In vitro conservation of Bacopa monnieri (L.) using mineral oil

Bacopa monnieri (L.) Wettst., a traditional Indian medicinal plant with high commercial potential, is used as a potent nervine tonic. A slow growth protocol was developed for medium-term conservation using mineral oil (MO) overlay. Nodal segments of B. monnieri (two genotypes; IC249250, IC468878) were conserved using MO for 24?months. Single node explants were implanted on MS medium supplemented with 0.2?mg?l^?1 BA and were covered with MO. Subculture duration could be significantly enhanced from 6 to 24?months, on the above medium. Normal plants regenerated from conserved cultures were successfully established in soil. On the basis of 20 random amplified polymorphic DNA and 5 inter-simple sequence repeat primers analyses and bacoside A content using HPLC, no significant reproducible variation was observed between the controls and in vitro-conserved plants. The results demonstrate the feasibility of using MO for medium-term conservation of B. monnieri germplasm without any adverse genetical and biochemical effects.     

Optimization of a <Emphasis Type="Italic">Bacopa monnieri</Emphasis>-based genetic transformation model for testing the expression efficiency of pathway gene constructs of medicinal crops

A fast regenerating Agrobacterium tumefaciens-mediated transformation protocol for Bacopa monnieri (L.) Wettst. was developed as a model system for heterologous expression of terpenoid indole alkaloid pathway genes from Catharanthus roseus (L.) G. Don. The direct regeneration of shoots from leaf explants co-cultured with A. tumefaciens resulted in the integration of a tryptophan decarboxylase (tdc) and strictosidine synthase (str) cassette (<hpt-<Tdc2-<Str-gus>) in the regenerated progeny. The highest transformation efficiency (83.88%) was achieved when leaf explants were infected on the adaxial laminar surface by manual pricking with 48- to 72-h-old suspensions (OD₆₀₀ = 0.5–0.6) of A. tumefaciens strain LBA1119 (carrying the binary vector pMOG22). The heterologous expression of tryptophan decarboxylase and strictosidine synthase genes that are otherwise not present in B. monnieri plants was confirmed through semi-quantitative PCR and metabolite quantification assays. The entire protocol duration from co-cultivation through regeneration of transgenic plants to their establishment in the glass house took 40–45 d. The developed B. monnieri model can be used to test expression cassettes carrying genes for plant secondary metabolic pathway engineering, especially those genes that are expressed in differentiated cell, tissue, or organs.     

Genetic and biochemical stability assessment of plants regenerated from cryopreserved shoot tips of a commercially valuable medicinal herb <Emphasis Type="Italic">Bacopa monnieri</Emphasis> (L.) Wettst

Shoot tips from four accessions (IC249250, IC 426442, IC 375976, and IC468878) of Bacopa monnieri (L.) Wettst., a commercially valuable memory revitalizing medicinal plant, were cryopreserved using a vitrification technique. Depending on the genotype, 0 to 20% plant regeneration without intermediary callus was achieved from cryopreserved shoot tips. Genetic stability of plants derived from cryopreserved shoot tips was assessed using biochemical and molecular markers. The regenerated plants from non-frozen controls and cryopreserved shoot tips exhibited morphological similarity to respective parental material when transferred to soil. On the basis of ten random amplified polymorphic DNA (RAPD) and bacoside A content using HPLC analysis, no significant reproducible variation was observed between the controls and in vitro-cryopreserved plants. Thus, after cryopreservation treatment, the regenerated plants exhibited molecular and biochemical genetic stability.     

Sulfur stress-induced antioxidative responses in leaves of Triticum aestivum L.

Antioxidative responses were investigated in leaves of wheat (Triticum aestivum L.) grown at varying S levels ranging from deficiency to excess (1, 2, 4, 6 and 8 mM S). Optimum yield was observed in plants supplied with 4 mM S. Wheat responded to S deficiency and excess supply by decreasing growth of root and shoot. Chlorosis in young leaves was observed after 15 days of deficient S supply. The biomass and concentration of photoassimilatory pigments decreased in plants grown at 1, 2, 6 and 8 mM S supply. The concentration of thiobarbituric acid reactive substances (TBARS), cysteine, nonprotein thiol and hydrogen peroxide (H₂O₂) increased in plants grown under S stress. Accumulation of TBARS and H₂O₂ in leaves indicated oxidative damage in S-deficient and S-excess plants. Deficient and excess levels of S showed an increase in the activities of antioxidative enzymes superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6), peroxidase (EC 1.11.1.7), ascorbate peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2).     

