Improved plant regeneration from wheat anther and barley microspore culture using phenylacetic acid (PAA)

Summary The effect of the auxin phenylacetic acid (PAA) on wheat anther and on barley anther/microspore culture was investigated. With PAA the induction response was not usually significantly different from controls but a significantly higher number of green plants were produced in wheat anther and barley microspore culture. For wheat anther culture 100 mg/L PAA was beneficial. For barley microspore culture the optimum levels were from 1 to 100 mg/L, depending on genotype. In barley anther culture there were no improvements using PAA. In wheat anther culture, 145 green plants/100 anthers were obtained with cultivar VeeryS, while the average response from twelve F₁ hybrids in the breeding program was 332 green plants/100 anthers. At least 1000 green plants were obtained using isolated microspores from 100 anthers in barley cv. Igri. With cv. Bruce, regeneration occurred only when 100 mg/L PAA was used. The influence of PAA appears at the embryogenic phase of the culture system. The possible mechanisms by which PAA may improve regeneration are discussed.     

Influence of photoperiod,temperature and host plant on the production of diapause eggs inPetrobia (Tetranychina) harti (Acari: Tetranychidae)

Petrobia harti (Ewing) diapauses in the egg stage. Adult females lay either diapause or nondiapause eggs. On the University of Thessaloniki campus (41°N), the mite was found to develop on leaves ofOxalis corniculata L. throughout the year, while no mites were found on leaves ofOxalis articulata Savigny growing in the same area. In the laboratory the mite could be maintained equally well on detached leaves of both plant species, kept on wet cotton-wool.Forty to 90% females laying diapause eggs (dlf) were produced when the mites developed under LD 1212 and 19±1 °C, or LD 168 and 19±1 °C or 25±1 °C on leaves ofO. articulata detached from plants grown in the open in various seasons. Under the same conditions, a very low to zero percentage ofdlf was produced onO. corniculata. By rearing certain feeding stages on one of these twoOxalis hosts, and the other feeding stages on the other host, various percentages ofdlf were obtained. These percentages were the net effect of the antagonistic action of the twoOxalis species.By rearing the mites at LD 8.515.5, LD 1212 or LD 168 and a temperature of 19±1 °C onO. articulata leaves renewed every 3 days, or every 16–18 days, or not at all, it could be shown that diapause induction or aversion is caused by the direct effect of photoperiod on the mites, and not by an effect through the host leaves.When wholeO. articulata plants were grown under LD 168 and 19±1 °C in the laboratory, or developed in the open during April and May, flowers were produced, while under LD 1212 no flowering occurred. In the laboratory under diapause-inducing conditions, higher percentages ofdlf were produced on leaves detached from flowering plants than on leaves detached from plants not flowering.OnO. articulata leaves at 20 °C, photoperiods with photophases equal to or longer than 12 h induced from 70 to 80%dlf, while photoperiods with photophases equal to or shorter than 10.9 h induced very low to zero percentages. By transferring different chrysalis stages from a diapause-inducing (LD 1212) to a diapause-averting (LD 8.515.5) photoperiod, and vice versa, it was found that the nymphochrysalis through deutonymph stages were sensitive to photoperiod, the deutochrysalis and deutonymph being the most sensitive.Under an LD 1212 photoperiod, a temperature of 20 °C induced diapause, whereas 25 °C, 30 °C, or a daynight thermoperiod of 25 °C18 °C suppressed it.     

