Plant regeneration of non-toxic Jatropha curcas—impacts of plant growth regulators, source and type of explants

Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel plant, however, oil and deoiled cake are toxic. A non-toxic variety of J. curcas is reported from Mexico. The present investigation explores the effects of different plant growth regulators (PGRs) viz. 6-benzyl aminopurine (BAP) or thidiazuron (TDZ) individually and in combination with indole-3-butyric acid (IBA), on regeneration from in vitro and field-grown mature leaf explants, in vitro and glasshouse-grown seedlings cotyledonary leaf explants of non-toxic J. curcas. In all the tested parameters maximum regeneration efficiency (81.07%) and the number of shoot buds per explants (20.17) was observed on 9.08 μM TDZ containing Murashige and Skoog’s (MS) medium from in vitro cotyledonary leaf explants. The regenerated 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 2.25 μM BAP and 8.5 μM IAA. 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 four days followed by transfer to growth regulators free half strength MS medium supplemented 0.25 mg/l activated charcoal. The rooted plants could be established in soil with more than 90% survival rate.     

Enhancing seedling growth of Jatropha curcas—A potential oil seed crop for biodiesel

The effects of aerosol smoke (AS), smoke-water (SW), potassium nitrate (KNO₃), naphthalene acetic acid (NAA) and indole-3-butyric acid (IBA) on germination and seedling growth of Jatropha curcas were investigated. Seed coat removal accelerated water imbibition and germination occurred within 48 h. Seeds subjected to AS failed to germinate over a 90 day period. There were no significant differences in germination percentage between the treatments and untreated control (intact- and shelled-seed). However, shelled-seeds had the shortest mean germination time (MGT). Seedlings developed from treated seeds were planted in trays under shade house conditions and growth traits measured after 3 months. Soaking intact-seeds in SW, KNO₃ and NAA (24 h) produced significantly heavier and longer seedlings, which resulted in higher vigour indices (VI) compared to the control treatments. These results provide empirical evidence of the stimulatory effect of SW, KNO₃ and NAA on J. curcas seedling growth and vigour and the continuation of the effect over time. The approach of treating intact-seeds of J. curcas with plant growth substances prior to planting will help in producing healthy seedlings and possibly improve crop productivity.     

Production of biodiesel from Jatropha curcas L. oil catalyzed by SO₄²⁻/ZrO₂ catalyst: effect of interaction between process variables

This study reports the conversion of Jatrophacurcas L. oil to biodiesel catalyzed by sulfated zirconia loaded on alumina catalyst using response surface methodology (RSM), specifically to study the effect of interaction between process variables on the yield of biodiesel. The transesterification process variables studied were reaction temperature, reaction duration, molar ratio of methanol to oil and catalyst loading. Results from this study revealed that individual as well as interaction between variables significantly affect the yield of biodiesel. With this information, it was found that 4h of reaction at 150°C, methanol to oil molar ratio of 9.88 mol/mol and 7.61 wt.% for catalyst loading gave an optimum biodiesel yield of 90.32 wt.%. The fuel properties of Jatropha biodiesel were characterized and it indeed met the specification for biodiesel according to ASTM D6751.     

An Efficient and Reliable Method of DNA Extraction from Meyna spinosa — A Traditional Medicinal Plant from North-East India

A simple, efficient and reliable CTAB method is standardized for genomic DNA isolation from fresh young leaves of a traditional medicinal plant Meyna spinosa. Key steps in the modified procedure include additional chloroform: isoamyl alcohol (24:1, v/v) extraction, addition of 4% PVP in the extraction buffer and an overnight isopropanol precipitation at room temperature. This procedure yields a high amount (46 μg DNA g^?1 fresh leaf tissue) of good quality DNA free from contaminants. The isolated DNA is suitable for digestion with EcoRI and HindIII restriction enzymes and can be used in other DNA manipulation techniques.     

