Somatic Embryogenesis and Synthetic Seed Production—a Biotechnological Approach for True-to-Type Propagation and In Vitro Conservation of an Ornamental Bulbaceous Plant Drimiopsis kirkii Baker.

An efficient plant regeneration protocol through indirect somatic embryogenesis pathway via callus had been developed from the leaf explant of an ornamental bulbaceous plant Drimiopsis kirkii. Optimum friable calli were induced on Murashige and Skoog (MS) basal medium supplemented with 3.0 mg/l of 2,4-dichlorophenoxyacetic acid and 1.0 mg/l of α-naphthalene acetic acid (NAA). On subculturing the callus on MS medium supplemented with 2.5 mg/l of thidiazuron (TDZ), 73.3 % of the cultures responded with 20.4?±?0.3 somatic embryos (SEs) per 500 mg callus at different stages of development after 6 weeks of culture. The highest response of 86.7 % with 28.3?±?0.5 embryos per 500 mg callus was observed on MS medium supplemented with 2.5 mg/l TDZ and 1.0 mg/l NAA. SEs were encapsulated in calcium alginate beads for the production of synthetic seeds (SSs) and their storability was investigated. The highest SS germination (93.3 %) was observed in 1.0 % sodium alginate followed by 86.7 % germination with 2.5 % sodium alginate. The SSs were stored at three different temperatures (4, 15, and 24?ºC) up to 6 months. The SSs kept at 15 °C showed 64.4 % germinability even after 4 months of storage. Both nonencapsulated and encapsulated SE-derived plants were successfully transferred to soil with 93.3 and 88.3 % survival rate accordingly. Randomly amplified polymorphic DNA (RAPD) analysis revealed that there were no somaclonal variations among the plants produced via somatic embryogenesis and they are true-to-type to their parental plant. These results confirmed the most reliable methods, which can be further used for genetic transformation studies as well as for mass propagation of ornamental D. kirkii at a commercial level.     

Piper nigrum: Micropropagation, Antioxidative enzyme activities, and Chromatographic Fingerprint Analysis for Quality Control

A reliable in vitro regeneration system for the economical and medicinally important Piper nigrum L. has been established. Callus and shoot regeneration was encouraged from leaf portions on Murashige and Skoog (MS) medium augmented with varied concentrations of plant growth regulators. A higher callus production (90 %) was observed in explants incubated on MS medium incorporated with 1.0 mg?L^?1 6-benzyladenine (BA) along with 0.5 mg?L^?1 gibberellic acid after 4 weeks of culture. Moreover, a callogenic response of 85 % was also recorded for 1.0 mg?L^?1 BA in combination with 0.25 mg?L^?1 α-naphthalene acetic acid (NAA) and 0.25 mg?L^?1 2,4-dichlorophenoxyacetic acid or 0.5 mg?L^?1 indole butyric acid (IBA) along with 0.25 mg?L^?1 NAA and indole acetic acid. Subsequent sub-culturing of callus after 4 weeks of culture onto MS medium supplemented with 1.5 mg?L^?1 thiodiazoran or 1.5 mg?L^?1 IBA induced 100 % shoot response. Rooted plantlets were achieved on medium containing varied concentrations of auxins. The antioxidative enzyme activities [superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX)] revealed that significantly higher SOD was observed in regenerated plantlets than in other tissues. However, POD, CAT, and APX were higher in callus than in other tissues. A high-performance liquid chromatography (HPLC) fingerprint analysis protocol was established for quality control in different in vitro-regenerated tissues of P. nigrum L. During analysis, most of the common peaks represent the active principle “piperine.” The chemical contents, especially piperine, showed variation from callus culture to whole plantlet regeneration. Based on the deviation in chromatographic peaks, the in vitro-regenerated plantlets exhibit a nearly similar piperine profile to acclimated plantlets. The in vitro regeneration system and HPLC fingerprint analysis established here brought a novel approach to the quality control of in vitro plantlets, producing metabolites of interest with substantial applications for the conservation of germplasm.     

