The Herbivore Assemblage, Herbivory and Leaf Chemistry of the Mangrove Kandelia obovata in Two Contrasting Forests in Hong Kong

The herbivore assemblage, intensity of herbivory and factors determining herbivory levels on the mangrove Kandelia obovata (previously K. candel, Rhizophoraceae) were studied over a 13-month period at two forests with contrasting growing conditions in Hong Kong. Mai Po was part of an eutrophic embayment in the Pearl River estuary and generally offered more favourable conditions for mangrove growth, whereas Ting Kok had a rocky substratum and oceanic salinity. Twenty-four insect herbivore species were recorded on K. obovata, with lepidopteran larvae that consume leaf lamina being the dominant species. While leaf litter production was similar at the two forests, herbivory level at Mai Po (mean = 3.9% in terms of leaf area loss) was more severe than that at Ting Kok (mean = 2.3%). Peak herbivory levels were found in summer at both locations (6.5% for Mai Po and 3.8% for Ting Kok). Young leaves of K. obovata at both locations were generally preferred by the herbivores from the period of late spring to summer. Concentrations of most feeding deterrents (ash, crude fibre, and total soluble tannins) were significantly higher in both young and mature leaves at Ting Kok, whereas leaf nutrients (total nitrogen and water) were the same at the two sites. Young leaves at Ting Kok contained about 30% more tannins than their counterparts at Mai Po. Significant differences in leaf chemistry also existed between young and mature leaves at either site. The differences were concomitant with the observed patterns of leaf herbivory on K. obovata, and suggest a potential relationship between environmental quality and plant defence against herbivory.     

Abscisic-acid contents and concentrations in protoplasts from guard cells and mesophyll cells ofVicia faba L.

The abscisic-acid (ABA) contents of isolated guard-cell protoplasts and mesophyll-cell protoplasts fromVicia faba were determined by high-pressure liquid chromatography followed by gas chromatography. The amounts of ABA found immediately after preparation of the protoplasts varied from 90 to 570 amol per guard-cell protoplast, and from 75 to 100 amol per mesophyll-cell protoplast. These contents correspond to concentrations between 36 and 230 mol per liter in guard-cell protoplasts and between 2.7 and 3.3 mol per liter in mesophyll-cell protoplasts. During exposure of protoplasts to betaine concentrations of 0.3, 0.5, and 0.8 mol·l^-1 at 0° and 20°C for 30 min, ABA contents as well as the fractions of ABA that leaked into the medium remained constant for both protoplast types. There was no evidence for net production of ABA in isolated protoplasts subjected to osmotic stress.Abbreviation ABA abscisic acid     

Plant regeneration from protoplasts of Enteromorpha intestinalis (Chlorophyta, Ulvophyceae) as seedstock for macroagal culture

A continuous micropropagation was established from protoplasts of thegreen alga Enteromorpha intestinalis. The effects of two differentcrude enzymes and the osmolarity at different concentrations of the enzymesolution on algal protoplast yields were tested. The optimal enzymecomposition for cell wall digestion and protoplast viability was 2%cellulase R 10 Onozuka and 2% Aplysie with 0.5 m mannitol. Largenumbers of Enteromorpha protoplasts were released (10.0 × 10⁶protoplasts from 1 g fresh thalli) and settled on a rangeof substrata. Regeneration of the protoplasts followed the normal patternfor this species. Conditions for pure cultures and efficient systems offloating supports with nets were determined to optimise the product qualityof plantlets of Enteromorpha. A promising storage process has beendeveloped which involves including protoplasts in beads of alginic acid gel.Plants regenerated from protoplasts may also be used as seedstock tofacilitate propagation for macroalgal culture.     

