Leaf Halves Research Articles

Uptake of the Herbicide Diuron as Visualised by the Fluorescence Imaging Technique

Abstract:A new fluorescence imaging system for monitoring the uptake of the PSII‐herbicide diuron (OCMU) was tested in tobacco leaves. UV‐laser‐induced (Λexc = 355 nm) fluorescence images were collected for blue fluorescence F440 (Λem = 440 nm), green fluorescence F520 (Λem = 520 nm), red chlorophyll fluorescence F690 (Λem = 690 nm) and for far‐red chlorophyll fluorescence F740 (Λem = 740 nm). Diuron‐treated leaf parts exhibited a higher red and far‐red chlorophyll fluorescence emission (F690 and F740) than untreated leaf halves, whereas the blue and green fluorescence, F440 and F520, remained unaffected. As a consequence, the fluorescence ratios blue/red (F440/F690) and blue/far‐red (F440/F740) significantly decreased in diuron‐treated leaf parts. The time course of diuron uptake into the leaf could be followed by fluorescence images taken 10 and 30 min after diuron application. The novel high resolution fluorescence imaging method supplies information on the herbicide uptake of each point of the leaf area. Its great advantage as compared to the point data fluorescence measurements applied so far is discussed.

Sectorial root growth in cuttings of Coleus rehneltianus in response to localized aerial defoliation

Asymmetries in root growth in response to localized aerial defoliation were examined in Coleus rehneltianus (Lamiaceae). We confirmed that assimilate transport was sectorial by examining the distribution of 14C-labeled carbohydrates following a 24-h chase period. Integrated physiological units (IPUs), or sectors, extended from the leaves into the roots, and this was reflected in the differential growth of roots following artificial defoliation of part of the leaf canopy. When defoliation was localized within leaves or leaf halves within sectors, roots grew asymmetrically, with decreased root growth in defoliated sectors. Three root populations were identified by their location and growth responses: stem side, stem corner, and bottom side roots, and asymmetric growth was observed in all three populations. Only the growth of stem corner roots, which made up 35–90% of dry mass of the total root population, was influenced by the pattern of aerial defoliation. In contrast, asymmetries in the growth of the other two root populations appeared to reflect the distribution of leaf biomass prior to defoliation.

Incomplete block design in plant tissue culture research

Most experiments in tissue culture employ either completely randomized or randomized complete block designs. The ability of orchardgrass (Dactylis glomerata L.) leaf halves and segments from individual leaves of chrysanthemum (Dendranthema grandiflora Tzvelev.) to respond similarly in vitro to identical treatments allows application of an alternative experimental design based on blocking by leaf. Incomplete block designs conserve experimental materials and reduce labor while maintaining the statistical power and efficiency of more conventional designs. They also allow combination with nesting (hierarchal designs) to monitor different sources of variability or with factorial studies. Application of incomplete block designs for orchardgrass (somatic embryogenesis) and chrysanthemum (shoot organogenesis) leaf cultures are presented.

Effects of the antifeedant polygodial on plant penetration by aphids, assessed by video and electrical recording

AbstractAdult apterous Myzus persicae (Sulz.) discriminated within 2 min between mature Chinese cabbage (Brassica pekinensis L.) leaf halves treated with (±)‐polygodial solution and solvent alone, and walked off leaf areas treated with polygodial faster than off the solvent‐treated areas. However, when aphids were attached to a fine gold wire and stylet penetration of cabbage leaves was recorded electrically, polygodial treatment did not affect the number or duration of electrically‐recorded penetrations, time taken to initiate a first penetration, or total penetration time. As the tethering of aphids causes behavioural restrictions which may negate the response to polygodial, indications of stylet penetration by freely‐moving insects were sought. Simultaneous electrical recording and video monitoring showed that stylet penetration duration could be accurately inferred from antennal and body movements, enabling assessment of penetration times without attachment to the wire tether. When freely‐moving M. persicae were video recorded during 15 min access to cabbage seedlings, polygodial treatment again had no apparent effect on stylet penetration. However, when aphids were presented with a choice of polygodial‐ and solvent‐treated sides of floating mature cabbage leaf discs, video recordings revealed that the insects spent less time and made fewer penetrations on the polygodial‐treated side. In addition to this rapid repellent effect, prolonged exposure to polygodial also produced behavioural changes. After being held for 24 h on polygodial‐treated leaves or green paper prior to behavioural examination, aphids penetrated seedlings fewer times but for longer periods. The relevance of the results to virus transmission studies is discussed.

