV absorption and fragmentation with EIMS. Based on the partial characterization of this activity, the production, secretion or accumulation of secondary metabolites in leaves of cytokinin producing PI-II-ipt N. plumbagini-folia plants appears to be responsible for the observed insect resistance.     

The expression of a heat-inducible chimeric gene in transgenic tobacco plants

Summary A chimeric gene under the control of the hsp70 promoter of Drosophila is heat regulated in roots, stems and leaves, but not in pollen of transgenic tobacco plants. For these and other parameters, it behaves similarly to plant heat-shock genes.     

Variability of organ-specific gene expression in transgenic tobacco plants

Variability of expression of introduced marker genes was analysed in a large number of tobacco regenerants from anAgrobacterium-mediated transformation. In spite of standardization of sampling, considerable variation of GUS and NPTII expression was observed between individual transformants at different times of analysis and in different parts of the same plant. Organ-specificity of root versus leaf expression conferred by the par promoter from the haemoglobin gene ofParasponia andersonii in front of thegus gene showed a continuous spectrum. GUS expression in roots was found in 128 out of 140 plants; expression in leaves was found in 46 plants, and was always lower than in the corresponding roots. NPTII expression regulated by the nos promoter also showed a continuous spectrum. Expression levels were generally higher in roots than in leaves. Plants with high GUS expression in leaves showed high NPTII activity as well. A positive correlation between the level of NPTII expression and the numbers of integrated gene copies was noted. Chromosomal position effects and physiological determination are suggested as triggers for the variations. The transformed regenerated tobacco plants were largely comparable to clonal variants.     

Ethylene formation and phenotypic analysis of transgenic tobacco plants expressing a bacterial ethylene-forming enzyme

A bacterial ethylene-forming enzyme (EFE) catalyzes oxygenation of 2-oxoglutarate to produce ethylene and carbon dioxide in contrast to a plant enzyme which uses 1-aminocyclopropane-1-carboxylic acid as a substrate. We constructed several lines of transgenic tobacco plants which expressed an EFE from Pseudomonas syringae pv. phaseolicola PK2. The gene encoding a chimeric protein consisting of EFE and beta-glucuronidase (GUS) was introduced into the tobacco genome using a binary vector which directs expression of the EFE-GUS fusion protein under the control of constitutive promoter of cauliflower mosaic virus 35S RNA. Two lines of transgenic plants produced ethylene at consistently higher rates than the untransformed plant, and their GUS activities were expressed in different tissues. A significant dwarf morphology observed in the transgenic tobacco displaying the highest ethylene production resembled the phenotype of a wild-type plant exposed to excess ethylene. These results demonstrate a potential use of bacterial EFE to supply ethylene as a hormonal signal via an alternative route using an ubiquitous substrate 2-oxoglutarate in plant tissues.     

Transactivation of a target gene using a suppressor tRNA in transgenic tobacco plants

The abundance of tRNAs, together with their central role in translation, has generated considerable interest in the use of tRNA genes for biotechnological applications. One such application is the use of suppressor tRNAs to transactivate target genes containing premature stop codons. Previous work has shown that such systems can work in transient expression experiments in plant protoplasts; here these experiments are extended to show that suppression of stop codons can occur in whole plants. Transgenic tobacco plants homozygous for a modified tRNA^Leu gene expressing a strong amber suppressor tRNA, and plants carrying a β-glucuronidase (gus) gene inactivated by a premature amber stop codon have been obtained. When the two types of plants are crossed, many of the F₁ hybrids show significant GUS activity. The GUS activity is dependent on the presence of both the suppressor tRNA gene and the gus gene. Tobacco plants carrying the suppressor tRNA gene are phenotypically normal, fertile and the gene shows normal Mendelian inheritance. The potential applications of such a system are discussed.     

Effect of lox-sites of the Cre lox recombination system on promoterless bar gene expression in transgenic plants

We demonstrate that localization of lox site between the right border of T-DNA and promoterless bar gene (RB-lox-bar-) led to its highly efficient expression in transgenic plants of Nicotiana tabacum and N. africana. Plasmid vectors used in gene integration experiments contained neomycin phosphotransferase II (npt II) gene under nos promoter as well. Transgenic plants were selected according to their capacity to grow on the medium with kanamycin and then they were tested on the selective medium containing phosphinothricin. 80% of transgenic plants expressed bar gene at the level similar to that in plants transformed with the bar gene under widely used constitutive promoter. Transformation of plants with the plasmid vector containing only promoterless bar gene near T-DNA right border (RB-bar-) and with the vector containing lox site and promoterless bar gene in the middle of the construction (-lox-bar-) led to obtaining no more than 4.5% of transgenic plants resistant to phosphinothricin. PCR analyses confirmed both the absence of tandem repeats and of plasmid recombination resulting in transference of bar gene under promoter in plasmid vector. Nos-terminator situated between the lox site and the right border of T-DNA did not decrease bar gene expression.     

