Irradiance-driven 20-hydroxyecdysone production and morphophysiological changes in Pfaffia glomerata plants grown in vitro

Pfaffia glomerata possesses potential pharmacological and medicinal properties, mainly owing to the secondary metabolite 20-hydroxyecdysone (20E). Increasing production of biomass and 20E is important for industrial purposes. This study aimed to evaluate the influence of irradiance on plant morphology and production of 20E in P. glomerata grown in vitro. Nodal segments of accessions 22 and 43 (Ac22 and Ac43) were inoculated in culture medium containing MS salts and vitamins. Cultures were maintained at 25 ± 2 °C under a 16-h photoperiod and subjected to irradiance treatments of 65, 130, and 200 μmol m⁻² s⁻¹ by fluorescent lamps. After 30 days, growth parameters, pigment content, stomatal density, in vitro photosynthesis, metabolites content, and morphoanatomy were assessed. Notably, Ac22 plants exhibited 10-fold higher 20E production when cultivated at 200 μmol m⁻² s⁻¹ than at 65 μmol m⁻² s⁻¹, evidencing the importance of light quantity for the accumulation of this metabolite. 20E production was twice as high in Ac22 as in Ac43 plants although both accessions responded positively to higher irradiance. Growth under 200 μmol m⁻² s⁻¹ stimulated photosynthesis and consequent biomass accumulation, but lowered carotenoids and anthocyanins. Furthermore, increasing irradiance enhanced the number of palisade and spongy parenchyma cells, enhancing the overall growth of P. glomerata.

Graphical abstract

     

Micropropagation of Capparis decidua (Forsk.) Edgew. — a tree of arid horticulture

A method for micropropagation of mature trees of Capparis decidua was developed. Multiple shoots were obtained from nodal explants on Murashige and Skoog's (1962) medium+0.1mgl^–1 NAA+5.0mgl^–1BAP+additives (50mgl^–1 ascorbic acid and25 mgl^–1 each of adenine sulphate, L-arginine and citric acid) at 28 ± 2°C, 12 h/dphotoperiod and 35–40 mol m^-2s^–1 photon flux density. The shoots were multiplied by (i) subculture of nodal shoot segments onto MS +0.1 mgl^-–1 IAA+1.0mgl^–1 BAPH+additives, and (ii) repeated transfer of original explant onto MS+ 0.1mgl^–1 IAA+mg l^–1 BAP+additives, at intervals of 3 weeks. Sixty to 70% of the shoots rooted when pulse treated with 100 mg l^–1 IBA in half strength MS liquid medium for 4h, and then transferred onto hormone-free half-strength agar-gelled MS basal saltmedium. Incubation in dark at 33 ± 2°C for 6d favoured root induction. In vitro hardened plants were transferred to pots.Abbreviations IAA Indole-3-aceticacid - IBA Indole-3-butyric acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - Kn 6-furfurylaminopurine - 2-ip Isopentenyl adenine - B₅ Gamborg et al. (1968) medium - MS Murashige and Skoog's (1962) medium - WP Woody plant medium (Lloyd and McCown 1981)     

Synergistic effect of BAP and GA3 on in vitro flowering of Guizotia abyssinica Cass.-A multipurpose oil crop

Apical and axillary buds of Guizotia abyssinica Cass., isolated from seedlings raised in vitro, were cultured. High frequency of shoot regeneration was achieved on MS medium with BAP (1 mgl⁻¹). Effect of BAP, Kn and GA₃ applied successively in culture on shoot regeneration and flower bud formation has been studied. The shoots differentiated in cultures elongated on this medium. These rooted subsequently on half strength MS medium. The shoots flowered in vitro on MS medium with a combination of BAP (0.1mgl⁻¹) + GA₃ (0.1 mgl⁻¹). The plantlets thus formed were successfully hardened with 90 % survival.     

