Ethylene action blockade and cold storage affect ripening of ‘Golden’ papaya fruit

The purpose of this work was to evaluate the effects of ethylene action blockade and cold storage on the ripening of ‘Golden’ papaya fruit. Papayas harvested at maturity stage 1 (up to 15% yellow skin) were evaluated. Half of the fruits, whether treated or not treated with 100 nL L⁻¹ of 1-methylcyclopropene (1-MCP), were stored at 23°C, while the other half were stored at 11°C for 20 days prior to being stored at 23°C. Non-refrigerated fruits receiving 1-MCP application presented a reduction in respiratory activity, ethylene production, skin color development and pectinmethylesterase activity. Even with a gradual increase in ethylene production at 23°C, fruits treated with 1-MCP maintained a high firmness, but presented a loss of green skin color. Cold storage caused a decrease in ethylene production when fruits were transferred to 23°C. The results suggest that pulp softening is more dependent on ethylene than skin color development, and that some processes responsible for loss of firmness do not depend on ethylene.     

Effect of Abscisic Acid on Banana Fruit Ripening in Relation to the Role of Ethylene

Abstract The role of abscisic acid (ABA) in banana fruit ripening was examined with the ethylene binding inhibitor, 1-methylcyclopropene (1-MCP). ABA (0, 10⁻⁵, 10⁻⁴, or 10⁻³ mol/L) was applied by vacuum infiltration into fruit. 1-MCP (1 μL/L) was applied by injecting a measured volume of stock gas into sealed glass jars containing fruit. Fruit ripening, as judged by ethylene evolution and respiration associated with color change and softening, was accelerated by 10⁻⁴ or 10⁻³ mol/L ABA. ABA at 10⁻⁵ mol/L had no effect. The acceleration of ripening by ABA was greater at 10⁻³ mol/L than at 10⁻⁴ mol/L. ABA-induced acceleration of banana fruit ripening was not observed in 1-MCP treated fruit, especially when ABA was applied after exposure to 1-MCP. Thus, ABA's promotion of ripening in intact banana fruit is at least partially mediated by ethylene. Exposure of ABA-treated fruit to 0.1 μL/L ethylene for 24 h resulted in increased ethylene production and respiration, and associated skin color change and fruit softening. Control fruit (no ABA) was unresponsive to similar ethylene treatments. The data suggest that ABA facilitates initiation and progress in the sequence of ethylene-mediated ripening events, possibly by enhancing the sensitivity to ethylene. Received 29 January 1999; accepted 16 January 2000     

Effect of 1-methylcyclopropene on postharvest physiological changes of ‘Zaohong’ plum

Plum is a highly perishable fruit and postharvest fruit softening limits its shelf life. The aim of this work was to study the specific effects of 1-methylcyclopropene (1-MCP) treatment on physiological changes in ‘Zaohong’ plums. Plums were treated with 500 nL L⁻¹ 1-MCP at 20°C for 18 h followed by 20°C storage. The results showed that 1-MCP treatment significantly reduced endogenous ethylene production and the activities of ethylene biosynthetic enzymes’ (1-aminocyclopropane-1-carboxylic acid synthase, ACS and 1-aminocyclopropane-1-carboxylic acid oxidase, ACO) in plum fruit during storage when compared with untreated fruit. Although 1-MCP treatment inhibited ethylene production and 1-aminocyclopropane-1-carboxylic acid (ACC) accumulation, it did not inhibit the accumulation of N-malonyl-ACC (MACC). Higher firmness was also found in 1-MCP-treated plums than in controls. During storage, superoxide anion (O₂^−·) and hydrogen peroxide (H₂O₂) levels decreased in 1-MCP-treated fruit. 1-MCP treatment also regulated superoxide dismutase (SOD) and catalase (CAT) activities during storage. Xylanase activity was upregulated while activities of polygalacturonase (PG), pectin methyl esterase (PME) and cellulase enzymes in the fruit were downregulated by 1-MCP treatment. In conclusion, 1-MCP might be a potent compound for extending both storage period and shelf life of ‘Zaohong’ plums by suppressing ethylene biosynthesis, regulating cell wall degradation enzymes and reducing fruit softening.     

Lignin Deposition and Effect of Postharvest Treatment on Lignification of Green Asparagus (Asparagus officinalis L.)

