Safe biotechnology (4). Recommendations for safety levels for biotechnological operations with microorganisms that cause diseases in plants

The Working Party on Safety in Biotechnology of the European Federation of Biotechnology has proposed a classification of microorganisms that cause diseases in plants. In this paper appropriate safety levels are proposed for these classes of microorganisms in order to ensure that research, development and industrial fermentation work with plant pathogens will limit the risk of outbreaks of diseases in crops that could result from work with such microorganisms when they are cultivated in laboratories, glasshouses and biotechnology installations.Co-opted: J. Dähne, J. Drozd, M. Lemattre, I. M. Smith , E. M. A. WaterschootA Report prepared by the Working Party on Safety in Biotechnology of the European Federation of Biotechnology (EFB)

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The risk/safety assessment of transgenic crops: the transportability of data

This paper presents the activities and publications of Organisation for Economic Co-operation and Developments (OECD’s) Working Group on Harmonisation of Regulatory Oversight in Biotechnology and the Task Force for the Safety of Novel Foods and Feeds. The main outputs of the work are the Series of “consensus documents” of the respective groups. These documents compile information which is intended to be used by those involved in the business of risk/safety assessment. These documents are one means of ensuring the transportability of data amongst authorities. An increasing trend in both the Working Group and Task Force is to consider crop species which are relevant to tropical regions and therefore to countries that are not necessarily members of the OECD. For example, the Working Group has recently published a consensus document on bananas and plantains while the Task Force has published a document on cassava. This trend towards crops of greater interest in the tropics is likely to continue into the future.     

Profile – Børge Diderichsen

Børge Diderichsen (Fig. 1) is the President of the European Federation of Biotechnology (EFB) and Vice President at Novo Nordisk (Denmark). After a master's degree in biochemistry, followed by a Ph.D. in microbiology, both from the University of Copenhagen, he joined Novo Industry as part of a small team of researchers who were to examine whether genetically modified organisms (GMOs) could be used in the production of insulin and industrial enzymes. Since then he has played an important role in Novo Nordisk's efforts to resolve many of the problems that arise in connection with the production of GMOs and was a pioneer in bringing some of the first industrial enzymes from GMOs onto the market. Diderichsen has many honorary titles and has served on a large number of boards and commissions. Most recently he was invited to join the Commissioner's Advisory Group for Life Sciences, Genomics and Biotechnology for Health corresponding to the 6th Framework Programme. For more than 20 years he has contributed to the public debate concerning genetic engineering and ethics, biotechnology and society, conditions for universities and public research among others. He has been a major force in reforming the EFB, of which he was elected president in January 2002.

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Fig. 1. Børge Diderichsen.     

Innovation and the regulation of products of agricultural biotechnology in the United States of America

The policy of the United States government is to seek regulatory approaches, consistent with applicable laws, that protect health and the environment while reducing unnecessary regulatory burdens and avoiding unjustifiably inhibiting innovation, stigmatizing new technologies, or creating unnecessary trade barriers [Adapted from the National Strategy for Modernizing the Regulatory System for Biotechnology Products, Product of the Emerging Technologies Interagency Policy Coordination Committee’s Biotechnology Working Group (OSTP 2016)]. U.S. agencies are focused on delivering health and environmental protection based on the best available science; establishing transparent, coordinated, predictable, and efficient regulatory practices across agencies; and promoting public confidence in the oversight of the products of biotechnology through clear and transparent public engagement [Adapted from the Task Force on Agriculture and Rural Prosperity Report (USDA 2017)]. U.S. agencies that regulate the products of agricultural biotechnology discuss regulatory approaches presented during the June 2018 OECD Conference on Genome Editing Applications in Agriculture, focusing on plants developed using genome editing.

     

OECD Best Practice Guidelines for Biological Resource Centers. OECD, 2007, 115 p.

The book is the first published collection of guidelines regulating operational activities of biological resource centers, approved by representatives of the 30 member nations of the Organization for Economic Cooperation and Development (OECD). The published standards were prepared as a result of multiple discussions held by expert groups of the OECD members, together with key experts of partner nations, under the auspices of a specialist task force established by the OECD Working Party on Biotechnology.     

