Agrobacterium-mediated genetic transformation of a phalaenopsis orchid

 Genetically transformed plants of a phalaenopsis orchid [Doritaenopsis Coral Fantasy×Phalaenopsis (Baby Hat×Ann Jessica)] were regenerated after cocultivation of cell clumps with Agrobacterium tumefaciens strains LBA4404 (pTOK233) and EHA101 (pIG121Hm) that harbored genes for β-glucuronidase (GUS) and hygromycin resistance. The efficiency of transformation was markedly increased by 10 h cocultivation of cell clumps with A. tumefaciens that had been induced with 200 μm acetosyringone, and by inclusion of 500 μm acetosyringone in the cocultivation medium. Hygromycin-resistant cell clusters (0.5–3 mm in diameter) were selected from the infected cell clumps after 4–6 weeks of culture on agar (8 g/l)-solidified new Dogashima medium (NDM) containing 20 g/l sucrose, 0.1 mg/l naphthaleneacetic acid, 1.0 mg/l benzyladenine (BA), 50 mg/l hygromycin and 300 mg/l cefotaxime. The cell clusters proliferated 4 weeks after transfer onto the same medium. To induce callus greening, the carbon source was changed from sucrose to maltose. The green calli obtained produced protocorm-like bodies (PLBs) after 4 weeks of culture on phytohormone-free NDM medium. Regeneration of transgenic plantlets was enhanced by incubating PLBs on NDM medium supplemented with 0.1 mg/l abscisic acid, followed by partial desiccation for 10–30 min. Successful transformation was confirmed by histochemical GUS assay, PCR analysis and Southern hybridization of transformants. With this transformation system, more than 100 hygromycin-resistant phalaenopsis plantlets were produced about 7 months following infection of the cell aggregates. Received: 10 November 1998 / Revision received: 4 June 1999 / Accepted: 22 June 1999     

PharmDB-K: Integrated Bio-Pharmacological Network Database for Traditional Korean Medicine

Despite the growing attention given to Traditional Medicine (TM) worldwide, there is no well-known, publicly available, integrated bio-pharmacological Traditional Korean Medicine (TKM) database for researchers in drug discovery. In this study, we have constructed PharmDB-K, which offers comprehensive information relating to TKM-associated drugs (compound), disease indication, and protein relationships. To explore the underlying molecular interaction of TKM, we integrated fourteen different databases, six Pharmacopoeias, and literature, and established a massive bio-pharmacological network for TKM and experimentally validated some cases predicted from the PharmDB-K analyses. Currently, PharmDB-K contains information about 262 TKMs, 7,815 drugs, 3,721 diseases, 32,373 proteins, and 1,887 side effects. One of the unique sets of information in PharmDB-K includes 400 indicator compounds used for standardization of herbal medicine. Furthermore, we are operating PharmDB-K via phExplorer (a network visualization software) and BioMart (a data federation framework) for convenient search and analysis of the TKM network. Database URL: http://pharmdb-k.org, http://biomart.i-pharm.org.     

Tumor-Targeting Salmonella typhimurium A1-R Arrests a Chemo-Resistant Patient Soft-Tissue Sarcoma in Nude Mice

A patient-derived nude-mouse model of soft-tissue sarcoma has been established and treated in the following groups: (1) untreated controls; (2) gemcitabine (GEM) (80 mg/kg, ip, weekly, 3 weeks); (3) Pazopanib (100 mg/kg, orally, daily, 3 weeks) and (4) Salmonella typhimurium A1-R (5 × 10⁷ CFU/body, ip, weekly, 3 weeks). The sarcoma was resistant to GEM (p = 0.879). Pazopanib tended to reduce the tumor volume compared to the untreated mice, but there was no significant difference (p = 0.115). S. typhimurium A1-R significantly inhibited tumor growth compared to the untreated mice (p = 0.001). S. typhimurium A1-R was the only effective treatment for the soft-tissue sarcoma nude mouse model among all treatments including a newly approved multiple tyrosine kinase inhibitor; Pazopanib. These results suggest tumor-targeting S. typhimurium A1-R is a promising treatment for chemo-resistant soft-tissue sarcoma.     

Secreted Wnt "inhibitors" are not just inhibitors: regulation of extracellular Wnt by secreted Frizzled-related proteins

Gradient formation and signaling ranges of secreted proteins are crucial problems to understand how morphogens work for positional information and patterning in animal development. Yet, extracellular behaviors of secreted signaling molecules remain unexplored compared to their downstream pathways inside the cell. Recent advances in bioimaging make it possible to directly visualize morphogen molecules, and this simple strategy has, at least partly, succeeded in uncovering molecular behaviors of morphogens, such as Wnt (wingless-type MMTV integration site family member) and BMP (bone morphogenetic protein) as well as secreted Wnt binding proteins, sFRPs (secreted Frizzled-related proteins), in embryonic tissues. Here, we review the regulation of Wnt signaling by sFRPs, focusing on extracellular regulation of Wnt ligands in comparison with other morphogens. We also discuss evolutionary aspects with comprehensive syntenic and phylogenetic information about vertebrate sfrp genes. We newly annotated several sfrp genes including sfrp2-like 1 (sfrp2l1) in frogs and fishes and crescent in mammals.     