To understand how lignin synthesis is regulated after harvest, detached green asparagus stalks (Asparagus officinalis L.) were treated with 1 μl l⁻¹ of 1-methylcyclopropene (1-MCP), 50 μg l⁻¹ gibberellic acid (GA₃), 2% (v:v) ethanol or 1 μl l⁻¹ ethylene. The results showed that lignin concentration in asparagus stalks stored at room temperature rapidly increased. Three conventional precursors of lignin, 4-hydroxycinnamic acid (coumaric acid), 3,4-dihydroxycinnamic acid (caffeic acid) and 4-hydroxy-3-mythoxycinnamic acid (ferulic acid), were found to be the major phenolics in the asparagus stalks. Furthermore, the concentrations of O₂⁻ in asparagus stalks steadily increased during the storage. Deposition of lignin in harvested asparagus was significantly reduced by treating the stalks with GA₃, 1-MCP or ethanol. The concentration of lignin in stalks treated with GA₃, 1-MCP or ethanol was 32, 20 or 27% lower, respectively, than in controls 3 days after treatment. Treating stalks with ethylene enhanced lignin synthesis (p<0.05). The concentration of total phenol in stalks was also significantly reduced by GA₃, 1-MCP and ethanol, but was enhanced by ethylene treatment. However, the concentration of active oxygen (O₂^−⋅) in stalks was significantly reduced by treatment with GA₃, 1-MCP and ethanol, but was enhanced by treatment with ethylene. Our study show that postharvest treatment with 1-MCP, GA₃ or ethanol may be applied to improve the quality of green asparagus.     

Assimilate export by leaves of Ricinus communis L. growing under normal and elevated carbon dioxide concentrations: the same rate during the day, a different rate at night

Castor bean (Ricinus communis L.) plants were grown for 5–7 weeks in a controlled environment at 350 μl l⁻¹ or 700 μl l⁻¹ CO₂. Carbon assimilation, assimilate deposition, dark respiration and assimilate mobilization were measured in leaves 2, 3 and 4 (counted from the base of the plant), and a balance sheet of carbon input and export was elaborated for both CO₂ concentrations. Carbon dioxide assimilation was nearly constant over the illumination period, with only a slight depression occurring at the end of the day in mature source leaves, not in young source leaves. Assimilation was ca. 40% higher at 700 μl l⁻¹ than at 350 μl l⁻¹ CO₂. The source leaves increased steadily in weight per unit area during the first 3 weeks, more at 700 μl l⁻¹ than at 350 μl l⁻¹ CO₂. On top of an irreversible weight increase, there was a large gain in dry weight during the day, which was reversed during the night. This reversible weight gain was constant over the life time of the leaf and ca. 80% higher at 700 μl l⁻¹ than at 350 μl l⁻¹. Most of it was due to carbohydrates. The carbon content (as a percentage) was not altered by the CO₂ treatment. Respiration was 25% higher in high-CO₂ plants when based on leaf area, but the same when based on dry weight. The rate of carbon export via the phloem was the same during the daytime in plants grown at 350 μl l⁻¹ and 700 μl l⁻¹ CO₂. During the night the low-CO₂ plants had only 50% of the daytime export rate, in contrast to the high-CO₂ plants which maintained the high export rate. It was concluded that the phloem loading system is saturated during the daytime in both CO₂ regimes, whereas during the night the assimilate supply is reduced in plants in the normal CO₂ concentration. Two-thirds of the carbon exported from the leaves was permanently incorporated as plant dry matter in the residual plant parts. This “assimilation efficiency” was the same for both CO₂ regimes. It is speculated that under 350 μl l⁻¹ CO₂ the growing Ricinus plant operates at sink limitation during the day and at source limitation during the night. Received: 2 February 1999 / Accepted: 19 April 1999     

CO<Subscript>2</Subscript>-enriched microenvironment affects sucrose and macronutrients absorption and promotes autotrophy in the <Emphasis Type="Italic">in vitro</Emphasis> culture of kiwi (<Emphasis Type="Italic">Actinidia deliciosa</Emphasis> Chev. Liang and Ferguson)

