Quercetin and silver nitrate modulate organogenesis in <Emphasis Type="Italic">Carissa carandas</Emphasis> (L.)

An in vitro organogenesis protocol for Carissa carandas L. was developed using an auxin transport inhibitor (quercetin) and silver nitrate (AgNO₃), an inhibitor of ethylene action, in association with cytokinins in the culture medium. This protocol produced the maximum number of shoots from aseptic seedling-derived shoot apex explants of C. carandas. The highest rate of shoot multiplication was recorded on MS medium containing 2.0 mg L^?1 6-benzylaminopurine; 0.5 mg L^?1 kinetin, and 0.75 mg L^?1 quercetin at after 4 wk of culture. Similar results were obtained when MS medium fortified with 2.0 mg L^?1 BAP, 0.5 mg L^?1 kinetin, and 1.5 mg L^?1 AgNO₃ was used. However, successful rooting was achieved on quarter strength MS medium with 0.5 mg L^?1 indole-3-acetic acid. In this study, an inhibitor of auxin transport and ethylene action maximized shoot multiplication in medium fortified with cytokinins. The established rapid micropropagation method could be used to conserve elite genotypes of C. carandas.     

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.     

Evaluation of fermentative potential of <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> ATCC 36907 in cellulosic and hemicellulosic sugarcane bagasse hydrolysates on xylitol and ethanol production

This paper evaluates the fermentative potential of Kluyveromyces marxianus grown in sugarcane bagasse cellulosic and hemicellulosic hydrolysates obtained by acid hydrolysis. Ethanol was obtained from a single glucose fermentation product, whereas xylose assimilation resulted in xylitol as the main product and ethanol as a by-product derived from the metabolism of this pentose. Fermentation performed in a simulated hydrolysate medium with a glucose concentration similar to that of the hydrolysate resulted in ethanol productivity (Qp?=?0.86 g L^?1 h^?1) that was tenfold higher than the one observed in the cellulosic hydrolysate. However, the use of hemicellulosic hydrolysate favored xylose assimilation in comparison with simulated medium with xylose and glucose concentrations similar to those found in this hydrolysate, without toxic compounds such as acetic acid and phenols. Under this condition, xylitol yield was 53.8 % higher in relation to simulated medium. Thus, the total removal of toxic compounds from the hydrolysate is not necessary to obtain bioproducts from lignocellulosic hydrolysates.     

Ribosome engineering and fermentation optimization leads to overproduction of tiancimycin A,a new enediyne natural product from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CB03234

Tiancimycin (TNM) A, a recently discovered enediyne natural product from Streptomyces sp. CB03234, showed rapid and complete killing of cancer cells and could be used as a payload in antibody drug conjugates. The low yield of TNM A in the wild-type strain promoted us to use ribosome engineering and fermentation optimization for its yield improvement. The Streptomyces sp. CB03234-R-16 mutant strain with a L422P mutation in RpoB, the RNA polymerase β-subunit, was obtained from the rifamycin-resistant screening. After fermentation optimization, the titers of TNM A in Streptomyces sp. CB03234-R-16 reached to 22.5 ± 3.1 mg L^?1 in shaking flasks, and 13 ± 1 mg L^?1 in 15 L fermentors, which were at least 40-fold higher than that in the wild-type strain (~ 0.3 mg L^?1). Quantitative real-time RT-PCR revealed markedly enhanced expression of key genes encoding TNM A biosynthetic enzymes and regulators in Streptomyces sp. CB03234-R-16. Our study should greatly facilitate the future efforts to develop TNM A into a clinical anticancer drug.     

