SIVB Student Awards

he following student awards were presented at the 2013 In Vitro Biology Meeting in Providence, Rhode Island. Information on additional awardees at the 2013 Meeting was presented in the previous 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, sivb@sivb.org.

THE 2013 SIVB STUDENT TRAVEL AWARD

Optimization of Cryopreservation Protocols for Shoot Meristem Tips of Sea Oats (Uniola paniculata)

Sea oats (Uniola paniculata) plantings serve as a stabilizer and builder of coastal dunes, which decrease the destructive forces of hurricanes and storms. Micropropagation of diverse sea oats genotypes from multiple Florida populations is limited due to labor costs and culture space requirements compounded by the loss of specific donor populations. Cryopreservation protocols were developed to reduce these limitations but still maintain genetic diversity. Cryopreservation, the ultra-low storage of plant tissues in liquid nitrogen (-196°C) is an effective technique for long-term plant germplasm storage. Protocols employing vitrification and encapsulation-dehydration of shoot meristem tips were optimized in this study. Shoot meristem tips could be cryopreserved using either method. Multiple studies were conducted to optimize significant cryopreservation steps. The vitrification protocol was optimized by inclusion of a 2M glycerol and 0.4M sucrose loading solution pretreatment, 40 minutes exposure to either PVS2 or A9, a modified PVS2 solution, and one rehydration step with 1.2M sucrose. The different vitrification steps screened resulted in regrowth ranging between 13% and 30% and averaged about 25%. Encapsulation-dehydration resulted in higher average regrowth. Meristem tips were encapsulated in a 0.75M sucrose and 3% alginic acid gel and polymerized into a bead using 100mM CaCl2. Beads were placed in 0.75M sucrose for 20 hours before drying in a laminar flow hood for 0, 2, 4, 8, or 12 hours. Regrowth (28.3%) occurred only after 4 hours of drying when a moisture content (MC) of 27.2% was obtained. Regrowth did not significantly decrease with subsequent drying to 19.6% MC. These studies show that cryostorage of sea oats shoot meristem tips can be successfully achieved.

James J. Sadler, Environmental Horticulture Department, PO Box 110675, University of Florida, Gainesville, FL 32611-0675. In Vitro Cellular and Developmental Biology, 49:S70-71, 2013

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THE 2013 SIVB STUDENT TRAVEL AWARD

Comparison of Larvicidal Effect of rhizomes of Tissue Cultured and Naturally Grown Plants of Kaempferia galanga (Zengiberaceae)

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:S71-72, 2013.

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THE 2013 JOHN S. SONG AWARD

Insights into the Role of α-Tocopherol in Plants: Multiple Abiotic Stress Tolerance via ROS Scavenging

Alpha (α)-tocopherol, the biologically most active form of vitamin E, is a major antioxidant that bulwarks the cells against oxidative damage. It constitutes a small fraction of the total tocopherol pool in most oilseed crops. We generated transgenic (TR) Brassica juncea plants with ~6 fold higher α-tocopherol levels compared to the wild type (WT) plants by overexpressing γ-tocopherol methyl transferase. This enzyme catalyzes a rate limiting step in the α-tocopherol biosynthetic pathway. To better understand the roles of different tocopherol forms in plants we compared the performance of TR plants under conditions of abiotic stresses induced by NaCl (salinity), CdCl2 (heavy metal) and mannitol (drought). This resulted in an increase in total tocopherol levels in both the WT and TR plants. Seed germination, shoot growth, and leaf disc senescence showed that TR B. juncea had enhanced tolerance to these stress and that induced by high temperature and methyl viologen. Damage caused by the induced stress was lower in TR plants compared to WT plants as assessed by their higher relative water content, lower MDA and H2O2 accumulation and lower electrolyte leakage. Lesser superoxide and H2O2 accumulation was observed in TR seedlings exposed to these stress. Enhanced levels of different antioxidant enzymes and molecules were present in TR plants when compared to WT plants under similar stress. Analysis of chlorophyll a fluorescence rise kinetics showed that there were differential effects of the applied stress on different sites of the photosynthetic machinery. These effects were found to be alleviated in TR plants. Thus, biofortification by metabolic engineering not only offers sustainable alternative to vitamin E supplementation for improvement of human health but also plays an important role in the alleviation of various environmental stress conditions in plants.