A submerged culture system for rapid micropropagation of the commercially important aquarium plant, ‘Amazon sword’ (Echinodorus ‘Indian Red’)

Echinodorus ‘Indian Red’ is an underwater plant, used worldwide for aquarium ornamentation. An efficient method for in vitro propagation and plantlet acclimatization of this popular aquarium plant was standardized. Surface-disinfected shoot-tips were cultured in submerged conditions in a solid–liquid bilayer medium, consisting of an upper, liquid layer (sterile distilled water) and a lower, solid layer Murashige and Skoog (MS) basal medium supplemented with 3.0% (w/v) sucrose, 0.8% (w/v) agar-agar, and plant growth regulators (PGRs) in different combinations and concentrations. The combination of 2.5 mg L⁻¹ 6-benzylaminopurine and 1.0 mg L⁻¹ α-naphthaleneacetic acid improved the multiplication rate to a maximum of 26.8 ± 0.51 shoots per explant after 60 d of culture. The number of multiplied shoots increased with each regeneration cycle, thus from only 26.8 ± 0.51 shoots per explant (first regeneration cycle), this number increased to 33.5 ± 0.58 (second regeneration cycle), and to 38.3 ± 0.62 for the third regeneration cycle with the same medium composition. The highest number of roots (8.3 ± 0.28) per shoot was induced in the presence of 1.0 mg L⁻¹ indole-3-butyric acid, but further growth of these roots was stunted. The best rooting was achieved on PGR-free ½-strength MS medium, where 6.1 ± 0.21 roots per shoot were induced with 5.8 ± 0.35 cm length after 30 d of culture. The regenerated plantlets were successfully acclimatized to submerged underwater conditions, with 100% survival rate. The present protocol is suitable for the commercial propagation of Echinodorus ‘Indian Red’ for aquarium-industries.

     

Factors affecting in vitro clonal propagation of Prosopis cineraria

Genotype, age of tree, nature of explant and size (length and diameter), season of explant collection, explant position on medium, plant growth regulators and certain additives (ascorbic and citric acids, adenine sulphate, L-arginine, glutamine and ammonium citrate), incubation conditions, and subculturing period greatly influenced the in vitro clonal propagation of P. cineraria. The maximum number of 10–12 shoots were induced from the nodal shoot segment from pruned thorny adult trees on Murashige and Skoog's (MS) medium containing 0.1 mgl^-1 indole- 3-acetic acid (IAA)+2.5 mgl^-1 benzylaminopurine (BAP)+additives. Higher temperature (31+-2°C) and mixed (fluorescent and incandescent) light of 50 mol m^-2 s^-1 photon flux density for 12 h per day photoperiod favoured shoot induction and subsequent growth. Explants from thornless trees produced 6–8 shoots per explant on MS medium containing 0.1 mgl^-1 IAA+5.0 mgl^-1 BAP + additives. Nodal shoot segments obtained from root and stump sprouts produced multiple shoots. Root segments differentiated into multiple shoots on MS medium containing 0.5 mgl^-1 indolebutyric acid (IBA)+2.5 mgl^-1 BAP.Differentiated shoots multiplied best on MS medium containing 0.1 mgl^-1 naphthalene acetic acid (NAA)+1.0 mgl^-1 BAP + additives. To yield multiple shoots the original explant was transferred 6 times on fresh medium after harvesting the differentiated shoots. Shoots were rooted by pulsing with 100 mgl^-1 IBA for 4 h and then culturing on hormone-free half strength MS medium. Initial dark incubation for 5 days at high temperature (33±2°C) was found essential for root induction from shoots which was 63% within two weeks. The rooted plantlets contained a consistent number of chromosomes (2n=28). It is suggested that the protocol developed could be useful for cloning of mature and tested trees of P. cineraria.     

Cloning of Maytenus emarginata (Willd.) Ding Hou — a tree of the Indian Desert,through tissue culture