Effects of culture conditions on isolated microspore response of barley cultivar Igri

Pretreatment of anthers in mannitol prior to isolation of microspores by glass rod homogenization was effective for in vitro induction of embryogenesis in barley cv. Igri. A procedure for separation of viable microspores using centrifugation on 20% maltose was developed. The concentration of microspores was important and greatly increased the number of developing structures. Initial culture of microspores on FHG medium containing 62 g l^-1 maltose, 4.4 M (1 mg l^-1) BA and 200 g l^-1 Ficoll-400 resulted in high frequencies of plant regeneration. Albino plant frequency was correlated to length of time in culture. Stock plant condition appeared to be a major factor influencing induction frequency. From 868 to 1738 green plants per 100 anthers were produced. The number of calli and embryos obtained and the number of green plantlets regenerated were improved by increasing the Ficoll concentration from 100 g l^-1 to 400 g l^-1 during the culture period compared to continuous culture on FHG Ficoll 200 g l^-1.Abbreviations BA benzyladenine     

Microspore culture of radish (Raphanus sativus L.): influence of genotype and culture conditions on embryogenesis

A number of factors influencing embryogenesis from isolated microspores of radish (Raphanus sativus) were examined. Of 11 genotypes evaluated, six produced embryos ranging from 8.3 embryos per 10⁵ microspores for Chugoku-ao to 0.2 for Tenshun, but five genotypes were not responsive. An initial culture period at elevated temperature before incubation at 25°C was essential for induction of microspore embryogenesis. However, the optimum period of the treatment varied among genotypes and/or experiments. Bud size also influenced microspore embryogenesis. Though optimum bud size was different between genotypes, the microspore populations represented in these buds contained uninucleate and binucleate microspores. Selection of embryogenic microspores using percoll density gradient resulted in up to 1.3-fold increase of embryo yield. Though almost all embryos failed to develop directly into plantlets, plants were obtained by multiple subcultures. The regenerated plants had hyperploid chromosome numbers.     

Direct embryogenesis and green plant regeneration from isolated microspores of hexaploid triticale (× <Emphasis Type="Italic">Triticosecale</Emphasis> Wittmack) cv. Bogo

The use of doubled haploids improves the efficiency of cultivar development in many crops and can be helpful in genetic and molecular studies. The major problem with this approach is the low efficiency of green plant regeneration. We describe here an efficient method for inducing embryos and regenerating green plants directly from isolated microspores of hexaploid triticale (× Triticosecale Wittmack) cv. Bogo. The absence of growth regulators in the induction medium was the most effective condition for the formation of embryo-like structures. The highest induction rates were observed at microspore densities of 1.5×10⁵ microspores and 2×10⁵ microspores per milliliter. Such cultures produced an average of 54.9 green plants per single donor spike. The frequency of albino plants ranged from 9.3% to 22.9%. Among the green progeny tested, 30.8% were spontaneously doubled haploids.Abbreviations BAP Benzylaminopurine - DAPI 4-6 Diamidino-2-phenylindole - 2,4-D 2,4-Dichlorophenoxyacetic acid - NAA -Naphthaleneacetic acid     

Interspecific hybrids between Brassica napus L. and B. oleracea L. developed by embryo culture

Summary Interspecific hybrids between Brassica napus and B. oleracea are difficult to produce, and previous attempts to transfer economic characters from one species to the other have largely been unsuccessful. In these studies, oilseed rape cv. Tower (2n38) (B. napus) was crossed with broccoli and kale (2n18) (B. oleracea), and hybrid plants were developed from embryos in culture by either organogenesis or somatic embryogenesis. In rape × broccoli, F₁ plants were regenerated from hybrid embryos and the plants produced viable selfed seeds. F₅ plants (2n38) homozygous for white flower colour were selected for high oil content (47%) and Line 15; a selection from these plants produced fertile hybrids with rape, broccoli and kale without embryo culture. In reciprocal crosses between oilseed rape cv. Tower and an aphid resistant diploid kale, 28 and 56 chromosome F₁ hybrid plants were regenerated from somatic embryos. The 56 chromosome plants were self-fertile and it was concluded from F₂ segregation ratios that a single dominant gene controls resistance to cabbage aphid in kale. The 28 chromosome F₁'s were self-sterile, but these and the 56 chromosome F₁'s could be backcrossed to rape and kale. A cross between the F₁ (2n56) and a forage rape resulted in the selection of a cabbage aphid (Brevicoryne brassicae L.) resistant line (Line 3). Both Line 15 and Line 3 can serve as bridges for gene interchange between B. campestris, B. napus and B. oleracea, which has not been possible hitherto. Hybridisations between rape and tetraploid kale produced F₁ plants with 37 chromosomes. One F₂ plant possessed coronal scales and the inheritance was shown to be controlled by a single recessive gene unlinked to petal colour.This paper is dedicated to Mr. T. P. Palmer, a colleague and close friend who retired from the DSIR as Assistant Director of the Crop Research Division in September 1984     

