Genetic transformation of alfalfa somatic embryos and their clonal propagation through repetitive somatic embryogenesis

Genetically transformed alfalfa (Medicago sativa L., cv. Zajearska 83) plantlets were obtained by inoculating somatic embryos with Agrobacterium tumefaciens strains A281/pGA472 and LBA4404/pBI121. Single somatic embryos, 5–7 mm long, were released from a repetitively embryogenic culture, wounded, and cocultivated with the bacteria. The agar-solidified culture medium contained mineral salts, vitamins, 40 g l^–1 sucrose, 1 g l^–1 yeast extract and 0.05 mg l^–1 BA. Five clones, transformed with A281/pGA472, and 4 clones transformed with LBA4404/pBI121, were selected for proliferation by repetitive somatic embryogenesis, on media containing 100 mg l^–1 of kanamycin. The transformation of kanamycin-resistant clones was confirmed by assaying the activity of neomycin phosphotransferase II and/or -glucuronidase enzymes, and by the Southern blot analysis. It is suggested that the transformation/regeneration system based on somatic embryogenesis may be suitable for establishing transgenic alfalfa lines. The relatively low frequency of embryo transformation is compensated for by abundant proliferation in secondary somatic embryogenesis.Abbreviations BA 6-benzyladenine - GUS -glucuronidase - Km kanamycin - NPTII neomycin phosphotransferase II - X-gluc 5-bromo-4-chloro-3-indolyl--glucuronic acid - BM basal medium     

An assessment of the annual mass balance of carbon,nitrogen, and phosphorus in Narragansett Bay

Narragansett Bay is a relatively well-mixed, high salinity coastal embayment and estuary complex in southern New England (USA). Much of the shoreline is urban and the watershed is densely developed. We have combined our data on C, N, and P inputs to this system, on C, N, and P accumulation in the sediments, and on denitrification with extensive work by others to develop approximate annual mass balances for these elements. The results show that primary production within the bay is the major source of organic carbon (4 times greater than other sources), that land drainage and upstream sewage and fertilizer are the major sources of N, and that landward flowing bottom water from offshore may be a major source of dissolved inorganic phosphorus. Most of the nutrients entering the bay arrive in dissolved inorganic form, though DON is a significant component of the N carried by the rivers. About 40% of the DIN in the rivers is in the form of ammonia. Sedimentation rates are low in most of Narragansett Bay, and it appears that less than 20% of the total annual input of each of these elements is retained within the system. A very small amount of C, N, and P is removed in fisheries landings, denitrification in the sediments removes perhaps 10–25% of the N input, and most of the carbon fixed in the system is respired within it. Stoichiometric calculations suggest that some 10–20% of the organic matter formed in the bay is exported to offshore and that Narragansett Bay is an autotrophic system. Most of the N and P that enters the bay is, however, exported to offshore waters in dissolved inorganic form. This assessment of the overall biogeochemical behavior of C, N, and P in the bay is consistent with more rigorously constrained mass balances obtained using large living models or mesocosms of the bay at the Marine Ecosystem Research Laboratory (MERL).     

Phosphorus limitation of forest leaf area and net primary production on a highly weathered soil

We tested the hypothesis that P was the nutrient limiting net primary production of a nativeMetrosideros polymorpha forest on a highly weathered montane tropical soil in Hawaii. A factorial experiment used all combinations of three fertilizer treatments: nitrogen (N), phosphorus (P) and a mix of other essential nutrients (OE), consisting primarily of mineral derived cations and excluding N and P. P addition, but not N or OE, increased leaf area index within 12 months, foliar P concentration measured at 18 months, and stem diameter increment within 18 months. Stem growth at 18 months was even greater when trees fertilized with P also received the OE treatment. N and P additions increased leaf litterfall and N and P in combination further increased litterfall. The sequence of responses suggests that increased available P promoted an increase in photosynthetic area which led to increased wood production. P was the essential element most limiting to primary production on old volcanic soil in contrast to the N limitation found on young volcanic soils.     

