Sensitive Detection of the Leaching-out from Membrane-surrounded Compartments: a New Approach Explored with Cells and Tissues of Nicotiana tabacum L.

In search of a simple method for testing the very early events of ozone damage to susceptible plants as well as complete destruction after threshold-exceeding treatments an over-all measurement of cell ingredients by their optical density in the UVB and UVC range was investigated. The cell particles were liberated after membrane permeabilization or after cell bursting. Uncontrolled results could be excluded. Furthermore, the results of the developed spectrophotometrical test could be, in the case of tissue samples (leaf discs), very well reproduced with an osmometrical measurement. The latter was less sensitive and not suitable for cellular samples because the protoplasts must be dissolved in a nearly isotonic medium which caused too large a background for this method but not for the UVS test. Contrary to the osmometric measurement, the photometric one cannot be used for determining the absolute amount of cell ingredients but only for relative measurement between samples in a given range of concentrations. Oxidative changes of the liberated ingredients do not influence their detection, which was demonstrated with ascorbate. The developed leaching test was also useful for determining the membrane damage caused by the detergent Triton X-100, although this was known to have UV absorbance by itself. It was noted that the far UV maximum is not only caused by absorbance and scattering is discussed as an additional explanation.     

Growth kinetics, polyamine pattern and biosynthesis in hairy root lines of Nicotiana tabacum

The possibility of a relation between the expression of root inducing (Ri) T-DNA genes of Agrobacterium rhizogenes and changes in polyamine metabolism has been explored in fast-growing tobacco hairy roots. Transformed root cultures have been established on hormone-fee medium; they came from transgenic plants of Nicotiana tabacum L. cv. Xanthi with different altered phenotypes, designated transformed (T) and supertransformed (T'). T and especially T' roots developed more rapidly both by elongation and lateral branching, and showed a higher growth rate than the untransformed control. After 3 weeks in culture, normal roots showed a very reduced meristematic zone, and flow cytometric analysis indicated that 2C nuclei were predominant in the apical parts in contrast to T and T' roots, in which endopolyploidisation also appeared. Putrescine, spermidine and traces of spermine were present in all the samples, both in free and in conjugated forms. Putrescine was the major polyamine detected in controls and in transformed roots. At the time of excision, the polyamine levels were similar in normal, T and T' roots. Significant differences were found during the progression of growth, particularly in the TCA-insoluble fraction in which polyamines varied differently according to the type of roots, increasing considerably in T roots on day 8, then decreasing. The lower polyamine contents found in growing transformed roots were concomitant to low arginine (EC 4.1.1.19) and ornithine (EC 4.1.1.17) decarboxylase activities. It is suggested that polyamine levels and related enzyme activities are linked to growth kinetics rather than being a consequence of foreign gene expression.     

Further studies on the effects of different auxins and cytokinins on flower formation in thin cell layers of Nicotiana tabacum: The effects of zeatin riboside, dihydrozeatin and dihydrozeatin riboside

Organogenesis in thin cell layers of Nicotiana tabacum L. was studied in relation to the effects of natural and synthetic auxins in combination with various cytokinins. All cytokinins tested, benzyladenine (BA), kinetin, zeatin (Z), zeatin riboside (ZR), N⁶-Δ²-isopentenyl) adenine (IPA), dihydrozeatin [(diH)Z] and dihydrozeatin riboside [(diH)ZR], seem to be active in flower bud formation. In addition to the initiation of flower buds, vegetative buds or roots were also formed on the explants in the presence of BA, Z or IPA as exogenous cytokinins. Only dihydrozeatin and its riboside stimulated the initation of flower buds alone (as is known for kinetin), especially if supplemented with indole-3-acetic acid (IAA) as exogenous auxin. A high number of explants with flower buds was also found with high cytokinin/2,4-D ratios. In these conditions the presence of (diH)Z yielded the higest number of flower buds per explant.     

Rare symmetric and asymmetric Nicotiana tabacum (+) N. megalosiphon somatic hybrids recovered by selection for nuclear-encoded resistance genes and in the absence of genome inactivation