Modification of the properties of a translocation in the German cockroach by selection

Summary A stock of Blattella germanica bearing the interchange T(3; 12)/3;12 was subjected to close inbreeding with selection for random disjunction at metaphase I. After 3–4 generations of selection, interchange quadrivalent chiasma frequency decreased, variability in free bivalent chiasma frequency increased sharply, and individuals with either random or directed disjunction were present in the stock. Random disjunction was modified from a ratio of 2112 (adj.-1; alt.-1; adj.-2; alt.-2) to a ratio of 1111. After 7–8 generations of selection, chiasma frequency appeared to stabilize at lower than normal levels and variability decreased for both quadrivalents and free bivalents. Directed disjunction was modified from a ratio of 2114 to 1112, and no individuals with the original high level of directed disjunction were detected. Chains-of-four tended to orient randomly, especially in individuals where the ring quadrivalents showed directed disjunction. Relaxation of inbreeding, but not selection, produced an increase in chiasma frequency and variability in both free bivalents and quadrivalents, but the modified ratios for both random and directed disjunction were retained. These results are discussed with respect to inbreeding effects and genetic control of chiasma frequency and metaphase I disjunction in interchange quadrivalents.     

Interaction of free fatty acids with the erythrocyte membrane as affected by hyperthermia and ionizing radiation

The interference of hyperthermia and ionizing radiation, respectively, with the effects of capric (100), lauric (120), myristic (140), oleic (cis-181) and elaidic (trans-181) acids on the osmotic resistance of human erythrocytes was investigated. The results are summarized as follows: (A) not only at 37°, but also at 42° and 47°C lauric acid (120) represents the minimum chain length for the biphasic behaviour of protecting against hypotonic hemolysis at a certain lower concentration range and hemolysis promotion at subsequent higher concentrations; (B) with increasing temperatures the protecting as well as the hemolytic effects occur at lower concentrations of the fatty acids; (C) the increase of temperature promotes the extent of hemolysis and reduces the extent of protection against hypotonic hemolysis; (D) Gamma-irradiation of erythrocytes selectively affects the concentration of oleic acid at which maximum protection against hypotonic hemolysis occurs, without altering the minimum concentration for 100% hemolysis.     

Structure of the majorO-glycosidic oligosaccharide of monkey erythrocyte glycophorin

Sialic acids and the majorO-glycosidic oligosaccharide of glycophorin MK from monkey (Japanese monkey,Macaca fuscata) erythrocyte membranes were characterized.N-Glycolylneuraminic acid (neu5Gc) was found as the major sialic acid, which was confirmed by gas-liquid chromatography-mass spectrometry as the trimethylsilyl methyl ester. ThreeO-glycosidic oligosaccharide units were obtained from a tryptic glycopeptide that contained all of the carbohydrate units in glycophorin MK by mild alkaline borohydride/borotritide treatment. Carbohydrate analyses of the oligosaccharides revealed that they were composed of Neu5Gc, galactose andN-acetylgalactosaminitol in the molar ratios of 111 (trisaccharide), 211 (tetrasaccharide) and 111 (pentasaccharide). The content of oligosaccharide units was estimated to be 1125 for penta-, tetra- and trisaccharide, respectively, based on the yields, the molecular weight, and the number of oligosaccharide attachment sites in the amino-acid sequence. The tetrasaccharide was the major oligosaccharide and its structure was proposed to be Neu5Gc2-3Gal1-3[Neu5Gc2-6]GalNAcol.     