Studies of cotyledon protoplast cultures from B napus,B. campestris and B. oleracea. II: Callus formation and plant regeneration

Cotyledons from twelve cultivars of Brassica; B. napus (Westar, Eureka, Global, Pivot and Narc 82); B. campestris: (Arlo, Sonja, Bunyip and Wonk Bok) and B. oleracea (Phenomenal Early, Sugar Loaf and Earliball) were used for protoplast isolation and culture in a comparative study of cell colony and callus formation, and plant regeneration. The formation of cell colonies and callus from protoplast cultures were significantly influenced by the light conditions of seed germination. All twelve cultivars showed callus formation from protoplast cultures derived from cotyledons of seedlings grown in dark for 3 days followed by 1 day dim light (dark/dim light-grown). Callus was obtained in all five liquid media used: modified K8P(1), modified K8P(2), modified MS, modified B and modified NN. In contrast, only six cultivars exhibited callus formation from the protoplasts isolated from cotyledons of seedlings germinated under light conditions for 7 days (light-grown) and in only three media: modified K8P(1), modified MS, modified B.Callus, derived from protoplast cultures isolated from dark/dim light-grown cotyledons and grown on K3 or MS series solid media for about 1 month, could develop shoots when further transferred onto MS series regeneration media. All five cultivars of B. napus, three of the four cultivars of B. campestris (Arlo, Sonja and Bunyip) and one of the three cultivars of B. oleracea (Sugar Loaf) exhibited shoot regeneration from protoplast cultures within 2–3 months after protoplast isolation. The frequency of shoot regeneration ranged among 1–22.5%. A high degree of reproducibility was observed in cultivars Westar, Eureka, Global, Arlo, Bunyip and Sugar Loaf. In contrast, among the six cultivars that formed callus in protoplast culture derived from light-grown cotyledons, only three cultivars from B. napus (Westar, Eureka, Global) exhibited shoot regeneration 5.5 months after protoplast isolation. Regenerated shoots from cultivars Westar, Eureka and Bunyip and Sugar Loaf, which derived from protoplasts of dark/dim light germinated seedling and were induced to root on rooting media, survived in soil and grew to produce silique and set seeds.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BA benzylaminopurine - EDTA ethylenediaminetetraacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - KT kinetin - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - PAR photosynthetically active radiation     

Plant regeneration from callus and protoplast cultures of Helianthus giganteus L.

Summary Methods of plant regeneration from callus and protoplasts of Helianthus giganteus L. are described. Embryogenic callus was obtained from leaf explants and plants were regenerated from these calli on MS media with different combinations of benzyladenine and naphtaleneacetic acid. Leaf protoplasts isolated from in vitro grown plants formed somatic embryos when cultured in agarose solidified droplets of V-KM medium containing benzyladenine and naphtaleneacetic acid. Embryos developed into plantlets on media with reduced auxin contents. Regenerated plants were successfully planted in soil.Abbreviations BA benzyladenine - IAA indoleacetic acid - MS Murashige and Skoog medium - NAA naphtaleneacetic acid - V-KM protoplast culture medium of Binding and Nehls     

Variation in Endogenous Gibberellins,Abscisic Acid,and Carbohydrate Content During the Growth Cycle of Colored <Emphasis Type="Italic">Zantedeschia</Emphasis> spp., a Tuberous Geophyte

Phenologic changes and variation in the level of endogenous gibberellins (GAs), abscisic acid (ABA), carbohydrate content, and α-amylase activity were examined in colored Zantedeschia spp. cv. Cala Gold. These changes were examined in the primary bud tissues and in the attached tuber tissue during the growth cycle. Dormant tubers were dry-stored at 20°C for 3 months, planted in a phytotron, and grown under 22/16 ± 1°C. Plant development was monitored under continued irrigation until leaf senescence and tuber dormancy. GAs and ABA were extracted from the primary bud tissues, fractionated by HPLC, and analyzed using GC-SIM. Starch, glucose, soluble protein, and α-amylase activity were monitored in the tuber tissue attached to the primary bud. Endogenous changes in GAs and ABA in the primary bud were correlated with endogenous changes in carbohydrate content and α-amylase activity in the attached tuber tissue. These correlations were observed during the rest and the growth periods and were associated with developmental changes in the plant, that is, bud dormancy relaxation, bud growth, and inflorescence differentiation. ABA content decreased and a transient pulse of GA was measured in the primary bud concomitantly with the onset of shoot elongation in dry tubers during storage, before planting. The sharp increase of GAs in the bud preceded inflorescence differentiation as observed in dissected apices by about 15 days, as well as the increase in α-amylase activity in the attached tuber tissue. A steep decrease in starch level was measured in the tuber after planting, concomitantly with massive plant growth. These findings suggest a possible involvement of gibberellin in the initiation of α-amylase activity during dormancy relaxation in colored Zantedeschia and in the autonomous induction of flowering.     