Regeneration-Based Quantification of Coumarins (Scopoletin and Scoparone) in <Emphasis Type="Italic">Abutilon indicum</Emphasis> In Vitro Cultures

Abutilon indicum exploited for its immense value has been propagated successfully through multiple shoot induction and somatic embryogenesis. Direct regeneration (8.20?±?0.83 shoots) was achieved from nodal explants using 0.5 mg/l kinetin (Kn) in MS media. The basal callus from nodal explants turned embryogenic on subsequent introduction of 0.2 mg/l TDZ into the Kn-supplemented media, giving rise to somatic embryos. The embryogenic potential of calli expressed in terms of embryo-forming capacity (EFC) increased from 8.15 EFC to 20.95 EFC after plasmolysis. The phytochemical analysis (HPLC) for the presence of scopoletin and scoparone has revealed a unique accumulation pattern, with higher levels of scopoletin during the earlier stages and scoparone in the later stages of development. The embryogenic calli contained the highest amount of coumarins (99.20?±?0.97 and 61.03?±?0.47 μg/gFW, respectively) followed by regenerated plant (9.43?±?0.20 and 36.36?±?1.19 μg/gFW, respectively), obtained via somatic embryogenesis. Rapid multiplication of A. indicum equipped with two potent coumarins is important in order to meet the commercial demand for combat against dreadful diseases, thereby providing a new platform for plant-based drugs and their manufacture on a commercial scale.     

A Comparative Study of Anti-Candida Activity and Phenolic Contents of the Calluses from Lythrum salicaria L. in Different Treatments

In the study, anti-Candida activity and phenol contents of Lythrum salicaria L. calli and wild species have been evaluated. The seeds of L. salicaria (Lythraceae), collected from Lahidjan City in the north of Iran, were cultured in Murashige and Skoog medium (MSM) with a supplement, gibberellin, to germinate. Callus inductions were performed from segments of seedling on MSM containing different concentrations of plant growth regulators, 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP). The activity of calluses extracts, wild plant, gallic acid, and 3,3′,4′-tri-O-methylellagic acid-4-O-β-d-glucopyranoside (TMEG) as the main phenolic compounds against Candida albicans was assessed using cup plate diffusion method. The total phenols contents of calli and wild plant extracts were analyzed using Folin–Ciocalteu reagent. The callus formation in MSM supplemented with various concentrations of 2,4-D and BAP were 0–100 %. Anti-Candida activity of callus extract which obtained from MSM supplemented with 2,4-D and BAP (1 mg?dm^?3) was similar to the wild plant extract. Minimum inhibitory concentration values of gallic acid and TMEG were obtained as 0.312 and 2.5 mg?cm^?3, respectively. Gallic acid equivalent values in all treatments were from 0 to 288 μg GAE mg^?1. Phenolic contents of plant aerial parts (331?±?3.7 μg GAE mg^?1) and the callus, which developed in MSM including 1 mg?dm^?3 of both 2,4-D and BAP, showed the same phenolic value and exhibited anti-Candida extract activity.     

An Improved Micropropagation of Arnebia hispidissima (Lehm.) DC. and Assessment of Genetic Fidelity of Micropropagated Plants Using DNA-Based Molecular Markers

An efficient and improved in vitro propagation method has been developed for Arnebia hispidissima, a medicinally and pharmaceutically important plant species of arid and semiarid regions. Nodal segments (3–4 cm) with two to three nodes obtained from field grown plants were used as explants for shoot proliferation. Murashige and Skoog’s (MS) medium supplemented with cytokinins with or without indole-3-acetic acid (IAA) or naphthalene acetic acid was used for shoot multiplication. Out of different PGRs combinations, MS medium containing 0.5 mg l^?1 6-benzylaminopurine and 0.1 mg l^?1 IAA was optimal for shoot multiplication. On this medium, explants produced the highest number of shoots (47.50?±?0.38). About 90 % of shoots rooted ex vitro on sterile soilrite under the greenhouse condition when the base (2–4 mm) of shoots was treated with 300 mg l^?1 of indole-3-butyric acid for 5 min. The plantlets were hardened successfully in the greenhouse with 85–90 % survival rate. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were employed to assess the genetic stability of in vitro-regenerated plants of A. hispidissima. Out of 40 (25 RAPD and 15 ISSR) primers screened, 15 RAPD and 7 ISSR primers produced a total number of 111 (77 RAPD and 34 ISSR) reproducible amplicons. The amplified products were monomorphic across all the micropropagated plants and were similar to the mother plant. To the best of our knowledge, it is the first report on the assessment of the genetic fidelity in micropropagated plants of A. hispidissima.     

Thidiazuron-Induced Changes in Biomass Parameters,Total Phenolic Content,and Antioxidant Activity in Callus Cultures of Artemisia absinthium L.

