Effect of activated charcoal and pruning of the taproot on the in vitro mycorrhization of Hevea brasiliensis Müll. Arg.

In vitro mycorrhization of Hevea brasiliensis under autotrophic culture conditions is a promising methodology to produce plantlets adapted to overcome stresses during acclimatization. However, to succeed in the in vitro production of mycorrhizal plantlets, root production and subsequent colonization by the mycorrhizal fungus need to be optimized. Plantlets of H. brasiliensis clone PB 260 were grown in contact with the extraradical mycelium network of the arbuscular mycorrhizal fungus Rhizophagus irregularis MUCL 41833. Addition of activated charcoal to the medium and pruning of the taproot were evaluated for their effects on root growth and colonization. None of the treatments stimulated the early formation of new roots. However, total root length, total root colonization, and production of arbuscules and intraradical spores/vesicles were significantly higher in plantlets grown in the presence of activated charcoal (especially after 13 wk of culture). In contrast, total root colonization was significantly lower in the pruned plantlets, while total root length and arbuscule formation were not affected. None of the treatments affected activities of succinate dehydrogenase and alkaline phosphatase measured in the extraradical mycelium of the fungus. It appeared that the addition of activated charcoal to the culture medium favored root growth and mycorrhization of rubber plantlets under in vitro culture conditions, while taproot pruning did not favor these parameters.     

In vitro mycorrhization of the rubber tree Hevea brasiliensis Müll Arg

In vitro cultivation systems of arbuscular mycorrhizal fungi are useful tools to study the interaction between plants and their fungal symbiont, and also to develop new biotechnologies. Plantlets of the latex-producing species Hevea brasiliensis clone PB 260 were grown in a dense extraradical mycelium network of the arbuscular mycorrhizal fungus Rhizophagus irregularis MUCL 41833 developed from a mycelium donor plant (Medicago truncatula A17). The factors indole-3-butyric acid (IBA), 2-morpholineoethanesulfonic acid monohydrate (MES) buffer, and carbon dioxide (CO₂) were tested on root development and colonization by the fungus. No colonization was observed in the presence of plantlets pre-treated with IBA. The highest levels of root colonization were obtained when plantlets were mycorrhized under a high CO₂ concentration (1,000 μmol?mol^?1) with MES (10 mM) added to the growth medium. Widespread root colonization (with presence of arbuscules, intraradical mycelium, and spores/vesicles) was predominantly observed in newly produced roots. Therefore, it appears essential to improve root initiation and growth for improving in vitro mycorrhization of H. brasiliensis. We demonstrated the potential of the “mycelium donor plant” in vitro culture system to produce colonized H. brasiliensis plantlets before their transfer to ex vitro conditions.     

Efficient Agrobacterium tumefaciens-mediated transformation of embryogenic calli and regeneration of Hevea brasiliensis Müll Arg. plants

An efficient procedure for producing transgenic Hevea brasiliensis callus and plant lines from clone PB 260 was established with Agrobacterium tumefaciens using strain EHA105 harbouring the vector pCAMBIA2301. Transformation capacity and competence of the embryogenic calli were improved after two cycles of cryopreservation. When the cocultivation temperature was reduced from 27 to 20°C, the duration of this phase could be increased up to 7 days, promoting an increase in GUS activity. These transformation conditions led to the isolation of 24 callus lines resistant to paromomycin, which is used as a selection agent. Nineteen of these lines revealed the existence of one to four copies of T-DNA by Southern-blot analysis. Nine of them were transferred for regeneration by somatic embryogenesis. Three hundred seventy-four transgenic plants have thus been generated from six independent lines bearing 1, 2 or 3 copies of T-DNA. The efficiency and reproducibility of this method means that functional characterization of genes involved in natural rubber production can be envisaged.     