In traditional in vitro culture, the low CO₂ concentration inside the vessels restricts photosynthesis and necessitates the addition of sucrose to the culture medium as the main energy source, thus bringing about changes in the absorption of mineral elements from the culture medium. In this study, we investigated macronutrient absorption and sugar consumption in Actinidia deliciosa Chevalier Liang and Ferguson cv. Hayward (kiwi), cultured on medium supplemented with varying amounts of sucrose (0, 10, and 20 g l⁻¹) under both heterotrophy and autotrophy, flushed with different concentrations of CO₂ (non-ventilation, 300, 600, and 2,000 μl l⁻¹). In ventilated systems with 20 g l⁻¹ of sucrose, sucrose absorption was less than under non-ventilation. The lowest rate of sucrose absorption was recorded when the explants were cultured on medium supplemented with 20 g l⁻¹ of sucrose and flushed with 600 μl l⁻¹ CO₂. Absorption of NO₃ ⁻, PO₄ ³⁻, and Mg²⁺ were high (maximum) at the end of the culture period (40 d) in explants flushed with 600 μl l⁻¹ CO₂ that have been cultured 20 d in the presence of sucrose and then transferred to a sucrose-free medium. These autotrophic conditions promoted maximum plant growth in terms of both fresh and dry mass as well as the length and number of shoots and leaves. The study shows that to maintain an optimum regime of mineral nutrition for prolonged culture of kiwi in vitro, an increased amount of these three ions should be supplemented in Murashige and Skoog’s medium.     

Effects of Chilling Temperatures on Ethylene Binding by Banana Fruit

Banana fruit are highly susceptible to chilling injury during low temperature storage. Experiments were conducted to compare ethylene binding during storage at chilling (3 and 8 °C) versus optimum (13 °C) temperatures. The skins of fruit stored at 3 and 8 °C gradually darkened as storage duration increased. This chilling effect was reflected in increasing membrane permeability as shown by increased relative electrolyte leakage from skin tissue. In contrast, banana fruit stored for 8 days at 13 °C showed no chilling injury symptoms. Exposure of banana fruit to the ethylene binding inhibitor 1-methylcyclopropene (1 l l^-1 1-MCP) prevented ripening. However, this treatment also enhanced the chilling injury accelerated the occurrence of chilling injury-associated increased membrane permeability. ¹⁴C-ethylene release assay showed that ethylene binding by banana fruit stored at low temperature decreased with reduced storage temperature and/or prolonged storage time. Fruit exposed to 1-MCP for 12 h and then stored at 3 or 8 °C exhibited lower ethylene binding than those stored at 13 °C. Thus, chilling injury of banana fruit stored at low temperature is associated with a decrease in ethylene binding. The ability of tissue to respond to ethylene is evidently reduced, thereby resulting in failure to ripen.     

Ethrel treatment enhanced isoflavonoids accumulation in cell suspension cultures of Pueraria tuberosa, a woody legume

The cell cultures of Pueraria tuberosa, a perennial leguminous lianas, were maintained in modified MS medium (KNO₃ 475 mg l⁻¹, thiamine 1 mg l⁻¹, biotin 1 mg l⁻¹, calcium pantothenate 1 mg l⁻¹) containing 0.1 mg l⁻¹ 2,4,5-trichloroacetic acid and 0.1 mg l⁻¹ kinetin. Isoflavonoids (puerarin, genistin, daidzein, genistein) accumulation in cell suspension cultures was increased by 14-fold to ~12 mg l⁻¹ after 48 h of adding 100 μM ethrel. Ethrel inhibitors (silver nitrate and silver thiosulfate) completely inhibited this effect in the presence of ethrel and isoflavonoids were not detected in the spent medium. The increase was dose dependent and can be explored to trigger high yield of isoflavonoids production.     

Role of ethylene and jasmonic acid on rhizome induction and growth in rhubarb (<Emphasis Type="Italic">Rheum rhabarbarum</Emphasis> L.)

Experiments were conducted to elucidate the hormonal induction and regulation of rhizome growth in rhubarb (Rheum rhabarbarum L.). It was found that ethylene is the key regulator of rhizome induction and development. The role of jasmonic acid (JA) and its interaction with ethylene in rhizome induction and growth were also examined. Both ethylene and JA have a significant effect on promoting rhizome formation in vitro. Conversely, the ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG) (1.5 μM) inhibited rhizome induction in multiple-shoot clumps in vitro, and suppressed the stimulatory effects of exogenously applied ethephon (1 mg l⁻¹) and JA (10 ng l⁻¹) in promoting mini-rhizome formation, further confirming the role of endogenous ethylene in the process. In addition, rhizome growth was significantly enhanced in the presence of both ethylene and JA (ethephon 1 mg l⁻¹ and JA 10 ng l⁻¹) compared to JA alone. These results suggest that endogenous ethylene is the key regulator of rhizome growth in rhubarb and JA promotes rhizome formation, possibly through inducing endogenous ethylene synthesis.     

