Bacoside production by suspension cultures of Bacopa monnieri (L.) Pennell

Cell suspension cultures of Bacopa monnieri (L.) Pennell, grown in modified MS medium, grew some 5–6 fold over 40 days. Selected cell lines produced the important saponin, bacoside A, up to 1 g/100 g dry wt after this time.     

Exiguobacterium oxidotolerans, a halotolerant plant growth promoting rhizobacteria, improves yield and content of secondary metabolites in Bacopa monnieri (L.) Pennell under primary and secondary salt stress

Brahmi (Bacopa monnieri), an integral component of Indian Ayurvedic medicine system, is facing a threat of extinction owing to the depletion of its natural populations. The present study investigates the prospective of exploitation of halotolerant plant growth promoting rhizobacteria (PGPR) in utilising the salt stressed soils for cultivation of B. monnieri. The effects of two salt tolerant PGPR, Bacillus pumilus (STR2) and Exiguobacterium oxidotolerans (STR36) on the growth and content of bacoside-A, an important pharmaceutical compound in B. monnieri, were investigated under primary and secondary salinity conditions. The herb yields of un-inoculated plants decreased by 48 % under secondary salinization and 60 % under primary salinization than the non salinised plants. Among the rhizobacteria treated plants, E. oxidotolerans recorded 109 and 138 %, higher herb yield than non-inoculated plants subjected to primary and secondary salinity respectively. E. oxidotolerans inoculated plants recorded 36 and 76 % higher bacoside-A content under primary and secondary salinity respectively. Higher levels of proline content and considerably lower levels of lipid peroxidation were noticed when the plants were inoculated with PGPR under all salinity regimes. From the results of this investigation, it can be concluded that, the treatments with salt tolerant PGPR can be a useful strategy in the enhancement of biomass yield and saponin contents in B. monnieri, as besides being an eco-friendly approach; it can also be instrumental in cultivation of B. monnieri in salt stressed environments.     

Chitinolytic microbes confer Meloidogyne incognita resistance and augment secondary metabolites in Bacopa monnieri (L.) Pennell

Meloidogyne incognita (Kofoid and White) Chitwood a calamitous plant pathogen affects almost all the commercial crops, especially medicinal plants viz. Bacopa monnieri L. In this study, the efficacy of indigenous chitinolytic microbes against M. incognita in B. monnieri was evaluated under sterilised and natural soil conditions. The plant growth was observed for various parameters viz. nutrient uptake, phytochemical activities and secondary metabolites (bacoside). In the natural soil under green house conditions, the isolates significantly (P ≤ 0.05) reduced M. incognita infestation (1.38–2.78-folds) with significant increase in secondary metabolites (1.28–1.88-fold) over the untreated control. The potential strains were identified through 16S-rRNA as Streptomyces sp., Microbacterium arabinogalactanolyticum, Cellulosimicrobium cellulans, Actinomycetales bacterium, Chitiniphilus sp. and Flavobacterium johnsoniae. These findings thus highlight the potential of indigenous chitinolytic microbes that can be utilised for overall fitness of the plant along with the reduced root-knot nematode infestation in B. monnieri.     

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.     

Effect of CCC and glycine betaine on growth and growth-substance content of primary leaves of dwarf Frenchbean (Phaseolus vulgaris L.)

CCC (2-chloroethyltrimethylammonium chloride) decreased growth of primary leaves and stem internodes above the hypocotyl of dwarf French bean, probably because it decreased the amount of auxin produced by interaction between tryptophan and phenolic esters in the primary leaves. Growth of leaves was less affected than that of stems, and the gibberellin content of the primary leaves, previously shown to be associated with their expansion, was unaffected by CCC. CCC delayed death of the primary leaves, the breakdown of chlorophyll, and the increase in auxin associated with death of bean leaves. CCC had less effect on the growth of leaf discs, probably because they are not sites of growth-substance production. Glycine betaine, a quaternary ammonium compound similar to CCC, did not affect growth of bean plants, their metabolism of growth substances or the longevity of their primary leaves, but inhibited growth of leaf discs, probably directly.     

