Development of a Polygonum minus cell suspension culture system and analysis of secondary metabolites enhanced by elicitation

Polygonum minus has been reported to contain valuable metabolites and to date, there is no report on using cell culture technique for metabolite production in P. minus. Naphthalene acetic acid (NAA) concentrations in the range of 2–6 mg L^?1 were used in a matrix of combinations with dichlorophenoxyacetic acid (2,4-D) concentrations in the range of 2–10 mg L^?1 as plant growth regulators (PGRs) to induce callus cultures. Media that were supplemented with 2 mg L^?1 2,4-D + 4 mg L^?1 NAA, 2 mg L^?1 2,4-D + 6 mg L^?1 NAA and 6 mg L^?1 2,4-D + 8 mg L^?1 NAA were effective for callus induction (93.3 % of the explants produced callus). To establish cell culture, the best growth was obtained from medium that was supplemented with 1 mg L^?1 2,4-D + 2 mg L^?1 NAA. From a 1-g inoculum size, the fresh weight increases exponentially after 5–10 days of culture, and a 26.71 g maximum fresh weight was obtained after 25 days of culture. The cell culture medium was then analyzed using gas chromatography–mass spectrometry (GC–MS). Jasmonic acid (100, 50, 25 and 5 μM), salicylic acid (100, 50, 25 and 5 μM), yeast extract (500, 250 and 100 mg L^?1) and glass beads were used in this research as elicitors. The cell cultures were then incubated with the different elicitors for 1, 2, 3 and 4 days. Several compounds with high peak area percentages were detected, including 2-furancarboxaldehyde, 5-hydroxymethyl, furfural, and 2-cyclopenten-1-one, 2-hydroxy. These results show the diversity of metabolites released by P. minus cell into the culture medium under control conditions.     

High efficient somatic embryogenesis development from leaf cultures of <Emphasis Type="Italic">Citrullus colocynthis</Emphasis> (L.) Schrad for generating true type clones

We report an efficient somatic embryogenesis and plant regeneration system using leaf cultures of Citrullus colocynthis (L.) and assessed the effect of plant growth regulators on the regeneration process. Initially leaf explants were cultured on Murashige and Skoog medium supplemented with different concentrations of auxins viz., 2,4-dichlorophenoxyacetic acid, 1-naphthaleneacetic acid, gibberellic acid alone and along with combination of 6-benzylaminopurine. The different forms of calli such as compact, white friable, creamy friable, brownish nodular, green globular and green calli were induced from the leaf explants on MS medium containing different concentrations of auxins and gibberellins. Subsequently initial callus was subcultured at 1.5 mg L^?1 BAP + 1.0 mg L^?1 2,4-D which resulted in 25 % somatic embryos from 85 % nodular embryogenic nodular callus that is highest percentage. Similarly the lowest percentage of somatic embryos was recorded at 2.5 mg L^?1 BAP + 0.5 mg L^?1 NAA from 55 % embryogenic globular callus i.e., 16 %. High frequency of embryo development takes place at intermittent light when compared with continuous light in the individual subcultures. The cotyledonary embryos were developed into complete platelets on MS medium. In vitro regenerated plantlets were washed to remove the traces of agar and then transferred to sterile vermiculite and sand (2:1) containing pot.     

Gibberellic acid mediated induction of salt tolerance in wheat plants: Growth,ionic partitioning,photosynthesis, yield and hormonal homeostasis

