The following student awards were presented at the 2014 World Forum on Biology in Savannah, Georgia. Information on additional awardees at the 2014 World Forum 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, sivb@sivb.org, or Dr. Pamela Weathers, Chair, Student Affairs and Awards Committee, at weathers@wpi.edu.
2014 WILTON R. EARLE AWARD AND 2014 SIVB STUDENT TRAVEL AWARD
Heterologous Expression of the Rice MicroRNA395h in Nicotiana tabacum Impairs the Sulfate Homeostasis
As a rudimental and essential element, sulfur participates in many important mechanisms and pathways of plant development. Inorganic sulfate (SO42-), the most common source of sulfur in soil, is absorbed into root tissue and distributed into aerial part through vasculature system, where it is reduced into sulfite and finally sulfide within the subcellular organs such as chloroplasts and mitochondria and used for cysteine biosynthesis. MicroRNAs (miRNAs) are involved in regulating many metabolisms by repressing their target gene expression. MiR395 family in Arabidopsis thaliana has been reported to be an important regulator involved in sulfate transport and assimilation, and a high-affinity sulphate transporter (SULTR2:1) and three ATP sulfurylases (APS1,3, and 4) were verified to be the target genes of miR395. We have studied the function of miR395 in rice, and hypothesized that rice miR395 may have similar functions to that in Arabidopsis. Our results indicated that transcripts level of rice miR395 genes increased in sulfate deficiency mediums, and two predicted target genes of miR395 were down regulated under the same conditions. Overexpression of the rice miR395h (Osa-miR395h) in tobacco (Nicotiana tabacum) impaired its sulfate homeostasis, but sulfate distribution was not impacted among leaves of different ages. One potential target gene of miR395 was identified in tobacco, which is predicated to be a sulfate transporter gene. These results indicate that the rice miR395 responds to sulfate deficiency by inducing degradation of two target genes in rice, and the function of miR395 is conserved in plant species. Our results suggest that miR395 expression could be manipulated to enhance the uptake and assimilation of sulfur, benefiting plant development.
Ning Yuan, Clemson University, Department of Genetics and Biochemistry, Clemson, SC 29631. In Vitro Cellular and Developmental Biology, 50:S47-48, 2014
2014 HOPE E. HOPPS AWARDS AND 2014 SIVB STUDENT TRAVEL AWARD
Constitutive Expression of Osa-miR528 Alters Plant Development and Enhances Plant Tolerance to Salinity Stress and Nitrogen Starvation in Transgenic Creeping Bentgrass
One of the adaptive mechanisms that plants have evolved in stress response is mediated by microRNAs (miRNAs). Large-scale expression analyses reveal that a conserved monocot-specific miRNA, miR528 has the potential of mediating multiple stress responses. So far, however, the experiment support is lacking. To determine the role miR528 plays in plant response to abiotic stress, and the underlying molecular mechanisms, we analyzed its impacts on a perennial monocot crop species, creeping bentgrass (Agrostisstolonifera L.). Our stem-loop RT-PCR analysis demonstrates that the expression of miR528 was up-regulated under salt and drought stresses, but down-regulated under N deficiency in the wild type (WT) creeping bentgrass. Furthermore, we generated transgenic creeping bentgrass plants overexpressing a rice miR528 (Osa-miR528). Our data indicate that both plant development and stress response have been altered in transgenic plants. Morphologically, transgenic plants display shorter internodes, more tillers and upright growth than WT controls. Resistance to salt stress and N deficiency was enhanced in transgenics. Improved salt stress resistance was associated with increased water retention, cell membrane integrity, and chlorophyll content, while enhanced tolerance to N deficiency was associated with increased biomass, total nitrogen and chlorophyll content. Gene expression analysis identified four putative target genes, AsASO, AsLAC11, AsCSD2, and AsLPR1, which function in oxidation-reduction. Additionally, the expression level of AsNir encoding for nitrite reductase is increased in transgenic plants compared to WT controls, which might contribute to enhanced N use efficiency. The data obtained indicate the potential of manipulating miR528 in improving plant abiotic stress resistance.
Shuanrong Yuan, Clemson University, C/O Hong Luo, 110 Biosystems Research Complex, Clemson, SC 29631. In Vitro Cellular and Developmental Biology, 50:S37-38, 2014
2014 JOSEPH F. MORGAN AWARD AND 2014 SIVB STUDENT TRAVEL AWARD
The Innate Antiviral Effects of Extracellular Viral DsRNA in Rainbow Trout Cell
Almost all viruses produce double-stranded (ds) RNA sometime in their replicative cycle. Viral dsRNA is a potent trigger of innate immune responses in animals; activating the keystones of innate immunity, type I interferons (IFNs) and interferon stimulated genes (ISGs). DsRNA molecules produced in a cell during a virus infection are released into the extracellular environment during lytic viral infections. The effects of extracellular dsRNA in mammals are just beginning to be elucidated and almost nothing is known of its effects in fish. The present study uses both commercially available dsRNA (poly IC) and in vitro transcribed dsRNA molecules, based on the viral hemorrhagic septicemia virus (VHSV) genome sequence, as stimuli to investigate the effects of dsRNA on IFN/ISG production and antiviral effects in fish cells. Using an ISG reporter system, functional IFN assay, antiviral assay, and RT-PCR we have found that extracellular dsRNA, both synthetic and in vitro transcribed, are able to induce innate antiviral responses in the fish cell line, RTG-2. High- and low- molecular weight poly IC and different lengths of in vitro transcribed dsRNA were used to explore length effects. Consistent with previous studies done in mammalian cells we saw a greater magnitude of immune response when cells were stimulated with longer dsRNA molecules.