Mannityl opine accumulation and exudation by transgenic tobacco

Three genes from the T_R region of pTi15955 were introduced into tobacco (Nicotiana tabacum L.) to direct the synthesis of the mannityl opines from hexose sugars and glutamine or glutamate. Opines were present in all tissue types tested and accumulated to levels of 100 to 150 micrograms per milligram dry weight in root, stem, and leaf tissues. Opine-producing plants appeared normal with respect to morphology and development. Transgenic plants grown for 60 days under sterile autotrophic conditions produced up to 540 micrograms of the mannityl opines per milligrams dry weight of tissue as root exudates. Opines were also detected in leaf and seed washes from soil-grown plants.     

Physiological,elemental, and stable isotope responses of the organs of mungbean to reduced atmospheric pressure

The effects of hypobaric conditions on stable isotope and mineral element concentrations during the germination of mungbean [Vigna radiata (Linn.) Wilczek] were evaluated. Mungbean seeds were cultured in lower atmospheric pressure (60 kPa) and normal air pressure (101 kPa) conditions, respectively. Oxygen and carbon dioxide partial pressures were maintained at 21 and 0.04 kPa, respectively. At 60 kPa, the fresh weight (FW) and dry weight (DW) of plants significantly increased by 5.41 and 9.62%, respectively, compared to those at 101 kPa after culturing for 7 d. Twelve mineral elements were compared among three organs (leaf, stem, and root) from seedlings grown under hypobaric and normal atmospheric conditions. This showed that lower air pressure generally improved element accumulation in the plant. A significantly lower value of δ ¹³C was observed at 60 kPa compared to that at 101 kPa. In addition, a significant increase in δ ¹⁵N value was detected in three different organs of plants grown under 60 kPa. Our survey provides a foundation for future field and laboratory studies on the influence of air pressure on plants, particularly in terms of stable isotope and mineral elements.     

Aspects of nitrogen metabolism in the rice seedling

The effects of nitrogen source NO₃⁻ or NH₄⁺ on nitrogen metabolism during the first 2 weeks of germination of the rice seedling (Oryza sativa L., var. IR22) grown in nutrient solution containing 40 μg/ml N were studied. Total, soluble protein, and free amino N levels were higher in the NH₄⁺-grown seedling, particularly during the 1st week of germination. Asparagine accounted for most of the difference in free amino acid level, in both the root and the shoot. Nitrate and nitrite reductase activities were present mainly in the shoot and were higher in the NO₃⁻-grown seedling, whereas the activity of glutamate dehydrogenase and glutamine synthetase in the root tended to be lower than that of the NH₄⁺-grown seedling during the 1st week of germination. Glycolate oxidase and catalase activities were present mainly in the shoot. Maximum activity of the above five enzymes occurred 7 to 10 days after germination. Differences in the zymograms of nitrate reductase, glutamate dehydrogenase, and catalase were mainly between shoot and root and not from N source. Nitrite reductase bands were observed only in plants grown in plants grown in NO₃⁻.     

Effects of CO2 and sugars on photosynthesis and composition of avocado leaves grown in vitro

The effects of micropropagation conditions on avocado (Persea americana Mill.) have been measured in leaves and plants cultured in vitro. The consequences of the type and concentration of sugar in the medium and of carbon dioxide concentration in the atmosphere on the rates of photosynthesis and amounts of ribulose 1,5-biphosphate carboxylase-oxygenase (EC 4.1.1.39; Rubisco) and total soluble protein (TSP) were measured. At the highest sucrose supply (87.6 mM), Rubisco content was substantially decreased in leaves, and even more when elevated CO₂ (1 000 μmol·mol⁻¹) was supplied. Maximum photosynthetic rate (P_max) was significantly decreased when plants developed in high sucrose and elevated CO₂. However, Rubisco concentration was significantly greater when glucose was supplied at the same molar concentration or when the concentration of sucrose was small (14.6 mM), and no differences were observed due to the CO₂ concentration in the air in these treatments. The ratio of Rubisco to total soluble protein (Rubisco/TSP) was dramatically decreased in plants grown in the highest concentration of sucrose and with elevated CO₂. Leaf area and ratio of leaf fresh weight/(stem + root) fresh weight, were greater in plants grown with CO₂ enriched air. However, upon transplanting, survival was poorer in plants grown on low sucrose/high CO₂ compared to those grown on high sucrose/high CO₂.     