In order to elucidate the GA₃-priming-induced physiochemical changes responsible for induction of salt tolerance in wheat, the primed and non-primed seeds of two spring wheat (Triticum aestivum L.) cultivars, namely, MH-97 (salt intolerant) and Inqlab-91 (salt tolerant) were sown in a field treated with 15 dS m⁻¹ NaCl salinity. Although all the three concentrations (100, 150 and 200 mg L⁻¹) of GA₃ were effective in improving grain yield in both cultivars, the effect of 150 mg L⁻¹ GA₃ was much pronounced particularly in the salt intolerant cultivar when under salt stress. Seed priming with GA₃ altered the pattern of accumulation of different ions between shoots and roots in the adult plants of wheat under saline conditions. Treatment with GA₃ (150 mg L⁻¹) decreased Na⁺ concentrations both in the shoots and roots and increased Ca²⁺ and K⁺ concentrations in the roots of both wheat cultivars. GA₃-priming did not show consistent effect on gaseous exchange characteristics and the concentrations of auxins in the salt stressed plants of both wheat cultivars. However, all concentrations of GA₃ reduced leaf free ABA levels in the salt intolerant, while reverse was true in the salt tolerant cultivar under saline conditions. Priming with GA₃ (150 mg L⁻¹) was very effective in enhancing salicylic acid (SA) concentration in both wheat cultivars when under salt stress. Treatment with GA₃ (100–150 mg L⁻¹) lowered leaf free putrescine (Put) and spermidine (Spd) concentrations in the plants of both wheat cultivars. The decrease in polyamines (Put and Spd) and ABA concentrations in the salt stressed plants of the salt intolerant cultivar treated with GA₃ suggested that these plants might have faced less stress compared with control. Thus, physiologically, GA₃-priming-induced increase in grain yield was attributed to the GA₃-priming-induced modulation of ions uptake and partitioning (within shoots and roots) and hormones homeostasis under saline conditions.     

Plant growth regulators affect biosynthesis and accumulation profile of isoflavone phytoestrogens in high-productive in vitro cultures of Genista tinctoria

The influence of plant growth regulators on biomass growth and the accumulation of medicinally-relevant isoflavone phytoestrogens, derivatives of genistein and daidzein (8 compounds including aglycones, glucosides and glucoside esters) in callus cultures of Genista tinctoria (Fabaceae) was examined. The experiments included 10 auxins [2,4-dichlorophenoxyacetic acid (2,4-D), p-chlorophenoxyacetic acid, indole-3-acetic acid, indole-3-butyric acid, indole-3-propionic acid, 1-naphthaleneacetic acid, β-naphthoxyacetic acid, picloram, 2,3,5-triiodobenzoic acid (TIBA), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)] and 7 cytokinins [6-benzylaminopurine, forchlorfenuron, 1,3-diphenylurea, 2-isopentenyladenine, kinetin (KIN), thidiazuron, zeatin] applied at 0.5 and 5.0 mg l^?1, jointly with 5.0 or 0.5 mg l^?1 KIN or 2,4-D (for auxins and cytokinins, respectively—36 phytohormone combinations in total). Statistical analysis of the relationships between callus growth [expressed as growth index (Gi)] and the accumulation of isoflavones showed positive correlation in the cytokinin group (r_xy values from 0.13 to 0.61) and negative correlation within auxins (r_xy values from ?0.31 to ?0.39). Among the cytokinins tested, the highest isoflavone content (6,436.26 mg/100 g dry weight) and the fastest biomass growth (Gi = 892.46 %) were obtained for 0.5 mg l^?1 KIN used jointly with 5.0 mg l^?1 2,4-D. In the group of auxins, the combination of 0.5 mg l^?1 TIBA and 5.0 mg l^?1 KIN provided the fastest culture growth (Gi = 983.07 %) and the isoflavone concentration of 10,474.23 mg/100 g dry weight, which is so far the highest amount of these metabolites achieved in callus cultures of higher plants.     

<Emphasis Type="Italic">In vitro</Emphasis> sucrose concentration influences microtuber production and diosgenin content in white yam (<Emphasis Type="Italic">Dioscorea rotundata</Emphasis> Poir)

