Phenotypically normal transgenic T-cyt tobacco plants as a model for the investigation of plant gene expression in response to phytohormonal stress

The tumour-inducing T-DNA gene 4 (T-cyt gene) of the nopaline Ti plasmid pTiC58 was cloned and introduced into tobacco cells by leaf disc transformation using Agrobacterium plasmid vectors. Tobacco shoots exposed to elevated cytokinin levels were unable to develop roots and lacked apical dominance. Using exogenously applied phytohormone manipulations we were able to regenerate morphologically normal transgenic tobacco plants which differed in endogenous cytokinin levels from normal untransformed plants. Although T-cyt gene mRNA levels, as revealed by dot-blot hybridization data, in these rooting plants were only about half those in primary transformed shoots the total amount of cytokinins was much lower than in crown gall tissue or cytokinin-type transformed shoots as reported by others. Nevertheless the cytokinin content in T-cyt plants was about 3 times greater than in control tobacco plants.Elevated cytokinin levels have been shown to change the expression of several plant genes, including some nuclear genes encoding chloroplast proteins. Our results show that the mRNA levels of chloroplast rbcL gene increase in cytokinin-type transgenic tobacco plants as compared with untransformed plants. Data obtained suggest that T-cyt transgenic plants are a good model for studying plant gene activity in different parts of the plant under endogenous cytokinin stress.     

In planta analysis of the Agrobacterium tumefaciens T-cyt gene promoter: identification of an upstream region essential for promoter activity in leaf,stem and root cells of transgenic tobacco

The promoter region of the Agrobacterium tumefaciens T-cyt gene was fused to a -glucuronidase (gusA) reporter gene and introduced into tobacco plants. Detection of gusA expression in transgenic F1 progeny revealed that the T-cyt promoter is active in many, if not all, cell types in leaves, stems and roots of fully developed plants. Developmental stage-dependent promoter activity was observed in seedlings. Analysis of 5-deleted promoter fragments showed that sequences located between positions–185 and –139 with respect to the T-cyt translational start codon are essential for T-cyt promoter activity in transfected tobacco protoplasts as well as in transformed tobacco plants.     

The <Emphasis Type="Italic">rolB</Emphasis> gene promotes rooting <Emphasis Type="Italic">in vitro </Emphasis>and increases fresh root weight <Emphasis Type="Italic">in vivo</Emphasis> of transformed apple scion cultivar ‘Florina’

Transgenic apple (Malus × domestica Borkh.) Florina plants were obtained by Agrobacterium-mediated transformation. The efficiency of gene transfer was 7.9%, calculated as a number of explants producing at least one transgenic shoot, after co-cultivation of leaf explants from in vitro-grown shoots in a thin layer of the A. tumefaciens C58C1 strain with the binary vector pCMB-B:GUS. Polymerase chain reaction revealed that all the clones contained the nptII and rolB genes, while four of them did not contain the gus gene. Southern blot analysis confirmed the integration of the nptII and rolB genes, with one to three copies per genome being present. All independent rolB-transgenic lines were able to produce roots in vitro on the hormone free medium, while the plants, transformed with the vector pIB16.1, or untransformed control plants did not root, and only half of shoots of MM106 rootstock rooted on this medium. The average root number in the rolB-transgenic clones ranged from 4 to 7.7. Pretreatment with indole-3-butyric acid caused root formation in all transgenic and control plants and significantly increased root number in the rolB-transgenic lines, compared to untransformed plants. RolB-transgenic plants, grown in vivo in greenhouse for 2 years, did not differ phenotypically from the wild type line with the exception of root parts. All rolB-transformed plants produced altered root systems containing more fine roots leading to significantly increased fresh root weight in five plant lines.     

Phenotypic changes in T-cyt-transformed potato plants are consistent with enhanced sensitivity of specific cell types to normal regulation by root-derived cytokinin

From over forty independently isolated potato lines transformed with wild-type and promoter-mutated T-cyt genes [12], a number of lines were selected for examination of phenotypic changes in growth and development for plants grown in soil in a controlled environment. The three lines chosen for most detailed examination showed a wide spectrum of phenotypic changes. In comparisons with control potato cv. Désirée, the plants of one line had a two- to three-fold increase in biomass production during early vegetative growth, advanced senescence and a shortened plant life-span. Another line showed abnormal cellulytic senescence. In two lines there were increases in tuber numbers and more skewed tuber size distributions which correlated with reduced shoot apical dominance and shortened dormancy of the stored tubers. None of the lines showed altered timing of onset of tuberization or flowering, although tuberization was consistently delayed when expressed as a function of increasing total plant weight. A hypothesis is proposed to explain the diverse phenotypes which postulates that (1) T-cyt transformation causes enhanced sensitivity to cytokinins in specific types of shoot cells which are already targets for regulation by normal root-derived cytokinins; (2) two distinct types of shoot target cells are present, one in shoot meristems and one in leaves; (3) the two types can acquire enhanced sensitivity, either separately or in combination depending on the particular T-cyt transformation event. The scope for using the transformed plants in subsequent physiological, biochemical and molecular studies, aimed at examining the molecular basis of the model or selected consequences of T-cyt transformation in altering regulation of potato plant growth and development, is discussed. The attention is drawn to the possible involvement at the subcellular level of sucrose phosphate synthase in mediating the phenotypic effects caused by T-cyt transformation.     

