Student and Post Doc Oral Presentation Competition Winners

Fredy Alpeter, coordinator of the Plant Graduate Student and Post Doc Competitions, announced this year’s winners. 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. Fredy thanked this year’s judges (Student Competition Judges: Kan Wang, Vincent Wingate, and John Finer; Post Doc Competition Judges: Paula Pijut, Vibha Srivastava, and Randall P. Niedz) for their time and encouragement of the student participants.

Winners of the Student Oral Presentation Competition were:

First Place

Expressing the Gibberllin Catabolizing Enzyme AT-GA-ox1 in a Low-input Turfgrass (Paspalum notatum Flugge) Improves Turf Quality and Field Performance

Bahiagrass (Paspalum notatum Flugge) is a popular forage and turf species in the southeastern US due to its persistence under low-input conditions. However, the turf quality of bahiagrass is limited by its open growth habit and prolific production of long seedheads. We recently reported improved turf characteristics of bahiagrass following constitutive expression of the gibberellin catabolyzing enzyme, GA 2-oxidase (AT-GA-ox1).  Here we describe a field evaluation of turf quality and drought tolerance of these transgenic bahiagrass lines. Transgenic bahiagrass and wildtype plants were established in 1 m x 1 m plots under USDA-APHIS permit 06-219-01r in a split-split-plot design. Following establishment plants were evaluated under two different mowing environments (weekly and biweekly) and three different irrigation regimes (full, moderate and no irrigation) in four replications. Turf was evaluated by comparing establishment, persistence, turf density, number of inflorescences, clipping weights, root and rhizome weight under different mowing and irrigation conditions. Statistical analysis was performed according to the randomization structure using the MIXED-procedure of SAS. Transgene expression under field conditions was evaluated with RT-PCR.  Bahiagrass over-expressing AT-GA-ox1 produced significantly more tillers than wildtype.  Transgenic plants also showed decreased stem length while root and rhizome biomass as well as drought tolerance and low input characteristics were not compromised. Delayed flowering and improved recovery from drought was also observed in some lines. These results suggest that suppression of bioactive GAs enhances tiller bud outgrowth, reduces apical dominance and delays inflorescence development in bahiagrass. Transgenic bahiagrass lines over-expressing the gibberellin catabolizing enzyme GA 2-oxidase (AT-GA-ox1) display improved turf quality without compromising its persistence and low-input qualities.

Paula Lomba, University of Florida-IFAS, Agronomy Department, Plant Molecular and Cellular Biology Program, Genetics Institute, University of Florida – IFAS, Gainesville FL-32611. In Vitro Cellular and Developmental Biology, 45:S34, 2009

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Second Place

Regeneration of Arachis paraguariensis Through Different Morphogenic Pathways

Several wild species in the Genus Arachis represent important sources of novel genes for improvement of cultivated peanut. Arachis paraguariensis Chodat & Hassl. is a wild peanut that is highly resistant to Cercospora arachidicola, the causal agent of early leaf spot disease. Attempts of conventional hybridization between cultivated peanut and A. paraguariensis have failed due to hybridization barriers. Tissue culture and biotechnology techniques offer potential routes for overcoming these barriers, but as with many legumes, the Arachis spp. are generally recalcitrant to tissue culture regeneration. This study investigated the roles and interactions of different genotypes, explant sources and growth regulators in tissue culture regeneration of A. paraguariensis. Deembryonated cotyledon and embryonic axis explants dissected from mature seeds were grown in vitro under continuous light in modified MS media that has been supplemented with auxin and cytokinins at different concentrations. Two factorial experiments in a complete randomized design led to the induction of embryogenic callus when combinations of 2,4-D and BA or 2iP were used. Somatic embryogenesis and direct shooting was also achieved with various combinations of TDZ and BAP or 2iP.  Analyses confirmed a tri-directional pathway via direct organogenesis, somatic embryogenesis and callus-mediated embryogenesis.  Deembryonated cotyledons explants produced higher number of shoots than the embryonic axis explants across all media treatments. Shoots were transferred into semi-solid MS medium containing either of IAA, NAA and IBA for adventitious root initiation before they were planted in soilless mix in the greenhouse. This regeneration system has been tested for six genotypes of A. paraguariensis and found to be reproducible; the procedure is genotype independent and can be utilized for improved introgression of genes from this wild species into the cultivated peanut.

Olubunmi O. Aina, Agronomy Department, University of Florida, 304 Newell Hall, PO Box 110500, Gainesville, FL 32611-0500. In Vitro Cellular and Developmental Biology, 45:S35-36, 2009

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Third Place

Highly Efficient Suppressor-dependent Protein Expression in Plants with a Foxtail Mosaic Virus Vector

A new viral vector based on foxtail mosaic potexvirus (FoMV) was constructed by eliminating the triple gene block and coat protein genes, reducing the viral genome more than 2-fold. The resulting FECT vector (Fomv Elimination of Coat protein and Triple gene block) is driven by a CaMV 35S promoter in a binary vector and was delivered via syringe agroinoculation of Agrobacterium tumefaciens to whole plants of Nicotiana benthamiana. Interestingly, agroinoculation of the vector alone results in only slight transient expression, whereas co-inoculation with silencing suppressor genes (carried in a separate agrobacterial strain) allows for highly efficient GFP expression of up to 40% TSP. Thus, the FECT vector provides high capacity expression coupled with a tight on-off switch which could be utilized in permanently transgenic plants. It is also a good model system for molecular biology and molecular virology studies.  FECT transient gene expression system are especially useful to rapidly confirm that the foreign molecule of interest is correctly assembled and retains its biological activity before generating stably transformed transgenic plants. Full-sized HC and LC components of an anti-langerin IgG, each carried by a separate FECT vector, were able to produce immunologically functional antibody upon co-inoculation. This vector also addresses many environmental safety concerns: i) its genome is reduced by more than half, ii) it does not replicate efficiently unless the plant immune system is suppressed, iii) it lacks a coat protein and cannot form a virion, and iv) it is derived from a virus that in most hosts causes only mild infections (no symptoms observed in N. benthamiana).