Improvement of somatic embryogenesis in Hevea brasiliensis (Müll. Arg.) using the temporary immersion technique

Summary A culture procedure using temporary immersion in a liquid medium was tested for somatic embryogenesis of Hevea brasiliensis (Müll. Arg.). Embryogenic callus was placed under regeneration conditions, either on a gelled medium (Phytagel, Sigma, St. Louis, MO) or in a container designed for temporary immersion. The latter technique has some advantages over the use of a gelled medium during both the early steps of somatic embryogenesis, i.e., embryo development, and later on, i.e., during maturation, desiccation and germination. Somatic embryo production in a liquid medium was three to four times greater than on a semi-solid medium: 400 embryos/g fresh weight under the best embryogenesis induction conditions. Somatic embryogenesis had to be initiated on a gelled medium before the embryogenic callus was transferred to temporary immersion, and the amounts of 3,4- dichlorophenoxyacetic acid and N⁶-benzyladenine had to be reduced. Temporary immersion resulted in substantially more consistent, synchronized somatic embryo development, reducing the number of abnormal embryos by half and stimulating germination. All of the late events could be carried out in the temporary immersion container. Effective drying conditions were achieved after 12 wk without immersion and without selection of the embryos. Temporary immersion during germination greatly stimulated root development (+60%) and epicotyl emergency (+35%), combined with increased synchronization and a substantially reduced workload.     

Differential carbohydrate metabolism conducts morphogenesis in embryogenic callus of Hevea brasiliensis (Müll. Arg.)

Somatic embryogenesis in Hevea is stimulated when the embryogenesis induction medium contains maltose, rather than glucose, fructose, or sucrose, in equimolarity (Blanc et al., 1999). Kinetic analyses were carried out on various physiological and biochemical indicators over the 8 weeks that the induction phase then expression of somatic embryogenesis can take. Embryogenesis induction in the presence of glucose, fructose or sucrose revealed strong callus growth in the first 3-4 weeks, associated with a high intra- and extracellular hexose content, a high starch content and a substantial decline in protein synthesis. In the presence of maltose, callus growth was slow and only half that seen with sucrose. This morphogenetic behaviour is associated with a drop in endogenous hexose and starch contents, and an increase in protein synthesis in the first three weeks of culture. The induction of embryogenesis in the presence of maltose was uniform and twice as fast as with sucrose supply. At the end of culture, peroxidase activity, antioxidant and membrane protein contents increased in these calluses; these characteristics may be associated with somatic embryo organization and with the maintenance of effective membrane integrity within a nutrient environment that has become limiting. These new results tally with data in the literature on the roles of sugars, and provide some precise information with regard to the 'carbohydrate deficit' hypothesis usually put forward to explain maltose action. An analysis of these results led to the hypothesis that regulation of endogenous hexose contents at a low level, through slow maltose hydrolysis, was a key element of the biochemical signal leading this callus towards somatic embryogenesis.     

Clonal Differences in Photosynthesis in Hevea Brasiliensis Müll. Arg.

The objective of the present investigation was to examine the extent of variations in single leaf net photosynthetic rate (P_N) and its relative dependence on stomatal conductance (g_s) and the mesophyll capacity to fix carbon in 12 clones of the natural rubber plant. There were significant variations in P_N measured at low and saturating photon flux density (PFD); the extent of variation was larger at low than at saturating PFD. The compensation irradiance (CI) and apparent quantum yield of CO₂ assimilation (φ_c) calculated from the P_N/PFD response curves showed significant variations among the clones. P_N at low irradiance was positively correlated with φ_c. Thus a clone with large P_N at low irradiance, high φ_c, and low CI may tolerate shade better and thus produce a high tree stand per hectare. A strong positive correlation existed between P_N saturated with radiant energy (P_sat) and carboxylation efficiency (CE) estimated from the response curves of P_N on intercellular CO₂ concentration (C_i), but g_s showed a poor correlation with P_sat High CO₂ compensation concentration (Γ) led to low CE in Hevea clones. A clone with large P_sat, high CE, low g_s, and low Γ is the one in which photosynthesis is more dependent on the mesophyll factors than stomata. Such a clone may produce relatively high biomass and maintain high water use efficiency. This revised version was published online in September 2006 with corrections to the Cover Date.     

Purification and characterization of NAD(P)H quinone reductase from the latex of Hevea brasiliensis Müll.-Arg. (Euphorbiaceae)