Callus culture of Artemisia absinthium L. was established for enhanced production of phenolics and higher antioxidant activity. Callus was induced from seed-derived leaf explants, incubated on to MS media supplemented with thidiazuron (TDZ; 0.5–5.0 mg/l) either alone or in combination with α-naphthalene acetic acid (NAA; 1.0 mg/l). These callus cultures were investigated for their growth kinetics, total phenolic content, and antioxidant activity on weekly basis for a period of 49 days. Maximum dry biomass accumulation of 8.73 g/l was observed on day 42 in response to 1.0 mg/l TDZ and 1.0 mg/l NAA. Furthermore, maximum level of total phenolic content of 8.53 mg GAE/g DW and highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of 72.6 % were observed in calli formed in response to 1.0 mg/l TDZ on day 42. The results showed a positive correlation of total phenolic content and DPPH radical scavenging activity in most of the callus cultures of A. absinthium L.     

Optimization of Factors Influencing Microinjection Method for Agrobacterium tumefaciens-Mediated Transformation of Tomato

A simple and efficient protocol for Agrobacterium-mediated genetic transformation of tomato was developed using combination of non-tissue culture and micropropagation systems. Initially, ESAM region of 1-day-old germinated tomato seeds were microinjected for one to five times with Agrobacterium inoculums (OD₆₀₀?=?0.2–1.0). The germinated seeds were cocultivated in the MS medium fortified with (0–200 mM) acetosyringone and minimal concentrations of (0–20 mg?L^?1) kanamycin, and the antibiotic concentration was doubled during the second round of selection. Bacterial concentration of OD₆₀₀?=?0.6 served as an optimal concentration for infection and the transformation efficiency was significantly higher of about 46.28 %. In another set of experiment, an improved and stable regeneration system was adapted for the explants from the selection medium. Four-day-old double cotyledonary nodal explants were excised from the microinjected seedlings and cultured onto the MS medium supplemented with 1.5 mg?L^?1 thidiazuron, 1.5 mg?L^?1 indole-3-butyric acid, 30 mg?L^?1 kanamycin, and 0–1.5 mg?L^?1 adenine sulphate. Maximum of 9 out of 13 micropropagated shoots were shown positive to GUS assay. By this technique, the transformation efficiency was increased from 46.28 to 65.90 %. Thus, this paper reports the successful protocol for the mass production of transformants using microinjection and micropropagation techniques.     

Optimization of Adventitious Root Culture for Production of Biomass and Secondary Metabolites in Prunella vulgaris L.

Adventitious root cultures of Prunella vulgaris L. were established in shaking flask system for the production of biomass and secondary metabolites. Adventitious root cultures were induced from callus cultures obtained from leaf explants on solid Murashige and Skoog (MS) medium containing combination of 6-benzyladenine (BA; 1.0 mg l^?1) and naphthalene acetic acid (NAA; 1.5 mg l^?1). Thereafter, 0.49 g inoculum was transferred to liquid MS medium supplemented with different concentrations of NAA (0.5–2.0 mg l^?1). Growth kinetics of adventitious roots was recorded with an interval of 7 days for 49 days period. Highest biomass accumulation (2.13 g/l) was observed in liquid medium containing 1.0 mg l^?1 NAA after 21 days of inoculation. However, other concentrations of NAA also showed similar accumulation pattern but the biomass gradually decreases after 49 days of inoculation. Adventitious roots were collected and dried for investigation of total phenolics (TP), total flavonoids (TF), and antioxidant activities. Higher TPC (0.995 GAE mg/g-DRB) and TFC (6.615 RE mg/g-DRB) were observed in 0.5 mg l^?1 NAA treated cultures. In contrast, higher antioxidant activity (83.53 %) was observed 1.5 mg l^?1 NAA treated cultures. These results are helpful in up scaling of root cultures into bioreactor for secondary metabolites production.     

Callus induction and plant regeneration from different explants of Actinidia deliciosa

In this study, an efficient procedure was developed for callus induction and regeneration of kiwifruit (Actinidia deliciosa) using different organs of shoots developed under in vitro conditions. Effects of explants source and media (M₁, 1.0 mg l⁻¹ BA + 2.0 mg l⁻¹ 2,4-D–M₂, 1.0 mg l⁻¹ NAA + 2.0 mg l⁻¹ 2,4-D) on initiation of callus were examined in order to obtain callus for organogenesis. The best callus for plant regeneration was obtained from leaf explants on Murashige and Skoog’s medium (MS) supplemented with M₂. Formation of callus from leaf of kiwifruit (A. deliciosa) was cultured in MS medium containing different concentration of N⁶-benzylaminopurin (BA; 0.0, 1.0, 2.0, 4.0, 6.0, 8.0 mg l⁻¹) for callus proliferation and plant regeneration. Although the first shoot formation was appeared in medium containing 6.0 and 8.0 mg l⁻¹ BA, the best shoots formation was obtained in medium with 4.0 mg l⁻¹ BA.     