Arsenic concentrations in water and fish from Chautauqua Lake,New York

Synopsis Arsenic persists in Chautauqua Lake, New York waters 13 years after cessation of herbicide (sodium arsenite) application and continues to cycle within the lake. Arsenic concentrations in lake water ranged from 22.4–114.81 g l^–1, = 49.0 ag l^–1. Well water samples generally contained less than 10 g l^–1 arsenic. Arsenic concentrations in lake water exceeded U.S. Public Health Service recommended maximum concentrations (10 g l^–1) and many samples exceeded the maximum permissible limit (50 g l^–1). Fish accumulated arsenic from water but did not magnify it. Fish to water arsenic ratios ranged from 0.4–41.6. Black crappie (Pomoxis nigromaculatus) contained the highest arsenic concentrations (0.14–2.04 g g^–1 ), X = 0.7 g g^–1) while perch (Perca flavescens), muskellunge (Esox masquinongy) and largemouth bass (Micropterus salmoides) contained the lowest concentrations (0.02–0.13 g g^–1). Arsenic concentrations in fish do not appear to pose a health hazard for human consumers.     

The contribution of size-fractionated plankton to biomass and primary production of phytoplankton in saline–alkaline ponds

Primary productivity, biomass and chlorophyll-a of size fractionated phytoplankton (<0.22 m, <3 m, <8 m, <10 m, <40 m, <64 m, <112 m and <200 m) were estimated in 6 ponds and 5 experimental enclosures. The results showed that the planktonic algae less than 10 m are important in the biomass and production of phytoplankton in saline–alkaline ponds. The production of size fractionated phytoplankton corresponding to <112 m, <10 m and <3 m in saline–alkaline ponds were 10.5 ± 6.6 , 8.6 ± 5.4 and 0.33 ± 0.1 mgC l^–1 d^–1, respectively. Mean community respiration rate was 1.80 ± 0.73, 1.69 ± 0.90 and 1.38 ± 1.12 mgC l^–1 d^–1, respectively. The average production of phytoplankton corresponding to micro- (10–112 m), nano- (3–10 m) and pico- (<3 m) were 1.61, 8.30 and 0.33 mgC l^–1 d^–1, respectively. The ratio of those to the total phytoplankton production was 15%, 79% and 3%, respectively. The mean respiration rate of the different size groups was 0.11, 0.31 and 1.38 mgC l^–1 d^–1; the ratio of those to total respiration of phytoplankton was 6%, 17% and 77%, respectively. The production of size-fractionated phytoplankton corresponding to <200 m, <10 m and <3 m in enclosures was 2.19 ± 1.63, 2.08 ± 1.75 and 0.22 ± 0.08 mgC l^–1 d^-1, respectively. Mean community respiration rates were 1.25 ± 1.55, 1.17 ± 1.42 and 0.47 ± 0.32 mgC l^–1 d^–1, respectively. The average production of phytoplankton corresponding to micro- (10–200 m), nano- (3–10 m) and pico- (<3 m) plankton was 0.11, 1.86 and 0.22 mgC l^–1 d^–1, respectively. The ratio of those to the total production of phytoplankton was 5%, 85% and 10%, respectively. The mean respiration rate of different size groups were 0.08, 0.72 and 0.46 mgC l^–1 d^–1, the ratio of those to total respiration of phytoplankton was 6%, 57% and 37%, respectively. The concentrations of chlorophyll-a of the phytoplankton in the corresponding size of micro- (10–112 m), nano- (3–10 m) and pico- (<3 m) plankton in the experimental ponds were 19.3, 98.2 and 11. 9 g l^–1, respectively. The ratio of those to the total chlorophyll-a was 15%, 76% and 9%, respectively. The concentrations of chlorophyll-a of phytoplankton micro- (10–200 m), nano- (3–10 m) and pico- (<3 m) plankton in enclosures were 1.7, 34.3 and 3.0 g l^–1, respectively. The ratio of those to the total chlorophyll-a was 4%, 88% and 8%, respectively.     