Effects of Cd and Zn on the development of annual xylem rings of young Norway spruce (Picea abies) plants

Three-year-old spruce (Picea abies) saplings were planted and cultivated for 2 years in pots with 3 1 substrate, consisting of a homogenized mixture of sand, peat and forest soil with a high organic content (volume ratio 11.52). This substrate was amended with 10–180 mol Cd [kg soil dry weight (DW)]^–1, 50–7500 mol Zn (kg soil DW)^–1 (determined with 1 M ammonium acetate extracts) or combinations of both elements. Annual xylem growth rings in stems of plants treated with 50 mol Cd (kg soil DW)^–1 or 7500 mol Zn (kg soil DW)^–1 were significantly narrower than in control plants. Growth reductions were more pronounced in the second year of the experiment. The contents of Cd and Zn in stem wood and needles were positively correlated with the substrate concentrations. The Mg contents of the spruce needles were inversely correlated with soil concentrations of Cd and Zn. Root development was impeded at moderate concentrations of Cd (50 mol kg^–1) or Zn (1000 mol kg^–1) in the substrate. The adverse effects of potentially toxic trace elements, like Cd or Zn, on xylem growth of spruce plants are discussed with regard to possible growth reductions in forest trees under field conditions.     

Light exposure stimulates arachidonic acid metabolism in intact rat retina and isolated rod outer segments

This paper presents evidence that short-term exposure to light increases synthesis of hydroxyei-cosatetraenoic acid (HETE) and prostaglandin D₂ (PGD₂) and stimulates the uptake and metabolism of 204 in phospholipids and triacylglycerols in rat retina. There was a time-dependent increase in incorporation of 1-¹⁴C-204 into glycerolipids in both dark-adapted and light-exposed groups. Exposure to light for 15 or 30 min enhanced the acylation of 204 into phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine and triacylglycerols. In the light-exposed groups there was a large increase in the conversion of 204 to leukotriene B₄, two diHETEs, 5-HETE, 15-HETE, and PGD₂. The stimulation of HETE synthesis by light could be due to early stages of light-induced lipid peroxidation in visual cells. To examine this, we studied peroxidation of 204 in isolated rod outer segments (ROS). There was more oxidation of 204 in light-exposed ROS, as compared to ROS incubated in the dark. Vitamin E and nordihydroguaiaretic acid inhibited the light-induced formation of some of these products. The data indicate that photo-oxidation of 204 in ROS is accompanied by enzymatic oxygenation that is stimulated by light. Increased production of HETEs and PGD₂ may be a consequence of the light-induced stimulation of the metabolism of 204 in membrane phospholipids in the retina.     

Culture conditions for induction of green plants from barley microspores by anther culture methods

Summary With barley a large variation in frequency of plant formation from microspores of spikes from the same plant has been observed. The highest frequency of plant formation was obtained when culturing anthers in the dark on a high Ficoll medium containing 2,4-D and kinetin to induce proembryo (or callus) formation. Subsequently the proembryos or calli were cultured in dim light on a high Ficoll-high sugar medium containing IBA and kinetin. Finally the embryos were transferred to a starch agar medium. A maximum of 13 green plants were obtained from microspores of a single anther.The ratios of green to albino microspore derived plants varied from 91 to 19 depending on culture conditions. Under anaerobic conditions, lactic acid and other organic acids may have damaged the organelles in the cells resulting in the formation of albino plants. Thus, direct embryogenesis by using a well-buffered, high Ficoll-high sugar medium and proper aeration are essential for obtaining high frequency of green plants from microspores.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - IBA 3 indolylbutyric acid     

Nitrogen and phosphorus requirements for raising mycorrhizal seedlings of Leucaena leucocephala in containers