Summary An in vitro method for cloning and mass multiplication of Maytenus emarginata, a highly drought resistant tree of the Indian Desert, has been developed. Shoot segments harvested from a plus tree (30-year-old) were cultured to produce multiple shoots (10–15 shoots/explant) on MS medium containing 0.1 mgl^–1 IAA and 2.5mgl^–1 BAP. In vitro produced shoots were cut into segments and cultured on shoot proliferation medium but with only 1.0 mgl^–1 of BAP to further multiply the shoots. Isolated individual shoots were cultured on a filter paper bridge in half strength MS liquid medium containing 25 mgl^–1 of IBA for 72 h in the dark at 28±2⁰ C for induction of root(s). About 70–80 percent of shoots rooted. The treelets developed were hardened and transferred to pots. Around 20,000 plants can be obtained from a single explant within a period of 6 months. The protocol is highly reproducible and efficient.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA -naphthalene acetic acid - NOA -naphthoxy acetic acid - BAP 6-benzylaminopurine - Kn 6-furfurylaminopurine - B5 Gamborg et al. (1968) medium - MS Murashige and Skoog (1962) medium     

Cultural behavior of protoplasts from different organs of eggplant (solanum melongena L.), and plant regeneration

Plants of Solanum melongena were propagated under in vitro conditions (27°C, 12h/day illumination at 62 Em^-2s^-1, 60% humidity) by subculture of terminal and lateral cuttings on MS medium +20 gl^-1 sucrose + Morel and Wetmore vitamins at 1/8 strength and 7 gl^-1 agar. Lamina, petioles and stems of 3-week-old cuttings were used as sources of protoplasts. The best mean yield of protoplasts was obtained from the lamina with 9,030×10³ protoplasts per gram of tissue. Petioles and stems yielded respectively 3,144×10³ and 1,220.4×10³ protoplasts per gram of tissue. first division of petiole and stem protoplasts occurred within 48 h, while lamina protoplasts underwent division after 3–4 days of culture in KM8p medium +2,4-D(0.2 gl^-1) + zeatin (0.5 mgl^-1) + NAA (1 mgl^-1) and 0.35M glucose as osmoticum. The highest percentage of dividing cells was obtained from petiole material, estimated at 33.4% after 7 days, compared to 23.8% and 19.4% respectively for stem and lamina protoplasts. When BAP replaced zeatin in KM8p, the division percentage of lamina protoplasts was reduced to 10–15%. When transferred to regeneration medium, all calli derived from KM8p + zeatin formed deep-green spots identified as embryo-like structures, while only few calli from KM8p + BAP underwent shoot organogenesis without formation of green spots. Some of embryo-like structure developed into plantlets with a frequency of 1–2 plantlets per callus especially on MS medium + zeatin (4 mgl^-1) + IAA (0.2 mgl^-1). Maintaining protoplast-derived calli on MS + BAP (0.5 mgl^-1) + NAA (0.5 mgl^-1) for more than 3 weeks resulted in a decrease and loss of cell totipotency.Abbreviations (IAA) Indol-3-acetic acid - (2,4-D) 2,4-dichlorophenoxyacetic acid - (NAA) naphthale-neacetic - (BAP) 6-benzylaminopurine - (MS) Murashige and Skoog basal medium - (CPW) Cell and Protoplast Washing solution     

Synergistic effect of cytokinins and auxins enables mass clonal multiplication of drumstick tree (Moringa oleifera Lam.): a wonder

The synergistic effect of plant growth regulators on axillary bud proliferation for mass clonal multiplication of Moringa oleifera Lam. (vern. drumstick) has been assessed for the first time. Treatment of decoated seeds with 1% (w/v) Bavistin for 60 min, 0.33% (w/v) streptocycline for 30 min, and 0.1% (w/v) HgCl₂ for 3.5 min resulted in complete removal of the surface contaminants. Maximum seed germination (89.13%) was obtained on quarter-strength Murashige & Skoog (MS) medium. Culture of nodal segments on MS + 6-benzyladenine (BA) at 3 mg L⁻¹ resulted in multiple shoot proliferation with ~ 18 shoots per explant. All combinations of indole-3-acetic acid (IAA) + kinetin (Kn) resulted in elongated shoots, while only lower concentrations of BA (0.5 mg L⁻¹), along with IAA (0.5 to 2 mg L⁻¹), or Kn (0.5 to 5 mg L⁻¹), showed significant synergy in the shoot morphogenesis. In addition, the maximum (100%) rooting efficiency was attained on half-strength MS medium supplemented with different concentrations of IAA and indole-3-butyric acid (IBA). The rooted plants were successfully established in the greenhouse for acclimatization. Clonality of the raised plants was assessed using 15 random primers of Operon® technologies (OPT and OPF series), and eight primers resulted in significant amplification with distinct, identical, and reproducible bands that confirmed clonality of the micropropagated plants. The present study provides a comprehensive analysis of the synergistic effect of plant growth regulators (PGRs) on in vitro shoot regeneration and proliferation for clonal mass multiplication disease-free plantlets, which can be utilized to maximize the yield of healthy and genetically identical plants of drumstick tree, which is considered to be a miracle multipurpose tree.