Direct somatic embryogenesis and encapsulation of somatic embryos for in vitro conservation of Bacopa monnieri (L.) Wettst

Direct somatic embryogenesis and shoot organogenesis were achieved from leaf explants excised from microshoots of Bacopa monnieri cultured on Murashige and Skoog medium containing N6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D). The maximum frequency of explants differentiated somatic embryos and shoot buds on MS medium supplemented with 12.5 µM BA and 1 µM 2,4-D. The frequency of explants differentiating somatic embryos decreased with increasing concentration of 2,4-D. Light and scanning electron microscopy revealed direct differentiation of somatic embryos and shoot buds from explants, and various developmental stages of the somatic embryos were observed. Somatic embryos and apical shoot tips were encapsulated in sodium alginate gel to produce synthetic seeds. The storage of synthetic seeds produced by encapsulation was studied at 4 and 25?°C (room temperature) for a period of 140 days. Encapsulated somatic embryos were found to retain viability after 140 days of storage at both temperatures, whereas encapsulated apical shoot buds failed to germinate even after 40 days when stored at 4?°C. The viability of synthetic seeds was higher when stored at 25?°C. All amplified markers scored by random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) were monomorphic for all the plants produced from synthetic seeds following different periods of storage, thus establishing the clonal fidelity of propagated plantlets.     

Influence of explant types,non-embryogenic synseed and reduced oxygen environment on in vitro conservation of Bacopa monnieri (L.) Wettst

In Vitro Cellular & Developmental Biology - Plant - The effect of using encapsulated shoot tips (ST) and nodal segment (NS) explants, in combination with mineral oil (MO) overlay, for in vitro...     

Effect of biologically synthesized silver nanoparticles on Bacopa monnieri (Linn.) Wettst. plant growth metabolism

In this present study, interactions of biologically synthesized silver nanoparticles on hydroponically grown Bacopa monnieri (Linn.) Wettst. plant growth metabolism were documented. Estimates of protein, carbohydrate, total phenols, in addition antioxidant enzymes, catalase and peroxidise were assayed in various parts of the plants grown in hydroponic solution. The silver nanoparticles used in this study were synthesized by treating AgNO₃ with aqueous leaves extracts of Acalypha indica Linn., a medicinal herb as a source of reductants. Enhanced peroxidase and catalase activity, simulated the stress conditions induced by the silver nitrate treatment. No severe toxic effects were observed in silver nanoparticles treated plants in the morphological studies under scanning electron microscopy (SEM) while structural aberrations were observed in the light microscopic evaluation of root and stem anatomy. Further, the uptake of silver in the root and stem tissues of B. monnieri (Linn.) Wettst. was confirmed using atomic absorption spectrophotometer (AAS).     

Effects of 24-epibrassinolide on seed germination, seedling growth, lipid peroxidation, proline content and antioxidative system of rice (Oryza sativa L.) under salinity stress

The effects of 24-epibrassinolide (24-epiBL) on seedling growth, antioxidative system, lipid peroxidation, proline and soluble protein content were investigated in seedlings of the salt-sensitive rice cultivar IR-28. Seedling growth of rice plants was improved by 24-epiBL treatment under salt stress conditions. When seedlings treated with 24-epiBL were subjected to 120 mM NaCl stress, the activities of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6) and glutathione reductase (EC 1.6.4.2) did not show significant difference, whereas the activity of ascorbate peroxidase (EC 1.11.1.11) significantly increased. Increased activity of peroxidase (EC 1.11.1.7) under NaCl stress showed remarkable decrease in the 24-epiBL+NaCl-applied group. Lipid peroxidation level significantly increased under salt stress but decreased with 24-epiBL application revealing that less oxidative damage occurred in this group (24-epiBL+NaCl). In addition, increased proline content in the NaCl-applied group was decreased by 24-epiBL application in the 24-epiBL+NaCl-applied group. Soluble protein content was increased by 24-epiBL application even under NaCl stress, being also higher than control conditions (no 24-epiBL or NaCl treatment). 24-epiBL treatment considerably alleviated oxidative damage that occurred under NaCl-stressed conditions and improved seedling growth in part under salt stress in sensitive IR-28 seedlings.     