SOMATIC EMBRYOGENESIS IN ORCHARDGRASS

Morphologically-faithful somatic embryos, as well as embryogenic calli, emerge directly from cultured leaf bases of selected orchardgrass clones. The highly embryogenic clone `Embryogen-P', released by the University of Tennessee, can be maintained in greenhouse pots as a ready source of material for laboratory exercises. To initiate embryogenic cultures, the basal 2 cm of the innermost three leaves are split in half longitudinally, surface disinfested in 25% bleach solution with a drop of detergent for 2 min, then rinsed twice in sterile water. The leaf halves are cut crosswise into 3-5 mm sections, which are placed on solidified Schenk and Hildebrandt's medium containing 3% sucrose and 30 μM dicamba. Cultures are incubated in dark After 3-6 weeks, small white somatic embryos grow directly on leaf surfaces and friable white embryogenic callus emerges from cut edges or from within leaf sections. Laboratory exercises to be described include a gradient embryogenic response observed in leaves and a unique “split leaf experimental design that is useful for decreasing experimental variation.

Storage of Wheat FoliagePrior to Enzyme‐Linked Immunosorbent Assay for Detection of Wheat Soil‐Borne Mosaic Virus

AbstractPlant samples, collected at various times during a growing season, are frequently stored prior to evaluating resistance to wheat soil‐borne mosaic virus (WSBMV) by enzyme‐linked immunosorbent assay (ELISA). Leaves of winter wheat cvs Sage and Vona, showing symptoms of WSBMV infection, were cut in half along the midrib. Each half was either: 1) refrigerated at 4 °C, 2) frozen at −20 °C, 3) frozen at –70 °C, or 4) desiccated with CaCl2. Relative virus antigen titres were evaluated for individual leaf halves by ELISA. ELISA absorbance means from desiccated leaf halves were consistently higher than absorbance means from corresponding leaf halves that had been frozen. This distinction suggests that virus antigen decreases during freezing but is retained during chemical desiccation. All 4 methods of storage were found to be suitable for short‐term storage prior to qualitative evaluations by ELISA, but chemical desiccation was the superior method for long‐term storage and for storage of foliar samples prior to quantitative evaluations by ELISA.

Increase of Free Space Solutes in Tobacco Leaves in Relation to the Localized Cellular Response Following Injections of a Bacterial Protein‐Lipopolysaccharide Complex*

AbstractVariations in pH, cell ultrastructure, conductivity, calcium ion molarity, reducing sugars, uronic acids and in protein of the intercellular washing fluid were studied at 0, 12, 24 and 48 h in tobacco leaf halves intercellularly injected with a bacterial pr‐LPS complex (250 μg‐ml−1). These injections induced a localized cellular reaction (LCR) in points opposite to intercellular structured deposits on the plant cell walls. The intercellular fluid pH was higher at 24 h than in the control, but not at 12 and 48 h. The conductivity, the calcium ion molarity, the free and hydrolyzed sugar content were higher at 12, 24 and 48 h than in the control tissue; the uronic acid content was higher at 24 and 48 h, but not at 12 h. There was a peak for, all 5 parameters at around 24 h, when LCR showed its highest activity. The protein content was significantly higher at 24 and 48 h than in the control intercellular fluid.The increase in conductivity, calcium ions, sugar, uronic acids and proteins in the intercellular fluids of the pr‐LPS injected tissue were interpreted as direct or indirect effects of the LCR, i.e. as an exocytosis induced by pr‐LPS injections. The associated high sugar and uronic acid content of the intercellular fluid at 12 and 24 h was not correlated to its capacity to prevent the hypersensitive confluent necrosis when injected intercellularly into tobacco tissue.

THE EFFECTS OF A POLYAMINE BIOSYNTHESIS INHIBITOR ON INFECTION OF VICIA FABA L. BY THE RUST FUNGUS, UROMYCES VICIAE‐FABAE (PERS.) SCHROET

The effects of the polyamine biosynthesis inhibitor, difluoromethylomithine (DFMO), on rust infection of the broad bean have been examined. DFMO was sprayed onto leaves at 0.025, 0.1 and 0.4 mM before and after inoculation. DFMO was also applied to leaf halves in order to examine the possible translocation of the inhibitor to untreated leaf halves (systemic treatment). DFMO gave good control of rust infection, irrespective of the treatment. DFMO applied after inoculation was more effective than a pre-inoculatory treatment when used in non-systemic tests. When used as a systemic treatment on leaf halves, pre-inoculatory application of DFMO was more effective. DFMO did not affect plant growth or endogenous concentrations of poly amines. The possible use of DFMO in the control of a range of plant diseases is discussed.