Phycoremediation potential,physiological, and biochemical response of <Emphasis Type="Italic">Amphora subtropica</Emphasis> and <Emphasis Type="Italic">Dunaliella</Emphasis> sp. to nickel pollution

Metal pollution can produce many biological effects on aquatic environments. The marine diatom Amphora subtropica and the green alga Dunaliella sp. possess a high metal absorption capacity. Nickel (Ni) removal by living cells of A. subtropica and Dunaliella sp. was tested in cultures exposed to different Ni concentrations (100, 200, 300, and 500 mg L^?1). The amount of Ni removed by the microalgae increased with the time of exposure and the initial Ni concentration in the medium. The metal, which was mainly removed by bioadsorption to Dunaliella sp. cell surfaces (93.63% of total Ni (for 500 mg Ni L^?1) and by bioaccumulation (80.82% of total Ni (for 300 mg Ni L^?1) into Amphora subtropica cells, also inhibited growth. Exposure to Ni drastically reduced the carbohydrate and protein concentrations and increased total lipids from 6.3 to 43.1 pg cell^?1, phenolics 0.092 to 0.257 mg GAE g^?1 (Fw), and carotenoid content, from 0.08 to 0.59 mg g^?1 (Fw), in A. subtropica. In Dunaliella sp., total lipids increased from 26.1 to 65.3 pg cell^?1, phenolics from 0.084 to 0.289 mg GAE g^?1 (Fw), and carotenoid content from 0.41 to 0.97 mg g^?1 (Fw). These compounds had an important role in protecting the algae against ROS generated by Ni. In order to cope with Ni stress shown by the increase of TBARS level, enzymatic (SOD, CAT, and GPx) ROS scavenging mechanisms were induced.     

Enhancing carotenoid production in <Emphasis Type="Italic">Rhodotorula mucilaginosa</Emphasis> KC8 by combining mutation and metabolic engineering

Rhodotorula mucilaginosa has been considered as a potential industrial yeast due to its unicellular and fast-growing characteristics, and its ability to produce carotenoids, including torularhodin. However, its low total carotenoid production limits its commercial application. In this study, mutation breeding and metabolic engineering were employed to enhance carotenoid production in the R. mucilaginosa strain KC8. After chemical–physical mutagenesis, R. mucilaginosa K4 with a 67% greater concentration of carotenoids (14.47 ± 0.06 mg L^?1) than R. mucilaginosa KC8 (8.67 ± 0.07 mg L^?1) was obtained. To further enhance carotenoid production, gene HMG1 encoding the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was introduced from another yeast, Saccharomyces cerevisiae, and overexpressed in R. mucilaginosa K4. The carotenoid production of HMG1-gene-overexpression transformant G1 reached 16.98 mg L^?1. To relieve the feedback inhibition of ergosterol, and to down-regulate ergosterol synthesis, ketoconazole, an ergosterol synthesis inhibitor, was added at a concentration of 28 mg L^?1. The carotenoid production of the transformant G1 reached 19.14 ± 0.09 mg L^?1, which was 121% higher than in R. mucilaginosa KC8. This suggests that a combination of chemical–physical mutagenesis, overexpression of the HMG1 gene, and adding ketoconazole is an effective strategy to improve carotenoid production.     

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.     

A rapid and efficient in vitro shoot regeneration protocol using cotyledons of London plane tree (<Emphasis Type="Italic">Platanus acerifolia</Emphasis> Willd.)

A rapid, prolific and reproducible protocol for in vitro shoot regeneration from mature cotyledons of Platanus acerifolia has been developed. The influences of different plant growth regulator (PGR) combinations and donor seedling ages on shoot regeneration were investigated. The results showed that the application of BA in conjunction with NAA was the most effective PGR combination for the induction of shoot regeneration. When cotyledon explants of 5-day-old seedlings were incubated on MS basal medium supplemented with 4.0 mg L^?1 BA and 0.2 mg L^?1 NAA, 67.6?±?4.9% of the cotyledon segments produced adventitious shoots. These regenerated shoots were initially formed as stunted rosette cluster forms and were encouraged to elongate to produce distinct shoots by transfer onto MS medium containing 0.5 mg L^?1 BA and 0.05 mg L^?1 NAA; the resulting mean number of adventitious shoots per explant was 5.81?±?0.36. The elongated shoots were readily induced to root (i.e. 89.3% of shoots) by incubation on ½-strength MS medium supplemented with 0.1 mg L^?1 IBA. This is the first report of an efficient in vitro shoot regeneration protocol for P. acerifolia through direct organogenesis using cotyledon explants. Hence, this provides a more efficient basis for the Agrobacterium-mediated genetic transformation of Platanus than previously available.     