Acorn mass and seedling growth in Quercus rubra in response to elevated CO2

Abstract. In order to explore whether seed size affects plant response to elevated CO₂, plants grown from red oak (Quercus rubra L.) acorns were studied for differences in their first year response to CO₂ concentrations of 350 and 700 μl/l. Overall, at final harvest, total biomass of plants grown in elevated CO₂ were 47 % larger than that of plants grown in ambient CO₂. There were significant interactions between CO₂ treatments and initial acorn mass for total biomass, as well as for root, leaf, and stem biomass. Although total biomass increased with increasing initial acorn mass for both high and ambient CO₂ plants, high CO₂ plants exhibited a greater increase than ambient CO₂ plants, as indicated by a steeper slope in high CO₂ plants. However, CO₂ levels did not affect biomass partitioning traits, such as root/shoot ratio, leaf, stem, and root weight ratios, and leaf area ratio. These results suggest that variation in seed size or initial plant size can cause intraspecific variation in response to elevated CO₂.     

三种湿地植物的生长及根系溶解性有机碳分泌物研究

研究了美人蕉(Canna indica Linn.)、风车草(Cyperus flabelliformis Rottb.)和水鬼蕉(Hymenocallis littoralis (Jack) Salisb.)3种湿地植物在人工气候室水培条件下的根系溶解性有机碳分泌物分泌量及其与生长的关系.结果表明,风车草和美人蕉的植...     

The influence of mycorrhizal inoculation and paclobutrazol on water and nutritional status of Arbutus unedo L.

The purpose of this study was to analyze morphological and physiological aspects of Arbutus unedo L. plants treated with paclobutrazol (PAC), compounds characterized by their double activity as plant growth regulators and fungicides, and the ectomycorrhizal fungus Pisolithus tinctorius (Pers.) Coker and Couch, which forms a special type of mycorrhizal colonization called arbutoid mycorrhiza. Native A. unedo L. seedlings were grown in a greenhouse and subjected to four treatments for 4 months: 0 or 60 mg of PAC and inoculated or not with P. tinctorius (Pers.). The arbutoid mycorrhizal inoculation increased in plants treated with PAC. Paclobutrazol reduced shoot and root biomass, plant height, internode length, stem diameter, leaf area, total root length and number of tips. P. tinctorius increased plant height and had a beneficial effect on the root system (increasing root diameter and the number of tips). PAC treatment led to an increase in ion levels in the leaf tissue, while mycorrhizal inoculation induced lower K and higher P contents in the roots. Leaf water potentials (at predawn and at midday) increased with the combined treatment. The absence of water deficit conditions meant there was no osmotic adjustment. Higher photosynthesis (P_n) values were associated with higher stomatal conductance (g_s) values in the mycorrhizal plants, which influenced water uptake from the roots. However, g_s decreased in the PAC-treated plants, reducing photosynthesis and, as a consequence, growth. The higher hydraulic conductivity (L_p) in the plants treated with PAC may have induced a better water energy status and good water transport. The combined treatment produced beneficial effects in the plants, improving their water and nutritional status.     