Pesticides and heavy metals in Danish streambed sediment

The role of streambed sediment as a sink for pesticides and heavy metals was investigated in 30 Danish lowland streams. The investigated streams drain catchments varying in hydrology, topography, soil type and land use. The <250 m newly accumulated fraction of the uppermost 1–2 cm layer of streambed sediment was analysed for 19 old and modern pesticides and 9 heavy metals. DDE was present in the sediment of all the streams. Of the herbicides, fungicides and insecticides currently in use, the most frequently detected was diuron (50.0%), fenpropimorph (66.7%) and lambda-cyhalothrin (6.7%), respectively. The pesticides detected in the highest concentration were fenpropimorph (1700 ng g^–1), propiconazole (130 ng g^–1) and isoproturon (110 ng g^–1). The heavy metals are listed in order of increasing median concentration: Cd (0.80 g g^–1), Co (9.1 g g^–1), As (12.0 g g^–1), Ni (19.0 g g^–1), Cr (19.2 g g^–1), Pb (19.7 g g^–1), Cu (20.1 g g^–1), V (28.5 g g^–1), Zn (103 g g^–1). The average number of pesticides detected in the 27 streams draining predominantly agricultural catchments was (3.7±2.0) being higher (p=0.077) than in the three streams draining non-agricultural catchments (1.7±0.6). Pesticides were significantly related to catchment size, soil type and hydrological regime. Several heavy metals (Cr, Cu, Pb, V and Zn) were related to urban activity and soil type.     

Analysis of primary food sources and trophic relationships of aquatic animals in a mangrove-fringed estuary,Khung Krabaen Bay (Thailand) using dual stable isotope techniques

Stable carbon (¹³C) and nitrogen (¹⁵N) isotopes were used to elucidate primary food sources and trophic relationships of organisms in Khung Krabaen Bay and adjacent offshore waters. The three separate sampling sites were mangroves, inner bay and offshore. The ¹³C values of mangrove leaves were –28.2 to –29.4, seagrass –10.5, macroalgae –14.9 to –18.2, plankton –20.0 to –21.8, benthic detritus –15.1 to –26.3, invertebrates –16.5 to –26.0, and fishes –13.4 to –26.3. The ¹⁵N values of mangrove leaves were 4.3 to 5.7, seagrass 4.3, macroalgae 2.2 to 4.4, plankton 5.7 to 6.4 , benthic detritus 5.1 to 5.3, invertebrates 7.2 to 12.2 , and fishes 6.3 to 15.9. The primary producers had distinct ¹³C values. The ¹³C values of animals collected from mangroves were more negative than those of animals collected far from shore. The primary carbon sources that support food webs clearly depended on location. The contribution of mangroves to food webs was confined only to mangroves, but a mixture of macroalgae and plankton was a major carbon source for organisms in the inner bay area. Offshore organisms clearly derived their carbon through the planktonic food web. The ¹⁵N values of consumers were enriched by 3–4 relative to their diets. The ¹⁵N data suggests that some of aquatic animals had capacity to change their feeding habits according to places and availability of foods and as a result, individuals of the same species could be assigned to different trophic levels at different places.     

The solution structure of rat Aβ-(1–28) and its interaction with zinc ion: insights into the scarcity of amyloid deposition in aged rat brain

The amyloid -peptide (A) is a major component of insoluble amyloid deposits in Alzheimers disease, and the ability of the -peptide to exist in different conformations is dependent on residues 1–28 [-(1–28)]. However, different from humans, no A amyloid deposition has been found in aged rats brains. Studying the three-dimensional solution structure of rat A-(1–28) and the binding circumstance of Zn²⁺ is beneficial to a clear understanding of the potential role of Zn²⁺ in Alzheimer-associated neuropathogenesis and to suggest why there is no amyloid deposition in aged rats brains. Here we used nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of rat A-(1–28) and the binding constant of Zn²⁺ to rat A-(1–28). Our results suggest that (1) the three-dimensional solution structure of rat A-(1–28) is more stable than that of human A-(1–28) in DMSO-d₆ and that a helical region from Glu16 to Val24 exists in the rat A-(1–28); (2) the affinity of Zn²⁺ for rat A-(1–28) is lower than that for human A-(1–28) and the NMR data suggest that Arg13, His6, and His14 residues provide the primary binding sites for Zn²⁺; and (3) the proper binding of Zn²⁺ to rat A-(1–28) can induce the peptide to change to a more stable conformation.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0556-xAbbreviations A amyloid -peptide - AD Alzheimers disease - hA-(1–28) human A-(1–28) - rA-(1-28) rat A-(1–28) - REM restrained energy minimization     

Light response of D1 turnover and photosystem II efficiency in the seagrassZostera capricorni

