Axillary shoot proliferation and plant regeneration in Euryodendron excelsum H. T. Chang, a critically endangered species endemic to China.

Euryodendron excelsum H. T. Chang is a single-type, rare and endangered woody plant unique to China. In this study, young stems were used as explants and cultured on Woody Plant Medium (WPM) supplemented with 5.0 µM 6-benzyladenine (BA), were subcultured for more than 15 times over a total of more than 3 years and finally an efficient axillary shoot proliferation and plantlet regeneration system was established in which one shoot could proliferate an average of 5.1 axillary shoots every 2 months on the medium supplemented with 5.0 µM BA and 0.5 µM α-naphthaleneacetic acid (NAA). Shoots rooted at a moderate frequencies (50.1%) on agarized WPM supplemented with 0.5 µM NAA but 100% of shoots rooted in agar-free vermiculite-based WPM after culture for 2 months. Plantlets, when transplanted to peat soil: vermiculite (1:1), showed the highest 95.1% survival within 1 month.

Shoot organogenesis and somatic embryogenesis from leaf and root explants of Scaevola sericea.

An efficient regeneration system via shoot organogenesis and somatic embryogenesis from in vitro leaf and root explants was established for Scaevola sericea for the first time. The highest axillary shoot proliferation coefficient (4.8) was obtained on Murashige and Skoog (MS) medium supplemented with 1.0 mg/L 6-benzyladenine (BA) and 0.1 mg/L α-naphthaleneacetic acid (NAA) every 45 days. Young in vitro leaves and roots, which were used as explants, were cultured onto medium supplemented with different plant growth regulators. Our results showed that only cytokinins BA and thidiazuron (TDZ), could induce adventitious shoots and somatic embryos from leaf and root explants. The optimal medium to achieve this was MS medium supplemented with 2.5 mg/L BA and which induced most adventitious shoots (2.7) and somatic embryos (17.3) from leaf explants within 30 days. From root explants, 1.1 adventitious shoots and 7.6 somatic embryos could be induced on MS medium supplemented with 2.5 mg/L TDZ. Histological observation showed that both somatic embryos and adventitious shoots were originated from homogeneous parenchyma and the development of somatic embryos was visible. Maximum rooting percentage (99.0%) was achieved on half-strength MS medium supplemented with 2.5 mg/L NAA. Well-rooted plantlets, which were transplanted into a substrate of pure river sand, displayed a high survival percentage of 91.7% after transplanting for 45 days while the best substrate for plantlet growth was river sand: coral sand (1:1).

[Preparation of monoclonal antibody against azithromycin and establishment of immunoassay for azithromycin].

Objective To prepare mouse monoclonal antibody (mAb) with haptens of azithromycin (AZM) and characterize its features. Methods The immunogen AZM-BSA and coating antigen AZM-OVA were synthesized by coupling AZM to the bovine serum albumin (BSA) and ovalbumin (OVA) with the activated ester method. Hybridoma cell line 50D10 was obtained by fusing mouse myeloma cells Sp2/0 with splenocytes from the BALB/c mice immunized with AZM-BSA. The cell line was then injected into BALB/c mice to prepare mAb against AZM, which was detected by ELISA using this mAb. Results The titer of AZM mAb was 1:4.0×105, which belonged to IgG1 type. The sensitivity and IC50 of the indirect competitive ELISA was 0.287 µg/L and 1.33 µg/L, respectively. The cross-reactivity rates of the mAb with erythromycin, roxithromycin, doramectin, clarithromycin, tilmicosin and tylosin were less than 0.1%. The recovery rates of AZM added into milk ranged from 79.6% to 104.1% with the coefficients of variation between 16.4% and 27.3%. Conclusion AZM mAb with high sensitivity and specificity has been prepared, which lays a foundation for establishing a method to rapidly detect AZM.

Adventitious shoot organogenesis from leaf explants of Portulaca pilosa L.

This study established, for the first time, shoot proliferation and plant regeneration protocols via shoot organogenesis from leaf explants of a medical and ornamental plant, Portulaca pilosa L. The optimal proliferation of axillary shoots was 6.2-fold within 30 days on Murashige and Skoog (MS) medium supplemented with 3.0 µM 6-benzyladenine (BA). Shoots could be induced directly from leaf explants, forming an average of 3.8 adventitious shoots per explant, on optimal MS medium supplemented with 1.0 µM thidiazuron (TDZ) and 0.1 µM α-naphthaleneacetic acid (NAA). A higher concentration of TDZ (3.0 µM), alone or in combination with 0.1 µM NAA, induced somatic embryo-like shoot buds and then developed into real shoots. Rooting was easier since roots were induced on all rooting media within one month. Half-strength MS medium free of plant growth regulators was best for rooting. Rooted plantlets were transferred to a sand: perlite (1:1, v/v) substrate, resulting in highest survival (90%). Plantlets showed more robust growth, however, on substrates of yellow mud: perlite (1:1, v/v) or peat soil: vermiculite: perlite (1:1:1, v/v).

