A Method for the Isolation of Stable Mesophyll Protoplasts from Tomato Leaves throughout the Year under Standard Conditions

A method is described, based on growing tomato plants under low light intensities and feeding with calcium nitrate, whereby stable protoplasts can be isolated throughout the year under standard conditions. Plants are grown in the equivalent of spring and winter light intensities (i. e. incoming solar radiation 2.52 to 10.8 MJ m^?2 day^?1 15 h day) in the glasshouse on peat compost supplied with a balanced fertilizer containing nitrate and urea as nitrogen sources and given a supplementary feed of calcium nitrate. Under these conditions, high yields of protoplasts can be isolated on incubation in the dark using low enzyme concentrations at 25°C for 2 h. Protoplasts isolated in this way can support tobacco mosaic virus replication and are capable of cell division.     

Importance of myo-inositol,calcium, and ammonium for the viability and division of tomato (Lycopersicon esculentum) protoplasts

Plants from four cultivars of Lycopersicon esculentum were grown under different conditions, in controlled environment chambers. Low light intensity, long photoperiod (16 h), 25° C/17°C temperature alternance (day/night) were found to be the most convenient conditions for obtaining viable protoplasts. The use of myo-inositol as an osmoticum in the digestion medium and the adjustment of the pH to 6.5, instead of the usual 5.8, for this medium increased the yield of viable protoplasts and enhanced their stability. Under these conditions neither pretreatment (dark and cold treatments), nor preplasmolysis of leaf tissues, were required before protoplast isolation. The concentrations of ammonium nitrate, calcium chloride, myo-inositol, and sucrose were found to be critical for the success of protoplast culture. A medium containing 5 mM ammonium nitrate, 40 mM calcium chloride, 10 mg l^-1 adenine sulfate, 0.5% myo-inositol and 6% sucrose gave sustained protoplast divisions. Under these conditions, plating efficiency ranged from 5% for the cultivar Lukulus to 15% for the cultivar Golden Sunrise.Abbreviations BA benzylaminopurine - CaCl₂ calcium chloride, 2,4,-D-2,4-dichlorophenoxyacetic acid - EDTA ethylene diamine tetraacetic acid - KCl potassium chloride - MES-2-N morpholino ethane sulfonic acid - MgCl₂ magnesium chloride - NH₄NO₃ ammonium nitrate - NAA naphthalene acetic acid, p-protoplasts     

Effect of cellulase fractionation on the viability of cultured barley (Hordeum vulgare) protoplasts

The age of the stock plants was important for the barley ( Hordeum vulgare L. cv. Perth) protoplast viability. Light conditions under which the stock plants were grown also affected the viability of the protoplasts. Greenhouse-grown plants yielded much higher number of protoplasts than dark-grown plants, but protoplast viability was better when protoplasts were isolated from etiolated plants. Light supplied during protoplast culture affected protoplast viability within the first 24 h of culture. Cellulase R-10 (Onozuka) was better than Cellulysin (Calbiochem) and Cellulase ⁺ Macerozyme R-10 (Onozuka) for barley mesophyll protoplast isolation. Cellulase R-10 (Onozuka) was fractionated on a G-75 Sephadex column. The eluted fractions were tested for their ability to release barley mesophyll protoplasts and for their toxicity towards the protoplasts. Only a small part of the Cellulase R-10 was necessary for protoplast isolation from barley leaves. When the fractionated cellulase was analysed by isoelectric focusing, this part of the cellolase appeared as a single band.     

Factors Influencing the Growth and Division of Pea Mesophyll Protoplasts

High yields of viable protoplasts were produced from pea leaves provided that only leaves of the same age were used in each preparation. The conditions under which the pea plants were grown and the age of the plants were also important. The protoplasts were cultured in a medium supplemented with 1 mg/1 2iP and 1 mg/1 2,4-D. They were able to regenerate cell walls within two days. After 5 days cell divisions were apparent and sustained divisions led to callus formation. Special emphasis has been given in this paper to the choice of leaf material for protoplast isolation.     