NAD(P)H quinone reductase [NAD(P)H-QR] present in the latex of Hevea brasiliensis Müll.-Arg. (Euphorbiaceae) was purified to homogeniety from the B-serum fraction obtained by freeze-thawing of the bottom fraction of ultracentrifuged fresh latex. The purification protocol involved acetone fractionation, heat treatment, ion exchange chromatography and affinity chromatography. The M(r) determined by SDS-PAGE for the protein subunit was 21 kDa, and the molecular mass of the native enzyme estimated by gel filtration was 83 kDa, indicating that the native enzyme is a homotetramer. The enzyme showed pH stability over a range of 6 to at least 10 (with an optimum at pH 8) and thermal stability up to 80 degrees C. High NAD(P)H-QR activity (70%) was still retained after 10 h of preincubation at 80 degrees C. A comparable substrate specificity for this enzyme was observed among menadione, p-benzoquinone, juglone, and plumbagin, with only duroquinone generating a lower activity. Positive correlations between latex NAD(P)H-QR activity and rubber yield per tapping [fresh latex (r=0.89, P<0.01), dry rubber (r=0.81, P<0.01)] together with flow time (r=0.85, P<0.01) indicated that enzyme activity could possibly be used as a marker to predict the yield potential of selected clones.     

Photosynthetic capacity and temperature responses of photosynthesis of rubber trees (Hevea brasiliensis Müll. Arg.) acclimate to changes in ambient temperatures

The aim of this study was to assess the temperature response of photosynthesis in rubber trees (Hevea brasiliensis Müll. Arg.) to provide data for process-based growth modeling, and to test whether photosynthetic capacity and temperature response of photosynthesis acclimates to changes in ambient temperature. Net CO₂ assimilation rate (A) was measured in rubber saplings grown in a nursery or in growth chambers at 18 and 28°C. The temperature response of A was measured from 9 to 45°C and the data were fitted to an empirical model. Photosynthetic capacity (maximal carboxylation rate, V _cmax, and maximal light driven electron flux, J _max) of plants acclimated to 18 and 28°C were estimated by fitting a biochemical photosynthesis model to the CO₂ response curves (A–C _i curves) at six temperatures: 15, 22, 28, 32, 36 and 40°C. The optimal temperature for A (T _opt) was much lower in plants grown at 18°C compared to 28°C and nursery. Net CO₂ assimilation rate at optimal temperature (A _opt), V _cmax and J _max at a reference temperature of 25°C (V _cmax25 and J _max25) as well as activation energy of V _cmax and J _max (E _aV and E _aJ) decreased in individuals acclimated to 18°C. The optimal temperature for V _cmax and J _max could not be clearly defined from our response curves, as they always were above 36°C and not far from 40°C. The ratio J _max25/V _cmax25 was larger in plants acclimated to 18°C. Less nitrogen was present and photosynthetic nitrogen use efficiency (V _cmax25/N _a) was smaller in leaves acclimated to 18°C. These results indicate that rubber saplings acclimated their photosynthetic characteristics in response to growth temperature, and that higher temperatures resulted in an enhanced photosynthetic capacity in the leaves, as well as larger activation energy for photosynthesis.     

Effects of Phyllanthus sellowianus Müll Arg. Extracts on the Rheological Properties of Human Erythrocytes

Phyllanthus sellowianus extracts have been used in Argentina since colonial times in the treatment of diabetes. The in vitro biorheological and hemoagglutinant action of different extracts of P. sellowianus bark on human erythrocytes (RBC) were studied. RBCs were incubated in vitro with four aqueous extracts: Maceration; Controlled Digestion (PD); Decoction; and Infusion. Biorheological parameters (deformability, membrane surface viscosity, elastic modulus, and dynamic viscolelasticity) were determined with an Erythrodeformeter, and erythrocyte adhesion was characterized by image digital analysis. Immunohematological assays in RBC incubated with all the extracts showed large globular aggregates and agglutination in human ABO blood group system. Isolated cell coefficient showed the increase of cell adhesion. Aggregated shape parameters were significantly higher than normal and they changed with the concentration, particularly of PD extracts. Rheological results showed that the extract biorheological action varies with the temperature used in the extract preparations. The results obtained are useful to study the action mechanism of extracts from P. sellowianus bark in order to evaluate its use as therapeutic agent in diabetes. Immunohematological Tests using ABO system showed its agglutinant power, which is of special interest in Immunohematology to be used as hemoclassifier.     