Anaerobic ethylene glycol degradation by microorganisms in poplar and willow rhizospheres

Although aerobic degradation of ethylene glycol is well documented, only anaerobic biodegradation via methanogenesis or fermentation has been clearly shown. Enhanced ethylene glycol degradation has been demonstrated by microorganisms in the rhizosphere of shallow-rooted plants such as alfalfa and grasses where conditions may be aerobic, but has not been demonstrated in the deeper rhizosphere of poplar or willow trees where conditions are more likely to be anaerobic. This study evaluated ethylene glycol degradation under nitrate-, and sulphate-reducing conditions by microorganisms from the rhizosphere of poplar and willow trees planted in the path of a groundwater plume containing up to 1.9 mol l⁻¹ (120 g l⁻¹) ethylene glycol and, the effect of fertilizer addition when nitrate or sulphate was provided as a terminal electron acceptor (TEA). Microorganisms in these rhizosphere soils degraded ethylene glycol using nitrate or sulphate as TEAs at close to the theoretical stoichiometric amounts required for mineralization. Although the added nitrate or sulphate was primarily used as TEA, TEAs naturally present in the soil or CO₂ produced from ethylene glycol degradation were also used, demonstrating multiple TEA usage. Anaerobic degradation produced acetaldehyde, less acetic acid, and more ethanol than under aerobic conditions. Although aerobic degradation rates were faster, close to 100% disappearance was eventually achieved anaerobically. Degradation rates under nitrate-reducing conditions were enhanced upon fertilizer addition to achieve rates similar to aerobic degradation with up to 19.3 mmol (1.20 g) of ethylene glycol degradation l⁻¹ day⁻¹ in poplar soils. This is the first study to demonstrate that microorganisms in the rhizosphere of deep rooted trees like willow and poplar can anaerobically degrade ethylene glycol. Since anaerobic biodegradation may significantly contribute to the phytoremediation of ethylene glycol in the deeper subsurface, the need for “pump and treat” or an aerobic treatment would be eliminated, hence reducing the cost of treatment.     

Efficacy of New Inhibitors of Ethylene Perception in Improvement of Display Quality of Miniature Potted Roses (<Emphasis Type="Italic">Rosa hybrida</Emphasis> L.)

1-Octylcyclopropene (1-OCP) and 1-Decylcyclopropene (1-DCP), ethylene receptor inhibitors, analogues to 1-MCP, substituted with longer carbon chain in the 1-position were investigated in miniature potted roses cultivar ’Lavender‘. All levels of both chemicals protected as compared to untreated plants. 1-OCP and 1-DCP were the most effective at concentrations 1000 and 1500 nl l⁻¹, which was five times higher than the concentration of 1-methylcyclopropene (1-MCP) (200 nl l⁻¹) used as a standard. The effectiveness of 1-OCP and 1-DCP was a function of time and temperature. At short (2 h) exposure times, the plants were highly sensitive to ethylene. Exposure time of 4 h for both 1-OCP and 1-DCP was sufficient to improve display life of miniature roses and longer exposures did not have any additional beneficial effect. Apparently, exposing miniature potted roses to various temperatures did not have an influence on the performance of both 1-OCP and 1-DCP while low temperature at 5 °C reduced their performance. The reasons for differences in the effects of these compounds are discussed.     

