The following student awards were presented at the 2013 In Vitro Biology Meeting in Providence, Rhode Island. Information on additional awardees at the 2013 Meeting will be presented in the next issue of the In Vitro Report. Information related to the available specific student awards can be found on the SIVB website (www.sivb.org) or by contacting the SIVB Business Office at (919) 562-0600, [email protected].
THE 2013 WILTON R. EARLE AWARD AND 2013 SIVB STUDENT TRAVEL AWARD
The Susceptibility of Walleye Cell Lines from the Spleen Stroma and Caudal Fin to Viral Hemorrhagic Septicemia Virus (VHSV)
Nguyen Vo
Viral hemorrhagic septicemia virus (VHSV) is a rhabdovirus that causes a significant disease (VHS) with high mortality in many fish species including walleye (Sander vitreus), leading to economic losses in aquaculture. In a susceptible fish species, severely infected individuals hemorrhage and die, whereas chronically infected individuals are often anemic. The anemia suggests that the virus could be impairing hematopoiesis, which in fish occurs in the spleen and head kidney. Hematopoiesis is supported in these tissues by stromal cells. One way of studying viral pathogenesis is to examine their impact on spleen stromal cells. Therefore, we developed a cell line (WES6S) from the walleye spleen and compared this cell line to a fibroblast cell line (W ECF11f) from the walleye caudal fin for susceptibility to VHSV IVb. Both cell lines produced the virus and showed a cytopathic effect (CPE), which was a loss of cell viability that was measured by fluorescent dyes Alamar Blue and CFDA-AM. However, virus production began earlier in WES6S as indicated by the earlier accumulation of viral proteins, development of CPE, and increase in viral titers. The induction of the antiviral polypeptide, Mx, by VHSV IVb was seen in WECF11f but not in WES6S. This result shows that the antiviral mechanisms are affected but regulated differently in response to VHSV infections in the caudal fin and spleen stroma of S. vitreus.
Nguyen Vo, University of Waterloo, Department of Biology, Waterloo, ON N2L 3G1, Canada. In Vitro Cellular and Developmental Biology, 49:S30, 2013
THE 2013 SIVB STUDENT TRAVEL AWARD
Induced Mutations for Enhancing Variability of Giant Miscanthus (Miscanthus x giganteus)
Dinum Perera
Giant miscanthus (Miscanthus x giganteus; Mxg) is a key candidate energy crop for use in biomass to liquid fuel production. Mxg is a natural sterile triploid lacking genetic variation, the basis for selection. Genetically uniform monocultures of Mxg are inherently vulnerable to pathogenic epidemics. Thus, induced mutation techniques are particularly important in Mxg. The aim of this study was to induce variation in Mxg through in vitro chemical mutagenesis. A previously developed in vitro propagation protocol for Mxg was utilized during the in vitro mutagenesis procedure. Mutagenic dosage was optimized using various concentrations (0.01%, 0.1%, 0.5%, 1%, and 3%) of ethyl methanesulfonate (EMS) and time periods (45 and 90 min) with 2% dimethyl sulfoxide (DMSO) incorporated into the mutagen solution. Mxg immature inflorescence explants (1-2 mm), embryogenic and shoot-forming calli (1-2 mm3) were treated with ten mutagenic dose treatments in order to determine the optimum mutagen dosage. Depending on the EMS concentration used, the mean survival rate of callus cultures decreased from 85% (0.1% EMS) to 32% (3% EMS); whereas with inflorescence explants, the mean survival rate decreased from 80% (0.1% EMS) to 29% (3% EMS) after treated with 3% EMS for 90 min. The dose at which 50% of the callus/explants died (LD50) was used as the optimum EMS dose. Mxg calli and inflorescence explants were treated with LD50, 2 x LD50, and 3 x LD50 EMS dosages and the regenerants were transferred to soil. Inter simple sequence repeat (ISSR) marker has been utilized to identify variants in the regenerated plants. Putative mutant population will be screened and evaluated in the field during 2013 for future selection.