A greenhouse study was undertaken to determine the nitrogen and phosphorus fertilization requirements for raising mycorrhizal seedlings in soil in containers. Seedlings of Leucaena leucocephala were grown for 40 days in dibble tubes containing fumigated or nonfumigated soil uninoculated or inoculated with Glomus aggregatum. The soil was fertilized with NH₄NO₃ solution to obtain 25–200 mg N kg^-1 soil, and with a KH₂PO₄ solution to establish target soil solution P concentrations of 0.015–0.08 mg P l^-1. At the end of 40 days, seedlings were transplanted into pots containing 5-kg portions of fumigated soil. Posttransplant vesicular arbuscular mycorrhizal fungal (VAMF) effectiveness, measured as pinnule P content, plant height, shoot dry weight and tissue N and P concentrations, was significantly increased by pretransplant VAMF colonization in both soils. The best posttransplant mycorrhizal colonization and mycorrhizal growth responses were observed if the nonfumigated pretransplant soil was amended with 50 mg N kg^-1 soil and 0.04 mg P l^-1 or if the fumigated pretransplant soil was amended with 100 mg N kg^-1 soil and 0.04 mg P 1^-1. There was no relationship between NP ratios of nutrients added to the pretransplant soil medium and shoot NP ratios observed after transplanting. Shoot NP ratio was also not correlated with root colonization level.Contribution from the Hawaii Institute of Tropical Agriculture and Human Resources Journal Series No. 4025     

Bicuculline induces free fatty acid release from phospholipids in Neuro-2A cells in culture

This study characterizes free fatty acid release in a neuroblastoma cell line (Neuro-2A), a potential model system for the study of factors that control phospholipase A₂ in neurons. Two compounds, bicuculline (an antagonist at -aminobutyric acid receptors), and A23187 (a Ca²⁺ ionophore), were examined. The release of endogenous fatty acids and the turnover of radiolabeled arachidonic and docosahexaenoic acids were measured. The cells actively incorporated radiolabeled fatty acids into various glycerolipid pools. Both endogenous fatty acids and radiolabeled fatty acids were released from glycerolipids in a time-dependent manner. Phosphatidylcholine was a major source of released fatty acids. Release of free fatty acids was markedly stimulated by both bicuculline and A23187. We conclude that the Neuro-2A cell contain phospholipase activity that is sensitive to Ca²⁺ ionophore and bicuculline, and may provide a good system for further studies on the regulation of phospholipase A₂ in neurons.Abbteviations 160 palmitic acid - 180 stearic acid - 181 oleic acid - 182 linoleic acid - 183 linolenic acid - 204 arachidonic acid - 226 docosahexaenoic acid - DG diacylglycerol - FAME fatty acid methyl ester - FFA free fatty acid - GABA -aminobutyric acid - PA phosphatidic acid - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - TG triacylglycerol     

Inheritance of high oleic acid content in the seed oil of soybean mutant M23

A mutant line, M23, of soybean [Glycine max (L.) Merr.] was found to have two fold increases in oleic acid content in the seed oil compared with the original variety, Bay. Our objective was to determine the inheritance of the high oleic acid content in this mutant. Reciprocal crosses were made between M23 and Bay. There were no maternal and cytoplasmic effects for oleic acid content. The F₁ seeds and F₁ plants were significantly different from either parents or the midparent value, indicating partial dominance of oleic acid content in these crosses. The oleic acid content segregated in the F₂ seeds and F₂ plants in a trimodal pattern with normal, intermediate and high classes, satisfactorily fitting a 121 ratio. The seeds of a backcross between M23 and F₁ segregated into intermediate and high classes in a ratio of 11. These results indicated that oleic acid content was controlled by two alleles at a single locus with a partial dominant effect. Thus, the allele in M23 was designated ol and the genotypes of M23 and Bay were determined to be olol and 0l0l, respectively. The oleic acid contents of the F₂ seeds and F₂ plants were inversely related with the linoleic acid content which segregated in a trimodal pattern with normal, intermediate and low classes in a 121 ratio. Thus, it was assumed that the low linoleic acid content in M23 was also controlled by the ol alleles. Because a diet with high oleic acid content reduces the content of low density lipoprotein cholesterol in blood plasma, the mutant allele, ol, would be useful in improving soybean cultivars for high oleic acid content.     