Effects of 1-MCP on the vase life and ethylene response of cut flowers

Pretreatment for 6 h with low concentrations of 1-MCP (1-Methylcyclopropene, formerly designated as SIS-X), a cyclic ethylene analog, inhibits the normal wilting response of cut carnations exposed continuously to 0.4 l·l^–1 ethylene. The response to 1-MCP was a function of treatment concentration and time. Treatment with 1-MCP was as effective in inhibiting ethylene effects as treatment with the anionic silver thiosulfate complex (STS), the standard commercial treatment. Other ethylene-sensitive cut flowers responded similarly to carnations. In the presence of 1 l·l^–1 ethylene, the vase life of 1-MCP-treated flowers was up to 4 times that of the controls.Abbreviations 1-MCP 1-Methylcyclopropene - STS silver thiosulfate     

Delay ripening of ‘Qingnai’ plum (Prunus salicina Lindl.) with 1-methylcyclopropene

‘Qingnai’ plum fruit were treated with 0, 250, 500 or 1000 nL L⁻¹ of 1-methylcyclopropene (1-MCP) for 6 h and stored at 20 °C. The fruit firmness, peel color, chlorophyll content, titratable acidity (TA), respiration rate and ethylene production, chlorophyllase, pectin methylesterase (PME) and polygalacturonase (PG) activities were monitored during postharvest ripening of ‘Qingnai’ plums. ‘Qingnai’ plums without 1-MCP treatment soften very rapidly at room temperature after harvest, showing a continuing decrease in hue angle, chlorophyll content, TA and increase in chlorophyllase, PME and PG activities during postharvest storage. In contrast, the 1-MCP-treated fruits showed reduced ethylene production and respiration rate and delayed softening, which was associated with the reduction in the activity of PME and PG. The 1-MCP treatment also significantly inhibited the chlorophyllase activity and peel color development in ‘Qingnai’ plums during postharvest ripening at 20 °C. These results suggest that 1-MCP treatment may be useful for maintaining the fruit quality and extending the postharvest shelf-life of ‘Qingnai’ plums.     

Exogenous gibberellic acid increases the fruit weight of ‘Comte de Paris’ pineapple by enlarging flesh cells without negative effects on fruit quality

In mainland China, the most popular pineapple cultivar is ‘Comte de Paris’. Gibberellic acids have been widely applied to enhance fruit growth in various species. To evaluate the effect of gibberellic acid (GA₃) on ‘Comte de Paris’ pineapple production and quality, pineapple fruits were sprayed with GA₃ at concentrations of 5, 20, 50, or 100 mg l⁻¹ at both 0 and 15 days after flowering (DAF). Fruits were sampled every 15 days from 0 to 60 DAF (maturation) for flow cytometric analysis and histological observation. The results showed that the treatments with the three highest concentrations of GA₃ significantly increased fruit weight, and the most effective concentration was 50 mg l⁻¹ GA₃, which increased the flesh weight by 20.3% compared to the control. Although treatment with GA₃ had little effect on the total soluble solids and fruit juice pH, it increased the vitamin C content. Although flow cytometric analysis showed that the 50 mg l⁻¹ GA₃ treatment had only a slight impact on the number of S phase cells, histological observations indicated that the increase of fruit volume and flesh weight under this GA₃ treatment was not due to the increase of cell number but a result of the increase of cell area in the fruit flesh.     

Primary Productivity and Water Balance of Grassland Vegetation on Three Soils in a Continuous CO2 Gradient: Initial Results from the Lysimeter CO2 Gradient Experiment

Field studies of atmospheric CO₂ effects on ecosystems usually include few levels of CO₂ and a single soil type, making it difficult to ascertain the shape of responses to increasing CO₂ or to generalize across soil types. The Lysimeter CO₂ Gradient (LYCOG) chambers were constructed to maintain a linear gradient of atmospheric CO₂ (~250 to 500 μl l⁻¹) on grassland vegetation established on intact soil monoliths from three soil series. The chambers maintained a linear daytime CO₂ gradient from 263 μl l⁻¹ at the subambient end of the gradient to 502 μl l⁻¹ at the superambient end, as well as a linear nighttime CO₂ gradient. Temperature variation within the chambers affected aboveground biomass and evapotranspiration, but the effects of temperature were small compared to the expected effects of CO₂. Aboveground biomass on Austin soils was 40% less than on Bastrop and Houston soils. Biomass differences between soils resulted from variation in biomass of Sorghastrum nutans, Bouteloua curtipendula, Schizachyrium scoparium (C₄ grasses), and Solidago canadensis (C₃ forb), suggesting the CO₂ sensitivity of these species may differ among soils. Evapotranspiration did not differ among the soils, but the CO₂ sensitivity of leaf-level photosynthesis and water use efficiency in S. canadensis was greater on Houston and Bastrop than on Austin soils, whereas the CO₂ sensitivity of soil CO₂ efflux was greater on Bastrop soils than on Austin or Houston soils. The effects of soil type on CO₂ sensitivity may be smaller for some processes that are tightly coupled to microclimate. LYCOG is useful for discerning the effects of soil type on the CO₂ sensitivity of ecosystem function in grasslands. Author Contributions: PF conceived study, analyzed data, and wrote the paper. AK, AP analyzed data. DH, VJ, RJ, HJ, and WP conceived study, and conducted research.     

