For the In Vitro Plant Biotechnology student competition, 23 students submitted abstracts. Entries were voted on by a core committee composed by Dr. Randal Niedz, Dr. Manjul Dutt and Dr. Ian Curtis based on the quality of their abstracts, originality of the work and accomplishments. The Oral presentation competition was held on Monday, June 2, 10:30 am – 12:30 pm, finalists were judged by Dr. Ajith Anand, Dr. Manjul Dutt and Dr. Ian Curtis based on the quality of the presentation, methods, conclusions and knowledge of the topic. All the presentations were highly professional and the students received many questions from the audience and the judges. The judges deliberated for a while to finally choose the winners. The first place winner was Jonathan D. Willis from the University of Tennessee, the second place winner was Yang Zhao, from the University of Florida and the third place winner was Jessica L. Rupp from Kansas State University. Certificates and cash awards were presented to these students at the PBS Business Meeting. We highly encourage all qualified students and to submit their abstracts for the 2015 competition.
Submitted by Cecilia Zapata
Development of Switchgrass (Panicum Virgatum L.) Transformable Cell Suspension Culture and a Screening System for Rapid Assessment of Cell Wall Genes for Improved Biomass for Biofuels
Transformation and chemical characterization of plant cell wall traits for switchgrass is arduous and time consuming. We have developed transformable switchgrass cell culture lines with corresponding chemical fingerprinting to rapidly screen cell walls with modified genes. Transgenic down-regulated lignin switchgrass plants (COMT and MYB) were used as donors for inflorescence meristem tissue to induce callus. COMT and MYB callus were added to a liquid culture system to produce aggregate and non-aggregate cells to be evaluated by spectral and chemical analysis for cell wall properties. Callus generated from wildtype plants were used to develop a liquid culture system. Wildtype aggregate and non-aggregate cell cultures were transformed using Agrobacterium tumefaciens harboring the pANIC vector carrying the COMT and MYB genes and cell wall traits were analyzed. Transgenic liquid cultures from transformed plants and transformed cultures from wildtype plants were analyzed by chemical (PyGC/MS) and spectral (FTIR/Flurolog) techniques to generate a prediction model for detecting cell wall changes. Development of this simple cell switchgrass culture is our first steps for developing a multiplex automatic genome engineering (MAGE) system for plants.
Jonathan Willis, University of Tennessee, 2431 Joe Johnson Dr, 252 Ellington Plant Sciences, Knoxville, TN 36996. In Vitro Cellular and Developmental Biology, 50:S31, 2014
Insulator Mediated Transgene Performance in Sugarcane
Prediction of transgene performance is difficult in sugarcane due to the random insertion into the large and highly polyploid sugarcane genome and associated position effects. Insulators have been identified which establish genomic barriers and block the activity of enhancers. These two properties make it desirable to include insulators in vector construction to provide predictable expression of transgenes, support the direct comparison of different promoters and/or facilitate the tissue specific expression of stacked transgenes. EXOB from bacteriophage λ and TBS from petunia were recently reported as effective insulators in model plants. Our objective is to explore the influence of these insulators on transgene expression in sugarcane. Constitutive nptII expression cassettes without insulators or flanked by insulators were bombarded into sugarcane leaf whorl explants. Plants were regenerated following selection with geneticin via direct somatic embryogenesis. Taqman ® qPCR, PCR with multiple primer combinations and Southern blot were performed for determination of transgene copy number and integration pattern. NPTII ELISA was conducted from replicated protein extracts of the mid section of the top visible dewlap leaf from single copy lines for quantification of transgene expression. The 11 single-copy lines with complete expression cassettes and without insulators displayed an average expression level of 9.1 ng/10μg soluble protein, ranging from 0.0 to 21.5 ng/10μg soluble protein. The 7 single-copy lines with insulators displayed an average expression level of 17.0 ng/10μg soluble protein, ranging from 14.5 to 20.5 ng/10μg soluble protein. The line to line variation of transgene expression appeared to be reduced in transgenic lines with insulators. The average transgene expression level of lines with insulators was almost two times higher than that of lines without insulators. Data from additional single copy lines with the different constructs are currently generated and will also be presented.
Yang Zhao, University of Florida, 1692 McCarty Drive, McCarty Hall D, RM 3062, Gainesville, FL 32611. In Vitro Cellular and Developmental Biology, 50:S32, 2014
RNAi Mediated Silencing of Endogenous Wheat Genes eIF4E2 and eIF4G Induces Resistance to Potyviruses Wheat Streak Mosaic Virus and Triticum Mosaic Virus
Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) are two viruses 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. Both of these viruses belong to the family Potyviridae and use host eukaryotic initiation factors in order to facilitate replication of their genomes. We evaluated the use of RNAi to silence eIFE2 and eIF4G to interrupt this process to induce resistance to these wheat viruses. RNAi expression vectors were independently created from the sequences of the wheat genes eIF4E2 and eIF4G. Immature embryos of the wheat cultivar ‘Bobwhite’ were independently co-transformed by biolistic particle delivery system with RNAi expression vectors and pAHC20, which contains the bar gene for glufosinate selection. Putative transformed plants were analyzed through PCR for the presence of the appropriate RNAi gene. Transgenic T1 seeds were collected and each line was tested for transgene expression via RT-PCR. To determine viral resistance, the progeny were also mechanically inoculated with the WSMV. Viral presence was established by ELISA. Transgenic lines were advanced to homozygosity. Although transgene silencing and deletion was detected with a few events in the T2 generation, a consistent stable resistance response was demonstrated in three transgenic lines of eIF4E2 construct and four transgenic lines of eIF4G. T3 progeny were independently subjected to inoculation with both WSMV and TriMV and were resistant. T4 progeny were co-infected with both viruses and were also demonstrated to be resistant to both potyviruses. The transgene expression in the T5 generation of either construct continued to exhibit high levels of viral resistance. This research provides evidence that a single transgene can provide resistance to multiple viruses and has great potential benefits to both breeders and producers.
Jessica Rupp, Kansas State University, 3004 Montana Ct, Manhattan, KS 66502. In Vitro Cellular and Developmental Biology, 50:S32, 2014