Immunological evidence for transfer of the barley nitrate reductase structural gene to Nicotiana tabacum by protoplast fusion

Summary A protoplast fusion experiment was designed in which the selectable marker, nitrate reductase (NR), also served as a biochemical marker to provide direct evidence for intergeneric specific gene transfer. NR-deficient tobacco (Nicotiana tabacum) mutant Nia30 protoplasts were the recipients for the attempted transfer of the NR structural gene from 50 krad -irradiated barley (Hordeum vulgare L.) protoplasts. Barley protoplasts did not form colonies and Nia30 protoplasts could not grow on nitrate medium; therefore, selection was for correction of NR deficiency allowing tobacco colonies to grow on nitrate medium. Colonies were selected from protoplast fusion treatments at an approximate frequency of 10^-5. This frequency was similar to the Nia30 reversion frequency, and thus provided little evidence for transfer of the barley NR gene to tobacco. Plants regenerated from colonies had NR activity and were analyzed by western blotting using barley NR antiserum to determine the characteristics of the NR conferring growth on nitrate. Ten plants exhibited tobacco NR indicating reversion of a Nia30 mutant NR locus. Twelve of 26 regenerated tobacco plants analyzed had NR subunits with the electrophoretic mobility and antigenic properties of barley NR. These included plants regenerated from colonies selected from 1) co-culturing a mixture of Nia30 protoplasts with irradiated barley protoplasts without a fusion treatment, 2) a protoplast fusion treatment of Nia30 and barley protoplasts, and 3) a fusion treatment of Nia30 protoplasts with irradiated barley protoplasts. No barley-like NR was detected in plants regenerated from a colony that grew on nitrate following selfed fusion of Nia30 protoplasts. Because tobacco plants expressing barley-like NR were recovered from mixture controls as well as fusion treatments, explanations for these results other than protoplast fusionmediated gene transfer are discussed.     

Accumulation of Oxalate in Tissues of Sugar-beet, and the Effect of Nitrogen Supply

In field-grown sugar-beet concentration of insoluble oxalatewas low in roots and high (about 12 per cent of ethanol insolublematerial) in leaves, and for a particular leaf the concentrationincreased continuously during its life. Of the insoluble oxalate,15–30 per cent was present as the magnesium salt and theremainder as the calcium salt. Oxalate contents of plants grownin culture solutions with nitrate as nitrogen source were similarto those of plants grown in soil, but when nitrogen was suppliedas ammonium sulphate or ammonium nitrate both soluble and insolubleoxalate were low. Plants grown in soil with regular additionsof ammonium sulphate or ammonium nitrate also had very low concentrationsof soluble oxalate although insoluble oxalate was only slightlylower than with nitrate nitrogen. Disks of root or leaf tissuewashed for several days in distilled water lost insoluble oxalatebut when washed in tap water insoluble oxalate increased morethan twofold. Addition of calcium and nitrate to the distilledwater caused an increase of insoluble oxalate, while additionof potassium caused a decrease. Use of ¹⁴C labelled oxalateand washing experiments showed that oxalate can be metabolizedby tissue disks and so is not necessarily a final product ofmetabolism. The accumulation of oxalate appears to be connectedwith the assimilation of nitrate and the preservation of thecation-anion balance of the plant.     

Plant regeneration from leaf protoplasts of Lycopersicon hirsutum f. hirsutum

A protocol is described for high frequency plant regeneration from isolated leaf protoplasts of several genotypes of the wild tomato species Lycopersicon hirsutum f. hirsutum based on modified tomato protoplast culture methods. Three to four week old seedlings exposed to a low light pretreatment yielded protoplasts capable of sustained divisions on modified Lycopersicon Culture Medium. Plating efficiencies varied from 7.2%–25.9%. Colonies were transferred to modified solid greening medium after 25–35 days. Developing calli that turned bright green and produced dark green bud primordia were transferred to shoot induction medium. Shoot formation efficiencies ranged from 60%–85%. Shoots rooted easily and regenerated plants grown to complete maturity showed only transient somaclonal variation.Abbreviations BA benzylamino purine - MES 2-(N-morpholino)-ethane sulfonic acid - NAA naphthalene acetic acid     

