2010 PBS Oral Presentation Competitions

Winners of the 2010 SIVB Plant Biotechnology Student and Post Doc Oral Competitions were announced during their respective sessions at the 2010 In Vitro Biology Meeting in St. Louis, Missouri. Cash awards of $250, $150, and $50 were provided for the first, second, and third place winners respectively. The awards were made possible by ad hoc donations from Monsanto Company. Coordinators Michael Bosela and Sukhpreet Sandhu  thank this year’s judges (Student Competition Judges: Michael Kane, Lisa Lee, and Ian Curtus; Post Doc Competition Judges: Jeffrey Adelberg, Maud Hinchee and Nancy Reichert) for their time and encouragement of the student participants.

PBS Student Competition    

                  
First Place

Enhancing Beta-carotene (Pro-Vitamin A) Content in Orange Fruits

Orange fruit contains high amounts of vitamin C and carotenoids, as well as other health promoting phytochemicals such as flavonoids and folic acid. Carotenoids are isoprenoid-derived molecules responsible for the attractive color of the peel and pulp of most citrus fruits and indispensable components of the human diet. In addition to being potent antioxidants, some of them, most importantly β-carotene, can be converted by the body to retinol and are called pro-vitamin A carotenoids. The most abundant carotenoids present in the peel and pulp of mature oranges are β-β-xanthophylls, which have null or low provitamin A activity. With the aim of increasing orange fruit nutritional value, we have attempted to enhance its content in β-carotene (provitamin A) by metabolic engineering of carotenoid biosynthesis. Recent advances in the identification and isolation of the genes responsible for the biosynthesis of carotenoids in citrus fruits have made this feasible. In this work, we blocked the expression of the endogenous β-carotene hydroxylase gene (β-CHX), involved in the conversion of β-carotene into xanthophylls, using either antisense or RNA interference (RNAi) technology. Transgenic orange fruits were obtained that showed important changes in carotenoid content and composition in both fruit peel and pulp, with β-carotene increases accompanied by a general decrease in the accumulation of downstream xanthophylls. This change in the carotenoid profile may have implications related with a putative enhanced capacity of transgenic oranges and their juice for protection against oxidative stress and/or degenerative diseases, due to a possible additive and synergistic effect of increased vitamin A content in a background matrix rich in vitamin C and other phytonutrients.

Elsa Pons, Centro de Proteccion Vegetal y Biotecnologia. Instituto Valenciano de Investigaciones Agrarias, Carretera Moncada-Náquera, kilometro 4.5, Moncada (Valencia), 46113, Spain.  Oral Presentation, Crop Biofortification II Symposia, Wednesday, June 9, 4:25 pm – 4:45 pm, 2010 In Vitro Biology Meeting held in collaboration with the 12th IAPB Congress.

Second Place

Virus Resistance Mediated by Artificial miRNA Against Wheat Streak Mosaic Virus in Transgenic Wheat Plants

Previous work of Nui et al. (2006) and Duan et al. (2008) highlighted the potential use of miRNAs as antiviral agents in plants. Genes encoding plant miRNAs are modified with complementary sequences to the genomic RNAs of plant viruses replacing the sequences which become native mature miRNA. On maturation, these ‘artificial’ miRNAs (amiRNAs) target the invading viruses and the plant becomes specifically immune to infection. We were encouraged to transform the more complex wheat genome with rice polycistronic miR395, modified with a number of 21 bp sequences from Wheat streak mosaic virus. The star-like secondary structure of miR395, consisting of multiple duplex arms, provided us the opportunity to target several areas of the WSMV genome. The synthetic miR395 construct was named “Fan Guard”. FG-miR395, driven by Ubiquitin promoter, was biolistically transformed into immature wheat embryos together with a separate plasmid carrying nptII for plant selection. The transgenic plants (T0) were analysed for the presence of transgene and selectable marker. T1 progeny from a number of transgenic events showed a high level of resistance segregating along with the transgene. Selection process of WSMV specific amiRNA and Molecular characterization of amiRNA expressing transgenic wheat plants will be discussed in the poster.