Sarah Poynter, Wilfiid Laurier University, 75 University Ave. W., Waterloo, ON N2L 3C5, Canada. In Vitro Cellular and Developmental Biology, 50:S42, 2014
2014 SIVB STUDENT TRAVEL AWARD
Evaluation of a Vitis-derived Reporter Gene System for Precision Breeding of Cold-hardy Grapevine Cultivars
Cisgenic and intragenic reporter gene systems are valuable tools for precision breeding of perennial fruit species including grapevine. To evaluate the development of a grapevine-derived reporter gene system, expression of the Vitis vinifera MybA1 gene, which is involved in the production of anthocyanin pigment, was compared with existing reporter genes including the green fluorescent protein (gfp) and β glucuronidase (gus) genes. Each reporter gene sequence was individually placed along with a nptII gene under the control of a CaMV35S promoter. Embryogenic cultures of ‘Bronx Seedless’ and ‘Thompson Seedless’ were initiated from in vitro-derived leaf explants. Somatic embryos at the mid-cotyledonary stage were co-cultivated with Agrobacterium harboring either the MybA1, gfp or gus genes. Transient and stable gene expression levels were recorded in co-cultivated embryogenic cultures. The number of independent embryo and plant lines obtained after transformation with individual reporter genes along with gene expression patterns was also recorded. Transient anthocyanin expression was evidenced by bright red spots on hypocotyl and cotyledonary regions of somatic embryos. Gene expression levels of cultures expressing VvMyBA1, gfp or gus reporter genes varied with cultivar. Transient and stable gene expression levels varied from 65-95% and 41-61%, respectively, whereas conversion of stable embryo lines ranged from 6 – 9%. Growth and development of stable anthocyanin embryo and plant lines was slower compared to GFP and GUS expressing lines, possibly due to the hyper-accumulation of anthocyanin in cell vacuoles. We are currently studying the possibility of using embryo specific promoters for anthocyanin expression solely in cell cultures. Grapevine-derived embryo specific promoters would enable selection of transformed tissues at the cell culture stage while allowing normal plant development following embryo germination.
Raju Kandel, University of Wyoming, 1358 Omarr Ave., Sheridan, WY 82801. In Vitro Cellular and Developmental Biology, 50:S47, 2014
2014 SIVB STUDENT TRAVEL AWARD
Improvement of Sunflower Transformation through the Use of Cotyledon Explants from Mature Seeds
Despite the high susceptibility of sunflower tissue to Agrobacterium, a reliable and efficient transformation protocol has not been available for routine gene characterization and manipulation in this plant. In order to use biotechnological tools for improvement of sunflower, sunflower transformation protocols must first be developed and optimized. A sunflower line RHA280 was identified as having high response to shoot induction, with an average of over 80 shoot primordia obtained from each of the 6 cotyledon explants, isolated from each seed. Sonication Assisted Agrobacterium-mediated Transformation (SAAT) was evaluated to improve the transformation efficiency, and a construct containing a gfp reporter gene driven by a sunflower polyubiquitin gene promoter (HaUbi) was used to monitor transgenic cell proliferation and shoot development. Imbibition of dry cotyledons in liquid medium for 1 d seemed to enhance their susceptibility to SAAT, by softening the dry cotyledon, leading to better wounding. A selection scheme in which hygromycin was applied immediately at the end of co-culture, instead of waiting 5 d after co-culture, increased the frequency of transgenic shoot production. Additionally, use of an intermediate level of hygromycin (15 mg/L) applied directly after co-culture yielded over 20% explants with transgenic shoot primordia, and over one transgenic shoots per explant on average. Due to the problems with shoot elongation and shoot rooting from cotyledon explants, a micrografting approach was also developed for the recovery of transgenic shoots, with a success rate over 30%. Transgenic shoots were also obtained from cotyledon tissue of cotyledon node explants which contained embryo shoot apex and the proximal end of cotyledon. The shoot primordia could elongate more easily than those from cotyledon explants even though the frequency of adventitious shoots was much lower, and rooting of these shoots remained problematic.
Zhifen Zhang, The Ohio State University, Dept of Horticulture & Crop Sciences, OARDC, 1680 Madison Ave., Wooster, OH 44961. In Vitro Cellular and Developmental Biology, 50:S36-37, 2014
2014 SIVB STUDENT TRAVEL AWARD
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, Dept of Plant Pathology, 4024 Throckmorton, Manhattan, KS 60502. In Vitro Cellular and Developmental Biology, 50:S31, 2014