Dinum Perera, Mississippi State University, Box 9555, Mississippi State, MS 39762. In Vitro Cellular and Developmental Biology, 49:S64, 2013
THE 2013 SIVB STUDENT TRAVEL AWARD
Engineering Drought and Salt Tolerant Soybeans by Overexpressing AVP1, the Vacuole Proton Pump
Marten Peterson
Salinization and drought conditions are major constraints to global crop production. The increased rate of desertification, irrigation with brackish water, and over use of fertilizers has increased these stresses, especially in developing nations. Development of resistant cultivars has become an important agronomic issue worldwide. AVP1, a simple pyrophosphate-driven proton pump, transports H+ ions from the cytosol into the plant cell vacuole. Overexpression of the AVP1 gene in several model plants was shown to create a unique system of ion gradients which confers drought and salt tolerance. The induced gradients increase cellular water absorption while also increasing auxin flux, which enhances root system development. These observations suggest that crops overexpressing AVP1 would be useful in regions struggling with salinization and drought conditions. When considering commercial and cultural impacts, a soybean cultivar that overexpresses AVP1 would have vast potential. Soybean crops are vulnerable to environmental stresses but prove to be an ideal source of protein, oils, and many of the nutrients and vitamins associated with malnutrition in developing nations. Agrobacterium was used to transform soybeans with the AVP1 overexpression cassette. Transformants undergo genetic and molecular characterization. Analysis of transgene stability and effectiveness will be further confirmed in T1 progeny by genetic testing, relative water content, and stress testing (drought/salt/cold). Transformation of soybeans in the academic field is unpopular due to difficulties in tissue culturing and explant preparation. In addition to implications in global agriculture, this research will be a vital addition to the literature describing these precise techniques.
Marten Peterson, Department of Natural Sciences, College of Agriculture and Technology, State University of New York, Cobleskill, NY 12034. In Vitro Cellular and Developmental Biology, 49:S72, 2013
THE 2013 SIVB STUDENT TRAVEL AWARD
Comparison of Larvicidal Effect of rhizomes of Tissue Cultured and Naturally Grown Plants of Kaempferia galanga (Zengiberaceae)
RM Uththara Sachinthanie Senarath
Kaempferia galanga (Zengiberaceae) commonly named as aromatic ginger or galangal is a medicinal plant used in folk medicine as well as in culinary purposes in South East Asia. It has been reported that the rhizomes of K. galanga contains chemicals that are potent insecticides and have potential in mosquito control. The plant has been overexploited and listed under threatened category in Sri Lanka and India. Therefore developing tissue culture techniques for plant regeneration of K. galanga would be beneficial. Evaluation of extracts of tissue cultured plants against larvae of Aedes aegypti , also has been studied which could give a preliminary idea about the presence of Kaempferol in tissue cultured plants and the possibility of tissue cultured plants in herbal industry. Objectives of the study were, to develop a protocol for tissue culture of Kaempferia galanga from axillary buds obtained from rhizome cuttings and to evaluate the insecticidal activity of extracts of tissue cultured plants comparing to natural plants. Rhyzomes of K. galangal has been wrapped in wet tissues and allowed the axillary buds to be elongated. Axillary buds were removed carefully and surface sterilized. Explants were cultured on Murashige and Skoog (MS) medium supplemented with BAP (Benzyl amino purine) and IAA (Indole-3-acetic acid) for shoot induction. For root induction elongated shoots were either continuously subcultured in the same medium or separated after 3 weeks and cultured in MS medium supplemented with lowered concentration of BAP and IAA. Rooted plantlets were carefully removed from the medium washed in luke water to remove all traces of agar and acclimatized in a potting mixture of 1: 1: 1 soil: sand : compost. Rhizomes were collected from tissue cultured plants as well as naturally grown plants. Kaempferol present was extracted separately in hexane, methanol and water and different concentrations were tested against the forth instar larvae of A. aegypti. Mortality was observed hourly and LD50 was calculated after 24h incubation at room temperature. MS medium supplemented with 2.0 mg/L BAP and 0.5 mg/L IAA found to be the best medium for shoot elongation and multiplication. Increased concentration of BAP affects negatively on shoot elongation and multiplication. Best root induction medium was MS medium supplemented with 1.0 IAA with no added cytokinin. For acclimatization 1: 1: 1 Sand: soil and compost mixture found to be the best. When consider about the larvicidal activity although it is a preliminary study, it indicated that the tissue cultured plants have higher concentrations of secondary metabolites than natural plants. Although the hexane extracts showed the highest activity against mosquito larvae water extracts also could be used for controlling mosquito larvae. As this is a preliminary study, improving the techniques for chemical extraction and testing larvicidal effect with further modifications are recommended.
RM Uththara Sachinthanie Senarath, Faculty of Arts and Sciences, Virgen Milagrosa University Foundation, PHILIPPINES. In Vitro Cellular and Developmental Biology, 49:S71-S72, 2013.