     

Tissue culture and plant regeneration from immature embryo explants of Calotropis gigantea (Linn.) R. Br.

Callus cultures were established from immature embryos of Calotropis gigantea (Linn.) R. Br. on a modified basal medium of Murashige & Skoog supplemented with 1 mgl^-1 2,4-D. In addition to 0.1 mgl^-1 of NAA the optimal BAP concentration for promoting shoot bud formation and growth was 2 mgl^-1. Rooting was induced when shoots were transferred to auxin-supplemented Bonner's solution or half-strength MS basal salt solutions.Abbreviations NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IBA indole-butyric acid - BAP 6-benzylaminopurine - Kin kinetin     

Shoot regeneration from cotyledonary leaf explants of <Emphasis Type="Italic">Jatropha curcas</Emphasis>: a biodiesel plant

A simple, high frequency, and reproducible method for plant regeneration through direct organogenesis from cotyledonary leaf explants of Jatropha curcas was developed using Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) or 6-benzyl aminopurine (BAP). Medium containing TDZ has greater influence on regeneration as compared to BAP. The induced shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM BAP, and 5.5 μM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA, and indole-3-butyric acid (IBA). MS medium with 2.25 μM BAP and 8.5 μM IAA was found to be the best combination for shoot elongation. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. Rooting was achieved when the basal cut end of elongated shoots were dipped in half strength MS liquid medium containing different concentrations and combinations of IBA, IAA, and NAA for 4 days, followed by transfer to growth regulators free half strength MS medium supplemented 0.25 mg l⁻¹ activated charcoal. Elongated shoot treated with 15 μM IBA, 5.7 μM IAA, and 11 μM NAA resulted in highest percent rooting. The rooted plants could be established in soil with more than 90% survival rate. The method developed may be useful in improvement of J. curcas through genetic modification.     

Plant regeneration in tissue cultures of pepper (Capsicum annuum L. cv. mathania)

Leaf, stem, hypocotyl, cotyledon, root, shoot tip and embryo explants of Capsicum annuum L. cv. mathania were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) or kinetin (Kin) alone or in combination with 3-indoleacetic acid (IAA), 3-indolebutyric acid (IBA), α-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). BAP (5.0 mgl⁻¹) in the medium was found to be the best growth regulator for shoot bud differentiation. Shoot buds cultured on 5.0 mgl⁻¹ BAP increased in number but did not elongate. For obtaining complete plantlets, shoot buds were placed on a medium with IBA or NAA (0.1 mgl⁻¹). Histological evidence revealed direct differentiation of buds from cotyledons. Regenerated plants were normal diploids. Unorganized callus could not be induced to differentiate shoot buds.     

Induction of callus and plant regeneration in Vicoa indica

Callus cultures were initiated from the stem and leaf explants of aseptically grown Vicoa indica. A simple method is described for plant regeneration from callus and the rapid multiplication of the plants thus obtained. Callus initiation was optimum in Gamborg B5 (B5) basal medium containing either 2.0 mg l^-1 naphthaleneacetic acid (NAA) with 0.2 mg l^-1 kinetin (Kn) or 2.0 mg l^-1 6-benzylaminopurine (BAP) with 0.2 mg l^-1 NAA. The calli initiated on B5 medium were able to proliferate on both Murashige and Skoog (MS) and B5 basal medium. Shoot primordia were obtained from greenish callus on passage to B5 basal medium containing 3.0 mg l^-1 BAP and 1.0 mg l^-1 Kn. On further subculture onto B5 medium containing 0.2 mg l^-1 Kn the shoot primordia developed into plantlets.     