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     

Effects of fusicoccin and abscisic acid on glucose uptake into isolated beetroot protoplasts

Uptake of glucose, 3-O-methylglucose and sucrose into beetroot protoplasts is considerably stimulated by 10^–6M fusicoccin. This effect is decreased in the presence of 10mM Na⁺ or K⁺, 2 mM Mg²⁺ or Ca²⁺. Whereas fusicoccin causes no change in the pH-optimum of the sugar uptake (pH 5.0), the apparent K_m of this uptake which obeys a biphasic kinetics is decreased by the action of fusicoccin. In the protoplast suspension, fusicoccin induces an acidification which is suppressed by uncoupling agents. Correspondingly, uncouplers as well as vanadate and diethylstilbestrol markedly inhibit the effect of fusicoccin on sugar uptake. The present data support the view that glucose uptake into beetroot protoplasts depend on the proton-pumping activity of the plasmalemma-ATPase. cis–Abscisic acid diminishes significantly the fusicoccin-enhanced glucose uptake. By using a radioimmunoassay, the internal abscisic acid content of the protoplast was estimated to be in the range of 10^–6 M. Protoplasts isolated from bundle tissue contain twice as much abscisic acid as those derived from storage parenchyma. Because protoplasts from the bundle tissue were shown to take up sugars much faster than those from the storage cells, the observed effect of abscisic acid might reflect an involvement of this hormone in the regulation of carbohydrate partitioning in the beet.Abbreviations ABA cis–abscisic acid - bundle protoplast protoplasts isolated from the conducting tissue of beetroots - DES diethylstilbestrol - FC fusicoccin - 3-OMG 3-O-methylglucopyranose - PCMBS p–chloromercuribenzenesulfonic acid - storage protoplasts protoplasts isolated from storage parenchyma     

Effects of ethylene and abscisic acid upon heterophylly in<Emphasis Type="Italic"> Ludwigia arcuata</Emphasis> (Onagraceae)

In this study, we examined the effects of ethylene and abscisic acid (ABA) upon heterophyllous leaf formation of Ludwigia arcuata Walt. Treatment with ethylene gas resulted in the formation of submerged-type leaves on terrestrial shoots of L. arcuata, while treatments with ABA induced the formation of terrestrial-type leaves on submerged shoots. Measurement of the endogenous ethylene concentration of submerged shoots showed that it was higher than that of terrestrial ones. In contrast, the endogenous ABA concentration of terrestrial shoots was higher than that of submerged ones. To clarify interactions of ethylene and ABA, simultaneous additions of these two plant hormones were examined. When L. arcuata plants were treated with these two plant hormones, the effects of ABA dominated that of ethylene, resulting in the formation of terrestrial-type leaves. This suggests that ABA may be located downstream of ethylene in signal transduction chains for forming heterophyllous changes. Further, ethylene treatment induced the reduction of endogenous levels of ABA in tissues of L. arcuata, resulting in the formation of submerged-type leaves. Thus the effects of ethylene and ABA upon heterophyllous leaf formation are discussed in relationship to the cross-talk between signaling pathways of ethylene and ABA.Abbreviations ABA abscisic acid - ACC 1-aminocyclopropane-1-carboxylic acid - L/W ratio ratio of leaf length to width - LN leaf number - GAs gibberellins     

Uptake of protoplasts from the filamentous fungiAspergillus nidulans andFusarium oxysporum into protoplasts from celery (Apium graveolens L.)