Dioscorea spp. is an important food crop in many countries and the source of the phytochemical diosgenin. Efficient microtuber production could provide source materials for farm-planting stock, for food markets, and for the production of high-diosgenin-producing cultivars. The first step in this study was optimizing the plant growth regulators for plantlet production, followed by a study of the effects of sucrose concentration on microtuber induction and diosgenin production. Significantly, more shoots (3.5) were produced at 4.65 μM (1 mg L^?1) kinetin (KIN), longer shoots (4.1 cm) were obtained at 2.46 μM (0.5 mg L^?1) indole-3-butyric acid (IBA), and root number (3.9) was significantly higher at 5.38 μM (1 mg L^?1) naphthalene acetic acid (NAA) than in other treatments. Increased sucrose concentrations in the optimized growth medium with 4.65 μM KIN and 5.38 μM NAA had significant effects on microtuber production (p < 0.01) and diosgenin content (p < 0.05). The most microtubers (6.2) were obtained with 100 g L^?1 sucrose, while those on 80 g L^?1 sucrose were the heaviest (0.7 g) and longest (7.4 mm). Microtubers formed in medium with 80 g L^?1 sucrose had significantly higher diosgenin content (3.64% [w/w]) than those in other sucrose treatments (< 2%) and was similar to that of field-grown parent tubers (3.79%). This result indicates an important role for sucrose in both microtuber growth and diosgenin production. Medium containing 4.65 μM KIN and 5.38 μM NAA is recommended for plantlet production, and medium containing 80 g L^?1 sucrose is recommended for microtuber and diosgenin production.     

Micropropagation, <Emphasis Type="Italic">in vitro</Emphasis> flowering,and tuberization in <Emphasis Type="Italic">Brachystelma glabrum</Emphasis> Hook.f., an endemic species

Brachystelma glabrum Hook.f. is an endemic plant species of Eastern Ghats, India. In this study, efficient protocols for in vitro micropropagation, flowering, and tuberization of this plant were developed. Sterilized shoot tip and nodal explants were cultured on Murashige and Skoog (MS) medium supplemented with different plant growth regulators (PGRs) and additives for shoot induction and multiplication. Both shoot tip and nodal explants showed the best response (90 and 100%, respectively) on MS medium supplemented with thidiazuron (TDZ) at 1.0 mg L^?1. The microshoots multiplied best on MS + TDZ (1.0 mg L^?1) in combination with α-naphthaleneacetic acid (NAA) at 0.5 mg L^?1 and coconut water (CW) at 25%. The highest number of in vitro flowers (4.0 flowers per microshoot) was observed on MS medium supplemented with a combination of N6-benzyladenine (BA) and indole-3-butyric acid (IBA), each at 1.5 mg L^?1. In vitro-derived shoots produced aerial tubers on MS + TDZ (2.0 mg L^?1) + IBA (0.5 mg L^?1) and basal tubers on MS + TDZ at 2.0 mg L^?1. In vitro shoots were best rooted on half-strength (½) MS + NAA at 0.5 mg L^?1. The rooted plantlets were successfully acclimatized in pots with 70% survival after a hardening period of 1 mo. This protocol provides an effective method for the conservation of this endemic plant species.     

Optimization of culture conditions for the production of polysaccharides and kinsenoside from the rhizome cultures of <Emphasis Type="Italic">Anoectochilus roxburghii</Emphasis> (Wall.) Lindl.

The present study designed two sets of experiments by using the uniform design method and investigated the effects of medium components on the accumulation of bioactive compounds (polysaccharide and kinsenoside) in rhizomes, in order to select a suitable culture medium for the rhizome suspension culture of Anoectochilus roxburghii (Wall.) Lindl. Among the combinations of Murashige and Skoog (MS) medium strengths and plant growth regulator (benzylaminopurine, BA; kinetin, KT; and α-naphthaleneacetic acid, NAA) concentrations, and the combinations of nitrogen, phosphorus, and sucrose concentrations, the maximum yield of polysaccharides and kinsenoside was achieved with 0.75 × MS?+?2.0 mg L^?1 BA?+?0.2 mg L^?1 KT?+?0.5 mg L^?1 NAA and 45 mM nitrogen?+?0.93 mM phosphorus?+?35 g L^?1 sucrose, respectively. Therefore, the optimal rhizome suspension culture medium was 0.75 × MS medium supplemented with 2.0 mg L^?1 BA, 0.2 mg L^?1 KT, 0.5 mg L^?1 NAA, and 35 g L^?1 sucrose. Yeast extract (YE) enhanced bioactive compound accumulation in rhizomes. The polysaccharide and kinsenoside production was significantly improved when 75 mg L^?1 YE was added to the culture medium after 30 d of rhizome suspension culture; 8.3 g L^?1 of polysaccharide and 6.1 g L^?1 of kinsenoside were obtained after 4 d of YE treatment. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of YE-treated rhizomes was higher than that of YE-untreated rhizomes, demonstrating enhanced antioxidant activity of the treated bioreactor-cultured rhizomes.     