Micropropagation of <Emphasis Type="Italic">eclipta alba</Emphasis> (L.) hassk—An important medicinal plant

Summary An efficient and reproducible protocol for mass propagation of Eclipta alba (L.) Hassk, an important medicinal plant, was standardized by culturing shoot tips and nodal segments taken from in vitro raised plants. Maximum shoot proliferation occurred when the explants were cultured on Murashige and Skoog (MS) medium supplemented with 1 mg l⁻¹ benzylaminopurine (BAP). The shoot buds formed were further multiplied and maintained on medium containing BAP (0.5 mgl⁻¹) and gibberellic acid (0.5 mgl⁻¹). Rooting was best achieved on MS medium supplement with 1 mg⁻¹ indole-3-butyric acid. Rooted plantlets attained maturity and flowered normally in the field.     

Sedimentary Organic Carbon and Nitrogen Sequestration Across a Vertical Gradient on a Temperate Wetland Seascape Including Salt Marshes,Seagrass Meadows and Rhizophytic Macroalgae Beds

Coastal wetlands are key in regulating coastal carbon and nitrogen dynamics and contribute significantly to climate change mitigation and anthropogenic nutrient reduction. We investigated organic carbon (OC) and total nitrogen (TN) stocks and burial rates at four adjacent vegetated coastal habitats across the seascape elevation gradient of Cádiz Bay (South Spain), including one species of salt marsh, two of seagrasses, and a macroalgae. OC and TN stocks in the upper 1 m sediment layer were higher at the subtidal seagrass Cymodocea nodosa (72.3 Mg OC ha⁻¹, 8.6 Mg TN ha⁻¹) followed by the upper intertidal salt marsh Sporobolus maritimus (66.5 Mg OC ha⁻¹, 5.9 Mg TN ha⁻¹), the subtidal rhizophytic macroalgae Caulerpa prolifera (62.2 Mg OC ha⁻¹, 7.2 Mg TN ha⁻¹), and the lower intertidal seagrass Zostera noltei (52.8 Mg OC ha⁻¹, 5.2 Mg TN ha⁻¹). The sedimentation rates increased from lower to higher elevation, from the intertidal salt marsh (0.24 g cm⁻² y⁻¹) to the subtidal macroalgae (0.12 g cm⁻² y⁻¹). The organic carbon burial rate was highest at the intertidal salt marsh (91 ± 31 g OC m⁻² y⁻¹), followed by the intertidal seagrass, (44 ± 15 g OC m⁻² y⁻¹), the subtidal seagrass (39 ± 6 g OC m⁻² y⁻¹), and the subtidal macroalgae (28 ± 4 g OC m⁻² y⁻¹). Total nitrogen burial rates were similar among the three lower vegetation types, ranging from 5 ± 2 to 3 ± 1 g TN m⁻² y⁻¹, and peaked at S. maritimus salt marsh with 7 ± 1 g TN m⁻² y⁻¹. The contribution of allochthonous sources to the sedimentary organic matter decreased with elevation, from 72% in C. prolifera to 33% at S. maritimus. Our results highlight the need of using habitat-specific OC and TN stocks and burial rates to improve our ability to predict OC and TN sequestration capacity of vegetated coastal habitats at the seascape level. We also demonstrated that the stocks and burial rates in C. prolifera habitats were within the range of well-accepted blue carbon ecosystems such as seagrass meadows and salt marshes.

     

Evaluation of the Impacts of Long-Term Enriched Artemia with Bacillus subtilis on Growth Performance,Reproduction, Intestinal Microflora,and Resistance to Aeromonas hydrophila of Ornamental Fish Poecilia latipinna