Circadian effects of hydrocortisone on the blood of the newt,Notophthalmus viridescens

Summary Differential counts of the leucocytes of newts,Notophthalmus viridescens, were made at four times of day (200, 900, 1400 and 2100), 72 hours after the injection of hydrocortisone acetate (experimentais) or distilled water (controls). At all times, increases in neutrophils and decreases in lymphocytes were observed in experimentais as compared to the controls (Table 1). The increases in neutrophils in the experimental newts were most pronounced at 1400, and the decreases in the lymphocytes were greatest at 2100. The least degrees of neutrophilia and lymphopenia occurred at 900. Consequently, circadian variations in response to the hydrocortisone are indicated. The possible mechanism of mediation of the variations is discussed.Supported in part by National Science Foundation grant, GY-7661, to Sweet Briar College.     

Inheritance of heat-stable resistance to Meloidogyne incognita in Lycopersicon peruvianum and its relationship to the Mi gene

Summary The inheritance of heat-stable resistance to the root-knot nematode, Meloidogyne incognita (Kofoid and White) Chitwood, was studied in crosses between different accessions and clones of Lycopersicon peruvianum L. F₁, F₂ and BC₁ generations were evaluated for their index of resistance based on numbers of eggs and infective second-stage juveniles (J₂) per gram of root, and the segregation ratios were determined in experiments carried out at constant soil temperatures of 25 °C and 30 °C. L. peruvianum P.I. 270435 clones 3 MH and 2R2 and P.I. 126443 clone 1 MH, all heatstable resistant, were crossed with L. peruvianum P.I. 126440 clone 9 MH, which is susceptible at both 25 °C and 30 °C. All F₁ progeny were resistant at 25 °C and 30 °C; F₂ and BC₁ generations at 25 °C gave resistant: susceptible (RS) ratios of 151 and 31, respectively, which suggests that resistance is conditioned by two independently assorting genes. However, at 30 °C, RS ratios of 31 and 11 were observed for the F₂ and BC₁ generations, respectively. These results indicate that heat-stable resistance is conferred by a single dominant gene expressed at 30 °C, while the second resistance gene is heat unstable and not expressed at 30 °C. P.I. 270435 clones 2R2 and 3 MH and P.I. 126443 clone 1 MH were crossed with P.I. 128657 clone 3 R4 (source of gene Mi), which is resistant at 25 °C but susceptible at 30 °C. All of the F₁ progeny were resistant at 25 °C and 30 °C.TC₁ progeny of 270435-2 R2 x 128657-3 R4, 270435-3 MH x 128657-3 R4 and 126443-1 MH x 128657-3 R4 crossed with susceptible 126440-9 MH were all resistant at 25 °C and segregated in a 11 ratio at 30 °C. These results also suggest that the heat-stable resistance is monogenic and that it is non-allelic to gene Mi. The non-segregation of TC₁ progenies at 25 °C, suggests that the heat-unstable resistance factor in L. peruvianum P.I. 270435 clones 2 R2 and 3 MH and in P.I. 126443 clone 1 MH is allelic to or the same as gene Mi. We propose the symbol Mi-2 for the gene in P.I. 270435 that confers heat-stable resistance to M. incognita.     