Seasonal flooding,nitrogen mineralization and nitrogen utilization in a prairie marsh

Flooding can be an important control of nitrogen (N) biogeochemistry in wetland ecosystems. In North American prairie marshes, spring flooding is a dominant feature of the physical environment that increases emergent plant production and could influence N cycling. I investigated how spring flooding affects N availability and plant N utilization in whitetop (Scolochloa festucacea) marshes in Manitoba, Canada by comparing experimentally spring-flooded marsh inside an impoundment with adjacent nonflooded marsh. The spring-flooded marsh had net N mineralization rates up to 4 times greater than nonflooded marsh. Total growing season net N mineralization was 124 kg N ha^–1 in the spring-flooded marsh compared with 62 kg N ha^–1 in the nonflooded marsh. Summer water level drawdown in the spring-flooded marsh decreased net N mineralization rates. Net nitrification rates increased in the nonflooded marsh following a lowering of the water table during mid summer. Growing season net nitrification was 33 kg N ha^–1 in the nonflooded marsh but < 1 kg N ha^–1 in the spring-flooded marsh. Added NO₃ ^–1 induced nitrate reductase (NRA) activity in whitetop grown in pot culture. Field-collected plants showed higher NRA in the nonflooded marsh. Nitrate comprised 40% of total plant N uptake in the nonflooded marsh but <1% of total N uptake in the spring-flooded marsh. Higher plant N demand caused by higher whitetop production in the spring-flooded marsh approximately balanced greater net N mineralization. A close association between the presence of spring flooding and net N mineralization and net nitrification rates indicated that modifications to prairie marshes that change the pattern of spring inundation will lead to rapid and significant changes in marsh N cycling patterns.     

Abundance of phytoseiid mites on Vitis species: effects of leaf hairs,domatia, prey abundance and plant phylogeny

We observed the number of predatory mites (Phytoseiidae:Typhlodromus caudiglans) on the foliage of 20 North American species of grapes (Vitis spp) plus the domesticated EuropeanVitis vinifera, all grown in a common garden. We found relatively few phytophagous mites. The numbers of phytophagous mites were not correlated with the plant characteristics that we measured. We found approximately five times as many predatory mites as phytophagous mites and the numbers of these phytoseiid predators were not affected by the availability of prey. Similarly, numbers of phytoseiids were unaffected by plant gender and, hence, the availability of pollen, another source of food. The numbers of phytoseiids were not clustered according to the taxonomic grouping of the tested plant species. Leaf surface characteristics explained over 25% of the variance in the numbers of phytoseiids. Numbers of phytoseiids were positively associated with the density of vein hairs, the density of bristles in leaf axils, and the presence of leaf domatia. These results suggest that sheltered habitats rather than food availability may limit the numbers of phytoseiid mites on grapevines.     

Fatty acid composition of vitellogenin from four teleost species

The fatty acid compositions of vitellogenin and liver from cod (Gadus morhua), rainbow trout (Oncorhynchus mykiss), turbot (Scophthalmus maximus) and wolffish (Anarhichas lupus) were determined. Vitellogenin was isolated from plasma of estradiol-17-treated fish by precipitation with EDTA-Mg²⁺ and distilled water or by high-performance ion-exchange chromatography. In all investigated species, vitellogenin contained 16–18% (w/w) lipid, in which polyunsaturated fatty acids, predominantly 20:5 (n-3) and 22:6 (n-3), comprised about 50% of the total fatty acids. The proportions of saturated, monounsaturated, polyunsaturated and (n-3) fatty acids in vitellogenin of the different species were generally similar, although the relative content of specific fatty acids was distinctive for each species. The distribution of fatty acids in total lipids of vitellogenin was highly consistent among individual females of each species. In contrast, liver fatty acid composition varied considerably, both within and between species. Altogether, the differences in the fatty acid composition of vitellogenin and liver from each species indicate that a specific selection of fatty acids occurs during the lipidation of vitellogenin.Abbreviations BHT butylated hydroxytoluene - E-17 estradiol-17 - EDTA ethylenedinitrilo tetra-acetic acid disodium salt dihydrate - FA fatty acids - FAME fatty acid methyl esters - HDL high density lipoproteins - PUFA polyunsaturated fatty acids - SD standard deviation - TLC thin-layer chromatography - VHDL very high density lipoproteins - VLDL very low density lipoproteins - v/v volume per volume - w/v weight per volume - w/w weight per weight     

In vitro investigation of α-amylase release from the digestive cells of the bivalve mollusc Pecten maximus: effect of second messengers and biogenic amines