Following protoplast fusion between Nicotiana tabacum (dhfr) and N. megalosiphon (nptII) somatic hybrids were selected on the basis of dual resistance to kanamycin and methotrexate. Despite strong selection for parental nuclear-encoded resistances, only nine N. tabacum (+) N. megalosiphon somatic hybrids were obtained. A preferential loss of the parental N. tabacum nuclear and organelle genome was apparent in some plants in spite of the lack of genomic inactivation by the irradiation or chemical treatment of the parental protoplasts. Only six of the nine hybrids recovered possessed both parental profiles of nuclear RFLPs and isoenzymes. The remaining three hybrids were highly asymmetric with two being identical to N. megalosiphon except for minor morphological differences and rearranged or recombined mitochondrial DNAs (mtDNA), while the other one was distinguishable only by the presence of a rearranged or recombined mtDNA, and was therefore possibly a cybrid. Overall, eight somatic hybrids possessed rearranged or recombined mtDNAs and chloroplast inheritance was non-random since eight possessed N. megalosiphon-type chloroplasts and only one had N. tabacum chloroplasts. In contrast, using the same selection approach, numerous morphologically similar symmetric somatic hybrids with nuclear RFLPs and isozymes of both the parental species were recovered from control fusions between N. tabacum and the more closely related N. sylvestris. In spite of the low frequency of recovery of symmetric N. tabacum (+) N. megalosiphon hybrids in this study, one of these hybrids displayed a significant degree of self-fertility allowing for back-crosses to transfer N. megalosiphon disease-resistance traits to N. tabacum. Plant Research Centre Contribution No. 1579     

Early season root production and zoospore infection of cultivars of flue-cured tobacco that differ in level of partial resistance to Phytophthora parasitica var. nicotianae

Root production of four cultivars of flue-cured tobacco was quantified in the field, greenhouse and phytotron. The cultivars ranged in level of partial resistance to the black shank pathogen, Phytophthora parasitica var. nicotianae, from susceptible to highly resistant. In the field, root-observation plates were installed approximately 10 cm from plants, and in greenhouse and phytotron studies, plants were grown in 4-liter containers with one sloping transparent side for root observation. Root growth was determined weekly for four weeks after transplanting in the field and daily up to 14 days after transplanting in the greenhouse and phytotron. Root tracings were made on acetate sheets placed against the sloping transparent side of the containers or against the transparent observation plates in the field following removal of soil from the outside of the observation plate. Root growth was quantified by retracing the root pattern on the acetate sheets over a digitizing tablet attached to a personal computer. Numbers of roots, root length, and mean and maximum rate of root growth were determined. Cultivars Hicks (susceptible) and K-326 (low level of resistance) had significantly larger root systems than moderately resistant G-28 or highly resistant NC 82. Differences in total root length were due to increased branching that resulted in development of significantly greater numbers of roots in Hicks and K-326. For example, between day 21 and 28, Hicks produced more than three times the number of new roots as NC 82 in the field. The mean rate of root extension observed (2.17 mm hr^–1) was similar in all four cultivars. Infection efficiency on the different cultivars was determined in the field by inoculating roots with zoospores of P. p. nicotianae. Lesions were visible as water soaked areas within 24 hr of inoculation. At 48 hr after inoculation, percentages of inoculations that resulted in lesion formation were 57, 46, 23, and 16% for Hicks, K-326, G-28 and NC 82, respectively. The possible role of rooting intensity as a mechanism of avoidance to P. p. nicotianae in tobacco cultivars is discussed.     

A novel wound-inducible extensin gene is expressed early in newly isolated protoplasts of Nicotiana sylvestris

A cDNA clone (6PExt 1.2) encoding a novel extensin was isolated from a cDNA library made from 6 h old mesophyll protoplasts of Nicotiana sylvestris. The screening was performed with a heterologous probe from carrot. The encoded polypeptide showed features characteristic of hydroxyproline-rich glycoproteins such as Ser-(Pro)₄ repeats and a high content in Tyr and Lys residues. The presence of four Tyr-X-Tyr-Lys motifs suggests the possibility for intramolecular isodityrosine cross-links whereas three Val-Tyr-Lys motifs may participate in intermolecular cross-links. The analysis of genomic DNA gel blots using both the N. sylvestris and the carrot clones as probes showed that the 6PExt 1.2 gene belongs to a complex multigene family encoding extensin and extensin-related polypeptides in N. sylvestris as well as in related Nicotianeae including a laboratory hybrid. This was confirmed by the analysis of RNA gel blots: a set of mRNAs ranging in size from 0.3 kb to 3.5 kb was found by the carrot extensin probe. The 6PExt 1.2 probe found a 1.2 kb mRNA in protoplasts and in wounded tissues as well as a 0.9 kb mRNA which seemed to be stem-specific. The gene encoding 6PExt 1.2 was induced by wounding in protoplasts, in leaf strips and after Agrobacterium tumefaciens infection of stems.     