In vitro bioaccessibility of metals from tape tea – A low-cost emerging drug

BackgroundAn in vitro physiologically relevant test based on the standard Unified Bioaccessibility Method (UBM) combined with inductively coupled plasma mass spectrometry was performed in this study to ascertain the elemental bioaccessibility pools of tape tea as emerging low-cost abuse drug under fasted conditions.MethodsElemental quantification in tape tea and body fluid extracts was performed by an inductively coupled plasma quadrupole mass spectrometer - ICP-MS, and for sample preparation of the bioaccessibility extracts prior to ICP-MS analysis, a microwave-assisted acid decomposition was applied by using a microwave oven. The Unified Bioaccessibility Method (UBM) was considered for investigation of elemental bioaccessibility in tape tea, required a full set of organic compounds, salts, and enzymes.ResultsConsidering total element evaluation through ICP-MS, Co, Ni, Mn, and Zn are found at the highest concentrations in the sample, namely 415 ± 36, 202 ± 55, 1389 ± 225 and 2397 ± 197 μg L⁻¹, respectively. Regarding the oral bioaccessibility test, after both gastric and gastrointestinal extractions Co, Ni, and Mn are fully bioaccessible while for Zn the bioaccessibility is ca. 66 %.ConclusionAccording to the first results in the literature proposed for these samples, the bioaccessibility results indicate an increment in day-to-day total element concentration and depending on the concentration of each element that an individual consumes in its usual diet, the total concentration can exceed the TDI. There are several possible toxic effects caused by the excess of Co, Ni and Mn, which might be expected by their high total concentrations.     

In vitro development of plantlets from axillary buds of Acacia auriculiformis — a leguminous tree

Multiple shoots have developed from axillary buds excised from in vitro grown seedlings of Acacia auriculiformis on Gamborg's (B5) basal medium supplemented with coconut milk (5 or 10%) and benzylaminopurine (10^-6M). These shoots, if transferred individually to indole-3-acetic acid (10^-7M) or naphthaleneacetic acid (10^-6 or 10^-7M) augmented B5 medium, produced roots at their base.Abbreviations B5 Gamborg's basal medium (BM) - CM coconut milk - BA 6-benzylaminopurine - Kn kinetin - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid     

In vitro pollination of maize (Zea mays L.) — Proof of double fertilization

Isolated ovules from the maize homozygous recessive brown midrib (bm₃) were in vitro pollinated by pollen carrying the dominant allele — and the purple embryo marker (PEM). The colour characters of the embryos and kernels corresponded to the results of control pollination and confirmed the process of double fertilization. The question whether the method is useful for obtaining haploids is discussed.     

Effects of Cadmium Stress on Leaf Chlorophyll Fluorescence and Photosynthesis of Elsholtzia argyi—A Cadmium Accumulating Plant

A hydroponic experiment was conducted to investigate the effects of cadmium (Cd) on chlorophyll fluorescence and photosynthetic parameters on a Cd accumulating plant of Elsholtzia argyi. Four weeks-seedlings of E. argyi were treated with 0 (CK) 5, 10, 15, 20, 25, 30, 40, 50 and 100 μmol L^?1 Cd for 21days. Fv/Fo, Fv/Fm, qP, ΦPSП, ETR and Fv′/Fm′ were significantly increased under low Cd (5–15 μmol L^?1 for Fv/Fo, Fv/Fm and qP, 5–10 μmol L^?1 for ΦPSП, ETR and Fv′/Fm′) stress, and these parameters were similar to control under Cd ≤ 50μmol L^?1. All above parameters were significantly decreased at 100 μmol L^?1 Cd. Compared with control, Pn was significantly (P < 0.05) increased under 5–30 μmol L^?1 Cd. However, 50 and 100 μmol L^?1 Cd significantly (P < 0.05) reduced it. Gs and Tr were substantially decreased at 50–100 and 40–100 μmol L^?1 Cd, respectively. Ci was significantly increased at 50 and 100 μmol L^?1 Cd. High Cd-induced decrease of Pn is not only connected to stomatal limitation but also to the inhibition of Fv/Fo, Fv/Fm, ΦPSП, qP, ETR and increase of NPQ. Maintain chlorophyll fluorescence and photosynthesis parameters under its Cd tolerance threshold were one of tolerance mechanisms in E. argyi.     