Effect of Salts (NaCl and Na2CO3) on Callus and Suspension Culture of Stevia rebaudiana for Steviol glycoside Production

Steviol glycosides are natural non-caloric sweeteners which are extracted from the leaves of Stevia rebaudiana plant. Present study deals the effect of salts (NaCl and Na₂CO₃) on callus and suspension culture of Stevia plant for steviol glycoside (SGs) production. Yellow-green and compact calli obtained from in vitro raised Stevia leaves sub-cultured on MS medium supplemented with 2.0 mg l^?1 NAA and different concentrations of NaCl (0.05–0.20 %) and Na₂CO₃ (0.0125–0.10 %) for 2 weeks, and incubated at 24?±?1 °C and 22.4 μmol m^?2 s^?1 light intensity provided by white fluorescent tubes for 16 h. Callus and suspension biomass cultured on salts showed less growth as well as browning of medium when compared with control. Quantification of SGs content in callus culture (collected on 15th day) and suspension cultures (collected at 10th and 15th days) treated with and without salts were analyzed by HPLC. It was found that abiotic stress induced by the salts increased the concentration of SGs significantly. In callus, the quantity of SGs got increased from 0.27 (control) to 1.43 and 1.57 % with 0.10 % NaCl, and 0.025 % Na₂CO₃, respectively. However, in case of suspension culture, the same concentrations of NaCl and Na₂CO₃ enhanced the SGs content from 1.36 (control) to 2.61 and 5.14 %, respectively, on the 10th day.     

Regeneration and Agrobacterium-mediated transformation of the apomictic species Eulaliopsis binata

Protocols for regeneration and Agrobacterium-mediated transformation of the apomictic species Eulaliopsis binata were developed. Initially, seeds of four genotypes of E. binata were incubated on a callus induction Murashige and Skoog (MS) basal medium supplemented with three concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D). It was found that 36.2 % of explants developed highly friable callus on medium containing 3.0 mg l^?1 2,4-D. Based on frequency of callus induction, the genotype Neixiang was selected for regeneration and transformation. Callus incubated on MS basal medium supplemented with 0.2 mg l^?1 α-naphthalene acetic acid and 6.0 mg l^?1 6-furfuryl-aminopurine developed shoots. Subsequently, Agrobacterium tumefaciens strain EHA105—harboring a plasmid pCAMBIA1381 carrying a hygromycin phosphotransferase (hpt) resistance gene and a synthetic green fluorescent protein (GFP) gene, both driven by the cauliflower mosaic virus 35S promoter—was used for transformation system. Putative transgenic callus was obtained following two cycles of hygromycin selection. Expression of the transgene(s) in putative transgenic callus was analyzed using the GFP detection. Molecular identification of putative transformed shoots was performed by polymerase chain reaction and Southern blot analysis to confirm presence and integration of the hpt gene.     

In Vitro Propagation,Encapsulation, and Genetic Fidelity Analysis of Terminalia arjuna: a Cardioprotective Medicinal Tree

The present study described an improved and reproducible in vitro regeneration system for Terminalia arjuna using nodal segment explants obtained from a mature plant. Shoot tips excised from in vitro proliferated shoots were encapsulated in 3 % sodium alginate and 100 mM CaCl₂?2H₂O for the development of synthetic seeds which may be applicable in short-term storage and germplasm exchange of elite genotype. Shoot multiplication was significantly influenced by a number of factors, namely types and concentrations of plant growth regulators, medium composition, repeated transfer of mother explants, subculturing of in vitro regenerated shoot clumps, agar concentrations, and temperature. Maximum numbers of shoots (16.50?±?3.67) were observed on modified Murashige and Skoog (MMS) medium containing 0.5 mg l^?1 of benzylaminopurine (BAP) and 0.1 mg l^?1 of naphthalene acetic acid (NAA). To shortening the regeneration pathway, rooting of micropropagated shoots under in vitro condition was excluded and an experiment on ex vitro rooting was conducted and it was observed that the highest percentage of shoots rooted ex vitro when treated with indole-3-butyric acid (IBA, 250 mg l^?1)?+?2-naphthoxy acetic acid (NOA, 250 mg l^?1) for 5 min. The well-developed ex vitro rooted shoots were acclimatized successfully in soilrite under greenhouse conditions with 80 % survival of plants. Randomly amplified polymorphic DNA (RAPD) analysis confirmed that all the regenerated plants were genetically identical to the mother plant, suggesting the absence of detectable genetic variation in the regenerated plantlets. To the best of our knowledge, this is the first report on synthetic seed production as well as ex vitro rooting and genetic fidelity assessment of micropropagated shoots of T. arjuna.     