Mutant induction through adventitious buds of Kohleria

Freshly cut leaves of Kohleria eriantha and K. x ‘Longwood’ were exposed to a wide range of gamma irradiation doses and allowed to root and form adventitious buds. K. Eriantha could not be successfully propagated from leaf half cuttings. ‘Longwood’ produced a small number of adventitious plantlets as compared to other Gesneriads. Colchicine treatments reduced leaf half survival in ‘Longwood’ by more than 50%. Leaf halves exposed to low and moderate doses of gamma irradiation showed increased overall plantlet production compared to nonirradiated leaf halves.

Translocation Patterns in Xanthium in Relation to Long Day Inhibition of Flowering

The nature of long day inhibition of flowering in the short day plant Xanthium strumarium L. was studied by correlating the flowering response with the translocation of (14)C-assimilates from induced leaves or parts thereof to the shoot tips.In contrast to an earlier study by Gibby and Salisbury (Plant Physiol 1971 47: 784-789) no inhibitory effect of an immature leaf in long day on the flower-promoting effect of an induced leaf was detected. When the stimulus moved in the basipetal direction, mature leaves in long day were inhibitory to flowering and at the same time reduced the amount of (14)C-photosynthate that accumulated in the receptor buds.Inhibition of flowering was observed when the apical half of a single leaf was induced and the basal half kept under long day conditions. Induction of a lateral leaf half, with the other half remaining in long day, resulted in a normal flowering response. More (14)C-photosynthate was translocated to the shoot tip from the basal than from the apical leaf half.Removal of the noninduced basal leaf half except for the midrib and major veins, cutting the basal half along the midrib, or keeping the basal half in darkness, resulted in normal flowering. In all three treatments the amounts of (14)C-assimilates that accumulated in the shoot tips also increased, presumably because competing export of nonlabeled assimilates from the basal leaf halves was diminished or eliminated.Autoradiography indicated that (14)C-assimilates produced in the apical half were channeled through the basal half in the major veins and midrib to the petiole without partitioning back into the mesophyll. Since the veins and midrib in the basal half were by themselves not inhibitory to flowering, it is unlikely that the floral stimulus was inactivated in the long day tissue. When the basal half was labeled with (14)CO(2), there was no indication in autoradiograms that (14)C-labeled assimilates moved in the blade in the apical direction.As a whole these results demonstrate that transmission of the floral stimulus and translocation of photosynthate are correlated, so that at least part of the long day inhibition in Xanthium can be explained in terms of translocation effects. However, the involvement of a transmissible inhibitor produced in long day tissue cannot be ruled out.

Permeability and membrane lipid metabolism of Phaseolus vulgaris infected with Uromyces phaseoli I. Changes in the efflux of cell constituents

The leakage of electrolytes, amino acids and sugars from rust-infected leaf halves and uninfected halves of the same leaves was determined and compared with the leakage from healthy primary leaves of bean plants. The infection brought about a significant increase in the leakage of ions, amino acids and sugars from the infected leaf halves. The leakage became evident at 4 days after inoculation and was further increased for electrolytes and amino acids during the remainder of the experimental period. No differences were observed between the uninfected leaf halves and the controls up to 8 days after inoculation, but afterwards (10 and 12 days after inoculation) the leakage from the uninfected leaf halves was higher.

Permeability and membrane lipid metabolism of Phaseolus vulgaris infected with Uromyces phaseoli II. Changes in lipid concentration and 32P incorporation into phospholipids

Phospholipids and glycolipids of rust-infected leaf halves and uninfected halves of the same leaves were quantitatively determined and compared with the lipids of healthy primary leaves of bean plants. The phosphatidyl-choline, phosphatidyl-ethanolamine, phosphatidylinositol and sulphoquinovosyl diglyceride (SL) content of the infected halves was the same as in the control plants. The infected tissue showed a decrease in monogalactosyl diglyceride (MGG), digalactosyl diglyceride (DGG) and phosphatidyl-glycerol (PG) and an increase in phosphatidyl-serine (PS) and phosphatidic acid content. These differences were localized mainly in the region of the pustule and not in the surrounding mycelium-free tissue. The amount of all phospholipids and glycolipids in the uninfected leaf halves was slightly lower than in the controls. The ungerminated and germinated uredospores of the parasite contained no MGG, DGG, SL or PG. The infected half leaves showed an increase in 32P incorporation into all phospholipids especially into PS. The differences in 32P-incorporation between the phospholipids of the uninfected half leaves and the controls were only small. These results were discussed in relation to permeability changes of rust-infected bean tissue.