Dimethyl Sulfoxide Is Feasible for Plant Tubulin Assembly In vitro： A Comprehensive Analysis

It is much more difficult for tubulin from plant sources to polymerize in vitro than tubulin from animal sources. Taxol, a most widely used reagent in microtubule studies, enhances plant microtubule assembly, but hinders microtubule dynamics. Dimethyl sulfoxide (DMSO), a widely used reagent in animal microtubule studies, is a good candidate for the investigation of plant microtubule assembly in vitro. However, proper investigation is lacking about the effects of DMSO on plant microtubule assembly in vitro. In the present study, DMSO was used to establish optimal conditions for the polymerization of plant tubulin. Tubulin, purified from lily pollen, polymerizes into microtubules at a critical concentration of 1.2 mg/mL in the presence of 10% DMSO. The polymers appear to have a normal microtubule structure, as revealed by electron microscopy. In the presence of 10% DMSO, microtubule polymerization decreases when the pH of the medium is increased from 6.5 to 7.4. Both the polymerization rate and the mass of the polymers increase as temperature increases from 25 to 40 ℃. Tubulin polymerizes and depolymerizes along with cycling of temperature, from 37 to 4 ℃, or following the addition to or the removal of Ca^2 from the medium. When incubated with nuclei isolated from tobacco BY-2 suspension cells, tubulin assembles onto the nuclear surface in the presence of 10% DMSO. Labeling lily pollen tubulin with 5- (and 6-) carboxytetramethyl-rhodamine succinimidyl ester (NHS-rhodamine) was performed successfully in the presence of 10% DMSO. Labeled tubulin assembles into a radial structure on the surface of BY-2 nuclei. The polymerization of lily pollen tubulin is also enhanced by microtubule-associated proteins from animal sources in the presence of 10% DMSO. All the experimental results indicate that plant tubulin functions normally in the presence of DMSO. Therefore, DMSO is an appropriate reagent for plant tubulin polymerization and investigation of plant microtubules in vitro.     

Amplectosporangium—A New Genus of Plant from the Lower Devonian of Sichuan,China

A new genus and species Amplectosporangium jiangyouense, is described from the Pingyipu Formation (Siegenian) of the Lower Devonian in Jiangyou district, Sichuan, China. The plant consists of naked dichotomizing erect axes with terminal fertile branch-systems. Sporangia are oval-shaped each with a short stalk and are arranged along the inner side of dichotomized axes in single row. By comparison with other known Devonian plants the genus is considered to represent a new morphological type of early land plants. Based on the theory of the telomic origin of the ovular integument, the plant may represent an early stage in the evolution of integument.     

In vitro morphogenic response of different explants of Gentiana kurroo Royle from Western Himalayas—an endangered medicinal plant

Micropropagation offers a great potential to produce millions of clonal individuals through tissue culture via induction of morphogenesis. The aim of this work was to obtain an efficient protocol for callus regeneration for Gentiana kurroo Royle. The morphogenic response of different explants (leaves, petioles, roots) varied and responded differently for regeneration according to combinations of growth regulators. The petiole explants were best responding for callus induction and subsequently for indirect and direct regeneration. The callus induction was achieved on MS basal + 1.0 mg/l benzyladenine (BA) and 3.00 mg/l naphthalene acetic acid (NAA). MS medium supplemented with 0.10 mg/l NAA and 1.0 mg/l thidiazuron (TDZ) was recorded as the best medium for indirect regeneration. However, for direct regeneration the maximum number of shoot emergence was observed on MS basal fortified with 0.10 mg/l NAA + 0.75 mg/l TDZ. Half strength MS basal supplemented with indole-3-butyric acid (IBA) 1.00 mg/l gave best response for root induction. Subsequently, the plantlets were transferred and 100 % survival rate was recorded only on autoclaved cocopeat. No morphological variations were recorded in the callus regenerated plantlets.     

A Rapid In Vitro Morphogenesis and Acclimatization Protocol for Balanites aegyptiaca (L) Del — a Medicinally Important Xerophytic Tree

The callus mediated regeneration system for Balanites aegyptiaca (L) Del is reported here. Different explants like apical buds, young thorns and cotyledon pieces from mature tree and root segments from in vitro raised seedlings were used for callus induction on MS medium supplemented with 2.23 μM 2,4-Dichlorophenoxyacetic acid. Seven to eight weeks old calli were transferred on hormone free MS medium in order to get regeneration. Shoot morphogenesis was achieved only from cotyledon-derived callus. The shoots so produced rooted well, when cultured on B5 medium supplemented with 9.84 μM Indole-3-butyric acid. Plantlets have been transferred to the field after two-phase hardening and are performing well.     

