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/awards/student-awards.html) or by contacting the SIVB Business Office at (910) 755-5431, email@example.com.
2015 HOPE E. HOPPS AWARDS AND 2015 SIVB STUDENT TRAVEL AWARD
Towards Targeting Multiple Expression Cassettes into a Pre-characterized Genomic Locus of Sugarcane for Predictable Transgene Performance
Sugarcane, a tropical C4 grass in the genus Saccharum (Poaceae), accounts for nearly 80% of sugar produced worldwide. Sugarcane is also an efficient and sustainable feedstock for commercial biofuel production. Sugarcane is a highly polyploid and frequently aneuploidy interspecific hybrid, making crop improvement by breeding and genetic transformation challenging. Future transgenic strategies will require gene stacking for incorporating several traits or entire pathways. However, transgenes integrate randomly into the genome and genomic regions that support high level and stable transgene expression seem to occur less frequent in sugarcane than in other plants. Therefore, it is highly desirable to develop strategies for site directed integration of transgenes into the sugarcane genome. Our goal is to enhance predictability of transgene performance by targeting transgene integration into a pre-characterized genomic locus by site-specific recombination (SSR). The series of experiments include: 1) Construct vector that introduces SSR target shielded by insulators into sugarcane. 2) Generate and characterize expression stability of single-copy target lines to select events for re-transformation. 3) Remove selectable marker from target line by SSR system. 4) Create and characterize elite events following SSR of donor vector mediated by recombinase and promoter trapping of promoter-less selectable marker. We will report on the generation of single-copy target lines with high and stable transgene expression, removal of the selectable marker gene and regeneration and initial characterization of potential elite events following re-transformation of target lines with the donor vector.
Yang Zhao, University of Florida – IFAS, Agronomy Department, Plant Molecular and Cellular Biology Program, Genetics Institute, Gainesville, FL 32611. In Vitro Cellular and Developmental Biology, 51:S50, 2015
2015 SIVB STUDENT TRAVEL AWARD
Expression Pattern and Activity Analysis of A Novel Tissue Specific Promoter
Promoter is a specific DNA sequence regulating the expression of downstream coding sequence or other noncoding expression cassettes. CaMV 35S (Odell, Nagy et al. 1985, Benfey and Chua 1990) and maize ubiquitin promoter (Cornejo, Luth et al. 1993) are two broadly used constitutive promoters in transgenic plants, but their strong and constitutive activities may cause many adversity consequences to transgenic plants such as growth suppression or ‘transgene silencing’ (Kooter, Matzke et al. 1999, Dietz-Pfeilstetter 2010). To avoid the disadvantages of constitutive promoters, we exploited a new tissue specific promoter that could be used to drive foreign gene expression in transgenic plants. We identified and cloned a promoter region of a leaf specific protein kinase gene Stress Responsive Factor 3 (SRF3) named Srf3 from Arabidopsis thaliana genomic DNA. To investigate the regulation pattern and evaluate the level of this promoter activity, we constructed a series of GUS reporter systems (β-glucuronidase), in which GUS gene is under the control of CaMV 35s, maize ubiquitin, Srf3a (-1536 to -12), Srf3b (-1033 to -12), or Srf3c (-395 to-12) promoters. Histochemical staining analysis of stable transgenic Arabidopsis plants harboring these constructs shows that all three Srf3 promoters have as strong activity as CaMV 35S and maize ubiquitin promoters in leaf tissue. The results of quantitative GUS activity assay indicate that unlike the two constitutive promoters, the activity of Srf3a is restricted to leaf tissue, and Srf3b and Srf3c are also leaf-predominant promoters. None of them are active in Arabidopsis seeds. Histochemical staining of transgenic tobacco harboring Srf3a/GUS construct show that Srf3a also exhibits a strong leaf-specific activity. Another binary vector was constructed, in which Srf3c promoter was used to drive an herbicide resistance gene bar. After treated with PPT, the transgenic Arabidopsis plants harboring Srf3c/bar survived, indicating that Srf3 could be used as a useful strong leaf specific promoter in agriculture for crop improvement.
Ning Yuan, Clemson University, Department of Genetics and Biochemistry, 105 Collings St., 104 BRC, Clemson, SC, 29634. In Vitro Cellular and Developmental Biology, 51:S34, 2015
2015 SIVB STUDENT TRAVEL AWARD
Development of Efficient Protocols for Somatic Embryogenesis and Regeneration of Fluted Pumpkin (Telfairia occidentalis Hook F.)