The present study investigated the effect of enriched Artemia with Bacillus subtilis on growth performance, reproductive factors, proximate composition, intestinal microflora, and resistance to Aeromonas hydrophila of ornamental fish, Poecilia latipinna. Using a completely randomized design, the experiment included three groups. The first group was fed with commercial food without any probiotic. The second group was fed with unenriched Artemia, and the last group consumed long-time enriched Artemia with Bacillus subtilis. The bacteria B. subtilis with a density of 1 × 10⁵ CFU mL⁻¹ was added daily to Artemia culture medium. The total microflora and Bacillus subtilis counts were significantly increased in enriched Artemia compared to the unenriched group (P < 0.05). In fish fed groups, growth factors did not show any significant difference (P > 0.05). The maximum relative fecundity (28.65 ± 2.52 egg number g⁻¹), fry production (62.93 ± 4.6 individual per female), and fry survival (70.97 ± 1.56%) obtained in the third group were found to be significantly more than those in the first and the second groups. Moreover, intestinal bacterial count for Bacillus revealed that the higher concentration of bacteria was significantly related to the third group (6.24 ± 0.11 log CFU g⁻¹) (P < 0.05). Maximum protein and fat contents were observed in fish fed with Bacillus-enriched Artemia; however, no significant difference was found between control and unenriched Artemia groups (P > 0.05). The highest amount of ash was observed in fish fed with commercial food without any probiotic (P < 0.05). At the end of the feeding period, each of the three groups along with positive group (oxytetracycline 100 mg kg⁻¹ of commercial food) was exposed to A. hydrophila (BCCM5/LMG3770) bacteria intraperitoneally. Based on the results, the lowest cumulative mortality was significantly found in group three (68.75 ± 3.6%) and positive group (62.5 ± 7.0%) compared to control and unenriched Artemia groups (P < 0.05). Hence, B. subtilis with a concentration of 1 × 10⁵ CFU mL⁻¹ during the period of Artemia culturing can improve the reproductive parameters, intestinal microflora, and resistance to pathogenic bacteria of Poecilia latipinna.

     

Hypermethioninemia induces memory deficits and morphological changes in hippocampus of young rats: implications on pathogenesis

The aim of this study was to investigate the effect of the chronic administration of methionine (Met) and/or its metabolite, methionine sulfoxide (MetO), on the behavior and neurochemical parameters of young rats. Rats were treated with saline (control), Met (0.2–0.4 g/kg), MetO (0.05–0.1 g/kg), and/or a combination of Met + MetO, subcutaneously twice a day from postnatal day 6 (P6) to P28. The results showed that Met, MetO, and Met + MetO impaired short-term and spatial memories (P < 0.05), reduced rearing and grooming (P < 0.05), but did not alter locomotor activity (P > 0.05). Acetylcholinesterase activity was increased in the cerebral cortex, hippocampus, and striatum following Met and/or MetO (P < 0.05) treatment, while Na⁺, K⁺-ATPase activity was reduced in the hippocampus (P < 0.05). There was an increase in the level of thiobarbituric acid reactive substances (TBARS) in the cerebral cortex in Met-, MetO-, and Met + MetO-treated rats (P < 0.05). Met and/or MetO treatment reduced superoxide dismutase, catalase, and glutathione peroxidase activity, total thiol content, and nitrite levels, and increased reactive oxygen species and TBARS levels in the hippocampus and striatum (P < 0.05). Hippocampal brain-derived neurotrophic factor was reduced by MetO and Met + MetO compared with the control group. The number of NeuN-positive cells was decreased in the CA3 in Met + MetO group and in the dentate gyrus in the Met, MetO, and Met + MetO groups compared to control group (P < 0.05). Taken together, these findings further increase our understanding of changes in the brain in hypermethioninemia by elucidating behavioral alterations, biological mechanisms, and the vulnerability of brain function to high concentrations of Met and MetO.

     

Somatic embryogenesis and plant regeneration in Heracleum candicans Wall

A protocol has been developed for achieving somatic embryogenesis and plant regeneration from petiole-derived callus of Heracleum candicans Wall. Callus was initiated on MS medium supplemented with 0.5 mg l^–1 2,4-D and 0.5 mg l^–1 BAP and subcultured on a medium containing double strength MS macrosalts, 1 mg l^–12,4-D and 0.25 mg l^–1 Kn. Numerous globular embryos were formed on the surface of the callus upon transfer to auxin-rich MS medium that lacked cytokinins. The globular embryos differentiated into mature embryos only when 2,4-D was removed from the medium. Mature embryo formation was significantly influenced by the pH of the medium and the addition of AgNO₃ and ABA. Eighty-five percent of the somatic embryos were converted into plantlets when transferred to a medium supplemented with 0.01 mg l^–1 BAP and 0.01 mg l^–1 IBA. The regenerated plants have been established in soil and appear to be identical to the parent plants in morphology and chromosome number. Received: 5 November 1997 / Revision received: 9 February 1998 / Accepted: 19 February 1998     