An improved protocol for somatic embryogenesis and plant regeneration in macaw palm (Acrocomia aculeata) from mature zygotic embryos

An improved protocol for plant regeneration via somatic embryogenesis was developed using mature macaw palm (Acrocomia aculeata) zygotic embryos as initial explant. For induction of the embryogenic callus (EC), two basic media (BM) were tested consisting of Murashige and Skoog and Eeuwens (Y3) salts with 30 g L^?1 sucrose, 0.5 g L^?1 glutamine and 2.5 g L^?1 Phytagel. The 3,6-dichloro-2-methoxybenzoic acid (dicamba), 4-amino-3,5,6-trichloro-picolinic acid (picloram) and 2,4-dichlorophenoxyacetic acid (2,4-D) auxins were added to the culture media at concentrations of 0, 1.5 or 3.0 mg L^?1. After 240 days, the embryogenic calli were transferred to the respective BM media with auxin concentrations reduced to 0.5 or 1.0 mg L^?1 in order to differentiate the somatic embryos (SEs). Plant regeneration was performed on the BM media without growth regulators. Embryogenic calli were observed after 180 days of culture and in all treatments with auxin. The Y3 medium showed the best EC formation results (60.8 %). These calli showed yellowish coloration, compact consistency and nodular aspect. After 60 days in differentiation medium, SEs were verified in different stages of development. Histological analysis showed that the SEs were formed from a nodular EC. The SEs generally presented unicellular origin with suspensor formation, and at the end of development, bipolar embryos were observed. The plant regeneration frequency reached levels up to 31.9 % when using induction medium consisting of Y3 associated to 1.5 mg L^?1 of 2,4-D and the subsequent auxin reduction to 0.5 mg L^?1 in the differentiation stage. Regenerated plants showed normal development, with root and aerial part growth.     

Conversion of oat (<Emphasis Type="Italic">Avena sativa</Emphasis> L.) haploid embryos into plants in relation to embryo developmental stage and regeneration media

Obtaining oat DH lines is only effective via wide crossing with maize. Seven hundred haploid embryos from 21 single F₁ progeny obtained from wide crosses with maize were isolated, divided into four groups according to their size (<0.5 mm, 0.5–0.9 mm, 1.0–1.4 mm, and ≥1.5 mm), and transferred into 190–2 regeneration medium with different growth regulators: 0.5 mg L^?1 kinetin (KIN) and 0.5 mg L^?1 1-naphthaleneacetic acid (NAA); 1 mg L^?1 zeatin (ZEA) and 0.5 mg L^?1 NAA; or 1 mg L^?1 dicamba (DIC), 1 mg L^?1 picloram (PIC), and 0.5 mg L^?1 kinetin (KIN). Among all isolated embryos, approximately 46.1% were between 1.0–1.4 mm, while the smallest group of embryos (7.1%) were those <0.5 mm. The ability of haploid embryos to germinate varied depending on oat genotypes and the size of embryos. Haploid embryos <0.5 mm were globular and did not germinate, whereas embryos ≥1.5 mm had clearly visible coleoptiles, radicles, and scutella, and were able to germinate. Germination of oat haploid embryos varied depending on growth regulators in the regeneration medium. Most haploid embryos germinated on medium with 0.5 mg L^?1 NAA and 0.5 mg L^?1 KIN, while the fewest germinated on medium with 1 mg L^?1 DIC, 1 mg L^?1 PIC, and 0.5 mg L^?1 KIN. One hundred thirty germinated haploid embryos converted into haploid plants. Fifty oat DH lines were obtained after colchicine treatment.     