Root restriction as a factor in photosynthetic acclimation of cotton seedlings grown in elevated carbon dioxide

Interactive effects of root restriction and atmospheric CO₂ enrichment on plant growth, photosynthetic capacity, and carbohydrate partitioning were studied in cotton seedlings (Gossypium hirsutum L.) grown for 28 days in three atmospheric CO₂ partial pressures (270, 350, and 650 microbars) and two pot sizes (0.38 and 1.75 liters). Some plants were transplanted from small pots into large pots after 20 days. Reduction of root biomass resulting from growth in small pots was accompanied by decreased shoot biomass and leaf area. When root growth was less restricted, plants exposed to higher CO₂ partial pressures produced more shoot and root biomass than plants exposed to lower levels of CO₂. In small pots, whole plant biomass and leaf area of plants grown in 270 and 350 microbars of CO₂ were not significantly different. Plants grown in small pots in 650 microbars of CO₂ produced greater total biomass than plants grown in 350 microbars, but the dry weight gain was found to be primarily an accumulation of leaf starch. Reduced photosynthetic capacity of plants grown at elevated levels of CO₂ was clearly associated with inadequate rooting volume. Reductions in net photosynthesis were not associated with decreased stomatal conductance. Reduced carboxylation efficiency in response to CO₂ enrichment occurred only when root growth was restricted suggesting that ribulose-1,5-bisphosphate carboxylase/oxygenase activity may be responsive to plant source-sink balance rather than to CO₂ concentration as a single factor. When root-restricted plants were transplanted into large pots, carboxylation efficiency and ribulose-1,5-bisphosphate regeneration capacity increased indicating that acclimation of photosynthesis was reversible. Reductions in photosynthetic capacity as root growth was progressively restricted suggest sink-limited feedback inhibition as a possible mechanism for regulating net photosynthesis of plants grown in elevated CO₂.     

The alleviation of zinc toxicity by silicon is related to zinc transport and antioxidative reactions in rice

The objective of this study is to elucidate the roles of silicon (Si) in enhancing tolerance to excess zinc (Zn) in two contrasting rice (Oryza sativa L.) cultivars: i.e. cv. TY-167 (Zn-resistant) and cv. FYY-326 (Zn-sensitive). Root morphology, antioxidant defense reactions and lipid peroxidation, and histochemical staining were examined in rice plants grown in the nutrient solutions with normal (0.15 μM) and high (2 mM) Zn supply, without or with 1.5 mM Si. Significant inhibitory effects of high Zn treatment on plant growth were observed. Total root length (TRL), total root surface area (TRSA) and total root tip amount (TRTA) of both cultivars were decreased significantly in plants treated with high Zn, whereas these root parameters were significantly increased when Zn-stressed plants were supplied with 1.5 mM Si. Supply of Si also significantly decreased Zn concentration in shoots of both cultivars, indicating lower root-to-shoot translocation of Zn. Moreover, superoxide dismutase (SOD), catalase (CAT), and asorbate peroxidase (APX) activities were increased, whereas malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) concentrations were decreased in Si-supplied plants of both Zn-sensitive and Zn-resistant rice cultivars exposed to Zn stress. These alleviative effects of Si, further confirmed by the histochemical staining methods, were more prominent in the Zn-resistant cultivar than in the Zn-sensitive one. Taken together, all these results suggest that Si-mediated alleviation of Zn toxicity is mainly attributed to Si-mediated antioxidant defense capacity and membrane integrity. The possible role of Si in reduction of root-to-shoot translocation of Zn can also be considered.     

5种红树科植物叶片的比较解剖及其生态适应研究

用石蜡切片法对红树（Rhizophora apiculata Bl.）、红海榄（R.stylosa Griff.）、秋茄（Kandelia candel(Linn.) Druce）、木榄（Bruguiera gymnorrhiza(Linn.) Savigny）和海莲（B.sexangula(Lour.) Poir.）等5种红树科植物的叶片进行解剖学的观察和研究,结果表明：5种红树科植物叶片的表皮均有厚的角质膜和下皮层,表皮和下皮层的细胞常含有单宁;上表皮无气孔器;栅栏组织多层;木质部非常发达。说明了红树科植物的叶片具有很强的耐旱能力;同属植物的叶片在解剖结构上有着相似的特征。     

Effects of root anaerobiosis on ethylene production, epinasty, and growth of tomato plants