In Vitro Adventitious Shoot Regeneration via Indirect Organogenesis from Inflorescence Explants and Peroxidase Involvement in Morphogenesis of Populus euphratica Olivier

The inflorescences as explants for rapid propagation in vitro remained unknown in Populus euphratica Olivier. Here, we reported that multiple shoots were initiation from calli of both male and female inflorescences. The optimum medium for shoot induction from male inflorescences was lactose sulfite medium containing 1.0?mg?L^?1 6-benzylaminopurine (BA) and 0.5?mg?L^?1 ??-naphthalene acetic acid (NAA) or Murashige and Skoog (MS) medium containing 0.5?mg?L^?1 BA and 0.2?mg?L^?1 NAA. The optimum medium of shoot induction from female inflorescence calli was the MS medium containing 0.5?mg?L^?1 BA and 0.2?mg?L^?1 NAA. Rooting of regenerated shoots was obtained on 1/2 MS medium supplemented with 0.5??1.0?mg?L^?1 indole-3-butyric acid (IBA) and the highest frequency rooting was on medium containing 0.5?mg?L^?1 IBA. No shoots were obtained on medium without BA and NAA. Peroxidase (POD) activity was measured by polyacrylamide gel electrophoresis during shoot induction and differentiation stages. The results showed that two bands of POD (2a and 2b) activity appeared lowest during the early 8?days at the dedifferentiation phase of leaves inducing calli, whereas POD 2a, 2b activity appeared to be increasing at the homeochronous dedifferentiation phase of inflorescence. Five most intensive bands, POD 1a, 1b, 1c, 2a, and ab, appeared in 8th and 28th days at the redifferentiation phase during shoot morphogenesis. These results demonstrated that the POD was involved in shoot morphogenesis from both leaf and inflorescence explants of Populus euphratica.     

Shoot organogenesis from root-derived callus of Rhinacanthus nasutus (L.) Kurz. and assessment of clonal fidelity of micropropagted plants using RAPD analysis

An efficient regeneration system was established for an ethnomedicinal shrub Rhinacanthus nasutus from root-derived callus organogenesis. The root segments were cultured on MS medium supplemented with various concentrations of Kn (1.0–4.0 μM) alone or in combination with IBA (0.2–0.6 μM) or 2, 4-D (0.5–1.5 μM). The optimum frequency (94 %) of callus induction was recorded on MS medium supplemented with 3.0 μM Kn and 0.4 μM IBA. For shoot regeneration from callus, MS medium supplemented with different concentrations (1.0–7.0 μM) of BA or TDZ alone or in combination with NAA (0.2–1.0 μm) was employed. The highest frequency of shoot regeneration (91 %) and mean number of shoots (28.3) were observed on MS medium supplemented with 5.0 μM BA and 0.7 μM NAA. The shoots were excised and cultured on MS medium with 4.0 μM IBA produced 3.4 roots per shoot in 88 % cultures. Of the 65 plants transferred to soil 54 survived (83 %). The plants were transferred to field after successful hardening. RAPD analysis of the regenerated plants showed high similarity with the mother plant.     