Zun Liu, Department of Biology, Baylor University, Waco, TX 76798-7388. In Vitro Cellular and Developmental Biology, 45:S35, 2009

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Winners of the Post Doctoral Oral Presentation Competition were:

First Place

Transposon Mutagenesis of Soybean (Glycine max) Using the Rice MITE mPing

The recently sequenced soybean genome is predicted to have ~ 65,000 genes.  Transposon tagging mutagenesis will facilitate the annotation and characterization of these genes.  mPing is a miniature inverted terminal repeat element (MITE) from rice (Oryza sativa) that acts as a natural mutagen when it transposes.  As a tagging tool it has the advantages of both the TNT (high activity and unlinked insertion) and the Ac/Ds (potential for activation tagging) mutagenesis systems.  Our objective is to determine whether mPing is suitable for transposon tagging of soybean.  mPing was previously shown to be mobilized in Arabidopsis when the Ping proteins, ORF1 and TPase, were expressed.  We transformed a similar construct into soybean somatic embryos and observed both excision and insertion of mPing for multiple transformation events.  We found that these transposition events produce unlinked insertions with the same T/A rich insertion preference observed for rice.  In addition, we found that for three independent lines, transposition is upregulated during late embryogenesis.  Together these results confirm the potential for mPing to produce useful mutations in soybean.  We hope to produce heritable mPing insertions by inducing transposition in the meristematic tissues that produce seeds.  Thus, we are testing additional promoters and a mutant TPase that shows increased transposition in Arabidopsis.  We hope to use the resulting plants to produce an mPing mutagenized soybean population for both forward and reverse genetic screens.

Charles N. Hancock, Dept. Crop and Soil Science University of Georgia, Athens, GA. In Vitro Cellular and Developmental Biology, 45:S37, 2009

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Second Place

Host-delivered RNAi: An Effective Strategy to Silence Nematode Genes in Transgenic Hairy Roots of Soybean

The soybean cyst nematode (SCN), Heterodera glycines, is the primary biotic factor limiting soybean production, accounting for 40% of total disease losses. Current methods to control this pest are not totally successful in part due to new SCN biotypes emerging that can overcome current resistant varieties. To control soybean cyst nematode (SCN) in soybean, our laboratory has been evaluating the expression of siRNAs against specific nematode genes in chimeric transgenic plants. Ten separate nematode genes were selected for this study. Gene fragments were cloned into siRNA expressing vectors by Gateway cloning strategy. The siRNA constructs of these ten genes were independently transformed into soybean using this hairy root systemmediated by Agrobacterium rhizogenes. Transgenic roots were confirmed via PCR and Southern-blot analysis. Transgene expression was surveyed by reverse transcription PCR. SCN bioassays resulted in up to 85% reduction in eggs g-1 root tissue, indicating that chimeric transgenic plants expressing specific RNA silencing vectors significantly suppressed the reproductive potential of H. glycines. Stable soybean transformation is in progress for some of the effective genes.

Jiarui Li, Department of Plant Pathology, Kansas State University, Manhattan, KS 66506.  In Vitro Cellular and Developmental Biology, 45:S37, 2009

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Third Place

TILLING for Peanut Improvement

Allergic reactions to peanuts (Arachis hypogaea) can cause severe symptoms and can even be fatal.  Avoidance is the best means to prevent allergic reactions, but accidental consumption is still of concern due to the prevalence of peanut-derived products in processed foods.  One strategy of reducing the allergenicity of peanuts is to alter the amino acid sequences of allergenic proteins in the seed, thus making them less reactive to IgE.  Mutagenized peanut populations have been generated using EMS.  Targeted Induced Local Lesions in Genomes (TILLING) is a reverse-genetics approach used to screen mutagenized populations for individuals carrying single-nucleotide mutations or small indels in specific genes.  Two similar copies of a major allergen, ara h 2, have been identified, one in each sub-genome.  The same situation has also been shown for major allergen ara h 1.  For TILLING these genes, a nested PCR approach is used in which both copies are amplified in the first round, and then IRDye-labeled primers specific to only one gene are used in the second round. Heteroduplexes are then formed and digested with CEL1 nuclease.  Cleaved PCR products representing mutations are detected on a Li-Cor DNA Analyzer.  Putative mutations are then confirmed by sequencing.  To date, we have identified several mutations in ara h 2.01, ara h 2.02, ara h 1A and ara h 1B; including a premature stop codon in ara h 1A.  This work represents the first steps toward the eventual goal of creating a peanut cultivar with reduced allergenicity.  This approach is also being used to alter seed oil composition through changes in fatty acid desaturases.  We have found potential mutations in both copies of AhFAD2, genes which control the ratio of oleic to linoleic acid in the seed.

Joseph E. Knoll, Dept. of Horticulture/NESPAL, University of Georgia-Tifton Campus, P.O. Box 748, Tifton, GA 31793.  In Vitro Cellular and Developmental Biology, 45:S37-38, 2009