Experiments were performed to determine the source(s) of ethylene-causing epinasty in flooded tomato plants (Lycopersicon esculentum Mill.). Simultaneous measurements were made of ethylene synthesized by the roots and shoots of tomato plants exposed to either aerobic or anaerobic atmospheres in the root zone. When the root zone was made anaerobic by a flowing stream of N₂ gas, petiole epinasty and accelerated ethylene synthesis by the shoots were observed. In soil-grown plants, ethylene synthesis by the root-soil complex increased under anaerobic conditions; but when grown in inert media under the same conditions, ethylene synthesis by roots remained constant or declined during the period of rapid epinastic growth by the petioles. Other characteristic symptoms of flooding, e.g. reduced growth and chlorosis, were also observed in plants with anaerobic roots. Pretreatment of plants with AgNO₃, an inhibitor of ethylene action, completely prevented epinasty, demonstrating that ethylene is the agent responsible for waterlogging symptoms. These results indicate that deprivation of O₂ to the roots is the primary effect of soil flooding, and that this is sufficient to cause increased ethylene synthesis in the shoot. The basis of the observed root-shoot communication is unknown, but root-synthesized hormones or specific ethylene-promoting factors may be involved.     

In Vitro Clonal Propagation and Medium Term Conservation of Brahmi [Bacopa monnieri (L) Wettst]

A protocol has been developed for in vitro clonal propagation leading to conservation of Bacopa monnieri (L) Wettst, a medicinal plant of high commercial potential with legendary reputation as a memory vitalizer. Single node explants when cultured on Murashige and Skoog’s medium supplemented with BA (0.2 mg l^-1), showed active shoot proliferation (22.2 shoots/ explant in 8 weeks) without callus formation. Rooting was achieved on the same medium. The generated shoots could also be conserved for 12 months with high survival rate (up to 100%). The regenerants upon transfer to soil showed no morphological variation as compared with the donor plants. The medium optimized in the present study was applied for culture establishment and conservation of a total of 15 Brahmi accessions procured from different regions.     

The manganese toxicity of cotton

Cotton plants (Gossypium hirsutum. Linn. var. Sankar 4) were grown at normal and toxic levels of substrate manganese, and the altered metabolism of manganese toxic plants was studied. The tissues of plants exposed to toxic levels of manganese had higher activities of peroxidase and polyphenol oxidase, and the activities of catalase, ascorbic acid oxidase, glutathione oxidase and cytochrome c oxidase were lowered. In addition, the high manganese tissue had lower contents of ATP and glutathione but higher amounts of ascorbic acid. The respiration of the partially expanded leaves and the growing tips of toxic plants were depressed when compared to that of the normal tissues. The metabolic changes of manganese toxicity of cotton are placed in the following order: accumulation of manganese in the leaf tissue; a rise in respiration; stimulation of polyphenol oxidase; the appearance of initial toxicity symptoms; the evolution of ethylene and stimulation of peroxidase; the presence of severe toxicity symptoms; the depression of terminal oxidases and respiration; abscission of the growing tip and proliferation of the stem tissue. The early stimulation of polyphenol oxidase may be used to detect potential manganese toxicity.     

Glycine-Glomus-Rhizobium Symbiosis: II. Antagonistic Effects between Mycorrhizal Colonization and Nodulation

Soybean (Glycine max [L.] Merr.) plants grown in pot cultures were inoculated with the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus mosseae (Nicol. & Gerd.) Gerd. and Trappe and Rhizobium japonicum strain 61A118 at planting (G₁R₁) or at 20 days (G₂₀R₂₀), or with one of the endophytes after the other has colonized the host root (G₁R₂₀, G₂₀R₁). Nodulated (PR₁) and VAM (G₁N) dipartite associations, or nonsymbiotic plants (PN) using nutrient solutions with N, P, or N + P concentrations providing endophyte-equivalent nutrient inputs were used as controls. The delayed tripartite associations received the appropriate N, P, or N + P amendment while one or both endophytes were absent during the first 20 days of growth. Prior inoculation with one endophyte significantly inhibited development of the other. Root hexose sugar concentrations were negatively correlated with VAM colonization (r = −0.89), nodule activity (r = −0.91), and root P content (r = −0.93). Nodule (r = 0.97) and root (r = 0.96) P content correlated positively with VAM colonization. Nodule weight or VAM-fungal biomass were significantly greater in associations grown with only one endophyte. Dry weights of the PN, G₁N, PR₁, and G₂₀R₂₀ plants were significantly greater than those of tripartite plants inoculated at planting with either or both endophytes. Interendophyte inhibition is attributed to competition for root carbohydrates, and this effect apparently also affects overall plant productivity. The objective of the study was to determine if the timing of endophyte introduction and establishment affected the development of the other symbiotic partners.     