Effect of salt stress on in vitro organogenesis from nodal explant of Limnophila aromatica (Lamk.) Merr. and Bacopa monnieri (L.) Wettst. and their physio-morphological and biochemical responses

Physiology and Molecular Biology of Plants - Salinity is one of the most severe abiotic stress factors that limit crop productivity by affecting the growth of plants. Therefore, it is significant...     

Effects of sucrose and pH levels on in vitro shoot regeneration from leaf explants of <Emphasis Type=Italic>Bacopa monnieri</Emphasis> and accumulation of bacoside A in regenerated shoots

Brahmi (Bacopa monnieri) is an important medicinal plant mainly used for the treatment of neurological disorders and depression. Recent investigations revealed that bacoside A is major chemical component shown to be responsible for memory facilitating action of brahmi. The current investigation was carried out to assess the potential for increasing biomass and the concentration of bacoside A in the in vitro regenerated shoots by varying sucrose and pH levels of shoot regeneration medium. The leaf explants were cultured on the Murashige and Skoog (MS) medium supplemented with 2 mg l⁻¹ kinetin (KN) and with varying concentrations of sucrose (0, 1, 2, 3, 4, 5 and 6% at pH 5.8) and pH (4.5, 5.0, 5.5, 6.0 and 6.5 with 2% sucrose) with the objective of verifying the effects of sucrose and pH level on shoot regeneration and to verify the accumulation of bacoside A in the regenerated shoots. The shoot biomass increased (150.50 ± 2.84 shoots per explant, fresh wt 6.31 ± 0.12 g and dry wt 250 ± 5.00 mg) on the medium supplemented with 2% sucrose and pH which was set at 4.5. The results of HPLC analysis indicate that increase in sucrose concentration (0, 1, 2, 3, 4, 5 and 6% at pH 5.8) lead to decrease in the bacoside A content (39.51, 22.43, 13.05, 12.17, 10.73, 9.56 and 8.93 mg g⁻¹ dry wt, respectively) in regenerated shoots. These findings provide evidence that stressful condition of inadequate supply of carbon elevated synthesis of bacoside A in brahmi shoots. However, 2% sucrose is found suitable for biomass accumulation. Therefore, medium supplemented with 2% sucrose and pH set at 4.5 was found suitable for both biomass (6.31 ± 0.12 g fresh wt and 250 ± 5.00 mg dry wt) and bacoside A accumulation (13.09 mg g⁻¹ dry wt).     

Effect of water stress on proline accumulation of genetically modified potatoes (Solanum tuberosum L.) generating fructans

This study aimed to investigate the effect of water stress on the accumulation of proline in the leaves of transgenic potato lines generating fructans. The results of the series of bifactorial experiments in 2002 and 2003 indicated an increase of the proline level in leaves of all potato lines examined under water deficit. In addition, an increase in proline concentration during plant development was observed. The proline content was related to leaf water potential and relative water content (RWC), which indicates that proline could be involved in osmoregulation of potato plants under the experimental conditions. Surprisingly, under water deficit, the proline level was lowest in most of the transgenic SST/FFT-lines, which generate fructan molecules with a high degree of polymerization. Therefore, a pleiotropic effect can not be excluded as the reason for the divergence in behavior of these transgenic lines. The present results suggest that the modification of carbohydrate metabolism, especially the high content of soluble carbohydrates, may affect water stress-induced proline accumulation.     

Correction to: Influence of explant types,non-embryogenic synseed and reduced oxygen environment on in vitro conservation of Bacopa monnieri (L.) Wettst

In Vitro Cellular & Developmental Biology - Plant - Readers should note the following error in the original version of this article:     

Calcium chloride and gibberellic acid protect linseed (Linum usitatissimum L.) from NaCl stress by inducing antioxidative defence system and osmoprotectant accumulation