Efficient in vitro regeneration of fertile plants from corm explants of Hypoxis hemerocallidea landrace Gaza—The “African Potato”

We present efficient protocols for the regeneration of fertile plants from corm explants of Hypoxis hemerocallidea Fisch. & C. A. Mey. landrace Gaza, either by direct multiple shoot formation or via shoot organogenesis from corm-derived calluses. The regeneration efficiency depended on plant growth regulator concentrations and combinations. Multiple direct shoot formation with high frequency (100% with 5–8 shoots/explant) was obtained on a basal medium (BM) supplemented with 3 mg/l kinetin (BM1). However, efficient indirect regeneration occurred when corm explants were first plated on callus induction medium (BM2) with high kinetin (3 mg/l) and naphthalene acetic acid (NAA 1 mg/l), and then transferred to shoot inducing medium (BM3) containing BA (1.5 mg/l) and NAA (0.5 mg/l). Shoot regeneration frequency was 100% and 30–35 shoots per explant were obtained. The regenerated shoots were rooted on a root inducing medium (BM4) containing NAA (0.1 mg/l). Rooted plantlets were transferred to the greenhouse. The regenerants were morphologically normal and fertile. Flow cytometric analyses and chloroplast counts of guard cells suggested that the regenerants were diploid. Efficient cloning protocols described here, have the potential not only to substantially reduce the pressure on natural populations but also for wider biotechnological applications of Hypoxis hemerocallidea—an endangered medicinal plant.     

In vitro regeneration in chile pepper (Capsicum annuum L.) from ‘half-seed explants’

An in vitro regeneration protocol has been developed from half-seed explants of a mild (cv. New Mexico-6) and a pungent (cv. Rajpur Hirapur) chile pepper (Capsicum annuum L). Imbibed seeds were cut into two parts such that one portion contained the cotyledons and a part of the hypocotyl (part A) while the other part had the proximal part of the hypocotyl and the radicle (part B). These explants were cultured on MS medium with or without cytokinins (KIN, BA, ZEA, 2iP). Cytokinins dramatically increased both the percentage of explants forming buds as well as the number of buds per explant, and also hastened the rate of bud production. The relative efficacy of cytokinins in inducing the formation of leafy buds varied in the two cultivars. However, the best response was observed with ZEA in both cultivars. The highest percentage of bud formation was recorded after presoaking part B explants for 72 hours. The elongation growth of leafy buds was severely inhibited in the continuous presence of high concentrations of cytokinins, and frequently the buds became quite thick, ill-defined and vitreous. Within 3–5 weeks of transfer to Magenta boxes containing vermiculite and soil (1:3), 70–85% of the rooted hypocotyls developed 1–2 elongated shoots. Following transfer to pots, these plantlets grew into normal plants.Abbreviations BA benzylamino purine - IAA indole-3-acetic acid - 2iP 6--dimethyl (allyl) amino purine - KIN kinetin - MS Murashige and Skoog medium - NAA napthaleneacetic acid - ZEA zeatin     

In vitro induction of tumor necrosis factor-α by ochratoxin A (OTA) from rat liver: role of Kupffer cells

Tumor necrosis factor-α (TNF-α) is released from blood-free perfused rat liver by the fungal metabolite ochratoxin A. Here we have identified Kupffer cells as the sole source of OTA-mediated cytokine release. If single cell preparation of Kupffer cells, hepatocytes, or sinusoidal endothelial cells were prepared from rat livers, only Kupffer cells released TNF-α upon incubation with 2.5 μmol/l OTA. OTA failed to induce TNF-α release in the blood-free perfused isolated rat liver when Kupffer cells were blockedin vitro by 15 μmol/l gadolinium chloride. When rats were pretreatedin vivo with the Kupffer cell depleting clodronate liposomes, OTA-mediated TNF-α release was abrogated in the isolated perfused liver model.     

In vitro regeneration through organogenesis of Meconopsis simplicifolia — An endangered ornamental species

Meconopsis simplicifolia (D.Don) Walp. could be propagated by induction of adventitious shoots from callus produced on hypocotyl, cotyledon and rosette leaf explants of 4-month-old seedlings. Callus was initiated on agar-solidified Murashige and Skoog medium supplemented with 1 M kinetin +10 M -naphthaleneacetic acid (NAA). Shoots formed when the callus was subcultured on medium supplemented with kinetin or benzyladenine (BA) in combination with NAA, indoleacetic acid, indolebutyric acid or gibberellic acid. Excised shoots were rooted on medium containing auxin with 10 M NAA producing the best rooting (55%).Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxy-acetic acid - FAA formalin-acetic acid-alcohol - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA -indolebutyric acid - NAA -naphthaleneacetic acid