DETERMINATION OF METHYLENE BLUE BIOSORPTION BY Rhizopus arrhizus IN THE PRESENCE OF SURFACTANTS WITH DIFFERENT CHEMICAL STRUCTURES

Methylene blue (MB) biosorption properties of Rhizopus arrhizus were investigated in the presence of surfactants. The effects of cationic and anionic surfactants on MB removal by dead biomass (1 g L^?1) were determined. MB removal was tested as a function of initial pH (2–12), contact time (5–1440 min), and dye (37.4–944.7 mg L^?1) and surfactant (0–10 mM) concentrations. The opposite charged anionic surfactant dodecylbenzenesulfonic acid sodium salt (DBS) enhanced sorption of cationic MB by biomass dramatically. Maximum biosorption capacity was 471.5 mg g^?1 at pH 8 with 0.5 mM DBS at 944.7 mg L^?1 MB concentration. The surfactant-stimulated fungal decolorization method may provide a highly efficient, inexpensive, and time-saving procedure in biological wastewater treatment technologies.     

Somatic embryogenesis and direct as well as indirect organogenesis in Lilium pumilum DC. Fisch., an endangered ornamental and medicinal plant

Somatic embryogenesis and organogenesis in Lilium pumilum were successfully regulated by picloram, α-naphthaleneacetic acid (NAA), and 6-benzyladenine (BA). In organogenesis, the highest shoot regeneration frequency (92.5%) was obtained directly from bulb scales on Murashige and Skoog (MS) medium containing 2.0 mg L^?1 BA and 0.2 mg L^?1 NAA, while organogenic callus (OC) formed from leaves on MS medium supplemented with 1.0 mg L^?1 BA and 0.5 mg L^?1 NAA. Following subculture, 76.7% of OC regenerated shoots. In somatic embryogenesis, the combination of picloram and NAA increased the amount of embryogenic callus (EC) that formed with a maximum on 90.7% of all explants which formed 11 somatic embryos (SEs) per explant. Differences between EC and OC in cellular morphology and cell differentiation fate were easily observed. SEs initially formed via an exogenous or an endogenous origin. The appearance of a protoderm in heart-shaped SE and the bipolar shoot–root development in oval-shaped SE indicated true somatic embryogenesis. This protocol provides a new and detailed regulation and histological examination of regeneration pattern in L. pumilum.     

Selection of Diazotrophic Bacterial Communities in Biological Sand Filter Mesocosms Used for the Treatment of Phenolic-Laden Wastewater

Agri effluents such as winery or olive mill wastewaters are characterized by high phenolic concentrations. These compounds are highly toxic and generally refractory to biodegradation. Biological sand filters (BSFs) represent inexpensive, environmentally friendly, and sustainable wastewater treatment systems which rely vastly on microbial catabolic processes. Using denaturing gradient gel electrophoresis and terminal-restriction fragment length polymorphism, this study aimed to assess the impact of increasing concentrations of synthetic phenolic-rich wastewater, ranging from 96 mg L^?1 gallic acid and 138 mg L^?1 vanillin (i.e., a total chemical oxygen demand (COD) of 234 mg L^?1) to 2,400 mg L^?1 gallic acid and 3,442 mg L^?1 vanillin (5,842 mg COD L^?1), on bacterial communities and the specific functional diazotrophic community from BSF mesocosms. This amendment procedure instigated efficient BSF phenolic removal, significant modifications of the bacterial communities, and notably led to the selection of a phenolic-resistant and less diverse diazotrophic community. This suggests that bioavailable N is crucial in the functioning of biological treatment processes involving microbial communities, and thus that functional alterations in the bacterial communities in BSFs ensure provision of sufficient bioavailable nitrogen for the degradation of wastewater with a high C/N ratio.     