Is it possible to manipulate root anchorage in young trees?

The optimal root system architecture for increased tree anchorage has not yet been determined and in particular, the role of the tap root remains elusive. In Maritime pine (Pinus pinaster Ait.), tap roots may play an important role in anchoring young trees, but in adult trees, their growth is often impeded by the presence of a hard pan layer in the soil and the tap root becomes a minor component of tree anchorage. To understand better the role of the tap root in young trees, we grew cuttings (no tap root present) and seedlings where the tap root had (?) or had not (+) been pruned, in the field for 7 years. The force (F) necessary to deflect the stem sideways was then measured and divided by stem cross-sectional area (CSA), giving a parameter analogous to stress during bending. Root systems were extracted and root architecture and wood mechanical properties (density and longitudinal modulus of elasticity, E _L ) determined. In seedlings (?) tap roots, new roots had regenerated where the tap root had been pruned, whereas in cuttings, one or two lateral roots had grown downwards and acted as tap roots. Cuttings had significantly less lateral roots than the other treatments, but those near the soil surface were 14% and 23% thicker than plants (+) and (?) tap roots, respectively. Cuttings were smaller than seedlings, but were not relatively less resistant to stem deflection, probably because the thicker lateral roots compensated for their lower number. Apart from stem volume which was greater in trees (+) tap roots, no significant differences with regard to size or any root system variable were found in plants (?) or (+) tap roots. In all treatments, lateral roots were structurally reinforced through extra growth along the direction of the prevailing wind, which also improved tap root anchorage. Predictors of log F/CSA differed depending on treatment: in trees (?) tap roots, a combination of the predictors stem taper and %volume allocated to deep roots was highly regressed with log F/CSA (R ² = 0.83), unlike plants (+) tap roots where the combined predictors of lateral root number and root depth were best regressed with log F/CSA (R ² = 0.80). In cuttings, no clear relationships between log F/CSA and any parameter could be found. Wood density and E _L did not differ between roots, but did diminish with increasing distance from the stem in lateral roots. E _L was significantly lower in lateral roots from cuttings. Results showed that nursery techniques influence plant development but that the architectural pattern of Maritime pine root systems is stable, developing a sinker root system even when grown from cuttings. Anchorage is affected but the consequences for the long-term are still not known. Numerical modelling may be the only viable method to investigate the function that each root plays in adult tree anchorage.     

Synthesis of silver nanoparticles using plant derived 4-N-methyl benzoic acid and evaluation of antimicrobial,antioxidant and antitumor activity

The present study is to investigate the antitumor, antioxidant and antibacterial potential of silver nanoparticles (Ag NPs) synthesized from a phenolic derivative 4-N-methyl benzoic acid, isolated from a medicinal plant (Memecylon umbellatum Burm F). The Bio-inspired nanoparticles (NPs) were analyzed by using UV–vis spectroscopy, FTIR, HRTEM, Zeta potential and XRD techniques. The UV–vis spectroscopy study at the band of 430 nm confirmed the nanoparticles formation. HRTEM report showed that the AgNPs synthesized were in the size range 7–23 nm. The harvested nanoparticles were subjected to anti-bacterial assay and a dose dependent inhibitory action was observed against the tested human pathogens. Among the tested bacteria, Acinetobacter baumannii was found to be highly sensitive to AgNPs (diameter of zone of inhibition was 31 mm). Further, the silver nanoparticles exhibited a good anti-tumor activity against the breast cancer cell line (MCF 7) with an IC₅₀ value of 42.19 µg/mL. As the present study confirmed a good antibacterial, antioxidant and antitumor activity in the nanoparticles synthesized using 4-N-methyl benzoic acid derived from a medicinal plant, the product can be further tested to formulate a good lead compound for biomedical applications.