Content of Oxalate in Actinidia Deliciosa Plants Grown in Nutrient Solutions with Different Nitrogen Forms

Kiwifruit plants (Actinidia deliciosa cv. Hayward) were grown in Hoagland nutrient solution with calcium nitrate, potassium nitrate, ammonium nitrate or ammonium chloride as the nitrogen source. Plants grown in the solution with nitrate nitrogen displayed a higher oxalate content, greater shoot length and leaf area, and higher content of ascorbic acid and NO₃ ^– ions in the leaves. Plants grown in the solution with ammonium nitrate, and particularly with ammonium chloride, showed low oxalate content, low content of ascorbic acid and NO₃ ^–, high content of Cl^– and Na⁺, low shoot length and leaf area. Oxalate formation appeared to be connected with the assimulation of nitrate, more precisely with nitrate reduction, while ammonium nitrogen assimilation did not induce the synthesis of oxalic acid.     

Observation of polarity induction by cytochemical localization of phenylalkylamine-binding sites in regenerating protoplasts of the moss Physcomitrella patens

Different external (e.g., light) and internal (e.g., auxin and calcium gradients) factors control differentiation of the moss protonema. The present investigations demonstrate that exogenously applied auxin, the pharmacological blockade of auxin efflux by naphthylphthalamic acid, and treatment with (-)bepridil, a calcium channel antagonist, inhibit protoplast division without affecting protoplast viability in the moss Physcomitrella patens. A fluorescently labelled phenylalkylamine (DM-Bodipy PAA), another calcium channel antagonist, was used as a probe for in vivo labelling of phenylalkylamine(PAA)-binding sites. The specificity of this binding was demonstrated by competition with (-)bepridil. Confocal laser scanning microscopy visualized PAA-binding sites on the plasma membrane and along the nuclear membrane as uniformly distributed clusters. During asymmetric division of P. patens protoplasts, however, fluorescence labelling particularly increases at the membrane invagination and later along the plate separating the new cells. Intracellular localization of PAA-binding sites, probably at the membranes of vesicles and vacuoles, significantly increases in the smaller daughter cell, destined to later form a polar outgrowth, the first chloronema cell. Thus, a system was established to visualize early events in P. patens protoplast polarization at the subcellular level.     

Correlation between chloroplast motility and elastic properties of tobacco mesophyll protoplasts

Translocations of chloroplasts induced by blue light were investigated in both leaves and protoplasts isolated from leaf mesophyll of Nicotiana tabacum. In the leaf tissue, the responses of chloroplasts were similar to those observed in other, higher and lower plant species. Weak and strong light induced movements of chloroplasts towards cell walls perpendicular and parallel to the light direction, respectively. Treatment with cytochalasin D, an actin-disturbing agent, blocked the movements. This shows that actin is involved in the motile system of chloroplast translocation in tobacco. By monitoring the response of chloroplasts to light in isolated protoplasts, we addressed the question whether the presence of the cell wall is necessary for the translocations of chloroplasts to occur. In control protoplasts (isolated at room temperature from unstressed leaves), no clear light intensity-dependent changes were observed in chloroplast distribution pattern. In contrast, in protoplasts obtained from plants treated with 4 °C for 8 h the chloroplasts maintained their responsiveness to light. Atomic Force Microscopy was used to measure elastic properties of the protoplasts. Young’s modulus, which reflects rigidity of the material, was 10 times higher for protoplasts of the coldstressed plants as compared to those isolated from the control plants. The rigidity of protoplasts isolated from the plants treated with low temperature was reduced four-fold by exposure to cytochalasin D. It appears that the status of protoplast actin is a factor responsible for elasticity of protoplasts. We speculate that unknown, cold stress-induced factors, maintain the orientational movements due to anchorage of the actin cytoskeleton in the plasma membrane despite the cell wall removal.     