Biodegradation of the Pyrethroid Pesticide Esfenvalerate by Marine-Derived Fungi

Esfenvalerate biodegradation by marine-derived fungi is reported here. Esfenvalerate (S,S-fenvalerate) and its main metabolites [3-phenoxybenzaldehyde (PBAld), 3-phenoxybenzoic acid (PBAc), 3-phenoxybenzyl alcohol (PBAlc), and 2-(4-chlorophenyl)-3-methylbutyric acid (CLAc)] were quantitatively analyzed by a validated method in triplicate experiments. All the strains (Penicillium raistrickii CBMAI 931, Aspergillus sydowii CBMAI 935, Cladosporium sp. CBMAI 1237, Microsphaeropsis sp. CBMAI 1675, Acremonium sp. CBMAI 1676, Westerdykella sp. CBMAI 1679, and Cladosporium sp. CBMAI 1678) were able to degrade esfenvalerate, however, with different efficiencies. Initially, 100 mg L^?1 esfenvalerate (Sumidan 150SC) was added to each culture in 3 % malt liquid medium. Residual esfenvalerate (64.8–95.2 mg L^?1) and the concentrations of PBAc (0.5–7.4 mg L^?1), ClAc (0.1–7.5 mg L^?1), and PBAlc (0.2 mg L^?1) were determined after 14 days. In experiments after 7, 14, 21, and 28 days of biodegradation with the three most efficient strains, increasing concentrations of the toxic compounds PBAc (2.7–16.6 mg L^?1, after 28 days) and CLAc (6.6–13.4 mg L^?1, after 28 days) were observed. A biodegradation pathway was proposed, based on HPLC-ToF results. The biodegradation pathway includes PBAld, PBAc, PBAlc, ClAc, 2-hydroxy-2-(3-phenoxyphenyl)acetonitrile, 3-(hydroxyphenoxy)benzoic acid, and methyl 3-phenoxy benzoate. Marine-derived fungi were able to biodegrade esfenvalerate in a commercial formulation and showed their potential for future bioremediation studies in contaminated soils and water bodies.     

Comparative analysis of the effects of chitosan and common plant growth regulators on <Emphasis Type="Italic">in vitro</Emphasis> propagation of <Emphasis Type="Italic">Ipomoea purpurea</Emphasis> (L.) Roth from nodal explants

This study aimed to evaluate the effects of partially N-acetylated chitosans with a degree of acetylation (DA) of 10% on in vitro propagation of an ornamental plant, Ipomoea purpurea, by emphasizing the importance of the degree of polymerization (DP) on in vitro plant development. The effects of either a chitosan oligomer mixture with a DP between 2 and 15 (5.0, 10.0, and 20.0 mg L^?1) or chitosan polymer with a DP of 70 were compared with commonly used cytokinins [6-benzylaminopurine (BAP) and kinetin (KIN) at 0.5, 1.0, 2.0, and 4.0 mg L^?1] and auxins [indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) at 0.25, 0.5, 1.0, and 2.0 mg L^?1]. The nodal explants used in this study were taken from donor plants obtained by germination of the seeds. The results indicated that all chitosan treatments had positive effects on the shoot induction, but only the oligomer mixture at 5 mg L^?1 gave the best results for mean shoot number, shoot length, and leaf number, compared to the other treatments and control. Also, all chitosan treatments increased mean number of roots and triggered adventitious root induction. However, root elongation was decreased in the presence of chitosan in the medium. The root elongation-inhibitory effects of chitosan become clearer in the presence of oligomer mixture. In general, chitosan had similar effects with tested cytokinins rather than auxins. The results of this study suggested that the application of chitosan oligomers—rather than polymers—can be an eco-friendly and effective alternative to synthetic cytokinins in horticulture.     

Complete and simultaneous removal of ammonium and m-cresol in a nitrifying sequencing batch reactor

The kinetic behavior, oxidizing ability and tolerance to m-cresol of a nitrifying sludge exposed to different initial concentrations of m-cresol (0–150 mg C L^?1) were evaluated in a sequencing batch reactor fed with 50 mg NH₄ ⁺-N L^?1 and operated during 4 months. Complete removal of ammonium and m-cresol was achieved independently of the initial concentration of aromatic compound in all the assays. Up to 25 mg m-cresol-C L^?1 (C/N ratio of 0.5), the nitrifying yield (Y-NO₃ ^?) was 0.86 ± 0.05, indicating that the nitrate was the main product of the process; no biomass growth was detected. From 50 to 150 mg m-cresol-C L^?1 (1.0 ≤ C/N ≤ 3.0), simultaneous microbial growth and partial ammonium-to-nitrate conversion were obtained, reaching a maximum microbial total protein concentration of 0.763 g L^?1 (247 % of its initial value) and the lowest Y-NO₃ ^? 0.53 ± 0.01 at 150 mg m-cresol-C L^?1. m-Cresol induced a significant decrease in the values of both specific rates of ammonium and nitrite oxidation, being the ammonium oxidation pathway the mainly inhibited. The nitrifying sludge was able to completely oxidize up to 150 mg m-cresol-C L^?1 by SBR cycle, reaching a maximum specific removal rate of 6.45 g m-cresol g^?1 microbial protein-N h^?1. The number of SBR cycles allowed a metabolic adaptation of the nitrifying consortium since nitrification inhibition decreased and faster oxidation of m-cresol took place throughout the cycles.     