Plantlet Regeneration from Callus Cultures of Selected Genotype of <Emphasis Type="Italic">Aloe vera</Emphasis> L.—An Ancient Plant for Modern Herbal Industries

Aloe vera L., a member of Liliaceae, is a medicinal plant and has a number of curative properties. We describe here the development of tissue culture method for high-frequency plantlet regeneration from inflorescence axis-derived callus cultures of sweet aloe genotype. Competent callus cultures were established on 0.8% agar-gelled Murashige and Skoog’s (MS) basal medium supplemented with 6.0 mg l⁻¹ of 2,4-dichlorophenoxyacetic acid (2,4-D) and 100.0 mg l⁻¹ of activated charcoal and additives (100 mg l⁻¹ of ascorbic acid, 50.0 mg l⁻¹ each of citric acid and polyvinylpyrrolidone, and 25.0 mg l⁻¹ each of l-arginine and adenine sulfate). The callus cultures were cultured on MS medium containing 1.5 mg l⁻¹ of 2,4-D, 0.25 mg l⁻¹ of Kinetin (Kin), and additives with 4% carbohydrate source for multiplication and long-term maintenance of regenerative callus cultures. Callus cultures organized, differentiated, and produced globular embryogenic structures on MS medium with 1.0 mg l⁻¹ of 2,4-D, 0.25 mg l⁻¹ of Kin, and additives (50.0 mg l⁻¹ of ascorbic acid and 25.0 mg l⁻¹ each of citric acid, l-arginine, and adenine sulfate). These globular structures subsequently produced shoot buds and then complete plantlets on MS medium containing 1.0 mg l⁻¹ of 6-benzylaminopurine and additives. A hundred percent regenerated plantlets were hardened in the greenhouse and stored under an agro-net house/nursery. The regeneration system defined could be a useful tool not only for mass-scale propagation of selected genotype of A. vera, but also for genetic improvement of plant species through genetic transformation.     

Role of TDZ in the Quick Regeneration of Multiple Shoots from Nodal Explant of Vitex trifolia L.—an Important Medicinal Plant

The effect of thidiazuron (TDZ) has been investigated in shoot multiplication for a simple, efficient, rapid, and commercially applicable regeneration protocol of an important medicinal plant, Vitex trifolia. Multiple shoots were induced in nodal explants obtained from a mature tree on Murashige and Skoog (MS) medium supplemented with TDZ in various concentrations (0.5, 1.0, 2.5, 5.0, 7.5, or 10.0???M). Prolonged exposure of the culture to TDZ had an adverse affect. To avoid this, the cultures were transferred to TDZ-free MS medium or MS medium fortified with various concentrations of 6-benzyladenine (BA) alone or in combination with ??-naphthalene acetic acid (NAA) to enhance multiplication, proliferation, and elongation of induced shoots. Optimum shoot multiplication and elongation was achieved when TDZ-exposed explants were repeatedly subcultured on MS media containing a combination of 1.0???M BA and 0.5???M NAA. The highest shoot regeneration frequency (90?%) and maximum number (22.3?±?0.2) of shoots per explant with shoot length of (5.2?±?0.2?cm) was recorded on MS medium fortified with 5.0???M TDZ. In vitro rooting of isolated shoots was achieved best in half-strength MS medium containing 0.5???M NAA. Properly rooted plantlets were successfully hardened off and acclimatized in thermocol cups containing sterile Soilrite. These plantlets were then transferred to pots containing different potting substrate; percentage survival of the plantlets was highest in vermiculite/garden soil mixture (1:1) and successfully transfer to greenhouse under sunlight.     

A sensitive and specific ELISA for determining a residue marker of three quinoxaline antibiotics in swine liver

Methyl-3-quinoxaline-2-carboxylic acid (MQCA) is a possible residue marker for three quinoxaline veterinary medicines (olaquindox, mequindox, and quinocetone). The wide application of mequindox/quinocetone or the illegal use of olaquindox leads to MQCA residue in animal’s original food, thereby threatening the safety of human food. The indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) with a specific coating antigen and monoclonal antibody (MAB) was established and optimized for detecting MQCA in swine liver. Samples were acidified with 2 mol?l^?1 hydrochloric acid, extracted with ethyl acetate–hexane–isopropanol (8?+?1?+?1, v/v/v) and then detected by IC-ELISA. The logarithm correlation of standards to OD values ranged from 0.2 to 200 μg?l^?1, with IC₅₀ of 6.46 μg?l^?1. Negligible cross-reactivity happened to five quinoxaline antibiotics (olaquindox, mequindox, quinocetone, carbadox, and cyadox) and the metabolite of carbadox and cyadox (quinoxaline-2-carboxylic acid). When spiked with 1 to 100 μg?kg^?1 of MQCA, the recoveries ranged from 85.44 to 100.02 %, with the intra-assay coefficient of variation (CV) of 6.64–10.57 % and inter-assay CV of 7.29–10.88 %. The limit of detection for MQCA was 1.0 μg?kg^?1 in swine liver. Furthermore, incurred samples were detected by the IC-ELISA and then conformed by a reported LC/MS/MS method, it shown that there was good correlation between the two methods. All these results indicated that the IC-ELISA method is appropriate for surveillance MQCA residue in animal tissues.