An envelope-shaped film culture vessel for plant cell suspension cultures and metabolite production without agitation

An envelope-shaped film culture vessel (named Culture Bag) made of fluorocarbon polymer film, which is much more permeable to oxygen, nitrogen and carbon dioxide than other films, was found to be suitable to grow plant cells in liquid medium without agitation. Proliferous BY-2 tobacco cells showed almost the same growth in a Culture Bag of 12.5 m-thick film as that in a shake flask; the growth was lower in a Culture Bag of a thicker film. Lithospermum erythrorhizon cells produced almost the same amount of red naphthoquinone pigments (shikonin derivatives) in a Culture Bag of 12.5 m-thick film as those in a shake flask although the productivity was suppressed as the film thickness increased. L. erythrorhizon cells in a Culture Bag produced much less abnormal stress metabolites (orange-colored benzoquinone derivatives) than those in a shake flask, suggesting that culturing cells in the Culture Bag was less stressful due to its stationary liquid environment.     

Effect of DNA fragment size on transformation frequencies in tobacco (Nicotiana tabacum) and maize (Zea mays)

During eukaryotic cell transformation, the transforming DNA must enter the host cell, traverse the cytoplasm and enter the nucleus before becoming stably integrated into the genome. The limiting step for plant protoplast transformation may lie at the cell membrane, the nuclear membrane, or at the integration step. We show here that the size of the DNA fragment containing the selectable marker used to monitor transformation can directly affect the efficiency of stable transformation. In both tobacco and maize protoplasts, the smallest DNA fragments gave the highest stable transformation frequencies.     

Developmental and pathogen-induced activation of an msr gene, str246C, from tobacco involves multiple regulatory elements

A family of genes, the so-called msr genes (multiple stimulus response), has recently been identified on the basis of sequence homology in various plant species. Members of this gene family are thought to be regulated by a number of environmental or developmental stimuli, although it is not known whether any one member responds more specifically to one stimulus, or whether each gene member responds to various environmental stimuli. In this report, we address this question by studying the tobacco msr gene str246C. Using transgenic tobacco plants containing 2.1 kb of 5 flanking DNA sequence from the str246C gene fused to the -glucuronidase (GUS) coding region, the complex expression pattern of the str246C promoter has been characterized. Expression of the str246C promoter is strongly and rapidly induced by bacterial, fungal and viral infection and this induction is systemic. Elicitor preparations from phytopathogenic bacteria and fungi activate the str246C promoter to high levels, as do wounding, the application of auxin, auxin and cytokinin, salicylic acid or copper sulfate, indicating the absence of gene specialization within the msr gene family, at least for str246C. In addition, GUS activity was visualized. histochemically in root meristematic tissues of tobacco seedlings and is restricted to roots and sepals of mature plants. Finally, analysis of a series of 5 deletions of the str246C promoter-GUS gene fusion in transgenic tobacco plants confirms the involvement of multiple regulatory elements. A region of 83 by was found to be necessary for induction of promoter activity in response to Pseudomonas solanacearum, while auxin inducibility and root expression are apparently not controlled by this element, since its removal does not abolish either response. An element of the promoter with a negative effect on promoter activation by P. solanacearum was also identified.Joint first authors     

Plant transformation by particle bombardment of embryogenic pollen

Summary Direct delivery of DNA into embryogenic pollen was used to produce transgenic plants in tobacco. A plasmid bearing the ß-glucuronidase (GUS) marker gene in fusion with the 35S-promoter was introduced by microprojectile bombardment into mid-binucleate pollen of Nicotiana tabacum that had been induced to form embryos by a starvation treatment. In cytochemical expression assays, 5 out of 10⁴ pollen grains were GUS⁺. Visual selection by staining with a non-lethal substrate for GUS was used to manually isolate transformed embryos. From the initial population of embryogenic GUS⁺ pollen, 1–5% developed into multicellular structures and 0.02% formed regenerable embryos. Two haploid transformants were regenerated. GUS expression was detected in different parts of the plants, and Southern analysis confirmed stable integration of the foreign DNA. Diploidisation was induced by injection of colchicine into the stem near adventitious buds. Offspring from selfings and backcrosses of one transformant were tested for GUS expression and by Southern blots. All F1-plants were transgenic, in accordance with Mendelian inheritance.Abbreviations GUS ß-glucuronidase - CaMV Cauliflower Mosaic Virus - MCS multicellular structure - NPTII neomycin phosphotransferase - PEG polyethylene glycol - X-gluc 5-bromo-4-chloro-3-indolyl glucuronide - DAPI 4,6-diamidino-2-phenylindole - Tris Tris(hydroxymethyl)aminomethane hydrochloride - EDTA ethylenedinitrilo tetraacetic acid, disodium salt dihydrate