Acetaldehyde kinetics of enological yeast during alcoholic fermentation in grape must

Acetaldehyde strongly binds to the wine preservative SO₂ and, on average, causes 50–70 mg l^?1 of bound SO₂ in red and white wines, respectively. Therefore, a reduction of bound and total SO₂ concentrations necessitates knowledge of the factors that affect final acetaldehyde concentrations in wines. This study provides a comprehensive analysis of the acetaldehyde production and degradation kinetics of 26 yeast strains of oenological relevance during alcoholic fermentation in must under controlled anaerobic conditions. Saccharomyces cerevisiae and non-Saccharomyces strains displayed similar metabolic kinetics where acetaldehyde reached an initial peak value at the beginning of fermentations followed by partial reutilization. Quantitatively, the range of values obtained for non-Saccharomyces strains greatly exceeded the variability among the S. cerevisiae strains tested. Non-Saccharomyces strains of the species C. vini, H. anomala, H. uvarum, and M. pulcherrima led to low acetaldehyde residues (<10 mg l^?1), while C. stellata, Z. bailii, and, especially, a S. pombe strain led to large residues (24–48 mg l^?1). Acetaldehyde residues in S. cerevisiae cultures were intermediate and less dispersed (14–34 mg l^?1). Addition of SO₂ to Chardonnay must triggered significant increases in acetaldehyde formation and residual acetaldehyde. On average, 0.33 mg of residual acetaldehyde remained per mg of SO₂ added to must, corresponding to an increase of 0.47 mg of bound SO₂ per mg of SO₂ added. This research demonstrates that certain non-Saccharomyces strains display acetaldehyde kinetics that would be suitable to reduce residual acetaldehyde, and hence, bound-SO₂ levels in grape wines. The acetaldehyde formation potential may be included as strain selection argument in view of reducing preservative SO₂ concentrations.     

Potential Outdoor Cultivation of Green Microalgae Based on Response to Changing Temperatures and by Combining with Air Temperature Occurrence

In this study, our working hypothesis was to examine whether temperature alters biomass and metabolite production by microalgae according to strain. We also addressed whether it is possible to choose a strain suitable for growing in each season of a given region. A factorial experiment revealed a significant interaction between chlorophylls a and b (Chl a and Chl b), carotenoid/Chl (a?+?b) ratio, biomass and total lipid productivity of six green microalgae (four Chlorella spp., Chlorella sorokiniana and Neochloris oleoabundans) after 15 days at four temperatures. At 39/35 °C, two Chlorella sp. strains (IPR7115 and IPR7117) showed higher total carotenoids/Chl (a?+?b) (0.578 and 0.830), respectively. N. oleoabundans had the highest Chl a (8210 μg L^?1) and Chl b (1909 μg L^?1) at 19/15 °C and highest maximum dry biomass (2900 mg L^?1), specific growth rate (0.538 day^?1) and total lipids (1003 mg L^?1) at 15/8 °C. We applied a method to infer the growth of these six green microalgae in outdoor ponds, as based on their response to changing temperatures and by combining with historical data on day/night air temperature occurrence for a given region. We conclude that the use of regionalized maps based on air temperature is a good strategy for predicting microalgal cultivation in outdoor ponds based on their features and tolerance to changing temperature.     