Effects of electric cell fusion treatment among leaf protoplasts of Populus alba and alnus firma on growth, leaf morphology, and rapd pattern of eleven acclimatized plants

Summary This study reports the characterization of 11 plants regenerated from electrically fused protoplasts between Populus alba and Alnus firma. Growth characteristics of five regenerated plants (AP-1-AP-5) in terms of shoot height and leaf color showed small differences compared with those of P. alba grown, in pots, and showed no difference in shoot height and diameter compared with those grown in nursery field. There was also no difference in the RAPD pattern between the plants regenerated from interfamilial protoplast fusion and P. alba. In contrast, the lately regenerated plants (AP-6-AP-11) grown in pots showed a marked difference in leaf morphology and RAPD pattern. There was a variation in the ratio of longitudinal to transverse length of leaves among the 11 plants from interfamilial fusions compared with that of protoclones and intraspecific fused protoplasts of P. alba.     

Improved efficiency of genotype-dependent regeneration from protoplasts of important potato cultivars

The regeneration of protoplasts from potato (Solanum tuberosum L.) cvs. Desiree and King Edward has been significantly improved. Different shoot culture media were required for the release of viable protoplasts from cvs. Maris Piper and Desiree, and the response of protoplasts to different culture conditions depended upon the cultivar genotype of the protoplast source. Using protoplast isolation media containing 6mM CaCl₂ improved protoplast viability and culture in enriched media lead to the reproducible and relatively efficient recovery of colonies from protoplasts of these cultivars. Over 70% of protoplast-derived calli from King Edward and Desiree regenerated shoots. Many shoots were grown to mature plants in soil. This is the first report of the regeneration of mature Desiree plants from protoplasts.Abbreviations NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - MES 2-(N-Morpholino)ethanesulphonic acid - CH Casein hydrolysate - CW Coconut water - Inos myo-Inositol - PABA p-Aminobenzoic acid     

Structure of Crystal Cells and Influences of Leaf Development on Crystal Cell Development and Vice Versa in Phaseolus vulgaris (Leguminosae)

The structure of cells with calcium oxalate crystals and their nelghbouring cells has been studied by light and transmission electron microscopy at different stages of bean leaf development. Plants were grown with varying calcium supply to identify a possible influence of calcium nutrition on cell structure. Crystals are formed inside the vacuole of already highly vacuolated cells of bundle sheath extensions. The membrane around the crystal vacuole is continuous with the plasmalemma. The crystal vacuole contains membraneous structures. In the fully expanded leaf the crystal becomes ensheathed by wall material. Chloroplasts of bundle sheath extension cells, with or without crystals, are smaller, with fewer membranes, and with much narrower stroma regions than those of the palisade parenchyma. There is a stage in the young leaf when only the bundle sheath extension cells without crystals have starch grains in their chloroplasts. As their number is lower in plants grown with high calcium supply this means that, in this case, less cells are competent for photosynthesis.     

An improved protocol for plant regeneration from leaf- and hypocotyl-derived protoplasts of carrot

An easy and effective regeneration system from leaf- and hypocotyl-derived protoplasts was established for carrot. The protoplast isolation efficiency after preplasmolytic treatment and digestion of source material in enzyme mixture consisted of 1% cellulase Onozuka R-10 and 0.1% pectolyase Y-23 reached on average 3 × 10⁶ and 10⁶ protoplasts per g of leaf and hypocotyl tissue, respectively. A modified thin alginate layer technique was applied for the protoplast culture. Direct somatic embryogenesis on a simplified Kao and Michayluk medium in the presence of 2,4-D and zeatin occurred during cultivation of both leaf- and hypocotyl-derived protoplasts for all accessions used. Morphologically normal plants were produced at very high efficiency within two months after initiation of the protoplast culture. Ninety three percent of in vitro derived plants were diploids. Pollen viability and seed set after self-pollination were similar to those of plants obtained from seeds.     