Summary Protoplasts isolated from celery cell suspension cultures, were mixed with fungal protoplasts, from either the saprophytic speciesAspergillus nidulans or the pathogenic speciesFusarium oxysporum. The incubation of protoplast mixtures with PEG caused close adhesion between plant and fungal protoplasts. Subsequent dilution of PEG resulted in the uptake of protoplasts from either fungal species into the plant protoplast cytoplasm. A range of PEG concentrations, incubation times and dilution rates were tested to maximise adhesion and uptake frequencies. Identification of uptake was achieved either by fluorescent staining of nuclei or by electron-microscopy. A maximum of 10% celery protoplasts had taken upA. nidulans protoplasts after PEG treatment. Fungal protoplasts were taken up into celery protoplast cytoplasm by endocytosis, and were maintained within vesicles; two bounding membranes were observed by electron microscopy. Plant protoplast viability was determined during prolonged incubation following fungal protoplast uptake. The presence ofA. nidulans protoplasts tended to maintain celery protoplast viability and although some morphological disintegration occurred intact celery protoplasts remained for at least 92 h after uptake. The uptake ofF. oxysporum protoplasts markedly depressed celery protoplast viability after 24 h incubation and greater celery protoplast disintegration occurred.Abbreviations PEG Polyethylene glycol - DAPI 4,6-diaminido-2-phenylindole - 2,4-D 2,4-dichlorophenoxyacetic acid     

The permeability of the epidermal cell plasma membrane of barley leaves to abscisic acid

Uptake of ³H-labelled (±)-abscisic acid (ABA) into isolated barley (Hordeum vulgare L.) epidermal cell protoplasts (ECP) was followed over a range of pH values and ABA concentrations. The present results show that ABA uptake is not always linearly correlated with the external concentration of undissociated ABA (ABAH). At pH 7.25, ABA uptake exhibited saturation kinetics with an apparent K _m value of 75 mmol·m^–3 to tal ABA. This saturable transport component was inhibited by pretreating the protoplasts with 1 mol·m^–3 p-chloromercuribenzenesulfonic acid at pH 8.0, conditions that minimized the uptake of this acid sulfhydryl reagent. Moreover, the rate of (±)-[^3]HABA uptake was reduced by addition of 0.1 mol·m^–3 (±)-ABA to 41%, whereas the same concentration of (±)-ABA was approximately half as effective (46% of the inhibitory effect). Thus, it was concluded that only (±)-ABA competes for an ABA carrier that is located in the epidermal cell plasma membrane. The permeability of the epidermal cell plasma membrane was studied by performing a Collander analysis. At pH 6 the overall plasma-membrane permeability of epidermal cells was similar to that of guard cells but was about two times higher than that of mesophyll cells.Abbreviations ABA abscisic acid - ABA^– anion of ABA - ABAH undissociated ABA - 2,4-D 2,4-dichlorophenoxyacetic acid - DMO 5,5-dimethyloxazolidine-2,4-dione - ECP deepidermal cell protoplast - K_r partition coefficient - M_r relative molecular mass - NEM N-ethylmaleimide - PCMBS p-chloromercuriben zenesulfonic acid - P_s permeability coefficient We are grateful to Barbara Dierich for expert technical assistance, to Prof. H. Gimmler (Lehrstuhl für Botanik I, Universität Würzburg, FRG) for helpful discussions and to the Deutsche Forschungsgemeinschaft (SFB 251, TP 3) for financial support.     

The monoclonal antibody JIM19 modulates abscisic acid action in barley aleurone protoplasts