Feeding elicitors and precursors enhance colchicine accumulation in morphogenic cultures of <Emphasis Type="Italic">Gloriosa superba</Emphasis> L.

Morphogenic cultures of Gloriosa superba were initiated on Murashige and Skoog’s medium fortified with 2 mg L^?1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg L^?1 naphthaleneacetic acid (NAA), 4% sucrose and 0.1% activated charcoal. To enhance the content of the alkaloid colchicine, morphogenic cultures were treated with different concentrations of abiotic elicitors like signalling compounds, metals, biotic elicitors, precursors and a combination of elicitors. Signalling molecules like acetyl salicylic acid (ASA) and sodium nitroprusside improved the production of colchicine. Abiotic elicitors have markedly (p?≤?0.05 or ≤?0.01) enhanced the colchicine content either at lower or higher concentrations. Among the metals, the highest amount of 11.67 mg of colchicine g^?1 dry wt was noticed at 60 mM rubidium chloride, followed by 60 mM NaCl (11.18 mg g^?1). Contrarily, in the presence of biotic elicitors such as Fusarium oxysporum, Alternaria solani, and Saccharomyces cerevisiae, colchicine content ranged only between 2 and 5.32 mg g^?1, but Bacillus subtilis repressed it. Among the aromatic amino acids, phenylalanine at 500 mg L^?1 influenced the highest accumulation of 19.48 mg g^?1 dry tissue, followed by tryptophan (12.47 mg g^?1), and tyrosine (9.87 mg g^?1), a direct precursor of colchicine biosynthesis, while intact tubers and leaves contained 4.65 and 4.16 mg of colchicine g^?1 dry tissue respectively. A combination of 10 µM AlCl₃ and 50 µM salicylic acid (SA) registered 17.34 mg g^?1 followed by 16.24 mg g^?1 tissue in presence of 1 µM HgCl₂ and 50 µM SA. The results suggest that the elicitor-stimulated colchicine accumulation was a stress response and can be exploited further for commercial production.     

Plantlet formation via somatic embryogenesis and LC ESI Q-TOF MS determination of secondary metabolites in <Emphasis Type="Italic">Butea monosperma</Emphasis> (Lam.) Kuntze

Medicinal properties of Butea monosperma (BM) and overexploitation of bark as a rich source of flavonoids for different biological activities, development of efficient method for high frequency somatic embryos and in vitro synthesis of bioactive secondary metabolites using plant tissue culture technology is important. Initially, callus was induced from leaf explants of BM on Murashige and Skoog (MS) medium containing 0.25 mg L^?1 2,4-d-dichlorophenoxyacetic acid (2,4-d) with 0.1 mg L^?1 kinetin (Kn) and ascorbic acid (AA). MS half strength macronutrients and full strength micronutrients containing 0.25 mg L^?1 2,4-d with 0.1 mg L^?1 Kn, and 0.5 mg L^?1 AA provided fragile callus with 84.0 ± 1.00 % optimal growth response. Shoot formation occurred via somatic embryogenesis through an intermediary callus phase. However, 2.1 mg L^?1 thidiazuron with 0.5 mg L^?1 AA provides high frequency (79.6 ± 2.02 %) of somatic embryogenesis within 5 weeks. Developed embryos when transferred to woody plant medium containing 0.5 mg L^?1 AA with 3.0 mg L^?1 Kn and 0.5 mg L^?1 α naphthalene acetic acid responded 44.0 ± 0.00 % embryo maturation, whereas 0.5 mg L^?1 Kn, 0.3 mg L^?1 indole-3-butyric acid, and 0.25 mg L^?1 AA induced rooting within 6 and 8 weeks, respectively. Liquid chromatography electro spray ionization quadrupole time of flight mass spectrometry (LC ESI Q-TOF MS) analysis of in vitro cultures showed similarity to those compounds identified in wild grown leaf samples known for osteogenic activity. Histological investigation through scanning electron microscopy demonstrates the developmental stages of somatic embryos, shoot bud formation, and induction of root primordial.     