Transcriptome Analysis of the SL221 Cells at the Early Stage during Spodoptera litura Nucleopolyhedrovirus Infection.

Spodoptera litura (S. litura) is one of the most destructive agricultural pests worldwide. There is urgent need for a nuclear polyhedrosis virus that is specific to S. litura. To date, there have been no reports regarding the responses of S. litura cells to early Spodoptera litura nucleopolyhedrovirus (SpltNPV) infection due to the lack of a reference genome and transcriptome for S. litura. In this study, a cell transcriptome from the host S. litura was assembled and used for Illumina strand-specific RNA sequencing (RNA-seq) to generate 99180 unigenes, representing the 18 hour infection cycle. More than 2000 S. litura genes were significant differentially regulated throughout the infection. The levels of viral mRNAs began to increase dramatically at 6 hpi, and this increase continued throughout the remainder of the infection. We focused on the expression of genes related to stress responses, apoptosis, metabolic enzymes and host cell innate immune system. A small subset of genes related to host stress response, especially for 62 ones being able to annotated as enzyme, ligand and receptor genes, were observed to be specifically differentially expressed at 6 hpi. At 18 hpi, 104 unigenes were continuously significantly changing from 0 hpi to 18 hpi, considered to be viral multiplication related genes, including 3 annotated SL221 unigenes and 81 viral genes, such as tetraspanin and iap gene. This information and further studies on the regulation of host gene expression by baculovirus infection at early stage will provide the tools needed to enhance the utility of this virus as an effective insecticide.

Comparative proteomics analysis of apoptotic Spodoptera frugiperda cells during p35 knockout Autographa californica multiple nucleopolyhedrovirus infection.

Infection with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) mutants lacking a functional p35 gene can induce host cell apoptosis, which provides the possibility to use the potential of these viruses in the biological control of pest insects. Nonetheless, the proteomics or the protein changes of Spodoptera frugiperda (Sf9) cells infected with p35 knockout AcMNPV have not yet been studied. To further improve the use of AcMNPV, we set out to analyze the protein composition and protein changes of Sf9 cells of different infection stages by isobaric tag for relative and absolute quantification (iTRAQ) techniques. A total of 4004 sf9 proteins were identified by iTRAQ. After comparation of the significantly expressed 483 proteins from p35koAcMNPV-infected Sf9 cells and the significantly expressed 413 proteins from wtAcMNPV-infected Sf9 cells, we found that 226 proteins were specific to p35koAcMNPV-infected Sf9 cells. The 226 proteins were categorized according to GO classification for insects and were categorized into: biological processes, molecular functions and cellular components. Of interest, the most up-regulated proteins related to Epstein-Barr virus infection, RNA transport, Calcium signaling pathway, cGMP-PKG signaling pathway, oxidative phosphorylation and N-Glycan biosynthesis. Determination of the protein changes in p35 knockout AcMNPV-infected Sf9 cells would facilitate the better use of this virus-host cell interaction in pest insect control and other related fields.

Comparative proteomics analysis of Spodoptera frugiperda cells during Autographa californica multiple nucleopolyhedrovirus infection.

BACKGROUND: Increasing evidence sugggest that in addition of balculovirus controling insect host, host cells also responds to balculovirus infection. However, compared to existing knowledge on virus gene, host cell responses are relatively poorly understood. METHODS: In this study, Spodoptera frugiperda (Sf9) cells were infected with Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The protein composition and protein changes of Spodoptera frugiperda (Sf9) cells of different infection stages were analysed by isobaric tag for relative and absolute quantification (iTRAQ) techniques. RESULTS: A total of 4004 Sf9 proteins were identified by iTRAQ and 413 proteins were found as more than 1.5-fold changes in abundance. The 413 proteins were categorised according to GO classification for insects and were categorised into: biological process, molecular function and cellular component. CONCLUSIONS: The determination of the protein changes in infected Sf9 cells would help to better understanding of host cell responses and facilitate better design of this virus-host cell interaction in pest insect control and other related fields.

Development of a direct competitive enzyme-linked immunosorbent assay using a sensitive monoclonal antibody for bisphenol A.

To set up an immunoassay-based method to detect Bisphenol A (BPA), we generated a monoclonal antibody (MAb) using a specially designed carboxyl derivative of BPA as the immunogen. BPA-HS was synthesized by reaction using BPA and succinic anhydride. The mice were immunized with the BPA-HS-BSA conjugate. The MAb was obtained from a hybridoma. In addition, we showed that the MAb was highly specific for BPA. The limit of detection was approximately 0.05 ng mL(-1) (ppb) in assay buffer and 0.1 ng mL(-1) (ppb) in water samples. The recoveries of BPA for water samples were from 90.8% to 114%, and coefficients of variation were from 15.6% to 39.4%. Thus, the ELISA method is a rapid and high throughput screening tool to detect BPA in water products.