Permeability and membrane lipid metabolism of Phaseolus vulgaris infected with Uromyces phaseoli III. Changes in relative concentration of lipid bound fatty acids and phospholipase activity

Fatty acids from membrane lipids and phospholipase activity of rust-infected half leaves and uninfected halves of the same leaves were determined and compared with healthy primary leaves. The following lipids were analysed for their fatty acid composition: monogalactosyl diglyceride (MGG), digalactosyl diglyceride (DGG), phosphatidyl-choline (PCH), phosphatidyl-ethanolamine (PE) and phosphatidyl-glycerol (PG). The major fatty acids detected in these lipids of both bean leaves and fungal uredospores were palmitic, stearic, oleic, linoleic, linolenic and in PG, trans-three-hexadecanoic acid. The PE and PCH isolated from the infected half leaves contained a higher amount of unsaturated fatty acids than the corresponding lipids from the two other tissues. The PE and PCH from fungal uredospores showed a higher degree of unsaturation than the lipids from bean leaves. The changes in fatty acid composition of PG, MGG and DGG were small and possibly not significant. The differences in fatty acid composition were localized mainly in the region of the pustule. The adjacent fungus free host tissue was affected only to a small extent. The infected tissue showed an increase in phospholipase activity as compared with the controls and the uninfected leaf halves. The results were discussed mainly in relation to permeability changes of rust-infected bean tissue.

Systemic fungicidal and insecticidal activities of 1- and 2-[bis (dimethylamido)phosphoryl]-3-alkyl-5-anilino-1,2,4-triazoles

The compounds referred to in the title have been investigated for fungicidal, insecticidal and acaricidal activities in laboratory and greenhouse tests. Several representatives of this class of compounds were active against powdery mildew on apple, cucumber, and barley, and against aphids and spider mites, both when applied to the leaves and when added to the nutrient solutions of test plants. Treatment of leaf halves resulted in protection of the entire leaves. A striking difference in pesticidal activity was observed between two series of isomers. Representatives of the series with the phosphoryl group in the 1-position showed much greater pesticidal activities than their corresponding isomers with the phosphoryl group in the 2-position. The optimum activity within the two homologous series (R-H, CH3 ... C6H13) was determined: generally the lower homologues (R-H, CH3, C2H5 and i-C3H7) showed the greatest pesticidal activity in the systemic tests. After leaf-application the influence of the length of R was less pronounced or even reversed.

An extractable agent, induced in uninfected tissues by localized virus infections, that interferes with infection by tobacco mosaic virus

Juice extracted from uninfected apical halves of leaves of Datura stramonium previously inoculated on their basal halves with tobacco mosaic virus (TMV) or tobacco necrosis virus, a procedure known to induce resistance to TMV in the uninfected apical halves, was found consistently to interfere with infection by TMV (when mixed with it just prior to inoculation of Nicotiana glutinosa, N. tabacum var. Samsun NN, or D. stramonium) to a much greater extent than did juice from apical halves of control leaves. In some tests, a standard TMV inoculum induced only 8% as many lesions when mixed with extracts from resistant half-leaves of Datura as it did when mixed with extracts from comparable control leaves. The difference in ability of the two juices to interfere with infection by TMV was found not to be due to differences in pH nor to inactivation of the virus in vitro. The differential effect of the two types of juice was eliminated by heating at 78°C for 15 minutes but not by heating at 68° for 15 minutes, by aging at 3° for 5 days, by dialysis, or by centrifugation at 93,000 g for 1 hour. Partial purification was effected by dialysis and use of an anion-exchange cellulose; after each step in the procedure, extracts from resistant half-leaves remained more inhibitory than did those from control leaves. Evidently, infection in basal leaf halves either induced the formation in apical halves of an interfering agent (or agents) not found in healthy leaves or stimulated the production of an inhibitor (or inhibitors) normally found in Datura leaves. Extracts from resistant half-leaves of Samsun NN tobacco were sometimes, but not always, more inhibitory than extracts from control leaves.