Solenostoma oblongifolium (K. Müll.) K. Müll. in Scotland—new to the British Isles

Abstract

To determine the responses of cryptogams to the variations in temperature, pH, and humidity, we studied the vegetation of Thermal Meadow, Hotsprings Island, Queen Charlotte Islands, B.C., Canada. Only seven cryptogamic taxa were present in the samples: five mosses, one liverwort, and one lichen. Humidity limited the distribution, with Racomitrium lanuginosum and Cladina ciliata preferring the warmer, drier sites and Pleurozium schreberi and Dicranum scoparium preferring cooler, more moist sites. Polytrichum formosum was the most frequent taxon, occurring under both cool, moist conditions and warm, dry conditions. Aulacomnium palustre occurred in the mid temperature range mostly where humidity was fairly high. The pH ranged 6.8–7.2, providing insufficient variation for us to delimit species preferences.     

The green fluorescent protein as an efficient selection marker for Agrobacterium tumefaciens-mediated transformation in Hevea brasiliensis (Müll. Arg)

An efficient genetic transformation procedure using a recombinant green fluorescent protein (GFP) has been developed in Hevea brasiliensis clone PB260. Transformation experiments have been performed using an Agrobacterium tumefaciens binary vector harbouring both uidA and S65T-GFP reporter genes in order to compare selection methods using glucuronidase assay (GUS activity) and paromomycin resistance, GFP activity and paromomycin resistance, or GFP activity only. At transient level, the number of spots showing GUS or GFP activities was similar for 4 and 5 days after coculture. After selection, stable transformation events were observed and led to the establishment of transgenic callus lines. A higher number of lines were generated with GFP selection compared to the GUS one. GFP selection is less time-consuming in terms of callus subculturing, and offers the possibility of producing antibiotic resistance marker-free transgenic plants.     

Natural rubber,Hevea brasiliensis (Willd. ex A. Juss.) Müll. Arg, germplasm collection in the Amazon Basin, Brazil: A retrospective

Hevea brasiliensis (Willd. ex A. Juss.) Müll. Arg., whose center of diversity is the Amazon basin, is the major source of commercial rubber in the world. In the 1970s, the need for new Hevea germplasm arose from the realization that there was a gradual erosion of the genetic variability of the rubber clones in many natural rubber plantations. This erosion occurred because most of the clones in cultivation were derived from the few surviving seeds collected in 1876 by Henry Wickham. Therefore, to broaden the genetic base of the major producers of natural rubber, it became necessary to undertake an expedition to the Amazon basin to obtain materials for the ‘gene pool’ in these populations. This expedition, called “Germplasm 81,” was organized in four stages. Initially, there was a 1976 seminar organized by the International Rubber Research and Development Board (IRRDB); then, in 1977, a Hevea workshop took place, also organized by the IRRDB. A preliminary mission by IRRDB officials to several countries in South America and the Caribbean followed in 1978. Finally, the Germplasm exercise by scientists from IRRDB countries (Cote d’lvoire, Indonesia, Malaysia, Nigeria, and Thailand), China, and Brazil took place from January to April of 1981. A ‘dummy run’ to test the viability of seeds from Manaus, Brazil, was undertaken in 1979. The Germplasm collection covered three Brazilian states: Acre, Mato Grosso, and Rondônia. Collection teams traveled into the remotest parts of the Amazon rain forests looking for high-yielding and disease-resistant trees from which to collect seeds, budwood, and seedlings growing around the trees. Three teams collected a total of 64,256 seeds, 1413 meters of budwood, and 1160 seedlings. Following agreements with the Brazilian government, collected seeds were divided on a 50-50 basis between IRRDB and Brazil Materials for Brazil were retained in Manaus, while those for the IRRDB were initially sent to Britain for phytosanitary treatment and then to Guadeloupe for further quarantine in 1982, before being sent to Asian and African reception centers. Scientists who participated in the exercise had to undergo quarantine in a temperate country for at least one week before returning to their research institutions. This quarantine was an attempt to avoid the spread of the deadly South American leaf blight (SALB) disease. This disease has prevented Brazil from becoming a major world producer of natural rubber, despite attempts made by Goodyear and Firestone Rubber Companies, as well as Henry Ford in the early 1900s     

An improved protocol for micropropagation of Mallotus repandus (Willd.) Müll.Arg.