Deepak Kumar, School of Life Sciences, Jawaharal Nehru University, New Delhi, INDIA. In Vitro Cellular and Developmental Biology, 49:S35, 2013

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THE 2013 HONOR B. FELL AWARD AND 2013 SIVB STUDENT TRAVEL AWARD

Characterization and Applications of Fundulus heteroclitus Embryonic Cell Lines to Ecotoxicology and Fish Health

The common killifish or mummichog (Fundulus heteroclitus) is a widely distributed estuarine teleost used as model organisms in comparative physiology, toxicology, endocrinology, immunology, embryology, and as sentinel organisms for aquatic health. Their ease of maintenance and ability to withstand a variety of physiological extremes, have made them popular research models. In vivo experiments with killifish have allowed the elucidation of many physiological and molecular events, and readily manipulable in vitro models (i.e. continuous killifish cell lines) could further aid in mechanistic studies. Cell lines are also critical for studying intracellular pathogens such as viruses and microsporidia. Microsporidia are emerging parasites of interest in fisheries and aquaculture, and evaluation of susceptibility in these sentinel species should be explored. In this study, we report on the development of 3 embryonal cell lines from killifish, KFE-1, KFE-3 & KFE-5 with distinct morphological and physiological characteristics. These have been maintained for almost two years and have been cryopreserved and thawed successfully. KFE-5 was derived from the mid-trunk region of a late stage embryo and has been maintained in Leibovitz-15 media with 10% fetal bovine serum at 26°C. KFE-5 have been authenticated by DNA barcoding as cells derived from F. heteroclitus, and have been passaged over 35 times to date. This cell line has characteristics of myoblasts, respond to model toxicants and hormones, and support growth of two species of microsporidia. KFE-1 and KFE-3 have distinct morphologies and detailed characterization is in progress. The potential applications of these cell lines in toxicology and parasitology are currently being investigated.

Sarah Gignac, Department of Biology, Wilfrid Laurier University, Waterloo, Ontario, CANADA. In Vitro Cellular and Developmental Biology, 49:S45, 2013

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THE 2013 JOSEPH F. MORGAN AWARD

Development of Cell Lines from the Walleye Cauda Fin and Their Ability to Survive and Support Viral Hemorrhagic Septicemia Virus (VHSV) Group IVb Replication at Low Temperatures

A unique Northern American genotype (IVb) of viral hemorrhagic septicemia virus (VHSV) emerged over the last 10 years in the Great Lakes, causing significant mortality in a wide range of fish, including walleye. However, cell lines from commercially important species native to this region that could be used to study the virus in these species are few. Therefore, we sought to develop cell lines from the walleye caudal fin and have developed one (WECF11-f) with a fibroblast-like morphology and another (WECF11-e) with an epithelial-like morphology. These survive at temperatures from 4 to 32 °C and provide an opportunity to compare VHSV IVb replication at 4 °C, which walleye experience through winter, with 14 °C, which is usually considered optimal for VHSV production. At 14 °C, VHSV IVb was produced in both cell lines and caused cell death. At 4 °C, the expression of VHSV IVb transcripts was slightly delayed in both cell lines. However, viral production and cell death were greatly delayed at 4 °C but after 2 weeks had occurred in WECF11-f cultures but still had not happened in WECF11-e cultures. Possibly VHSV IVb has a tissue tropism that is most prominently expressed at low temperatures.

Aaron Bender, Department of Biology, University of Waterloo, Waterloo, ON, CANADA. In Vitro Cellular and Developmental Biology, 49:S30, 2013

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THE 2013 PHILIP R. WHITE AWARD

RNAi Mediated Viral Resistance in Transgenic Wheat

Wheat streak mosaic virus (WSMV) and is one of three viruses of the wheat mosaic complex affecting wheat in the Great Plains of the United States. The current disease management strategy incorporates the deployment of resistant varieties, mite vector control and various cultural practices, however, it is not fully effective. As an alternative strategy, we evaluated the use of interference RNA to generate resistance to these wheat viruses. RNAi expression vector was created from the sequence of the 6K2-NIa protein of WSMV and immature embryos of the wheat cultivar ‘Bobwhite’ were ico-transformed by biolistic particle delivery system with the RNAi expression vector and pAHC20, which contains the bar gene for glufosinate selection. After tissue culture and plant recovery, putative transformed plants were analyzed through PCR for the presence of the appropriate RNAi 6K2-NIa gene. Transgenic T1 seeds were collected and each line was tested for transgene expression via RT-PCR. To determine viral resistance, T1 progeny were mechanically inoculated with the corresponding virus. Viral presence was established by ELISA. In the T1 generation, resistance was seen in up to 80% of the plants evaluated for the 6K2-NIa construct. These plants have undergone single plant selection up to T4 and continue to show high level of resistance when challenged with the virus.

Jessica L. Rupp, Department of Plant Pathology, Kansas State University, Manhattan, KS 66506. In Vitro Cellular and Developmental Biology, 49:S62, 2013