Animal‐borne video systems provide insight into the reproductive behavior of the Asian black bear

Previous studies on the mating system of the Asian black bear (Ursus thibetanus) have been limited to observations of captive populations and estimations of multiple paternities. Hence, the mating system of wild bears remains poorly understood. Animal‐borne camera systems (i.e., cameras mounted on animals) provide novel tools to study the behavior of elusive animals. Here, we used an animal‐borne video system to record the activities of wild bears during the mating season. Video camera collars were attached to four adult Asian black bears (male “A” and “B,” and female “A” and “B”) captured in Tokyo, central Japan, in May and June 2018. The collars were retrieved in July 2018, after which the video data were downloaded and analyzed in terms of bear activity and mating behavior. All the bears were found to interact with other uniquely identifiable bears for some of the time (range 9–22 days) during the deployment period (range 36–45 days), and multiple mating in males was documented. Both males and females exhibited different behaviors on social days (i.e., days when the bear interacted with conspecifics) compared with solitary days (i.e., days with no observed interactions with conspecifics). Compared with solitary days, the bears spent a lower proportion of time on foraging activities and higher proportion of time on resting activities on social days. Our results suggest that Asian black bears have a polygamous mating system, as both sexes consort and potentially mate with multiple partners during a given mating season. Furthermore, bears appeared to reduce their foraging activities on social days and engaged more in social interactions.     

Production of transgenic potato exhibiting enhanced resistance to fungal infections and herbicide applications

Potato (Solanum tuberosum L.), one of the most important food crops, is susceptible to a number of devastating fungal pathogens in addition to bacterial and other pathogens. Producing disease-resistant cultivars has been an effective and useful strategy to combat the attack of pathogens. Potato was transformed with Agrobacterium tumefaciens strain EHA101 harboring chitinase, (ChiC) isolated from Streptomyces griseus strain HUT 6037 and bialaphos resistance (bar) genes in a binary plasmid vector, pEKH1. Polymerase chain reaction (PCR) analysis revealed that the ChiC and bar genes are integrated into the genome of transgenic plants. Different insertion sites of the transgenes (one to six sites for ChiC and three to seven for bar) were indicated by Southern blot analysis of genomic DNA from the transgenic plants. Expression of the ChiC gene at the messenger RNA (mRNA) level was confirmed by Northern blot analysis and that of the bar gene by herbicide resistance assay. The results obviously confirmed that the ChiC and bar genes are successfully integrated and expressed into the genome, resulting in the production of bialaphos-resistant transgenic plants. Disease-resistance assay of the in vitro and greenhouse-grown transgenic plants demonstrated enhanced resistance against the fungal pathogen Alternaria solani (causal agent of early blight).     

Production of bialaphos-resistant Nierembergia repens by electroporation

Transgenic plants with the herbicide-resistance gene (bar gene) were obtained via organogenesis from isolated mesophyll protoplasts of Nierembergia repens after applying electroporation. Transient β-glucuronidase (GUS) activity of electroporated protoplasts assayed 2 days after applying an electric pulse showed that optimum condition (transient GUS activity 319 pmol 4 MU/mg per min and plating efficiency 2.43%) for electroporation was 0.5 kV/cm in field strength and 100 μF in capacitance. The protoplasts electroporated with the bar gene at this condition initiated formation of microcolonies on medium after 2 weeks. After 4 weeks of culture, equal volume of fresh 1/2-strength Murashige and Skoog (MS) medium containing 0.2 mg/l bialaphos was added for selection of transformed colonies. After 6 weeks of culture, growing colonies were transferred onto regeneration medium containing 1.0 mg/l bialaphos, on which they formed adventitious shoots 1–2 months after electroporation. The adventitious shoots rooted easily after transfer onto MS medium with bialaphos lacking plant-growth regulators. Transformation of these regenerants with the bar gene was confirmed by Southern analysis. Some of the transformants showed strong resistance to the application of bialaphos solution at 10.0 mg/l.     

Ti- and cryptic-plasmid-borne virulence of wild-type Agrobacterium tumefaciens strain CNI5 isolated from chrysanthemum (Dendranthema grandiflora Tzvelev)

The genetic similarity between pTiBo542 and pTiCNI5, which are harbored, respectively by the supervirulent Agrobacterium tumefaciens strain A281 and by the highly tumorigenic wild-type strain CNI5 isolated from chrysanthemum was investigated by Southern hybridization. pTiCNI5 and pTiBo542 exhibited highly similar hybridization patterns in both BamHI- and EcoRI-digested plasmids by using four vir-region-specific probes, whereas similarity in these two plasmids was not observed by probing with five TL-DNA-specific probes. The characteristics related to tumor formation of cryptic plasmids carried by strain CNI5 were investigated by using single-plasmid-cured derivatives. pTiCNI5-cured derivatives predictably failed to utilize agropine and mannopine and failed to induce tumors on chrysanthemum and tobacco leaf explants, while pAtCNI5a-, pAtCNI5c- and pAtCNI5d-cured derivatives could utilize these opines similar to the parent strain CNI5. Interestingly, pAtCNI5c- and pAtCNI5d-cured derivatives showed low tumorigenicity in comparison with strain CNI5 or with the pAtCNI5a-cured derivative. These results suggest that the highly virulent phenotype of strain CNI5 may be due to one or more vir genes, which exhibit cartographic similarity to those of pTiBo542. The results also suggest that the gene(s) related to tumor formation of strain CNI5 may exist not only on pTiCNI5 but also on cryptic plasmids pAtCNI5c and pAtCNI5d.