Engineering rTCA pathway and C4-dicarboxylate transporter for <Emphasis Type="SmallCaps">l</Emphasis>-malic acid production

l-Malic acid is an important component of a vast array of food additives, antioxidants, disincrustants, pharmaceuticals, and cosmetics. Here, we presented a pathway optimization strategy and a transporter modification approach to reconstruct the l-malic acid biosynthesis pathway and transport system, respectively. First, pyruvate carboxylase (pyc) and malate dehydrogenase (mdh) from Aspergillus flavus and Rhizopus oryzae were combinatorially overexpressed to construct the reductive tricarboxylic acid (rTCA) pathway for l-malic acid biosynthesis. Second, the l-malic acid transporter (Spmae) from Schizosaccharomyces pombe was engineered by removing the ubiquitination motification to enhance the l-malic acid efflux system. Finally, the l-malic acid pathway was optimized by controlling gene expression levels, and the final l-malic acid concentration, yield, and productivity were up to 30.25 g L^?1, 0.30 g g^?1, and 0.32 g L^?1 h^?1 in the resulting strain W4209 with CaCO₃ as a neutralizing agent, respectively. In addition, these corresponding parameters of pyruvic acid remained at 30.75 g L^?1, 0.31 g g^?1, and 0.32 g L^?1 h^?1, respectively. The metabolic engineering strategy used here will be useful for efficient production of l-malic acid and other chemicals.     

Nutrient removal from high strength nitrate containing industrial wastewater using <Emphasis Type="Italic">Chlorella</Emphasis> sp. strain ACUF_802

the research aim of this study was to characterize an isolated native strain of Chlorella sp. ACUF_802, well adapted to a high nitrate concentration environment and to investigate its potential to nitrate and phosphate removal from industrial wastewaters with the minimal addition of chemical reagents and energy. The isolated strain was identified and evaluated for its capability to support biomass growth and nutrient removal from synthetic wastewater in batch tests using different concentrations of carbon and nitrogen, different carbon sources and N:P ratios. The strain was isolated via the plating method from the settler of a pilot scale moving bed biofilm reactor performing a nitrification process. The strain was identified using molecular analysis with rDNA primers. Using sodium bicarbonate as carbon source, the batch productivity (71.43 mg L^?1 day^?1) of the strain Chlorella sp. ACUF_802 was calculated with a logistic model and compared to the values reported in the literature. Assays on the effect of the N:P ratio indicated that the productivity was increased 36% when the N:P ratio was close to 1 (111.96 mg L^?1 day^?1), but for a complete phosphorus removal a 5:1 N:P ratio with nitrate concentrations ≤125 mg?L^?1 is recommended. The isolated microalgae strain Chlorella sp. ACUF_802 showed versatility to grow in the synthetic industrial wastewaters tested and can be considered as an appropriate organism for nitrogen removal from industrial wastewaters in the presence of an organic or inorganic carbon source.     

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.     

<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.     

Effect of over-expression of <Emphasis Type="Italic">Linum usitatissimum</Emphasis> PINORESINOL LARICIRESINOL REDUCTASE (<Emphasis Type="Italic">LuPLR</Emphasis>) gene in transgenic <Emphasis Type="Italic">Phyllanthus amarus</Emphasis>

Shoot tip explants of Phyllanthus amarus were cocultivated with Agrobacterium tumefaciens strain LBA 4404 carrying plasmid pCAMBIA 2301 harbouring genes coding for betaglucuronidase (gus), kanamycin (kan), and neomycin phosphotransferase II (nptII) along with a gene coding for Linum usitatissimum PINORESINOL LARICIRESINOL REDUCTASE (Lu-PLR). Transformed shoot tip explants were maintained in a Murashige and Skoog (MS) medium containing TDZ 1.54 mg l^?1, kan 50 mg l^?1 and cephotaxime 62.5 mg l^?1. The optimum medium for regeneration of multiple shoots was MS supplemented with TDZ 1.54 mg l^?1, kan 50 mg l^?1. Efficient and effective rooting of plantlets was achieved by culturing the in vitro regenerated shoots on liquid ½ MS medium containing 0.7 mg l^?1 indole 3-butyric acid (IBA) and 5 mg l^?1 kan. Rooted plants were acclimatized in the mixtures of vermiculite and soil. The transformation of kan-resistant plantlets regenerated from shoot-tip explants was confirmed by GUS and polymerase chain reaction (PCR) analysis. Southern blot and reverse transcribed PCR (RT-PCR) analysis confirmed successful integration and expression of Lu-PLR gene. Quantitative analysis of phyllanthin performed on transgenic and wild plants using high-performance liquid chromatography (HPLC) revealed that transgenic lines contained higher phyllanthin content (0.3–0.81% w/w) than wild plants (0.09% w/w). The highest yield of phyllanthin was detected in transgenic lines was up to 1.16, 1.22 and 1.23 folds higher than that of wild plant. This report highlights the transgenic approach to enhance the contents of phyllanthin and hypophyllanthin.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration,production, and storage of artificial seeds in <Emphasis Type="Italic">Ceropegia barnesii</Emphasis>, an endangered plant