A panel of hybridoma products generated against pea (Pisum sativum L.) guard-cell protoplasts has been assayed for anti-abscisic acid (ABA) biological activity in barley (Hordeum vulgare L.) aleurone protoplasts. The effects of the antibodies on ABA-induced accumulation of mRNA transcribed from RAB-16, a gene responsive to ABA, were determined. Most of the antibodies, and culture medium, had no effect, but five monoclonal antibodies (MAbs) were found to inhibit ABA-induced RAB-16 gene expression and one MAb enhanced it. The effects of one inhibitory MAb, JIM19, were studied in some detail. These effects were specific to ABA-induced events, as incubation with JIM19 had no effect on the expression of a constitutively-expressed gene, GAPDH, encoding glyceraldehyde-3-phosphate dehydrogenase, and only a slight effect on the production of -amylase induced by gibberellic acid. Increasing concentrations of ABA in the incubation medium partly overcame the inhibitory effect of JIM19. Immunolabelling and biological activity remained together during immuno-purification of JIM19 from hybridoma culture supernatant. Immunoblotting of JIM19 to membrane preparations from barley aleurone protoplasts revealed that JIM19 recognised a number of proteins.Abbreviations ABA abscisic acid - GA gibberellin - GA₃ gibberellic acid - GAPDH gene encoding glyceraldehyde-3-phosphate dehydrogenase - GCP guard-cell protoplast - MAb monoclonal antibody - RAB (gene) responsive to ABA We thank the Agricultural and Food Research Council and The Nuffield Foundation for financial support, Professor Keith Roberts (John Innes Institute, Norwich, UK) for advice and generous use of his laboratory and Jan Peart (John Innes Institute) for animal cell culture. S.J.N. is grateful to Professor Colin Hawkes (University of the West of England, Bristol) for his continued support of this project.     

Isolation of cells and protoplasts from leaves of in vitro propagated peach (Prunus persica) plants

Yields of 10⁶–10⁸ peach mesophyll cells and protoplasts · gfw^-1 were obtained depending on factors such as digesting enzymes, and leaf size. Onozuka R-10 (2%) in combination with Macerase (0.5%) was found best for protoplast isolation and mediocre for cell isolation among several enzyme combinations tested. Viability was 90% for protoplasts and 60% for cells. Pectolyase Y23 was found to be ineffective in our investigation. Small leaves, 4–10 mm in length, were a superior source for protoplast isolation than medium or big expanded leaves, 22–30 mm in length. The high yields of protoplasts could be obtained only when keeping the ratio of leaf biomass to volume of digesting enzyme solution under 20 mg ml^-1. Purification of protoplasts on a sucrose gradient yielded about 10⁷ protoplasts · gfw^-1, however, the preparation was still contaminated by intact cells. Protoplasts were cultured under different growth regulators and physical conditions. Limited growth and division of protoplasts embedded in agarose drops were observed.Abbreviations BA 6-benzyladenine - IBA indolebutyric acid - FDA fluorescein diacetate - MES 2-M-morpholinoethane sulphonic acid - MS Murashige and Skoog - NAA -naphthaleneacetic acid - PVP polyvinylpyrrolidone     

Protoplast isolation and regeneration from <Emphasis Type="Italic">Gracilaria changii</Emphasis> (Gracilariales,Rhodophyta)

The tropical agarophyte Gracilaria changii has been much researched and documented by the Algae Research Laboratory, University of Malaya, especially with regards to its potential as a seaweed bioreactor for valuable compounds. Protoplast regeneration of this seaweed was developed following the optimization of protoplast isolation protocol. Effect of the concentration and combination of isolating enzymes, incubation period, temperature, enzyme solution pH, tissue source on the protoplast yields were used to optimize the isolation protocol. The enzyme mixture with 4% w/v cellulase Onozuka R-10, 2% w/v macerozyme R-10 and 1 unit mL^-1 agarase was found to produce the highest yield of protoplast at 28°C and 3 h incubation period. Thallus tips gave higher yields of protoplasts than middle segments. Freshly isolated G. changii protoplasts were cultured in MES medium. Regeneration of protoplast cell walls after 24 h was confirmed by calcofluor white M2R staining under UV fluorescence microscopy. The protoplasts with regenerated cell walls then underwent a series of cell division to produce callus-like cell masses in MES medium. Following this, juvenile plants of G. changii were obtained.     