Micropropagation of ‘Holy Basil’ (Ocimum sanctum Linn.) from young inflorescences of mature plants

In vitro micropropagation of holy basil (Ocimum sanctum L.), an Indian medicinal herb, has been accomplished on Murashige and Skoog (MS) medium utilizing young inflorescence explants. MS supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) or thidiazuron (TDZ) produced only non-morphogenetic callus. Direct multiple shoots differentiated within 2--3 weeks when explants were cultured on MS containing 6-benzyl aminopurine (BAP). Of the various levels of BAP tested, MS + BAP (1.0 mgl^–1) produced the maximum number of shoots. Incorporation of indole-3-acetic acid (IAA) (0.05 mgl^–1) along with BAP (1.0 mgl^–1) in the culture medium showed a marked increase in the number of shoots. About 92% of the in vitro regenerated shoots rooted on MS hormone-free medium within 2--3 weeks of culture and 85% of the micropropagated plantlets could be successfully established in soil, where they grew normally.     

Plant regeneration through the direct induction of shoot buds from petiole explants of Jatropha curcas: a biofuel plant

An efficient and reproducible method for the regeneration of Jatropha curcas plants has been developed. The method employed direct induction of shoot buds from petiole explants, without the formation of an intervening callus using a Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (58.35%) and the number of shoot buds per explant (10.10) were observed when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 µM TDZ after 6 weeks. The induced shoot buds were transferred to MS medium containing 10 µM kinetin (Kn), 4.5 µM 6-benzyl aminopurine (BAP) and 5.5 µM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA and indole-3-butyric acid (IBA). MS medium supplemented with 2.25 µM BAP and 8.5 µM IAA was found to be the best combination for shoot elongation and 3.01–3.91 cm elongation was achieved after 6 weeks. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. The orientation (horizontal or vertical) and source (in vitro or in vivo) of explants also significantly influenced plant regeneration. The elongated shoots could be rooted on half-strength MS medium supplemented with 2% sucrose, different concentrations and combinations of IBA, IAA and NAA, and 0.25 mg L⁻¹ activated charcoal. Half-strength MS medium supplemented with 2% sucrose, 15 µM IBA, 5.7 µM IAA, 5.5 µM NAA and 0.25 mg L⁻¹ activated charcoal was found to be the best for promoting rooting. The rooted plants could be established in soil with more than 90% survival.     

Impacts of exposure to mine tailings on zooplankton hatching from a resting egg bank

In the last five years, two colossal environmental disasters involving iron-enriched mine tailings have occurred in Brazil, affecting many aquatic ecosystems over the short, medium and long-terms. This study investigated whether these iron-enriched mine tailings affect the main biotic strategy to restore zooplankton populations affected by severe stress, i.e., hatching of dormant stages. A 30 day hatching experiment was conducted, using a resting egg bank from a natural lake, exposed to 3 concentrations of mine tailings: control (0 g), T25 (25 g) and T50 (50 g). A total of 22, 15 and 16 species hatched in the control, T25 and T50, respectively. Conochilus sp., Filinia terminalis, Hexartha mira, Bosmina longirostris and Ceriodaphnia silvestrii hatched only in the control, which suggests that these species are sensitive to any concentration of mine tailings. A gradual decrease in richness and hatchling abundance was recorded, from the control (8.0?±?1.0 SE species and 1597?±?73.9 hatchlings) to T25 (4.6?±?1.2 species and 1279?±?136.5 hatchlings) and then to T50 (2.3?±?1.2 species and 603.3?±?61.9 hatchlings). Our results suggest that exposure of zooplankton resting eggs to iron-enriched mine tailings may negatively impact these egg banks in natural ecosystems, with potential impacts on the restoration of zooplankton communities after even short-term exposures.

Graphic abstract