<Emphasis Type="Italic">In vitro</Emphasis> preservation and micropropagation of <Emphasis Type="Italic">Oreocharis mileense</Emphasis> (W.T. Wang) M. Möller &amp; A. Weber (Gesneriaceae) through shoot organogenesis

Oreocharis mileense (W.T. Wang) M. Möller & A. Weber is endemic to China and was considered to be extinct because it had not been seen in the wild since the first collection in 1906. In 2006, the species was rediscovered in Shilin County, Yunnan Province. Oreocharis mileense was considered critically endangered for its narrow geographic range and extremely small population. An efficient method to preserve plant germplasm by in vitro culturing of O. mileense has not been reported. In this study, an orthogonal array with three factors (6-benzyladenine, BA; α-naphthaleneacetic acid, NAA; and sucrose), at four levels was performed, and shoot induction as well as shoot proliferation were recorded. The results were analyzed to determine the most significant components and the optimum combination for micropropagation of O. mileense. The results showed that: (1) organogenesis was easily induced by different combinations of plant-growth regulators and sucrose; (2) NAA and sucrose had the most significant effect on shoot induction and shoot multiplication, and (3) the optimum induction and proliferation media were 0.5 mg L^?1 BA + 0.2 mg L^?1 NAA?+?30 g L^?1 sucrose and 1 mg L^?1 BA + 0.1 mg L^?1 NAA?+?30 g L^?1 sucrose, respectively.     

Sulphur application improves the growth, seed yield and oil quality of canola

Sulphur (S) nutrition is very important for harvesting potential seed and oil yield of rapeseed. This study evaluated response of foliage applied thiourea on the performance of two canola cultivars Shiralee and Dunkeld. Sulphur was applied to soil (40 kg ha^?1) or foliage (500 and 1,000 mg L^?1) at rosette, bud initiation and flowering stages using elemental S or thiourea as source, respectively; no S application was taken as control. Among all the treatments, soil application of S improved the crop growth, yield and oil quality in both cultivars and was followed by foliar application of thiourea at 1,000 mg L^?1 compared with no application. Soil applied S and foliar thiourea (1,000 mg L^?1) delayed the flowering and maturity. Soil and foliar applied S significantly improved leaf area index, crop growth rate, net assimilation rate and chlorophyll contents. Plant height, number of branches, siliqua per plant, seed number per siliqua, 1,000-seed weight, biological and seed yield were also increased by soil applied S and foliage applied thiourea (1,000 mg L^?1). Nonetheless, improvement in harvest index, seed oil, protein and glucosinolate contents was only observed from foliage applied thiourea (1,000 mg L^?1). Response of cv. Shiralee to sulphur application was better than cv. Dunkeld. In conclusion, foliar applied thiourea (1,000 mg L^?1) can have potential to improve growth, yield and oil quality in canola and can be economically viable and attractive alternative source.     

Effect of β-lactam antibiotics on plant regeneration in carrot protoplast cultures

Protoplasts of three carrot cultivars were isolated from in vitro-grown plantlets by overnight incubation in an enzyme mixture composed of 1% (w/v) cellulase Onozuka R-10 and 0.1% (w/v) pectolyase Y-23. After cell immobilization in modified thin alginate layers, three types of β-lactam antibiotics (cefotaxime, carbenicillin, or timentin) at five different concentrations (100, 200, 300, 400, or 500 mg L^?1) were added to the culture medium. In 20-d-old cultures, a different number of cell colonies had formed and varied on average from 27 to 56% in carbenicillin- and cefotaxime-containing media, respectively. Supplementation of the culture media with antibiotics at concentrations higher than 100 mg L^?1 resulted in a decrease in plating efficiency in comparison with the controls. However, from all antibiotic treatments, except carbenicillin at concentrations of 400–500 mg L^?1, efficient plant regeneration occurred. For this reason, we believe that cefotaxime and timentin in the concentrations analyzed here may be used in complex in vitro procedures or valuable carrot cultures as a prophylactic agent for prevention against occasional contaminations.