The (neuro)endocrine control of enzyme release from invertebrate digestive cells remains poorly understood. A tissue dissociation procedure was developed to investigate the regulatory mechanisms of -amylase discharge from the cells of the stomach-digestive gland complex of the scallop Pecten maximus. The validity of the experimental system was tested by increasing the intracellular concentration of second messenger analogues (N ⁶,2-o-dibutyryl-adenosine-3,5 cyclic monophosphate and the ionophore A23187) known to mimic the activity of naturally occurring secretagogues in vertebrates: N ⁶,2-o-dibutyryl-adenosine-3,5 cyclic monophosphate increased the time and dose-dependent release of -amylase in a similar way as in vertebrates. A23187 was also very effective in inducing enzyme discharge. Since the in vitro bioassay was shown to be functional and because axon terminals were previously seen in close contact to -amylase secreting cells, the effect of some classic neurotransmitters was explored. Only the cholinergic agonist carbachol and dopamine evoked a secretory response. Maximal stimulation of -amylase release was reached at 10^-5 mol·l^-1 carbachol; at the same concentration dopamine was less effective than carbachol. By contrast, serotonin was totally inactive. The in vitro bioassay should prove useful for the identification of regulatory molecules involved in the control of enzyme discharge and to study stimulus secretion coupling mechanisms in scallop digestive cells.Abbreviations DBcAMP N ⁶, 2-O-dibutyryl-adenosine-3,5 cyclic monophosphate - cAMP adenosine-3,5 cyclic monophosphate     

Food dependent color patterns inThamnocephalus platyurus Packard (Branchiopoda: Anostraca); a laboratory study

Coloration of phyllopods varies from place to place and from one life stage to another. It ranges from translucent or whitish through gray, blue, green, orange, and reddish. Here, we present experimental evidence for a food- dependent color pattern inThamnocephalus platyurus Packard. The presence or absence of the synthetic pigment trans — — carotene in a baker's yeast diet was the controlling factor. All the 24 old larvae used in the experiment were whitish in color. From day 6 until the end the experiment (day 11), 100% of the shrimps under a diet with synthetic trans — — carotene (treatment 1) exhibited a characteristic color pattern which consisted of an orange color in the cercopods, and in all theracopods; the rest of the body exhibited no particular color. In comparison, 100% of the shrimps under a diet without synthetic trans — — carotene (treatment 2) were whitish throughout the body. In females from treatment 1, the ovaries and oocytes were green-bluish, while in females from treatment 2 the ovaries and oocytes were whitish. No significant differences in survival and growth were found, except that at day 9, there was a significant difference in growth, the females with the synthetic trans — — carotene group growing faster.     

Land/water ecotone effects in reservoirs on the fish fauna

The morphology and function of one tropical and 25 temperate reservoirs are examined in relation to their effect upon the nature of the land/water interface and, further, to what extent the features of these ecotones satisfy the ecological requirements of the reservoir fish species throughout their life cycle during spawning, larval, juvenile and adult stages. The two main conclusions are that (1) reservoir fish species are especially dependent upon land/water ecotones during their early life history and (2) there exists a strong relationship between the extent of the littoral area and the nature of the fish stocks. Several examples are given to show that manipulation of the land/water ecotone is a major tool for the management of reservoirs advantageously for their major functions.     

Effects of ambient temperature and of temperature acclimation on nitrogen excretion and differential catabolism of protein and nonprotein resources in the intertidal snails,Littorina saxatilis (Olivi) and L. obtusata (L.)

Nitrogenous excretion in two snails, Littorina saxatilis (high intertidal) and L. obtusata (low intertidal) was studied in relation to temperature acclimation (at 4° and 21°C), including total N excretion rates, the fraction of urea in N excretion, corresponding O:N ratios and the partitioning of deaminated protein between catabolic and anabolic processes at 4°, 11° and 21°C. Aggregate N excretion rates in both species showed no significant compensatory adjustments following acclimation. Total weight specific N excretion rates at 21°C were higher in standard 3 mg L. saxatilis (739 ng N mg⁻¹ h⁻¹) than standard 5 mg L. obtusata (257 ng N mg⁻¹ h⁻¹) for snails acclimated to 21°C. Comparisons of Q₁₀ values of total weight specific N excretion to Q₁₀ values for weight specific oxygen consumption ({xxV}O₂) between 4° to 11 °C and 11° to 21°C indicated that, while total rates of catabolic metabolism ({xxV}O₂) and protein deamination in L. obtusata were essentially parallel, the relationship between N excretion and {xxV}O₂ in L. saxatilis revealed the partitioning of a larger share of deaminated protein carbon into anabolism at 4° and 21°C than at 11°C. Urea N accounted for a larger share of aggregate N excreted in L. saxatilis than in L. obtusata, but in both species urea N is a greater proportion of total N excreted when acclimated at 4°C (urea N: ammonia N ratio range: 1 to 2.15) than in snails acclimated to 21°C (urea N: ammonia N ratio range: 0.46 to 1.39). Molar O:N ratios indicate that the proportion of metabolism supported by protein catabolism is greater in L. saxatilis (O:N range: 2.5–8.4) than in L. obtusata (O:N range: 7.3–13.0). In both species, regardless of acclimation temperature, the O:N ratios are generally lowest (high protein catabolism) at 4°C and highest at 21°C.