Stimulated N2O flux from intact grassland monoliths after two growing seasons under elevated atmospheric CO2

Long-term exposure of native vegetation to elevated atmospheric CO₂ concentrations is expected to increase C inputs to the soil and, in ecosystems with seasonally dry periods, to increase soil moisture. We tested the hypothesis that these indirect effects of elevated CO₂ (600 μl l⁻¹ vs 350 μl l⁻¹) would improve conditions for microbial activity and stimulate emissions of nitrous oxide (N₂O), a very potent and long-lived greenhouse gas. After two growing seasons, the mean N₂O efflux from monoliths of calcareous grassland maintained at elevated CO₂ was twice as high as that measured from monoliths maintained at current ambient CO₂ (70 ± 9 vs 37 ± 4 μg N₂O m⁻² h⁻¹ in October, 27 ± 5 vs 13 ± 3 μg N₂O m⁻² h⁻¹ in November after aboveground harvest). The higher N₂O emission rates at elevated CO₂ were associated with increases in soil moisture, soil heterotrophic respiration, and plant biomass production, but appear to be mainly attributable to higher soil moisture. Our results suggest that rising atmospheric CO₂ may contribute more to the total greenhouse effect than is currently estimated because of its plant-mediated effects on soil processes which may ultimately lead to increased N₂O emissions from native grasslands. Received: 11 September 1997 / Accepted: 20 March 1998     

Treatment of phenol in an anaerobic fluidized bed reactor (AFBR): continuous and batch regime

Results of this study describe the feasibility of anaerobic treatment of highly concentrated phenol synthetic wastewater using an anaerobic fluidized bed reactor (AFBR) in both continuous and batch modes. Wastewater with a maximum load of 2,100 mg C·l⁻¹ was prepared using phenol (maximum concentration of 1,600 mg C·l⁻¹) as substrate and a mixture of acetic, propionic and butyric acids (500 mg C·l⁻¹) as co-substrate. AFBR reached total organic carbon (TOC) and phenol removal efficiency over 95% treating the highest organic loading rate (OLR) containing phenol studied for this kind of reactor (5.03 g C·l⁻¹·d⁻¹). The phenol loading rate rise caused volumetric biogas rate increase up to 4.4 l·l⁻¹·d⁻¹ (average yield of 0.28 l CH₄·g⁻¹ COD_removed) as well as variation in the biogas composition; the CO₂ percentage increased while the CH₄ percentage decreased. Morphological examination of the bioparticles at 4.10 g C·l⁻¹·d⁻¹, revealed significant differences in the biofilm structure, microbial colonization and bacterial morphological type development. The five batch assays showed that phenol degradation may be favoured by the presence of volatile fatty acids (VFAs) (co-metabolism), whereas VFAs degradation may be inhibited by phenol. AFBR reached initial phenol degradation velocity of 0.25 mg C·l⁻¹·min⁻¹.     

Elicitor-induced accumulation of stilbenes in cell suspension cultures of Cayratia trifolia (L.) Domin

Cell cultures of Cayratia trifolia (Vitaceae), a tropical lianas, were maintained in Murashige and Skoog’s medium containing 0.25 mg l⁻¹ NAA, 0.2 mg l⁻¹ kinetin and casein hydrolysate 250 mg l⁻¹. Cell suspension cultures of C. trifolia accumulate stilbenes (piceid, resveratrol, viniferin, ampelopsin), which on elicitation by any of 500 μM salicylic acid, 100 μM methyl jasmonate, 500 μM ethrel and 500 mg l⁻¹ yeast extract, added on the 7th day, were enhanced by 3- to 6-fold (5–11 mg l⁻¹) by the 15th day.     