Biochemical and biophysical parameters, including D1-protein turnover, chlorophyll fluorescence, oxygen evolution activity and zeaxanthin formation were measured in the marine seagrassZostera capricorni (Aschers) in response to limiting (100 mol·m^–2·^–1), saturating (350 mol·m^–2·s^–1) or photoinhibitory (1100 mol·m^–2·s^–1) irradiances. Synthesis of D1 was maximal at 350 mol·m^–2·s^–1 which was also the irradiance at which the rate of photosynthetic O₂ evolution was maximal. Degradation of D1 was saturated at 350 mol·m^–2·s^–1. The rate of D1 synthesis at 1100 mol·m^–2·s^–1 was very similar to that at 350 mol·m^–2·s^–1 for the first 90 min but then declined. At limiting or saturating irradiance little change was observed in the ratio of variable to maximal fluorescence (Fv/Fm) measured after dark adaptation of the leaves, while significant photoinhibition occurred at 1100 mol·m^–2·s^–1. The proportion of zeaxanthin in the total xanthophyll pool increased with increasing irradiance, indicative of the presence of a photoprotective xanthophyll cycle in this seagrass. These results are consistent with a high level of regulatory D1 turnover inZostera under non-photoinhibitory irradiance conditions, as has been found previously for terrestrial plants.We would like to thank Professor Peter Böger (Department of Plant Biochemistry, University of Konstanz, Germany) for the kind gift of D1 antibodies. This work was partly supported by a University of Queensland Enabling Grant to CC.     

Comparison of cyclopropene, 1-methylcyclopropene, and 3,3-dimethylcyclopropene as ethylene antagonists in plants

A comparison has been made of cyclopropene (CP), 1-methylcyclopropene (1-MCP), and 3,3-dimethyl-cyclopropene (3,3-DMCP) in their ability to protect plants against ethylene. In bananas, both CP and 1-MCP are effective around 0.5 nL L^–1, and 3,3-DMCP was effective at 1 L L^–1. Bananas treated with CP and 1-MCP again become sensitive to ethylene at 12 days and those treated with 3,3-DMCP at 7 days. Mature green tomatoes are protected by 5–7 nL L^–1 of 1-MPC for 8 days at 25°C and tomatoes treated with 3,3-DMCP at 5–10 L L^–1 are protected for 5 days. Carnation flowers are protected with CP or 1-MCP after exposure to 0.5 nL L^–1 for 24 hours and by 1 L L^–1 of 3,3-DMCP. The display life of Campanula flowers is increased from 3.3 to 5.4 days by 10 L L^–1 of 3,3-DMCP and to 9 days by 20 nL L^–1 of 1-MCP. Ethylene inhibition of pea seedlings is reduced by treatment with 1-MCP at 10 L L^–1 of ethylene but as ethylene is increased to 3000 L L^–1 growth inhibition increases. 3,3-DMCP treatment causes very little reduction of the ethylene effect even at very low concentrations.     

Influence of inorganic microelements on the production of camptothecin with suspension cultures of <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

The effects of eight microelements (I^–, BO₃ ^3–, MoO₄ ^2–, Co²⁺, Cu²⁺, Mn²⁺, Fe²⁺, Zn²⁺) on the biosynthesis of camptothecin and the growth of suspension cultures of Camptotheca acuminata were studied. The increase of I^– to 25 M l^–1, Cu²⁺ to 1 M l^–1, Co²⁺ to 2 M l^–1 and MoO₄ ^2– to 10 M l^–1 in Murashige and Skoog (MS) medium resulted in 1.66, 2.84, 2.53 and 2.04 times higher of camptothecin yield than that in standard MS medium respectively. Combined treatment of I^– (25 M l^–1), Cu²⁺ (1 M l^–1), Co²⁺ (2 M l^–1) and MoO₄ ^2– (10 M l^–1) lead to improve cell dry weight, camptothecin content, and camptothecin yield to 30.56 g l^–1, 0.0299%, and 9.15 mg l^–1, respectively, which were 20.2, 208.9 and 273.8% increment respectively when compared with those of control.     