Changes in translatable poly(A) RNA from differentiated potato tissues transformed with shoot-inducing Ti TL-DNA of Agrobacterium tumefaciens

Summary Two dimensional gel electrophoresis was used to examine differences in steady state total poly(A) RNA from untransformed potato (Solanum tuberosum cv. Maris Bard) and potato transformed with shoot-inducing T_L-DNA from A. tumefaciens. RNA was compared from phenotypically very distinct in vitro cultured shoots, more similar grafted plants and tubers. In each case between 200–400 translation products were identified representing the more abundant poly(A) mRNA's. In general, poly(A) RNA from the transformed tissues gave more high molecular weight products. This increase was most evident in poly(A) RNA from shoot cultures. Depending on the tissue examined, 1–5% of the translation products with a molecular weight <43 KD were observed to increase or decrease in abundance. The influence of T-DNA on cellular gene expression in the different transformed potato tissues is discussed in relation to previously determined changes in T-DNA gene expression (particularly of the T-DNA cytokinin gene) and the corresponding changes in endogenous hormone concentrations. It is concluded that some of the specific changes in low molecular weight products are either directly caused by the increased cytokinin levels or are indirectly involved in maintaining the transformed phenotype. re]19850530 rv]19851206 ac]19851210     

TL-DNA from Agrobacterium rhizogenes plasmid pRi1855 reduces the osmotic pressure in transformed plants grown in vitro

Growth, water content, osmotic pressure and solute content were examined for normal potato (Solanum tuberosum L. cv. Desiree) and a derivative (line D9X8a), which was genetically transformed with T_L-DNA from Agrobacterium rhizogenes. Plants were grown (i) in vitro, (ii) in a growth chamber and (iii) in the field. In vitro, the transformed potato plants produced more biomass than the untransformed plants, partly because they had a higher water content. Potassium concentration and osmotic pressure were lower in cell sap extracted from the transformed potato shoots. In some cases the difference was as much as 50%. These differences were less clear, absent or reversed in plants from a growth chamber or from the field. In the field, however, transformed potato senesced early. It is suggested that a cellular basis for these observations may be changes induced by Ri T_L-DNA expression products in plant membrane properties.Abbreviations Ri root inducing - Ti tumour inducing - T-DNA transferred DNA     

Regulation of Agrobacterium tumefaciens T-cyt gene expression in leaves of transgenic potato (Solanum tuberosum L. cv. Désirée) is strongly influenced by plant culture conditions

The promoter region of the Agrobacterium tumefaciens T-cyt gene was linked in a translational fusion to the coding DNA of the reporter gene uidA (for -glucuronidase or GUS protein; EC 3.2.1.31) and to nos 3 flanking DNA. The chimaeric gene was introduced by Agrobacterium transformation into potato (Solanum tuberosum L. cv. Désirée). In nine transgenic lines, the average GUS levels were highest in extracts from stems and roots of in vitro grown plants (ca. 11 000 GUS activity units per pmol MU per mg protein per min) but lower in leaves of the in vitro grown plants (ca. 7000 units). GUS activity was intermediate in stems and roots of plants grown in soil as well as in in vitro crown galls (ca. 3000 units). Activity was low in tubers, irrespective of whether these developed in vitro or in soil (both ca. 100 units), and lowest of all in leaves of soil-grown plants (ca. 10–15 units). However, in shoot cultures reestablished from soil-grown plants, GUS activity in the leaves increased to that determined in the original shoot cultures. Hence, plant culture conditions strongly influenced the expression of the T-cyt-uidA-nos gene. In particular, it was silenced in leaves of soil-grown plants. The results are compared with previous analyses of the promoter region of the wild-type T-cyt gene and with the growth properties of a large number of crown gall cell lines and crown-gall-derived plants, including over forty S. tuberosum cv. Désirée cell lines isolated in the present study that were transformed with the wild-type T-cyt gene and six promoter-mutated derivatives. A number of implications are discussed for crown gall formation and for control of expression of plant genes which contain Activator or G-box type 5 expression control sequences.     