Halophilic and salts tolerant protoplast cultures of mangrove plants, Sonneratia alba and Avicennia alba

The effects of sea salts, NaCl, KCl, MgCl₂, MgSO₄, and CaCl₂, on the growth of protoplast cultures of two mangrove species, Sonneratia alba and Avicennia alba, were investigated using 96-well culture plates. Plants of these two species naturally grow at the seaward side of a mangrove forest. Cotyledon protoplasts of S. alba showed halophilic nature to NaCl, KCl, and MgCl₂ at low concentrations (10–50 mM) when cultured in Murashige and Skoog’s (MS) medium containing 0.6 M mannitol. CaCl₂ at a concentration higher than 25 mM was inhibitory to cell growth. On the other hand, in protoplast culture of A. alba suspension cells, which were induced from cotyledon tissues, in the modified amino acid (mAA) medium containing 1.2 M sorbitol, tolerance to NaCl, MgCl₂ and MgSO₄ were observed at a wide range of concentrations up to 400 mM. CaCl₂ was always inhibitory for cell divisions in A. alba, but stimulatory for spherical enlargement of cells. However, no difference in cell enlargement was observed among other salts. Similarity and difference in reactivity to salts between protoplasts and suspension cells from our previous studies were discussed in relation to the site of salt tolerance or halophilic adaptation within mangrove cells. For protoplast cultures, the site(s) for response of S. alba and A. alba are located in the cytoplasm and/or the cell membrane.     

Protoplast culture and paclitaxel production by Taxus yunnanensis

Viable protoplasts of Taxus yunnanensis were isolated from friable, light yellow callus. Protoplast yield was dependent on callus age, with a maximum from 20-day-old callus. Protoplasts were induced to undergo sustained divisions and to form cell colonies when cultured in medium consisting of B5 salts, KM vitamin and organic components, 0.45 M fructose, 3.0 mg l^-1 2,4-dichlorophenoxyacetic acid and 0.1 mg l^-1 kinetin. The planting density was 2.5–3.0×10⁵ protoplasts per ml of culture medium. Cell-free extract from callus enhanced protoplast division and the highest plating efficiency was about 7%. Protoplast-derived colonies showed significant variations in both growth and paclitaxel content. A negative correlation existed between paclitaxel accumulation in colonies and their growth to some extent (r = −0.4485). Among 70 colonies isolated from the heterogeneous protoplast cultures, colony TY-7 accumulated the highest paclitaxel content. Paclitaxel accumulation in colony TY-7 was not great enough to produce paclitaxel for commercial purposes, however, success in inducing colony formation from T. yunnanensis protoplasts provides an opportunity to obtain cell lines with high paclitaxel productivity from mutagenized protoplast cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Biomass and aboveground net primary production in a subtropical mangrove stand of Kandelia obovata (S., L.) Yong at Manko Wetland, Okinawa, Japan

Biomass and aboveground net primary production (ANPP) in a monospecific pioneer stand of a mangrove Kandelia obovata (S., L.) Yong were quantified. The estimated biomasses in leaves, branches, stems, roots, aboveground and total were 5.61 (3.68%), 28.8 (18.9%), 46.1 (30.2%), 71.8 (47.2%), 80.5 (52.8%) and 152 Mg ha⁻¹ (100%), respectively. Stem phytomass increment per tree was estimated using allometric relationships and stem analysis. Stem volume without bark of harvested trees showed a strong allometric relationship with D _0.1² H (D _0.1, diameter at a height of one-tenth of tree height H) (R ² = 0.924). Annual stem volume increment per tree showed a strong allometric relationship with D _0.1² H (R ² = 0.860). Litterfall rate ranges from 3.87 to 56.1 kg ha⁻¹ day⁻¹ for leaves and 0.177 to 46.2 kg ha⁻¹ day⁻¹ for branches. Seasonal changes of litterfall rate were observed, which showed a peak during wet season (August–September). Total annual litterfall was estimated as 10.6 Mg ha⁻¹ year⁻¹, in which 68.2% was contributed by the leaves. The ANPP in the K. obovata stand was 29.9–32.1 Mg ha⁻¹ year⁻¹, which is ca. 2.8–3.0 times of annual litterfall. The growth efficiency (aboveground biomass increment/LAI) was 5.35–5.98 Mg ha⁻¹ year⁻¹. The low leaf longevity (9.3 months) and high growth efficiency of K. obovata makes it a highly productive mangrove species.     