Muhammad Fahim, CSIRO and The Australian National University, MUHAMMAD FAHIM, PhD Student, L2E Discovery Plant Industry, Blackmountain Labs, CSIRO, Canberra ACT 2601, Australia. Oral Presentation, Biotic Stress III Symposium, Thursday, June 10, 2:35 pm – 2:55 pm, 2010 In Vitro Biology Meeting held in collaboration with the 12th IAPB Congress.

Third Place

Rapid Functional Characterization of Soybean Promoters in Transiently- and Stably-transformed Tissues

Promoters are DNA sequences located upstream of gene coding regions and are largely responsible for controlling gene expression. Discovery and analysis of novel promoters will lead to a better understanding of gene regulation in native contexts and new promoters are useful for the development of transgenic crops. Despite their importance and the high value of soybean, only a few soybean promoters have been characterized. For this research, we have characterized 10 Gmubi (Glycine max Ubiquitin) and 10 GmERF (G. max Ethylene Response Factor) promoters using two distinct validation tools, which were optimized for this research. Promoters from these two distinct families were identified from the soybean genome, PCR-cloned and fused to the gfp gene. For the first validation tool, transient GFP expression was quantified using image analysis following introduction of DNAs into lima bean cotyledons via particle bombardment. For the second validation tool, GFP expression in stably-transformed soybean hairy roots was also evaluated. Soybean cotyledon explants, inoculated with Agrobacterium rhizogenes harboring promoter constructs, showed numerous GFP-positive independent hairy root events 14-20 days after inoculation. GFP quantification in hairy roots revealed considerable variation in GFP expression among different hairy root events but expression was consistent for roots derived from individual events. GFP intensity in hairy roots seemed to be associated with transgene copy number. Strength of promoter activity was generally consistent using transient expression and stable transformation. The most highly expressing soybean promoters were Gmubi3 followed by Gmubi1, Gmubi5, Gmubi9, Gmubi7 and GmERF10.

Carlos Hernandez-Garcia, Department of Horticulture and Crop Science, OARDC/The Ohio State University, 1680 Madison Ave., Williams Hall # 225, Wooster OH 44691, USA. Abstract number P-070, 2010 In Vitro Biology Meeting held in collaboration with the 12th IAPB Congress.

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PBS Post Doc Competition

First Place

The Role of Genetic Background in Determining Qualitative and Quantitative Carotenoid Profiles in Corn Endosperm

Lutein and zeaxanthin are major carotenoids in the human retinal macula. Their yellow pigmentation helps to absorb blue and ultraviolet light and there is evidence that they protect against age-related macular degeneration (ARMD). Neither molecule can be synthesized de novo, but whereas lutein is abundant in the human diet there are only a few good sources of zeaxanthin, including certain varieties of corn. In these varieties, the β-carotene/zeaxanthin branch of the carotenoid pathway is favored over the α-carotene/lutein branch due to a high ratio of β:ε lycopene cyclase activity. We previously described a transgenic white corn (Zea mays) line expressing the early enzymes in the carotenoid pathway and accumulating high levels of carotenoids. Here we demonstrate that introgressing the induced (transgenic) mini carotenoid pathway in this transgenic line into a wild-type yellow-endosperm variety known for its high β:ε ratio gives rise to a novel hybrid line producing zeaxanthin at an unprecedented 56 µg/g dry weight. We also demonstrate that introgressing the same pathway from this line into an alternative yellow corn line with a low β:ε ratio results in a very different carotenoid profile in the resulting hybrid. We discuss the role of the genetic background in determining carotenoid profiles in corn and we identify further rate limiting steps in the pathway in view of our recent data.

Changfu Zhu, Departament de Produccio Vegetal i Ciencia Forestal, Universitat de Lleida, Av. Alcalde Rovira Roure, 191, E-25198 Lleida, Spain.  Oral Presentation, Crop Biofortification II Symposium, Wednesday, June 9, 4:05 pm – 4:25 pm, 2010 In Vitro Biology Meeting held in collaboration with the 12th IAPB Congress.