Salinity stress affects many metabolic facets of plants and induces anatomical and morphological changes resulting in reduced growth and productivity. To overcome the damaging effects of salinity, different strategies of the application of nutrients with plant hormones are being adopted. The present study was carried out with an aim to find out whether application of calcium chloride (CaCl₂) and gibberellic acid (GA₃) could alleviate the detrimental effects of salinity stress on plant metabolism. Fifteen days old plants were supplied with (1) 0 mM NaCl + 0 mg CaCl₂ kg^?1 sand + 0 M GA₃ (control, T0); (2) 0 mM NaCl + 10 mg CaCl₂ kg^?1 sand + 0 M GA₃ (T1); (3) 0 mM NaCl + 0 mg CaCl₂ kg^?1 sand + 10^?6 M GA₃ (T2); (4) 150 mM NaCl + 0 mg CaCl₂ kg^?1 sand + 0 M GA₃ (T3); (5) 150 mM NaCl + 10 mg CaCl₂ kg^?1 sand + 0 M GA₃ (T4); (6) 150 mM NaCl + 0 mg CaCl₂ kg^?1 sand + 10^?6 M GA₃ (T5); (7) 150 mM NaCl + 10 mg CaCl₂ kg^?1 sand + 10^?6 M GA₃ (T6). To assess the response of the crop to NaCl, CaCl₂ and GA₃, plants were uprooted randomly at 60 days after sowing. The presence of NaCl in the growth medium decreased all the growth and physio-biochemical parameters, except electrolyte leakage, proline (Pro) and glycine betaine (GB) content, thiobarbituric acid reactive substances (TBARS), H₂O₂ content, activities of superoxide dismutase (SOD) and catalase (CAT) and leaf Na content, which exhibited an increase of 37.6, 29.3, 366.9, 107.5, 59.1, 17.1, 28.4 and 255.2%, respectively, compared to the control plants. However, application of CaCl₂ in combination with GA₃ appears to confer greater osmoprotection by the additive role with NaCl in Pro and GB accumulation. Although the activities of antioxidant enzymes (SOD, CAT and POX) were increased by salt stress, the combined application of CaCl₂ and GA₃ to salt-stressed plants further enhanced the activities of these enzymes by 25.1, 6.7 and 47.8%, respectively, compared to plants grown with NaCl alone. The present study showed that application of CaCl₂ and GA₃ alone as well as in combination mitigated the adverse effect of salinity, but combined application of these treatments proved more effective in alleviating the adverse effects of NaCl stress.     

Effect of nickel on antioxidative enzyme activities, proline and chlorophyll contents in wheat shoots

Effect of two Ni concentrations (10 and 200 μM) on growth, Ni accumulation, chlorophyll and proline contents, relative water content (RWC) as well as the activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and glutathione S-transferase (GST) were studied in shoots of wheat plants. Treatments caused a considerable accumulation of Ni in the shoots. However, exposure of plants to 10 μM Ni did not lead to significant alterations in shoot growth except for a slight increase in fresh mass. The other parameters studied were not affected by treatment of plants with 10 μM Ni. In contrast, 200 μM Ni caused inhibition of shoot growth, a decline in RWC and chlorophyll content, accumulation of proline and occurrence of visible symptoms of Ni toxicity. The activities of SOD and CAT decreased in response to 200 μM Ni. Conversely, several-fold enhancements of POD and GST activities were observed following the 3^rd day of 200 μM Ni treatment.     

Effect of short-term heat stress on growth, physiology and antioxidative defence system in wheat seedlings

An experiment was conducted to find out the effect of short-term heat stress on morpho-physiological characters and antioxidants in 10 diverse wheat genotypes. Seed were aseptically grown in test tubes containing filter paper whose lower half was dipped in one-fourth MS media. Heat stress conditions were created by exposing the seedlings at 45 °C for 2 h after 7 days of their germination. Measurements were taken after 3 days of treatment. Heat stress significantly reduced the shoot dry mass, root dry mass, shoot length and root length in all the genotypes. The chlorophyll content and membrane stability index decreased, whereas proline content increased in heat-treated plants. There was significant increase in the activity of catalase, guaiacol peroxidase and superoxide dismutase under stress conditions. The genotypic variations were also significant. On the basis of a coordinated simulation of all these parameters, wheat genotypes Raj 4037 and PBW 373 were identified as tolerant to high temperature stress. The study provides evidence that the tolerant genotypes were equipped with better management of physiological processes along with an efficient antioxidative defence system, sensitivity of which can be evaluated to a sufficient level of certainty at seedling stage.     