Enhanced regeneration of haploid plantlets from microspores of <Emphasis Type="Italic">Brassica napus</Emphasis> L. using bleomycin,PCIB, and phytohormones

The effects of three periods of incubation (10, 20 and 30 min) at different levels of bleomycin (0, 0.1, 0.2, 0.3, 0.4 and 0.5 μg ml⁻¹), as well as three periods of exposure (12, 24 and 48 h) to different levels of the anti-auxin p-chlorophenoxyisobutyric acid (PCIB), including 1, 2, 3, 4 and 5 mg l⁻¹, on microspore embryogenesis of rapeseed cv. ‘Amica’ were investigated. Microspore embryogenesis was significantly enhanced following 20 min treatment with 0.2 μg ml⁻¹ bleomycin compared with untreated cultures. Highest embryo yield (163 embryos Petri dish⁻¹) was observed with 24 h treatment of 4 mg l⁻¹ PCIB. The highest percentage of secondary embryogenesis was observed on B₅ medium containing 0.15 mg l⁻¹ of gibberellic acid (GA₃) and 0.2 mg l⁻¹ 6-benzyladenine (BA) in 4–6 mm microspore-derived embryos (MDEs). Most callus formed on B₅ medium containing 0.15 mg l⁻¹ GA₃, 0.1 mg l⁻¹ BA and 0.1 mg l⁻¹ indole-3-acetic acid (IAA) when 4–6 mm embryos were used. Regeneration was highest on B₅ medium containing 0.05 mg l⁻¹ GA₃ or 0.1 mg l⁻¹ BA and 0.2 mg l⁻¹ IAA with 2–4 mm embryos. Microspore embryogenesis and plant regeneration could be improved by both bleomycin and PCIB when the appropriate MDE length and phytohormone level were selected.     

The effect of elevated CO2 concentrations on K+ and anion channels of Vicia faba L. guard cells

The effects of elevated CO₂ concentrations on stomatal movement, anion- and K⁺-channel activities were examined in guard cells from epidermal strips of Vicia faba. Membrane voltage was measured using intracellular, double-barrelled microelectrodes and ion-channel currents were recorded under voltage clamp during exposure to media equilibrated with ambient (350 μl · l⁻¹), 1000 μl · l⁻¹ and 10 000 μl · l⁻¹ CO₂ in 20% O₂ and 80% N₂. The addition of 1000 μl · l⁻¹ CO₂ to the bathing solution caused stomata to close with a halftime of approx. 40 min, and with 10 000 μl · l⁻¹ CO₂ closure occurred with a similar time course. Under voltage clamp, exposure to 1000 μl · l⁻¹ and 10 000 μl · l⁻¹ CO₂ resulted in a rapid increase (mean, 1.5 ± 0.2-fold, n = 8; range 1.3- to 2.5-fold) in the magnitude of current carried by outward-rectifying K⁺ channels (I_K,out). The effect of CO₂ on I_K,out was essentially complete within 30 s and was independent of clamp voltage, but was associated with 25–40% (mean, 30 ± 4%) decrease in the halftime for current activation. Exposure to CO₂ also resulted in a four-fold increase in background current near the free-running membrane voltage, recorded as the instantaneous current at the start of depolarising and hyperpolarising voltage steps, and a decrease in the magnitude of current carried by inward-rectifying K⁺ channels (I_K,in). The effect of CO₂ on I_K,in was generally slower than on I_K,out; it was allied with a transient acceleration of its activation kinetics during the first 60–120 s of treatment; and it was associated with a negative shift in the voltage-sensitivity of gating over a period of 3–5 min. Measurements carried out to isolate the background currents attributable to anion channels (I_Cl), using tetraethylammonium chloride and CsCl, showed that CO₂ also stimulated I_Cl and dramatically altered its relaxation kinetics. Within the timeframe of CO₂ action at the membrane, no significant effect was observed on cytosolic pH, measured using the fluorescent dye 2′,7′-bis-(2-carboxyethyl)-5,6-carboxyflourescein (BCECF) and ratio fluorescence microphotometry. These results are broadly consistent with the pattern of guard-cell response to abscisic acid, and indicate that guard cells control both anion and K⁺ channels to achieve net solute loss in CO₂. By contrast with the effects of abscisic acid, however, the data indicate that CO₂ action is not mediated through changes in cytosolic pH and thereby implicate new and, as yet, unidentified pathway(s) for channel regulation in the guard cells. Received: 8 January 1997 / Accepted: 28 February 1997