THE 2013 HOPE E. HOPPS AWARD AND 2013 SIVB STUDENT TRAVEL AWARD
Engineering Hydroxyproline-O-Glycosylated Peptide Motifs in Hairy Roots for an Enhanced Bioproduction Platform
Ningning Zhang
Hydroxyproline-O-glycosylation involves post-translational hydroxylation of proline to hydroxyproline (Hyp) and subsequent glycosylation, a modification unique to plants. Evidence has showed that Hyp-O-glycosylated peptide motifs, e.g. tandem repeats of “Ser-Pro”, engineered in plant cells could function as a “carrier” to significantly excrete tagged proteins into culture media and stabilize the proteins. However, the precise process of Hyp-O-glycosylation in plants has yet been elucidated. Hairy root cultures are among the most attractive plant-based production systems for recombinant proteins as they combine merits of both suspension cell culture and whole-plants cultivation. The purpose of this research is in two aspects: 1) to test if the Hyp-O-glycosylation technology can be applied to an alternative production system-hairy root; 2) to study the Hyp-O-glycosylation process with hairy root culture system. Two major types of HRGP motifs, an extensin consisting of tandem repeats of “Ser-Hyp-Hyp-Hyp-Hyp” motif, and an arabinogalactan protein (AGP) consisting of tandem repeats of “Ser-Hyp” motif, were respectively engineered in tobacco hairy roots as fusion with a reporter protein, enhanced green fluorescence protein (EGFP). As in plant cell cultures, significantly enhanced secretion of the target protein (EGFP) carried by the Hyp-O-glycosylated motifs (by more than 10 folds) was detected in hairy root cultures. While fully glycosylated extensin motif was identified in both the culture media and roots, interestingly, two types of glycoforms of the AGP motif were observed; they were completely segregated with the partially glycosylated form (presumably with single galactose) retained inside the roots while the fully glycosylated form (with arabinogalactan polysaccharides) was recovered in the culture media. This research demonstrated high potential of engineering Hyp-O-glycosylated motifs in hairy roots for an enhanced production platform, and that hairy root culture may provide a unique platform to study the Hyp-O-glycosylation process.
Ningning Zhang, Arkansas Biosciences Institute, Arkansas State University, Jonesboro, AR 72401. In Vitro Cellular and Developmental Biology, 49:S38-S39, 2013
THE 2013 CELLULAR TOXICOLOGY AWARD
Regulation of RNA Polymerase III Transcription by EGCG in Lungs
 Jana Suchtova |
 Nicole Nole |
RNA polymerase III (RNA pol III) is responsible for the transcription of small, untranslated RNAs involved in essential metabolic processes such as translation (tRNA) and mRNA processing (U6 snRNA). RNA pol III transcription plays a contributing role in dictating the biosynthetic capacity of a cell. Proper initiation by RNA pol III requires TFIIIB. In mammals two forms of TFIIIB have been identified. Proper initiation from gene internal RNA pol III promoters requires TBP, Bdp1 and Brf1. Gene external promoters require a different form of TFIIIB containing TBP, Bdp1 and Brf2. TFIIIB activity has been shown to be regulated by tumor supressors and oncogenes. Deregulation of TFIIIB activity has been demonstrated to play a role in oncogenesis. Namely, the TFIIIB subunit Brf2 has been characterized as an oncogene in lung squamous cell carcinomas. We have previously demonstrated that the chemopreventative EGCG, a major component of green tea, negatively regulates RNA pol III transcription in HeLa cells. Thus, we sought to determine if EGCG regulates RNA pol III transcription in lung cancer, both in vitro and in vivo. A lung cancer cell line was treated with 25 uM EGCG and using qPCR revealed that Brf1, Brf2 and 7SK expression was negatively regulated by EGCG. Furthermore, the observed decreases were statistically significant. These data suggest that EGCG negatively regulates RNA pol III transcription in lung cancer cells. Subsequently, we investigated the possibility that EGCG regulates RNA pol III transcription in C57BL/6J mice, specifically lung tissue. C57BL/6J mice were orally administered 0.01% EGCG for 3 weeks. Using qPCR we determined that oral administration of 0.01% EGCG negatively regulated the expression of the TFIIIB subunits Brf1 and Brf2, as well as U6 snRNA in lung tissue. Levels of expression of Maf1, a known negative regulator of TFIIIB activity, remained relatively unchanged in mice treated with EGCG. This indicates that the observed negative regulation of RNA pol III transcription by EGCG may occur independently of MAF1. Taken together, these data suggest we have developed an in vivo model to investigate the regulation of RNA pol III transcription by EGCG.
Nicole Nole and Jana Suchtova, Department of Biological Sciences, St. John’s University, 8000 Utopia Parkway, Queens NY 11439. In Vitro Cellular and Developmental Biology, 49:S42, 2013