Production of camptothecin in the elicited callus cultures of <Emphasis Type="Italic">Nothapodytes nimmoniana</Emphasis> (J. Graham) Mabberly

The biotechnological approach of in vitro cultures elicitation offers an alternative strategy for the production of camptothecin (CPT) in Nothapodytes nimmoniana to mitigate indiscriminate harvest of the endangered natural population for the alkaloid. Yeast extract (YE) and vanadyl sulfate (VS) elicitors were used to enhance the biosynthesis of CPT in hypocotyl-derived callus cultures of N. nimmoniana by cultivation using solid and liquid Murashige and Skoog (MS) medium amended with NAA + BAP (2.0 + 1.0 mg L^?1). Effects of the two elicitors on biomass and CPT production at 6.25, 12.5, 25, 50 and 75 mg L^?1 concentrations using callus cultures from three cell lines were evaluated after 15, 30 and 45 days culture. Yeast extract elicitor treatments showed a linear enhancement effect on biomass and CPT production up to 50 mg L^?1 YE and beyond the concentrations, no significant effect was observed. Enhanced biomass and CPT production were achieved with VS elicitor up to 25 mg L^?1 concentrations but, 50 and 75 mg L^?1 VS had minimal effects on biomass and CPT production in callus sources and incubation duration-dependent manner. The intracellular yield of CPT in liquid media-cultivated cultures at concentrations of the two elicitors was lower when compared to solid media treatments relative control due to the extracellular accumulation but, higher overall production. Accumulation of the biomass showed association with produced CPT in the elicitor treatments and control cultures.     

Kinetics and Thermodynamics of Ethanol Production by Saccharomyces cerevisiae MLD10 Using Molasses

In this study, we have used ultraviolet (UV) and γ-ray induction to get a catabolite repression resistant and thermotolerant mutant with enhanced ethanol production along with optimization of sugar concentration and temperature of fermentation. Classical mutagenesis in two consecutive cycles of UV- and γ-ray-induced mutations evolved one best catabolite-resistant and thermotolerant mutant Saccharomyces cerevisiae MLD10 which showed improved ethanol yield (0.48?±?0.02 g g^?1), theoretical yield (93?±?3 %), and extracellular invertase productivity (1,430?±?50 IU l^?1 h^?1), respectively, when fermenting 180 g sugars l^?1 in molasses medium at 43 °C in 300 m³ working volume fermenter. Ethanol production was highly dependent on invertase production. Enthalpy (ΔH*) (32.27 kJ M^?1) and entropy (ΔS*) (?202.88 J M^?1 K^?1) values at 43 °C by the mutant MLD10 were significantly lower than those of β-glucosidase production by a thermophilic mutant derivative of Thermomyces lanuginosus. These results confirmed the enhanced production of ethanol and invertase by this mutant derivative. These studies proved that mutant was significantly improved for ethanol production and was thermostable in nature. Lower fermentation time for ethanol production and maintenance of ethanol production rates (3.1 g l^?1 h^?1) at higher temperature (43 °C) by this mutant could decrease the overall cost of fermentation process and increase the quality of ethanol production.     

Determination of Benzoyl Peroxide Levels in Wheat Flour and Pharmaceutical Preparations by Differential Pulse Voltammetry in Nonaqueous Media

Abstract

Benzoyl peroxide (BP) was determined by differential pulse voltammetry (DPV) using a glassy carbon electrode in a dichloromethane‐acetic acid (1.5×10^?2 mol l^?1) solution and tetrabutyl ammonium perchlorate (0.01 mol l^?1) as the supporting electrolyte. The peak potential was ?0.045 V (vs. Ag/AgCl). There was a good linear relationship between the peak current and the benzoyl peroxide concentration in the range of 2.5×10^?6–1.0×10^?4 mol l^?1. The detection limit of the method was 2.5×10^?7 mol l^?1. The recovery was 94.8–106.0%. The samples of wheat flour and the pharmaceutical preparations for the treatment of acne vulgaris were directly detected with desired results. The reaction mechanism of benzoyl peroxide on the electrode was also discussed, which was two electrons and two protons irreversible reaction.