Approaches for in vitro regeneration and fabrication of synthetic seeds were formulated to support restoration in the wild and genetic manipulation of Ceropegia barnesii (categorized as endemic and endangered). MS medium augmented with 4 mg L^?1 benzyl adenine was most advantageous for the production of multiple shoots from nodal explants. Fabrication of synthetic seeds was accomplished by sodium alginate encapsulation of nodes from microshoots. The most favorable medium combination for the induction of multiple shoots from synthetic seeds was MS medium complemented with 4 mg L^?1 benzyl adenine and 1 mg L^?1 gibberelic acid. Following root induction promoted by half strength MS basal medium augmented with indolebutyric acid, multiple shoots were subjected to hardening. Influence of vesicular-arbuscular mycorrhizal fungi on the hardening trials was investigated and it was observed that dual inoculation of Glomus aggregatum and G. intraradices enhanced the survival rate. The encapsulated nodes of C. barnesii were tested for their capability to endure different temperatures during storage and the optimal temperature for storage was found to be 4°C. A methodology for initiation of somatic embryogenesis from C. barnesii is also reported here, but embryos could not be induced to develop further. The micropropagated plants were reintroduced in to their natural habitat. This is the first report on micropropagation of C. barnesii.     

Micropropagation of <Emphasis Type="Italic">Glossonema varians</Emphasis> (Stocks) Benth. ex Hook.f.—a rare Asclepiadeae of Indian Thar Desert

In the present study, an in vitro regeneration protocol for Glossonema varians (Stocks) Benth. ex Hook.f. of family Asclepiadaceae was optimized. Cotyledonary nodes of in vitro cultured seedlings were used as explants for activation of axillary shoot buds. Axillary shoot buds were initially activated on 0.1 mg L^?1 6-benzylaminopurine (BAP) and then multiplied on 0.05 mg L^?1 BAP. Shoots were rooted in vitro on 1/4 strength Murashige and Skoog medium containing 0.1 mg L^?1 2-naphthoxyacetic acid and 100 mg L^?1 activated charcoal. The cultures were maintained in a 12 h photoperiod at 40–50 μmol m^?2 s^?1 spectral flux photon, 25–30?±?2°C, and 60% relative humidity (RH). Up to 80% of in vitro regenerated plantlets were acclimatized on soilrite in cotton-plugged culture tubes in the greenhouse. This protocol can be a useful method to mass propagate and conserve this rare plant to balance biodiversity in the desert ecosystem.     

Iron homeostasis in tropical <Emphasis Type="Italic">indica</Emphasis> rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) cultivars having contrasting grain iron concentration

Iron homeostasis was studied in two tropical indica rice cultivars viz. Sharbati (high Fe) and Lalat (low Fe) having contrasting grain Fe concentration. Plants were hydroponically grown with 5 concentrations of Fe (0.05, 2, 5, 15, 50 mg L^?1) till maturity. The effect of incremental Fe treatment on the plant was followed by analyzing accumulation of ferritin protein, activities of aconitase enzyme, enzymes of anti-oxidative defense and accumulation of hydrogen peroxide and ascorbic acid. Plant growth was adversely affected beyond 15 mg L^?1 of Fe supplementation and effects of Fe stress (both deficiency and excess) were more apparent on the high Fe containing cultivar Sharbati than the low Fe containing Lalat. Level of ferritin protein and aconitase activity increased up to 5 mg L^?1 of Fe concentration. Lalat continued to synthesize ferritin protein at much higher Fe level than Sharbati and the cultivar also had higher activities of peroxidase, superoxide dismutase and glutathione reductase. It was concluded that the tolerance of Lalat to Fe stress was because of its higher intrinsic ability to scavenge free radicals of oxidative stress for possessing higher activity of antioxidative enzymes. This, together with its capacity to sequester the excess Fe in ferritin protein over a wider range of Fe concentrations made it more tolerant to Fe stress.     