Phosphorus composition of upland soils polluted by long-term atmospheric nitrogen deposition

Atmospheric N deposition can enhance biological P limitation in terrestrial ecosystems and increase the importance of organic P to plants and microorganisms. We used NaOH–EDTA extraction and solution ³¹P NMR spectroscopy to determine the P composition of soils in the Upper Teesdale National Nature Reserve, northern England, an upland region influenced by such deposition for at least 150 years. Three characteristic soil types were sampled on three occasions during an annual cycle: blanket peat (318 mg g^–1 total C, 607 g g^–1 total P, pH 3.9); acid organic soil under grassland (354 mg g^–1 total C, 1190 g g^–1 total P, pH 3.7); calcareous soil under grassland (140 mg g^–1 total C, 649 g g^–1 total P, pH 7.3). Between 58 and 99% of the total P in soil and litter layers was extracted by 0.25 M NaOH + 0.05 M EDTA. Extracts of all soils were dominated by organic P, mainly in the form of orthophosphate monoesters (43–69% extracted P). The two acidic soils also contained large proportions of orthophosphate diesters (6–19% extracted P) and phosphonates (7–16% extracted P), suggesting that these compounds become stabilised at low pH. However, a seasonal trend of increasing orthophosphate monoester-to-diester ratios, most evident in the calcareous grassland soil, indicated the preferential degradation of orthophosphate diesters during the growing season. Orthophosphate was the major inorganic P compound (17–34% extracted P), and all soils contained pyrophosphate (1–5% extracted P). However, orthophosphate determined in the NaOH–EDTA extracts by solution ³¹P NMR spectroscopy was substantially greater than that determined by molybdate colourimetry, suggesting that orthophosphate occurred in complexes with humic compounds that were not detected by conventional procedures. Our results suggest that organisms able to use recalcitrant soil organic P may have a competitive advantage in environments under enhanced atmospheric N deposition.     

Bioavailability of zinc in the sediment to the estuarine amphipod <Emphasis Type="Italic">Grandidierella</Emphasis><Emphasis Type="Italic">japonica</Emphasis>

The utility of interstitial water concentrations of metals and simultaneously extracted metals/acid-volatile sulfide differences (SEM–AVS) in two seasons were investigated to explain the biological availability of zinc in sediments to benthic organisms exposed in the laboratory. The amphipod Grandidierella japonica was exposed, in 10-day acute toxicity tests, to clean sediment spiked with zinc to obtain nominal treatments ranging from 0.25 to 74.4 mol g^–1 dry weight with respect to the molar difference between SEM_Zn and AVS. When the molar difference between SEM_Zn and AVS (i.e., SEM–AVS) was <0 mol g^–1, the concentration of zinc in the sediment interstitial water was low and few adverse effects were observed for any of the biological endpoints measured. Conversely, when SEM_Zn–AVS exceeded 0 mol g^–1, the concentration of zinc in the interstitial water and amphipod mortality increased. These data compare favorably with observations made in short-term exposures and thus support the use of AVS as a normalization phase for predicting toxicity in metal-contaminated sediments in different season.     

Is there a “dopaminergic glial cell”?

Intracellular cAMP increased 9-fold in cerebral hemisphere primary cultures after incubation with dopamine (10^–4M). The effect was dose- and time-dependent (10^–6 M-10^–4M; 2–10 minutes). It was mimicked, to some extent, by the partial agonist apomorphine (10^–5 M-10^–4 M) and antagonized by fluphenazine (10^–5 M-10^–4 M). The elevation of cAMP caused by dopamine was incompletely antagonized by propanolol (10^–5 M-10^–4 M), obviating an interaction with -adrenergic receptors. A -adrenergic effect was antagonized by propranolol but only slightly by fluphenazine. The effect of dopamine on cAMP-level was more pronounced in a subpopulation of the hemisphere culture, i.e. in astroglial cultures from the striatum, 12-fold compared with controls at 10^–4 M. No dopamine stimulated formation of cAMP was found in primary cultures from brain-stem. The results demonstrated some heterogeneity among astroglial cells. The cultures used contained mainly astroglial-like cells, as judged from immnohistochemical localization mainly astroglial-like cells, as judged from immunohistochemical localization of the glial specific proteins S 100 and GFA (-albumin). No mature neurons or oligodendroglial cells have so far been demonstrated in the cultures.     

Enzymatic transformation of desacetyl-lanatoside A into digitoxin by β-glucosidase action in cyclodextrin solutions

Summary The enzymatic transformation of desacetyl-lanatoside A (DLA) to its secondary glycoside, digitoxin, in solutions of -and -cyclodextrins is effected using of -glucosidase from barley. Due to the interaction of cyclodextrins (CyDs) with desacetyl-lanatoside A, an increase in solubility of the latter of 24.5 and 230 times was observed for -cyclodextrin and -cyclodextrin, respectively. Kinetic studies of the enzymatic transformation gave for -glucosidase the values K_M=3.3×10^–4 mol. dm^–3 and V_max=0.557 mol mg^–1 min^–1 when the substrate was the deacetyl-lanatoside A complex with -cyclodextrin, while in the case of the complex with -cyclodextrin these values were K_M=5.45×10^–4 mol dm^–3 and V_max=0.896 mol mg^–1 min^–1.     