Fluted pumpkin (Telfairia occidentalis Hook. F.) is a leafy vegetable popularly consumed in Africa. It is traditionally propagated by seeds that are characterized with sporadic germination, and poor rooting of plants. Efficient protocols were developed for the induction of somatic embryos (SE) and its regeneration into plantlets using cotyledons from mature seeds. This study specifically evaluated the effect of plant growth regulators (mg/l); 2,4-D (0, 0.01, 0.05, 0.1 and 0.5) and Kinetin (0, 0.1, 0.5, 1 and 2) on the induction of SE, and IAA (0 and 0.01), 2,4-D (0 and 0.01) with Kinetin (0, 0.02 and 0.1) on its regeneration into plantlets. A significantly (p < 0.001) higher number of SE (381) were formed on MS medium with 0.5 mg/l 2,4-D and 0.1 mg/l Kinetin after 4 weeks of culture. All SE were obtained through an intermediary callus. For the production of SE-derived plantlets, treatments with 0.01 mg/l IAA + 0.02 mg/l Kinetin) had significantly (p < 0.001) higher plantlets than other treatments. With such a high number of SE achieved through this study, and its regeneration into rooted plants, it is now possible to mass produce Telfairia, a genus with narrow genetic diversity. This is the first report of its kind to the best of our knowledge. The regenerated plantlets which had broad leaves and high vigour were morphologically similar to the parent. A cryopreservation technique for somatic embryos can now be developed for the long term storage of this species to reduce the problems associated with erratic germination of seeds in storage.
Dotun Awosika, Dept. of Agronomy, University of Ibadan, Oyo State, NIGERIA. In Vitro Cellular and Developmental Biology, 51:S61, 2015
2015 CELLULAR TOXICOLOGY AWARD
Effect of Flavonoids and Phenolic Acids on Bioavailability of Artemisinin from Dried Leaves of Artemisia annua Using Simulated Digestion and Caco-2 Intestinal Epithelial Cells
Treating and preventing malaria has proven to be difficult in developing countries due to lack of infrastructure and economic hardship. Recently, research studying treatment using orally consumed dried leaves of Artemisia annua has shown promise. Previously we showed that various dietary constituents such as common fats and grains can alter artemisinin bioavailability when ingested with the dried leaves. Here we used the same simulated digestion method, to determine the effect of common protein-rich dietary constituents on artemisinin bioavailability and observed that digestion with two protein-rich dietary constituents, peanut butter and dried milk, decreased artemisinin bioavailability each by 33%. However, pure protein alone was not sufficient to reduce artemisinin bioavailability. In another earlier study using mice, oral treatment using A. annua dried leaves yielded 40 times more artemisinin in the blood of the mice than oral treatment with pure artemisinin. We used a Caco-2 model of the intestinal epithelium to determine if secondary metabolites found in A. annua increased artemisinin transport across the intestine and, thus, its bioavailability. Cells were grown in 12 well plates on cell culture inserts for 3 weeks to allow for differentiation into mature enterocytes before experiments were performed. We observed that neither of the flavonoids, quercetin and rutin, altered transport of artemisinin across the intestinal border. Ongoing experiments are investigating the effect of other secondary metabolites, such as phenolic acids, at different concentrations and aim to elucidate their effects on the transport of artemisinin. These results will help explain the enhanced bioavailability of artemisinin when delivered from dried leaves vs. as the pure drug.
Matt Desrosiers, Worcester Polytechnic Institute, Dept. of Biology and Biotechnology, 100 Institute Road, Worcester, MA 01609. In Vitro Cellular and Developmental Biology, 51:S42-S43, 2015
2015 JOHN S. SONG AWARD
RNAi Mediated Silencing of Triticum mosaic virus coat protein gene induces resistance to virus in transgenic wheat
Triticum mosaic virus (TriMV) is one of three viruses of the wheat mosaic complex affecting wheat in the Great Plains of the United States. Currently, there are no resistant commercial varieties. The disease management strategy incorporates 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. A RNAi expression vector was created from the sequence of the coat protein of TriMV and immature embryos of the wheat cultivar ‘Bobwhite’ were co-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 TriMV CP 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 TriMV. Viral presence was established by ELISA. In the T1 generation, resistance was seen in up to 80% of the plants evaluated for the TriMV CP construct. These plants have undergone single plant selection up to the T6 generation and continue to show high level of resistance when challenged with the virus. Crosses have been made with the virus susceptible winter wheat, ‘Overley.’ Real-time PCR results show a decrease in viral titer up to 20-fold in the T6 transgenic lines, the F1 crosses, and the BC1F1 compared to control plants. This research provides evidence that this RNAi silencing construct can provide stable resistance to TriMV 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, 2015