An efficient protocol for <Emphasis Type="Italic">in vitro</Emphasis> regeneration and conservation of Shirui lily (<Emphasis Type="Italic">Lilium mackliniae</Emphasis> Sealy): a lab-to-land approach to save the rare endangered Asiatic lily species

An efficient protocol for direct and indirect shoot regeneration and proliferation from bulb scales of Shirui lily (Lilium mackliniae Sealy), an endangered Asiatic lily species endemic to the Shirui hill peak, Manipur, India, has been developed. Bulb scales were isolated from mature bulbs and cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of 6-benzylaminopurine (BAP), kinetin (KIN), or thidiazuron (TDZ). For direct shoot regeneration from bulb scale explants, 0.5 mg L^?1 BAP yielded the highest shoot induction (3.5 shoots per scale; a 96.7% response). For indirect de novo organogenesis, optimum callus induction was achieved with 2.0 mg L^?1 2,4-dichlorophenoxyacetic acid (2,4-D), and shoot organogenesis was higher (16.2) when subcultured onto 0.5 mg L^?1 BAP medium. Multiple shoot regeneration and pseudo-bulb formation protocols were assessed; the highest shoot proliferation (10.1) occurred with 0.5 mg L^?1 BAP and 1.0 mg L^?1 gibberellic acid (GA₃). Rooting response was 96% with 0.5 mg L^?1 1-naphthalene acetic acid (NAA), with multiple roots per shootlet. Plantlet survival was increased to 92.5% during the hardening-off process by using hydroponics with Hoagland’s solution in a mist chamber. Clonal fidelity was assessed through random amplified polymorphic DNA (RAPD) analysis comparing the mother plant and regenerated plantlets. After confirming genetic uniformity, the pseudo-bulblets with four to six leaves and three to four roots were successfully established at the Shirui hills peak. This in vitro regeneration and ex vitro conservation approach could be helpful to save this rare endangered species in a sustainable way.     

Exploring potential applications of a novel extracellular polymeric substance synthesizing bacterium (<Emphasis Type="Italic">Bacillus licheniformis</Emphasis>) isolated from gut contents of earthworm (<Emphasis Type="Italic">Metaphire posthuma</Emphasis>) in environmental remediation

The aim was to isolate, characterize, and explore potentials of gut bacteria from the earthworm (Metaphire posthuma) and imply these bacteria for remediation of Cu(II) and Zn(II). An extracellular polymeric substance (EPS) producing gut bacteria (Bacillus licheniformis strain KX657843) was isolated and identified based on 16S rRNA sequencing and phylogenetic analysis. The strain showed maximum tolerance of 8 and 6 mM for Cu(II) and Zn(II) respectively. It removed 34.5% of Cu(II) and 54.4% of Zn(II) at 25 mg L^?1 after 72 and 96 h incubation respectively. The bacteria possessed a great potential to produce indole acetic acid (38.49 μg mL^?1) at 5 mg mL^?1 l-tryptophan following 12 days incubation. The sterilized seeds of mung beans (Vigna radiata) displayed greater germination and growth under bacterium enriched condition. We observed that the bacterial strain phosphate solubilization ability with a maximum of 204.2 mg L^?1 in absence of Cu(II) and Zn(II). Endowed with biosurfactant property the bacterium exhibited 24% emulsification index. The bacterium offered significant potential of plant growth promotion, Cu(II) and Zn(II) removal, and as such this study is the first report on EPS producing B. licheniformis KX657843 from earthworm which can be applied as powerful tool in remediation programs of Cu(II) and Zn(II) contaminated sites.