Characterization of a Light-Controlled Anion Channel in the Plasma Membrane of Mesophyll Cells of Pea

In leaf mesophyll cells of pea (Pisum sativum) light induces a transient depolarization that is at least partly due to an increased plasma membrane conductance for anions. Several channel types were identified in the plasma membrane of protoplasts from mesophyll cells using the patch-clamp technique. One of these was an anion channel with a single-channel conductance of 32 picasiemens in symmetrical 100/100 KCl solutions. In asymmetrical solutions the reversal potential indicates a high selectivity for Cl- over K+ at high cytoplasmic Cl-. At negative membrane voltages the channel openings were interrupted by very short closures. In the open channel conductance several substrates were identified. At a cytoplasmic negative logarithm of Ca concentration higher than 6.3, no channel openings were observed. When the protoplast was illuminated in the cell-attached configuration, at least one channel type had a higher opening probability. This channel can tentatively be identified as the above-described anion channel based on conductance and the characteristic short closures at negative membrane potentials. This light activation of the 32-picasiemen anion channel is a strong indication that this channel conducts the light-induced depolarizing current. Because channel activity is strongly Ca2+-dependent, a role of cytoplasmic Ca2+ concentration changes in the light activation of the conductance is discussed.     

Citrate-permeable channels in the plasma membrane of cluster roots from white lupin

White lupin (Lupinus albus) is well adapted to phosphorus deficiency by developing cluster roots that release large amounts of citrate into the rhizosphere to mobilize the sparingly soluble phosphorus. To determine the mechanism underlying citrate release from cluster roots, we isolated protoplasts from different types of roots of white lupin plants grown in phosphorus-replete (+P) and phosphorus-deficient (-P) conditions and used the patch-clamp technique to measure the whole-cell currents flowing across plasma membrane of these protoplasts. Two main types of anion conductance were observed in protoplasts prepared from cluster root tissue: (1) an inwardly rectifying anion conductance (IRAC) activated by membrane hyperpolarization, and (2) an outwardly rectifying anion conductance (ORAC) that became more activated with membrane depolarization. Although ORAC was an outward rectifier, it did allow substantial inward current (anion efflux) to occur. Both conductances showed citrate permeability, with IRAC being more selective for citrate3- than Cl- (PCit/PCl = 26.3), while ORAC was selective for Cl- over citrate (PCl/PCit = 3.7). Both IRAC and ORAC were sensitive to the anion channel blocker anthracene-9-carboxylic acid. These currents were also detected in protoplasts derived from noncluster roots of -P plants, as well as from normal (noncluster) roots of plants grown with 25 microm phosphorus (+P). No differences were observed in the magnitude or frequency of IRAC and ORAC currents between the cluster roots and noncluster roots of -P plants. However, the IRAC current from +P plants occurred less frequently than in the -P plants. IRAC was unaffected by external phosphate, but ORAC had reduced inward current (anion efflux) when phosphate was present in the external medium. Our data suggest that IRAC is the main pathway for citrate efflux from white lupin roots, but ORAC may also contribute to citrate efflux.     

Cytologic control of protoplast formation and regeneration in Streptomyces erythraeus, strain BTCC-2

Experiments on protoplast formation and regeneration in S. erythraeus, strain BTCC-2 (Saccharopolyspora erythrae) were performed under microscopic control at all the stages. It was shown that the highest protoplast titer was provided by the mycelium grown in one step in the absence of glycine. For characterizing the protoplasts formed by the mycelium grown under different conditions, their regeneration capacity was estimated by microscopic examination of the protoplasts after 15-20-hour growth in microchambers and evaluation of the regeneration efficiency 7-10 hours later. Of interest was the fact of spontaneous development of colonies consisting of the protoplast-like cells (L-cells) in 15-20 hours. Such colonies were formed only by the protoplasts grown from the mycelium incubated in one step in the absence of glycine or in the presence of 0.1 per cent of glycine. Such conditions provided also the maximum efficiency of the protoplast regeneration. The long-term storage of protoplasts led to a decrease in their viability.     