 

Second Place

An Argonaute Family Gene, Lobbed Leaflet1, Regulates Leaf, Flower and Root Development Through the TAS3 Trans-Acting siRNA Pathway in Medicago Truncatula

Small RNAs (sRNAs) have been shown to play important roles in regulating plant development. Trans-acting siRNAs (ta-siRNAs) are endogenous siRNAs that act in trans and direct the cleavage of complementary mRNA targets. Several key components, such as SGS3, RDR6, DCL4 and AGO7 are required for the biogenesis of ta-siRNAs. However, only TAS3-derived ta-siRNAs depend on AGO7. Recent studies show that AGO7 is involved into different processes of plant development. To further understand the roles of AGO7 homolog in development in legumes, we identify and characterize a leaf development mutant, lobbed leaflet1 (lol1), from a Tnt1 retrotransposon-tagged mutant population of Medicago truncatula. lol1 mutant exhibits strong defects in leaf, flower and root development. Further analyses show that LOL1 is the ortholog of Arabidopsis AGO7 which is required for the biogenesis of TAS3 ta-siRNAs. Microarray data indicate that three MtARF genes (Auxin Response Factor) are negatively regulated by the TAS3 ta-siRNA pathway. Moveover, we find that MtNAC1 which is the homolog of Arabidopsis NAC1, together with three target MtARF genes, are involved into the development of root in M. truncatula. In conclusion, LOL1 is required for the TAS3 ta-siRNAs in M. truncatula. The phenotype is caused by overexpression of its targets MtARF genes which are conserved between eudicots and monocots. Compared with the observation that Arabidopsis ago7 mutanthas no phenotype in root, the defects of root system in lol1 mutant suggest that TAS3 ta-siRNAs processing by LOL1 has a broader role in M. truncatula development.

Chuanen Zhou, The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK, USA.  Abstract number P-208, 2010 In Vitro Biology Meeting held in collaboration with the 12th IAPB Congress.

 

Third Place

Altering Lignin Content in Bahiagrass (Paspalum Notatum Flugge) by Down Regulation of 4-coumarate-CoA Ligase

Bahiagrass is the primary warm season forage grass in the south-eastern USA. In Florida alone it is grown on more than 5 million acres to support the beef cattle industry. However, the high lignin content of the bahiagrass biomass significantly reduces its forage quality. RNAi suppression of lignin biosynthetic enzymes in various plant species resulted in reduced lignin content that was accompanied with increased digestibility of forage biomass. The objective of this study is the improvement of the forage quality of bahiagrass by RNAi mediated suppression of its total lignin content. The 4-coumarate-CoA ligase (4CL) is a key enzyme in the lignin biosynthetic pathway. Four partial genes coding for 4CL were cloned from tetraploid bahiagrass cv. `Argentine`. RNAi vector targeting highly conserved domains of two bahiagrass 4CL genes was constructed using 200 bp of the coding sequences. The 4CL-RNAi construct was subcloned under transcriptional control of three different promoters: the constitutive e35S promoter, the OsC4H promoter from rice for xylem specific expression or the ZmdJ1 promoter from corn for expression in the green tissue. Each of these cassettes was co-transferred with the neomycin phosphotransferase (npt-II) selectable marker expression cassette to bahiagrass callus by biolistic gene transfer. Following selection regenerated plants were confirmed as transgenic events by PCR and Southern blot analysis. Significant reduction of 4CL gene expression was confirmed in several transgenic lines by Northern blot analysis. RNAi suppression of 4CL was more effective under transcriptional control of the xylem specific OsC4H promoter than under control of the e35S or ZmdJ1 promoters. Klason lignin analysis was conducted on transgenic lines growing in the green house in 2m x 1m x 0.6m soil bins in a randomized block design of three replications. Data correlating the 4CL gene expression level with the lignin content and in-vitro digestibility analysis will be presented.