The use of Agrobacterium rhizogenes transformed roots to obtain transgenic shoots of the apple rootstock Jork 9

The apple rootstock Jork 9 was transformed using four different Agrobacterium rhizogenes virulent strains. The mannopine strain 8196 gave the best results in the production of chimeric plants compared to two agropine strains (A4 and 15834) and one cucumopine strain. Shoot regeneration was performed on both untransformed and transformed roots. Optimum combination and concentration of thidiazuron (TDZ) and -naphtaleneacetic acid (NAA) was different between untransformed and transformed roots. From the transformed roots seven shoots were obtained and propagated as individual clones. All shoots from these clones rooted on a hormone-free medium contrary to untransformed shoots that did not root under similar culture conditions. Differences in the morphology of the leaves and stems were observed between the clones. The transformed status of the different clones was verified with mannopine tests, PCR and Southern blot analyses. Five clones contained the mas1', the ORF 13 and the rolB genes, whereas two clones contained only the rolB gene.     

In vitro plantlet regeneration from cotyledon, hypocotyl and root explants of hybrid seed geranium

In vitro plantlet regeneration systems for the seed geranium (Pelargonium x hortorum Bailey) using cotyledon, hypocotyl and root explants were optimized by studying the influence of seedling age, growth regulators and excision orientation on organogenesis. Indole-3-acetic acid combined with zeatin yielded the highest rate of shoot production on cotyledon explants (0.2–2 shoots per explant). More shoots were produced on explants cut from the most basal region of cotyledons from 2 to 4-day-old seedlings than from older seedlings or more distal cut sites. Hypocotyl explants produced the highest number of shoots, up to 40 shoots per explant, on indole-3-acetic acid (2.8–5.6 mM) + zeatin (4.6 mM) or thidiazuron (4.5 mM). Maximum shoot formation (0.3–1.4 shoots per explant) on root explants occurred when they were cultured on medium containing zeatin. Regenerated shoots rooted best on a basal medium containing no growth regulators. There were substantial differences among cultivars in shoot formation from each of the explant systems.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - TDZ thidiazuron     

The Expression of a Cytosolic Cyclophilin Promoter from Periwinkle in Transgenic Tobacco Plants

The cloning of a 465 bp fragment from the 5-flanking region of the gene encoding a cytosolic cyclophilin from periwinkle was achieved through inverse polymerase chain reaction. The DNA fragment was fused to a gusA-intron marker then introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. Histochemical analysis of the transgenic shoot cultures demonstrated that the construct was able to drive GUS expression in stomata guard cells, but not in mesophyll cells when shoots were still attached to the callus from which they were initiated. In separated transgenic shoots and in seedlings, GUS was expressed in external and internal phloem and root hairs, respectively. GUS activity in transgenic tobacco seedlings was also investigated by fluorimetric assays. Treatments with NaCl or ABA decreased promoter activity whereas treatment with yeast extracts increased it.     

Regeneration and characterization of plants from potato root lines transformed by Agrobacterium rhizogenes

Summary Transformed root lines were obtained after infection of leaf segments and tuber discs of tetraploid potato cvs Bintje and Desirée with Agrobacterium rhizogenes. In response to shoot induction, about 10% of the root lines produced shoots through callus formation. The tests for opine suggest that all 26 shoot lines of cv Bintje (Ri-Bintje) and 13 of Desirée (Ri-Desirée) were transformed. All shoot lines were tetraploid except for one octoploid subshoot line of cv Desirée; no aneuploids were observed. With the exception of two shoot lines derived from the same root line, all other Ri-Bintje plants showed a pattern of phenotypic variation, generally observed among transformed plants. In contrast, the phenotype of Ri-Desirée plants was uniform and normal; variation was observed in tuber form and size. Phenotypic variation observed among Ri-plants appeared to be mainly root line-dependent, particularly for height of plants and tuber size and form. Variation was also observed within root and shoot lines and was more pronounced among the Ri-Bintje plants. Segregation of phenotypic characteristics was observed among transformed plants, resulting in the occurrence of phenotypes resembling the control. Chromosomal stability and the frequent reversion to normal phenotype of Ri-plants make A. rhizogenes particularly suitable as a virulence vector in the binary transformation system for the transfer of desirable genes.     

Genetic manipulation in cultivars of oilseed rape (Brassica napus) using Agrobacterium

Summary The response of oilseed rape cultivars to infection with Agrobacterium tumefaciens and A. rhizogenes and the possibility of regenerating genetically transformed oilseed rape plants were examined. The frequency at which Agrobacterium induced galls or hairy-roots on in vitro cultured plants ranged from 10% to 70%, depending on the cultivar. From galls induced by the tumorigenic strain T37, known to be strongly shoot inducing on tobacco, roots developed frequently. Occasionally, shoots formed and some of these produced tumour cell specific nopaline. Attempts to grow the transformed shoots into plants have so far been unsuccessful. Whole plants transformed with Ri-T-DNA, however, were regenerated. These had crinkled leaves and abundant, frequently branching roots that showed reduced geotropism, similar to previously isolated Ri T-DNA transformed tobacco and potato plants. The transformed oilseed rape plants flowered, but failed to form seeds.     