Differences in chemical composition of plants grown at constant relative growth rates with stable mineral nutrition

Summary Leaf chemistry of a willow clone (Salix aquatica Smith) differed significantly when grown at constant relative growth rates depending upon the relative availability of nutrients and light. Concentration of amino acids and nitrate were high in plants grown with a relative surplus of nutrients. Concentrations of starch, tannin, and lignin, on the other hand, were high in plants grown with a relative surplus of carbon. Photosynthetic rates, expressed per unit leaf area, were similar when plants were grown under high light conditions, regardless of nutrient availability. Dark respiration was much higher in plants supplied with abundant nutrients than in those with a more limited supply, reflecting differences in nitrogen concentration of the tissue. The experimental approach allows plants to be grown to a standard size with differing, but highly uniform chemistry. Plants grown in such a manner may provide good experimental material to evaluate interactions between herbivores or pathogens and their hosts.     

Leaf shape variation and herbivore consumption and performance: a case study with Ipomoea hederacea and three generalists

The effect of leaf shape variation on plant-herbivore interactions has primarily been studied from the perspective of host seeking behavior. Yet for leaf shape to affect plant-herbivore coevolution, there must be reciprocal effects of leaf shape variation on herbivore consumption and performance. We investigated whether alternative leaf morphs affected the performance of three generalist insect herbivores by taking advantage of a genetic polymorphism and developmental plasticity in leaf shape in the Ivyleaf morning glory, Ipomoea hederacea. Across four experiments, we found variable support for an effect of leaf shape genotype on insects. For cabbage loopers (Trichoplusia ni) and corn earworms (Helicoverpa zea) we found opposing, non-significant trends: T. ni gained more biomass on lobed genotypes, while H. zea gained more biomass on heart-shaped genotypes. For army beetworms (Spodoptera exigua), the effects of leaf shape genotype differed depending on the age of the plants and photoperiod of growing conditions. Caterpillars feeding on tissue from older plants (95 days) grown under long day photoperiods had significantly greater consumption, dry biomass, and digestive efficiency on lobed genotypes. In contrast, there were no significant differences between heart-shaped and lobed genotypes for caterpillars feeding on tissue from younger plants (50 days) grown under short day photoperiods. For plants grown under short days, we found that S. exigua consumed significantly less leaf area when feeding on mature leaves than juvenile leaves, regardless of leaf shape genotype. Taken together, our results suggest that the effects of leaf shape variation on insect performance are likely to vary between insect species, growth conditions of the plant, and the developmental stage and age of leaves sampled. Handling editor: May Berenbaum.     

Cytokinin-dependent expression of the ARR5::GUS construct during transgenic arabidopsis growth

Growth and glucuronidase (GUS) activity were followed in the cotyledons and rosette leaves of Arabidopsis thaliana (L.) Heynh (ecotype Wassilewskija) plants transformed with the GUS gene under the control of the cytokinin-dependent promoter of the ARR5 gene. The presence of active cytokinins in plant tissues was assessed from GUS activity. Plants were grown for three weeks on the nitrate-or ammonium-containing nutrient medium. In plants grown on ammonium nutrition, cotyledon and leaf growth was substantially suppressed as compared with plants feeding with nitrates. In correspondence with this growth inhibition, GUS activity was markedly lower in plant leaves grown on the ammonium-containing medium. This indicated a reduction in these leaves of active cytokinin forms capable of activation of the promoter for the ARR5 gene. On both nitrogen sources, GUS activity increased during leaf growth and dropped sharply after growth ceasing. This indicated that leaf growth depended on the cytokinin content in them. High GUS activity was detected in petioles and leaf conductive system, indicating leaf providing with cytokinins along the conductive vessels. A sharp drop in the GUS activity after leaf growth stoppage coincided in time with GUS activation in the leaf positioned above this leaf. This indicated possible cytokinin redistribution in the plant; its content could be a limiting factor for leaf growth. A higher growth rate in plants on nitrate nitrogen nutrition and corresponding high GUS activity in them are discussed in terms of cytokinin signaling role in leaf growth regulation mediated by nitrate.