Ectopic expression of a UDP-glucose:phenylpropanoid glucosyltransferase leads to increased resistance of transgenic tobacco plants against infection with Potato Virus Y

Transgenic tobacco plants over-expressing a salicylate- and pathogen-inducible glucosyltransferase (TOGT) acting on various phenylpropanoids show enhanced resistance against infection with potato virus Y (PVY). The transgenic plants are characterized by a several-fold increased glucosyltransferase activity in leaves as well as in roots. Under non-infectious conditions profiles of phenylpropanoids in leaves of transgenic lines were similar to that of controls. Feeding experiments with leaf-discs demonstrated a higher capacity for glucosylation of the coumarin scopoletin. After inoculation with PVY the transgenic lines showed similar formation of necrotic leaf lesions but significantly decreased levels of virus coat-protein when compared with control plants. Thus, our results imply that the activity of TOGT and the subsequent accumulation of glucosylated coumarins represent an important step in the cascade of events resulting in confinement of viral pathogens.     

Expression of tandem gene fusions in transgenic tobacco plants.

We have studied the expression of four sets of tandem gene fusions in transgenic tobacco plants. This was to determine if the problem of between-transformant variability in expression of introduced genes could be overcome by using a linked reference gene as a co-ordinately expressed control. Tandem gene fusions containing identical 5' flanking regions (SSU301-ocs with either SSU301-cat or SSU301-SSU911) were not co-ordinately expressed in the transgenic tobacco plants whereas the tandem gene fusions containing similar but not identical 5' flanking regions (SSU301-ocs with SSU911-cat or SSU911-SSU301) were co-ordinately expressed. The lack of co-ordinate expression of some of the tandem gene fusions appears to be partially explained by absence of the corresponding genomic DNA segments in the transgenic plants.     

Influence of flanking sequences on variability in expression levels of an introduced gene in transgenic tobacco plants.

The petunia rbcS gene SSU301 was introduced into tobacco using Agrobacterium tumefaciens-mediated transformation. The time at which rbcS expression was maximal after transfer of the tobacco plants to the greenhouse was determined. The expression level of the SSU301 gene varied up to 9 fold between individual tobacco plants which had been standardized physiologically as much as possible. The presence of adjacent pUC plasmid sequences did not affect the expression of the SSU301 gene. In an attempt to reduce the between-transformant variability in expression, the SSU301 gene was introduced into tobacco surrounded by 10kb of 5' and 13 kb of 3' DNA sequences which normally flank SSU301 in petunia. The longer flanking regions did not reduce the between-transformant variability of SSU301 gene expression.     

The effect of auxin on cytokinin levels and metabolism in transgenic tobacco tissue expressing an ipt gene

The ipt gene from the T-DNA of Agrobacterium tumefaciens was transferred to tobacco (Nicotiana tabacum L.) in order to study the control which auxin appears to exert over levels of cytokinin generated by expression of this gene. The transgenic tissues contained elevated levels of cytokinins, exhibited cytokinin and auxin autonomy and grew as shooty calli on hormone-free media. Addition of 1-naphthylacetic acid to this culture medium reduced the total level of cytokinins by 84% while 6-benzylaminopurine elevated the cytokinin level when added to media containing auxin. The cytokinins in the transgenic tissue were labelled with ³H and auxin was found to promote conversion of zeatin-type cytokinins to ³H-labelled adenine derivatives. When the very rapid metabolism of exogenous [³H]zeatin riboside was suppressed by a phenylurea derivative, a noncompetitive inhibitor of cytokinin oxidase, auxin promoted metabolism to adenine-type compounds. Since these results indicated that auxin promoted cytokinin oxidase activity in the transformed tissue, this enzyme was purified from the tobacco tissue cultures. Auxin did not increase the level of the enzyme per unit tissue protein, but did enhance the activity of the enzyme in vitro and promoted the activity of both glycosylated and non-glycosylated forms. This enhancement could contribute to the decrease in cytokinin level induced by auxin. Studies of cytokinin biosynthesis in the transgenic tissues indicated that trans-hydroxylation of isopentenyladenine-type cytokinins to yield zeatin-type cytokinins occurred principally at the nucleotide level.Abbreviations Ade adenine - Ados adenosine - BA 6-benzylaminopurine - C control - Con A concanavallin A - CP cellulose phosphate - IPT isopentenyl transferase - NAA 1-naphthylacetic acid - NP normal phase - NPPU N-(3-nitrophenyl)-N-phenylurea - RIA radioimmunoassay - RP reversed phase We wish to thank Dr. J. Zwar for supplying phenylurea derivitives.