Node and internode explants of Mallotus repandus were precultured on basal medium (BM: Murashige and Skoog (MS) medium with 3% sucrose and 0.55% Agargel) for 0–18 d before culture on shoot induction Medium (SIM: BM added with 4.44 μM of benzylaminopurine) for 4 wk. The cultures were subsequently transferred to BM for 4 wk for shoot elongation. Node explants precultured on BM for 14 d before incubation on SIM were at an optimum for shoot regeneration with the response rate of 95%, compared to a 21% response for the control without preculture. Internode explants precultured on BM for 16 d responded with an optimal shoot formation response rate of 69%, whereas the control response rate was 6%. The maximum shoot regeneration rates were 3.1 ± 0.3 and 2.7 ± 0.4 shoots/responding explant in node and internode explants, respectively. This study demonstrates for the first time that shoot organogenesis can be induced from internode explants of M. repandus. Furthermore, the results suggest that the explants need to acquire competence before shoot organogenesis. Rooting was obtained by incubation of regenerated shoots on half-strength MS with 10.74 μM of 1-naphthylacetic acid for a week before culture on half-strength MS for 4 wk. Regenerated plants were successfully transferred to soil.     

Use of Arbuscular Mycorrhiza and Organic Amendments to Enhance Growth of Macaranga peltata (Roxb.) Müll. Arg. in Iron Ore Mine Wastelands

Macaranga peltata (Roxb.) Mull. Arg. is a disturbance tolerant plant species with potential in mine wasteland reclamation. Our study aims at studying the phyto-extraction potential of M. peltata and determining plant-soil interaction factors effecting plant growth in iron ore mine spoils. Plants were grown in pure mine spoil and spoil amended with Farm Yard Manure (FYM) and Vermicompost (VC) along with arbuscular mycorrhizal (AM) species Rhizophagus irregularis. Pure and amended mine spoils were evaluated for nutrient status. Plant growth parameters and foliar nutrient contents were determined at the end of one year. FYM amendment in spoil significantly increased plant biomass compared to pure mine spoil and VC amended spoil. Foliar Fe accumulation was recorded highest (594.67μg/g) in pure spoil with no mortality but considerably affecting plant growth, thus proving to exhibit phyto-extraction potential. FYM and VC amendments reduced AM colonization (30.4% and 37% resp.) and plants showed a negative mycorrhizal dependency (–30.35 and –39.83 resp.). Soil pH and P levels and, foliar Fe accumulation are major factors determining plant growth in spoil. FYM amendment was found to be superior to VC as a spoil amendment for hastening plant growth and establishment in iron ore mine spoil     

The production of β-carotene inMortierella ramanniana var.ramanniana M29: the effect of changes in the environment upon growth and pigmentation

The production of -carotene in a strain ofMortierella ramanniana var.ramanniana (M29) has been shown to have many features characteristic of secondary biosynthetic processes. It is formed at the end of the primary growth phase in amounts greater than those reported inPhycomyces blakesleeanus. It has no apparent function in primary growth. The production has an optimum temperature lower than that of the primary growth phase. The amount of pigment formed is greatest with those carbon sources which do not support maximum growth within a given incubation period. Changes in the initial carbon, nitrogen, thiamine, phosphate, magnesium, trace elements, hydrogen ion concentration or oxygen tension of the medium did not abolish or delay the production of the pigment. It is suggested that the production of -carotene in this strain is an example of secondary metabolite production and that a mutation could be responsible for the production in this one strain.The technical assistance of Mr. D. J. Trott is gratefully acknowledged.     

Temperature effects on energy metabolism in banded and unbanded morphs of the snail Cepaea hortensis Müll

Summary Energy metabolism (oxygen consumption) in acclimated juvenile yellow banded and unbanded Cepaea hortensis Müll. was measured at 5°, 10°, 15°, 20°, and 25°C with Englemann respirometers. Metabolic rates were also measured after abrupt temperature changes from 5° to 20° and from 25° to 10°C.Energy metabolism in the two morphs was significantly different at 5° and 20°C. The metabolism/temperature curve in the banded morph was relatively flat with reduced regulating power at 20°C. The yellow unbanded form had a lower metabolism at 5°C, but between 15° and 25°C the curve was flat and indicated good regulating ability. A sudden decrease in experimental temperature gave significant response in both morphs. The effect of abrupt temperature increase was significant only in unbanded snails. Banded and unbanded snails thus displayed different adaptation strategies.Differential heating because of different pigmentation is discussed. The increase in metabolic rate with increased temperature was highest in the yellow unbanded morph. The effects were small and probably overridden by the influences of acclimation and behavior.The use of energetic parameters in ecological genetics is considered. It is suggested that the cohort producing most progeny with lowest energetic cost per energy unit of offspring is the better adapted to the local environment.