Aggregated Beta Amyloid Peptide 1–40 Decreases Ca2+- and Cholinergic Receptor-Mediated Phosphoinositide Degradation by Alteration of Membrane and Cytosolic Phospholipase C in Brain Cortex

The effects of full-length amyloid protein, A (1–40), on phosphoinositide-specific phospholipase C (PLC) were investigated in synaptic plasma membranes (SPM) and cytosol prepared from the cerebral cortex of adult rats. Moreover, the role of A (1–40) on the activation of lipid peroxidation was evaluated. The activity of phospholipase C (PLC) acting on phosphatidylinositol (PI) and phosphatidylinositol-4, 5-bisphosphate (PIP₂) was determined using exogenous labeled substrates. The subcellular fractions were the source of enzyme(s). The radioactivity of lipid messengers derived from degradation of [¹⁴C- arachidonoyl] PI was also determined. The stable aggregated form of -amyloid peptide (1–40) at 25 M concentration exerted reproducible effects. The aggregated form of A (1–40) inhibited Ca²⁺-regulated PI and PIP₂ degradation by SPM and cytosolic enzymes. Aggregated A also decreased significantly the level of diacylglycerol, the product of PLC. This additionally supports the inhibitory effect of A on membrane-bound and cytosolic PLC. Moreover, A (1–40) significantly decreased the basal activity of the PIP₂-PLC in SPM and the enzyme activity regulated through cholinergic receptors. However, in spite of the lower enzyme activity, the percentage distribution of inositol (1,4,5) P₃ radioactivity (IP₃) in the total pool of inositol metabolites was not significantly changed. The aggregated neurotoxic fragment, A (25–35), mimicked the effect of full-length A (1–40). A (1–40) enhanced the level of malondialdehyde indicating an activation of free radical stimulated membrane lipid peroxidation that may be involved in alteration of phospholipase(s) activity. Our results indicated that aggregated A (1–40) alters Ca²⁺-dependent phosphoinositide degradation affecting synaptic plasma membrane and cytosolic phospholipase(s) activity. Moreover, this peptide significantly decreased the phosphoinositide-dependent signal transduction mediated by cholinergic receptors. The effect of aggregated A (1–40) is more pronounced than that of the neurotoxic fragment A (25–35). Our study suggests that the deposition of aggregated A may alter phosphoinositide signaling in brain.     

Macronutrient input from pollen in two regenerating pine stands in southeast Korea

This study examined macronutrient input from pollen in two naturally regenerating pine stands in southeast Korea. Durham gravity pollen collectors were used to measure pine pollen deposition and the macronutrients in the collected pine pollen were analyzed. In 1998, pine pollen deposition began just before 18 April and lasted for approximately 2weeks. Total pine pollen deposition differed between the two sampling sites; 27.5kgha^–1 was collected from the mature stand and 17.7kgha^–1 was collected from the young stand. The values for nutrient deposition from pine pollen are 549gha^–1 N, 78gha^–1 P, 240gha^–1 K, 45gha^–1 S and 22gha^–1 Mg at the mature stand and 353gha^–1 N, 51gha^–1 P, 151gha^–1 K, 27gha^–1 S and 14gha^–1 Mg at the young stand, suggesting that nutrients from pine pollen contribute to forest nutrient cycling. The pine pollen deposition values obtained from our study (17.7–27.5kg^–1ha^–1year^–1) are approximately 1/115–180-fold that of pine litterfall in Korea. If we take pollen nutrients into account, the contribution rate of pollen to the annual nutrient input is very high in our study (N 1/30, P 1/5, K 1/9 that of litterfall). Macronutrient deposition from pine pollen is concentrated temporally in spring. Although the annual contribution of nutrient mass by pollen is small compared to that of litterfall, the rapid turnover rate of pollen nutrients combined with episodic deposition suggests that pollen may play a disproportionate role in temperate pine forest nutrient cycling.     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy