The following student awards were presented at the 2015 In Vitro Biology Meeting in Tucson, Arizona. Information on additional awardees at the 2015 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 (910) 755-5431, firstname.lastname@example.org.
2015 WILTON R. EARLE AWARD AND 2015 SIVB STUDENT TRAVEL AWARD
Elevating the Lipid Content in Vegetative Sugarcane Biomass by Metabolic Engineering
Sugarcane is one of the highest yielding biomass crops used for production of table sugar and fuel ethanol. Diverting carbon flux for the production of oil in vegetative biomass has emerged as a promising strategy for increasing energy density and biofuel yield. Storage lipids are mainly composed of energy dense glycerol esters of fatty acids, also known as triacylglycerol (TAG). We investigated the TAG accumulation in vegetative tissues of sugarcane following over-expression and, or RNAi suppression of several candidate genes. Single or multiple gene expression/suppression cassettes were co-delivered with the selectable nptII expression cassette by biolistic gene transfer into sugarcane callus. Plants were regenerated on geneticin containing culture medium and analyzed for presence and expression of target constructs by PCR and RT-PCR, respectively. Plants expressing single or multiple constructs or displaying suppression of target genes were analyzed for TAG content by GC-MS. These results demonstrate the feasibility of engineering sugarcane for accumulation of lipids in vegetative biomass and will open new prospects for biofuel applications.
Saroj Parajuli, University of Florida, Agronomy Department, Plant Molecular and Cellular Biology Program, Genetics Institute, IFAS, Gainesville, FL. In Vitro Cellular and Developmental Biology, 51:S60, 2015
2015 SIVB STUDENT TRAVEL AWARD
Wheat Gene Expression Differences Induced by Six Races of Puccinia triticina
Puccinia triticina, the casual agent of wheat leaf rust, is a devastating disease that can cause up to 40% yield loss. During fungal infection the host plant recognizes pathogen effectors, which trigger a host defense response. Changes in the pathogen effectors due to host selection pressure are responsible for the rapid development of new rust races and make durable resistance hard to obtain. The objectives of this study are to identify and characterize wheat genes that are utilized by races differently throughout infection and to understand functions of these genes using gene silencing. Six races of leaf rust were inoculated on a susceptible wheat variety and tissue was collected at six days post inoculation. RNA was sequenced and 63 wheat genes were identified that showed varying expression in response to the six races. 54 of these genes were evaluated in a time course study from zero days to six days post inoculation with the same six races. Real-time PCR was then used to analyze the timing of expression during early infection. The characterized genes have proposed functions involved in plant defense and stress, energy and metabolism, protein transport, replication, and RNA binding. Majority of the candidate genes showed three main expression patterns. However, race specific expression was found in three wheat genes that are affected by race shifts on Lr2A, Lr2C, and Lr17A. Sixteen potential susceptibility genes were also identified. Host susceptibility genes could be altered to provide durable resistance. RNAi was used to silence seven wheat genes to further understand their roles in leaf rust infection. T0 and T1 plants have been obtained and confirmed for the gene of interest. T2 plants were inoculated and observed for changes in susceptibility.
Kerri Neugebauer, Kansas State University, Department of Plant Pathology, 4024 Throckmorton, Manhattan, KS 66506. In Vitro Cellular and Developmental Biology, 51:S32, 2015
2015 HONOR B. FELL AWARD
Generation of Induced Pluripotent Stem Cell-derived Choroidal Endothelial Cells for the Treatment of AMD
Age-related macular degeneration (AMD), the most common cause of incurable blindness in the western world, involves the dysfunction and eventual death of choroidal endothelial (CEC), retinal pigment epithelial (RPE), and photoreceptor cells. Induced pluripotent stem cell (iPSC)-based strategies designed to replace both photoreceptor and RPE cells are currently a major scientific focus. For some patients, success of these approaches may also require replacement of CECs. The purpose of this study was to generate CECs from two Tie2 GFP iPSC reporter lines to develop efficient differentiation and transplantation protocols. Dermal fibroblasts from the Tie2 GFP mouse (carrying a GFP reporter gene under the control of the EC-specific Tie2 promoter) were isolated and reprogrammed into two iPSC lines via viral transduction of the Yamanaka factors Oct4, Sox2, Klf4, and c-Myc. iPSC potency was characterized via RT-PCR, immunocytochemistry (ICC), and teratoma formation assays. iPSC-CECs were differentiated using a co-culture method with either the RF6A CEC line or primary mouse CECs. iPSC-CECs from both lines were characterized via RT-PCR and ICC for various EC- and CEC-specific markers. The two iPSC-derived CEC lines expressed the EC markers CD31, CD34, Tie2, VE-Cadherin, and VWF, as determined by RT-PCR, expressed the CEC-specific markers carbonic anhydrase IV and ICAM1, as determined by RT-PCR and ICC, and were morphologically indistinguishable from native CECs that expressed ZO-1 and VE-Cadherin in cell-cell adhesions. Thus, we successfully generated murineiPSCs that can be efficiently differentiated into CECs. This work has paved the way for future studies focused on investigating AMD pathophysiology and CEC replacement.
Allison Songstad, University of Iowa, University of Iowa, Stephen A. Wynn Institute for Vision Research, Carver College of Medicine, Department of Ophthalmology, 375 Newton Rd., 4156 MERF, Iowa City, 52242. In Vitro Cellular and Developmental Biology, 51:S26-27, 2015
2015 PHILIP R. WHITE AWARD
In Vitro and in Planta Expression of Thermostable Endo-arabinase for Generating Functional Oligosaccharides from Plant Cell Wall for Colon-specific Health Benefits
Lignin-deficient (LD) plant fibers, e.g., sugar beet pulp and rice bran, are rich sources of functional cell wall polysaccharides. Used primarily as low-value animal feed, these established feedstock can be capitalized on for generation of value-added bioproducts, which will improve the economic viability and competitiveness of sugar beet and rice industry. The overall goal of this project is to develop an efficient enzymatic platform for generating functional oligosaccharides, specifically, feruloylated arabino-oligosaccharides (FAOs), from LD plant fibers. FAOs may be used in food and feed applications for healthful colon functioning through prebiotic, anti-inflammatory and mucosal immunomodulatory activities. FAOs of defined structure can be released from LD fibers through selectively cleaving the arabinan chain of cell wall polysaccharides by a key glycohydrolase – arabinase (ABN). In this study, a thermostable endo-1,5-α-L-ABN from Bacillus thermodenitrifican was expressed in yeast (Pichia pastoris) to produce significant quantity of enzyme for testing its activity of digesting plant fibers. Recombinant enzyme was secreted into culture media at a yield of ~70 mg/L and showed a specific enzymatic activity of 350 U/mg. ABN was also expressed in planta (in tobacco) to assess its function in post-harvest modification of cell wall polysaccharides to facilitate in situ FAOs releasing. Transgenic tobacco accumulated ~20 μg/gFW active ABN, and were not phenotypically different from wild-type plants. Finally, T84 epithelial cell culture was established to assay the anti-inflammatory activities of FAOs. Preliminary study indicated that FAOs isolated from rice bran significantly enhanced the expression of occludin, a biomarker of epithelial cells’ immune response to FAOs.
Ningning Zhang, Arkansas Biosciences Institute, Arkansas State University, Jonesboro, AR 72401. In Vitro Cellular and Developmental Biology, 55:S59, 2015