Biochemical responses to drought stress in mulberry (<Emphasis Type="Italic">Morus alba</Emphasis> L.): evaluation of proline,glycine betaine and abscisic acid accumulation in five cultivars

Five popularly grown mulberry cultivars (K-2, MR-2, TR-10, BC2-59 and S-13) were subjected to drought stress by withholding irrigation, to obtain leaf water potentials (Ψ_w) ranging from −0.75, −1.50 and −2.25 MPa. Accumulation of proline, glycine betaine and abscisic acid (ABA) were quantified in control and water stressed mulberry leaves. The activities of enzymes involved in proline accumulation including glutamate dehydrogenase (EC1.4.1.2-4), pyrroline-5-carboxylate synthetase (EC 1.2.1.41), pyrroline-5-carboxylate reductase (EC1.5.1.2), ornithine transaminase (EC 2.6.1.13) were significantly enhanced in the leaves of all the cultivars with decreasing leaf water potentials, while the activities of proline dehydrogenase (EC 1.5.1.2) were reduced with progressive increase in water stress. Accumulation of proline, glycine betaine and abscisic acid was relatively higher in S-13 and BC2-59 compared to K-2, MR-2 and TR-10 under water deficit conditions. Our results demonstrate that S-13 and BC2-59 have superior osmoprotectant mechanisms under water-limited growth regimes.     

Effect of sodium chloride on gas exchange, antioxidative defense mechanism and ion accumulation in different cultivars of Indian jujube (Ziziphus mauritiana L.)

An experiment was conducted to study the effect of NaCl (electric conductivity of 0, 4, 8, 12, and 16 dS m^?1) on growth, gas exchange parameters, water status, membrane injury, chlorophyll stability index and oxidative defense mechanisms in two cultivars (Gola and Umran) of Indian jujube (Ziziphus mauritiana). Results showed that the dry mass and leaf area reduced linearly with increasing levels of salinity. Net photosynthetic rate (P _N), transpiration (E), and stomatal conductance (g _s) were comparatively lower in Umran which further declined with salinity. Leaf relative water content, chlorophyll (Chl) stability and membrane stability also decreased significantly under salt stress, with higher magnitude in Umran. Superoxide dismutase (SOD), peroxidase (POX) and catalase (CAT) activities were higher in Gola whereas hydrogen peroxide (H₂O₂) accumulation and lipid peroxidation (MDA content) were higher in control as well as salttreated plants of Umran. The Na⁺ content was higher in the roots of Gola and in the leaves of Umran, resulting in high K⁺/Na⁺ ratio in Gola leaves. Thus it is suggested that salt tolerance mechanism is more efficiently operative in cultivar Gola owing to better management of growth, physiological attributes, antioxidative defense mechanism, and restricted translocation of Na⁺ from root to leaves along with larger accumulation of K⁺ in its leaves.     

Growth, proline accumulation and ion content in sodium chloride-stressed callus of date palm

Summary Growth and physiological responses of date palm. Phoenix dactylifera L. cv. Barhee, callus to salinity stress were examined. Callus induced from shoot tips of offshoots was cultured on Murashige and Skoog medium supplemented with NaCl at concentrations ranging from 0 to 225 mM, in consective increments of 25 mM. Data obtained after 6 wk of exposure to salt have shown a significant increase in callus proliferation in response to 25 mM NaCl the lowest level tested, beyond which callus weight decreased. At 125 mM NaCl and higher, callus growth was nearly completely inhibited. Physiological studies on callus exposed to salt stress have shown an increase in proline accumulation in response to increased salinity. Proline accumulation was correlated to callus growth inhibition. Furthermore, increasing the concentration of NaCl in the culture medium generally resulted in a steady increase in Na⁺ and reduction in K⁺ concentrations. However, at 25 mM NaCl, the only level at which callus growth was significantly enhanced, an increase in K⁺ content was noted, in comparison to the NaCl free control. In response to increasing external NaCl level, the Na⁺/K⁺ ratio increased The Na⁺/K⁺ ratio was positively correlated to proline accumulation and hence callus growth inhibition. This study provides, an understanding of the response of date palm callus to salinity, which is important for future studies aimed at developing strategies for selecting and characterizing somaclonal variants tolerant to salt stress.