Colony formation and plant regeneration from mesophyll protoplasts of Solanum etuberosum

A procedure for the culture of Solanum etuberosum mesophyll protoplasts with subsequent shoot regeneration is described. Several factors affected protoplast yield, colony formation, and shoot regeneration from in vitro plants. A protoplast isolation medium with 0.6 M sucrose produced twice the yield as one with 0.3 M sucrose. uowever, a higher concentration of osmoticum was inhibitory to colony development unless it was diluted into a lower osmoticum medium in a bilayer system. A 16 hour light/8 hour dark photoperiod for stock plants allowed twice the protoplast yield compared to plants grown under continuous light but no effect was found on subsequent colony formation or shoot regeneration. The concentrations of four major salts in the protoplast plating medium were critical for a high frequency of colony formation from protoplasts. Levels of 0.25 × or 1 × were considerably better than 4 ×. Fast colony formation, but at a lower efficiency, was obtained with a monolayer plating method. A bilayer plating system allowed a higher efficiency but colonies developed more slowly. For the best treatments, the frequency of colony formation from protoplasts ranged from 2.4 to 3.6 × 10^-3 with 37% to 66% of the colonies producing shoots ten weeks after protoplast isolation.Cooperative investigation of the USDA-ARS and the Wisconsin Agric. Exp. Stn.     

Gibberellins do not act against abscisic acid in the regulation of bulb dormancy of Allium wakegi Araki

Abscisic acid (ABA) is involved in bulb dormancy of Alliumwakegi Araki. We examined the antagonistic role of gibberellins(GAs)against ABA in the regulation of this dormancy. The concentrations of ABA andGAs in the basal leaf sheaths or bulbs of A. wakegi cv.Kiharawase were investigated during growth in the field and postharveststorage.The concentration of ABA in the basal leaf sheaths began to increase about onemonth before they began to swell, reached a maximum shortly after bulbharvesting, and decreased during postharvest storage. The plants showed bulbdormancy accompanied with the change in ABA concentration. GA1,GA3, GA4, GA12, GA15, GA19, and GA20 were identified in the basal leaf sheaths of A. wakegi from Kovats retention indices (KRI) andfull-scan mass spectra by gas chromatography - mass spectrometry (GC-MS)analysis. The concentrations of all classes of GAs in the basal leaf sheathsestimated by the dwarf rice micro-drop assay increased transitorily shortlybefore they began to swell, and decreased rapidly during bulb development. Bulbdormancy had already been induced when the concentration of the GAs becamemaximum. All the GAs in the bulbs remained at a low level during postharveststorage, when bulbs were gradually released from dormancy. The concentrationsof GA1+3, GA4, GA15, and GA20 inthe bulbs increased after sprouting of the bulbs planted in moist vermiculite.Hence, the state of bulb dormancy is considered to be independent of the GAconcentrations of in the basal leaf sheaths or bulbs of A.wakegi.     

Morphogenetic responses from protoplasts and tissue culture of Laminaria digitata (Linnaeus) J. V. Lamouroux (Laminariales, Phaeophyta): callus and thalloid-like structures regeneration

The regeneration of meristematic tissues from sporophytes of Laminaria digitata was studied by protoplast and tissue culture. Sequential treatment of explants in sterile seawater with 1% Betadine for 5 min, 1% commercial bleach for 1–2 min and 2% antibiotic treatment supplemented with 1 μM GeO₂ overnight enabled viable explants as high as 55%. Different morphogenetic responses were observed from tissue culture on media supplemented with plant growth regulators alone or in combination, mainly filamentous calluses up to 50% according to the media. Dark green compact calluses were observed on two combinations: 4 μM Pi + 2 μM N-(2-chloro-4-pyridyl)-N’-phenylurea (CPPU) and 0.04 μM Pi + 0.44 μM 6-benzylaminopurine. Thalloid-like structures comparable to adventitious buds were regenerated on medium supplemented with 4 μM Pi + 0.45 μM zeatin but at low frequency suggesting a strong genotypic effect. Friable calluses were developed from protoplasts in enriched medium with polyamines and containing 0.40 μM CPPU + 0.45 μM 2,4-dichlorophenoxyacetic acid. In order to produce protoplasts, a one-step enzymatic protocol was developed and yields reached 22 × 10⁶ protoplasts per gram of fresh weight.