Study of different antibiotic combinations for use in the elimination of Agrobacterium with kanamycin selection in carnation

The effect of several β-lactam antibiotics on shoot regeneration, growth and rooting of carnation (Dianthus caryophyllus L.), and their use in combination with kanamycin in Agrobacterium-mediated genetic transformation studies, was determined. Carbenicillin, cefotaxime and ticarcillin increased the regeneration rate when added alone in non-inoculated explants; whereas, with inoculated explants, this effect was only observed in ticarcillin-containing medium. Cefotaxime inhibited root growth in both transgenic and non-transgenic shoots. Rooting of non-transgenic shoots was completely inhibited in all culture media containing kanamycin. The different antibiotics used, alone or in combination, did not prevent the occurrence of false positive shoots, but it was possible to select transgenic shoots when rooting was induced in a kanamycin + ticarcillin-containing medium. Regenerated transformed shoots were free of Agrobacterium after culturing in rooting medium, as was proven by the PCR analysis for the nptI gene, the antibiogram and the culture of tissue pieces of transgenic shoots on LB broth. The use of kanamycin and timentin with or without carbenicillin, was very useful in the transformation procedure, for the elimination of Agrobacterium in regenerated shoots before their transfer to greenhouse conditions and also in the selection of transgenic versus false-positive shoots. This revised version was published online in June 2006 with corrections to the Cover Date.     

Developmental phases and STM expression during Arabidopsis shoot organogenesis

Shoot organogenesis in Arabidopsis thaliana wasstudied with regard to the timing of key developmental phases and expression ofthe SHOOTMERISTEMLESS (STM) gene.Shoot regeneration in the highly organogenic ecotype C24 was affected byexplanttype and age. The percentage of C24 cotyledon explants producing shootsdecreased from 90% to 26% when donor seedlings were more than 6 dold, but 96% of root explants produced shoots regardless of the age of thedonorplant. Using explant transfer experiments, it was shown that C24 cotyledonexplants required about 2 days to become competent and another 8-10 days tobecome determined for shoot organogenesis. A C24 line containing the promoterofthe SHOOTMERISTEMLESS (STM) genelinked to the -glucuronidase(GUS) gene was used as a tool for determining the timingofde novo shoot apical meristem (SAM) development incotyledon and root explants. Cotyledon and root explants from anSTM:GUS transgenic C24 line were placed on shoot inductionmedium and GUS expression was examined after 6-16 days ofculture. GUS expression could be found in localizedregionsof callus cells on root and cotyledon explants after 12 days indicating thatthese groups of cells were expressing the STM gene, hadreached the key time point of determination, and were producing an organizedSAM. This was consistent with the timing of determination as indicated byexplant transfer experiments. Root explants from anSTM:GUStransgenic Landsberg erecta line and a two-step tissue culture method revealedasimilar pattern of localized GUS expression duringde novo shoot organogenesis. This is the first studydocumenting the timing and pattern of expression of theSTMgene during de novo shoot organogenesis.     

Arbuscular mycorrhiza decreases cadmium phytoextraction by transgenic tobacco with inserted metallothionein

The effect of arbuscular mycorrhiza (AM) on the phytoextraction efficiency of transgenic tobacco with increased ability to tolerate and accumulate cadmium (Cd) was tested in a pot experiment. The tobacco plants bearing the yeast metallothionein CUP1 combined with a polyhistidine cluster were compared to non-transgenic tobacco of the same variety at four Cd concentrations in soil, non-inoculated or inoculated with two isolates of the AM fungus Glomus intraradices. Mycorrhizal inoculation improved the growth of both the transgenic and non-transgenic tobacco and decreased Cd concentrations in shoots and root to shoot translocation. Differences were found between the two AM fungal isolates: one isolate supported more efficient phosphorus uptake and plant growth in the soil without Cd addition, while the other isolate alleviated the inhibitory effect of cadmium on plant growth. The resulting effect of inoculation on Cd accumulation was dependent on Cd level in soil and differed between the more Cd tolerant transgenic plants and the less tolerant non-transgenic plants. Mycorrhiza mostly decreased the phytoextraction efficiency of transgenic plants while increased that of non-transgenic plants at Cd levels in soil inhibitory to tobacco growth. Mechanisms of the observed effects of inoculation on growth and Cd uptake are discussed as well as the possible implications of the results for the exploitation of AM in phytoextraction of heavy metals from contaminated soils.