Substrate and inhibitor specificity of anion exchangers on the brush border membrane of rabbit ileum

Summary In previous studies we have found that several anions can be transported by an exchange process in rabbit ileal brush border membranes. We demonstrated exchanges of Cl for OH or HCO₃, SO₄ for OH, oxalate for OH, and oxalate for Cl. The purpose of these studies was to determine the number of distinct carriers mediating these exchanges. We utilized substrate and inhibitor specificity studies to distinguish between different anion exchange transporters. We conclude that SO₄OH and oxalate: OH exchange occur on the same carrier because: (i) pH-gradient stimulated transport of both¹⁴C-oxalate and³⁵SO₄ were equally sensitive tocis-inhibition by unlabeled SO₄ or oxalate; and (ii) both were inhibited equally by K. We conclude that oxalate: OH and oxalate: Cl exchanges occur on different carriers because: (i) Cl or SO₄ caused unequalcis-inhibition of these two exchanges; and (ii) as compared to oxalate: Cl exchange, oxalate: OH exchange was more sensitive to inhibition by probenecid and K and less sensitive to inhibition by bumetanide. Finally, we conclude that oxalate: Cl exchange and ClHCO₃ exchange occur on different carriers because: (i) ClHCO₃ exchange was almost completely insensitive tocis-inhibition by oxalate; and (ii) oxalate: Cl exchange was more sensitive to inhibition by DIDS and bumetanide than ClHCO₃ exchange. Thus we have found that there are at least three separate anion exchangers on rabbit ileal brush border: (i) a ClHCO₃ exchanger; (ii) a SO₄OH exchanger, which also transports oxalate; and (iii) an oxalate: Cl exchanger.     

Distribution and composition of particulate organic matter in the Ross Sea (Antarctica)

The biochemical composition and spatial distribution of particulate organic matter (POM) were studied in the Ross Sea (Antarctica) in summer 1989 to assess the quantitative role of organic carbon fractions in the cycling of organic matter in the water column. Large differences in chemical composition were observed between surface and deep layers. The results indicated that, despite large geographical differences, POM was quite homogeneous, of phytoplankton origin and mostly detrital. Different ratios were used to investigate the changes in biochemical composition of particulate organic matter in relation to the ice-melting: CN (organic carbonorganic nitrogen ratio) and C-POMPOC (sum of carbohydrate, protein and lipid carbontotal organic carbon ratio) were used to analyse the percentage of refractory organic material. PPRTPCHO (proteincarbohydrate ratio) were used to establish POM age and RNADNA ratios as a relative measure of particulate activity; POCChl a and N-PPRTChl a ratios were used to estimate the autotrophic contribution to the suspended particulate organic matter. Despite its low caloric value (5.3 Kcal g POM^–1), an high caloric content in the photic layer (1.6 Kcal m^–3 of POM and 2.5 Kcal m^–3 of POC) was found thus indicating that a large amount of food was available to higher trophic levels.     

Plant regeneration from callus-derived protoplasts of Pelargonium x domesticum

A protocol for regenerating plants from callus-derived protoplasts of Pelargonium x domesticum (rega l geranium cv. Melissa) has been developed. Protoplasts were isolated from leaf-derived callus tissue on MS medium supplemented with 3.0 mg/l naphthalene acetic acid, 2.0 mg/l 6- benzylaminopurine, and 3.0% sucrose. This callus yielded 2.7×10⁵ protoplasts/gram of tissue after a 6 hr incubation in an enzyme solution consisting of 2.0% cellulysin, 0.5% macerase, and 0.5 M sucrose. Protoplasts were plated at 1×10⁵ protoplasts/ml in a mixture (11 v/v) of KMP8/KP liquid medium layered on the same medium solidified with 0.6% agarose. Protoplast division was initiated within 2 days, and colonies of 15 to 50 cells developed 8 wk after plating. P-calli 1–2 mm³ developed 15 wk after plating, and plants regenerated from the p-calli have been transferred to the greenhouse.Abbreviations NAA naphthaleneacetic acid - 6-BAP 6-benzylaminopurine - CW Calcofluor White - FDA fluorescein diacetate