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2011 IN VITRO BIOLOGY MEETING 2011 Meeting of the Society for In Vitro Biology

June 4 – 8, Raleigh, North Carolina

LATE SUBMISSION ABSTRACTS

The following abstracts will be included in an upcoming issue of In Vitro Cellular and Developmental Biology: SYMPOSIUM ABSTRACTS SINGLE MOLECULE SEQUENCING TECHNOLOGY: THE APPLICATIONS AND IMPLICATIONS FOR

BIOLOGICAL RESEARCH - Sunday, June 5, 2011, 1:30 pm J-2 Dealing with the Threads of Life: A Singular View of the Genome David C. Schwartz. University of Wisconsin-Madison

CURRENT TOPICS IN SYSTEMS BIOLOGY – Wednesday, June 8, 2011, 8:00 am PS-10 Fish Nets and Ecotoxicology: Network Methods for Exploring Perturbations of the Hypothalamic Pituitary

Gonadal Axis Lyle D. Burgoon, US Environmental Protection Agency ANIMAL POSTER ABSTRACTS IN VITRO ANIMAL CELL SCIENCES POSTERS A-3000 Insect-derived Antifreeze Peptides for Cryopreservation of Cell and Tissues: A Cryomicropscopy Evaluation

L. H. Campbell, Cell and Tissue Systems, D. O. Halwani, K. G. M. Brockbank, and J. G. Duman A-3001 Organ-cultured Human Colon Tissue Growth Optimized for Stroma: Isolated Mesenchymal Cells Respond

to Calcium and Lanthanide Metals with Increased Proliferation and Differentiation M. K. Dame, University of Michigan Medical School, M. Naik, I. Veerapaneni, and J. Varani A-3002 Development and Characterization of a Pituitary Derived Cell Line from Adult Atlantic Salmon, Salmo salar Lucy E. J. Lee, Wilfrid Laurier University, Nguyen T. K. Vo, Mike S. Mikhaeil, Una Adamcic-Bistrivoda, Heather E.

Braid, and Robert H. Hanner A-3004 Cryopreservation of Cells with Recombinant Insect-derived Anti-freeze Proteins L. H. Campbell, Cell & Tissue Systems, Inc., L. Freeman, H. Kershaw, D. Halwani, J. G. Duman, and K. G. M.

Brockbank A-3005 Generation and Analysis of a New Immortalized Human Corneal Epithelium Cell Line H. Kojima, National Institute of Health Sciences, N. Yamamoto, K. Hirano, M. Sumitomo, H. Yamashita, M.

Nakamura, K. Hara, A. Tanikawa, M. Horiguchi, and K. Taniguchi A-3006 Rational Design of Engineered Botulinum Neurotoxin B-insensitive VAMP-2 Mutants Adam Swartz, USAMRICD, Kaylie Tuznik, and Patrick McNutt SILENT ABSTRACT A-3003 Prostaglandins Alter Protein Phosphorylation Levels in the Insect Cell Line BCIRL-HzAM1 Cynthia L. Goodman, USDA-ARS-BCIRL, David Stanley, and Qisheng Song

PLANT POSTER ABSTRACTS BIOTECHNOLOGY P-3000 Preliminary Safety Assessment of Two Genetically Engineered Corn Lines Expressing Carotenoid and Other

Vitamin Biosynthetic Genes G. Arjó, Universitat de Lleida, T. Capell, C.Piñol, and P. Christou

P-3001 Evaluation of a Cyst Nematode Parasitism Gene in Arabidopsis thaliana J. M. Chiera, North Carolina State University, N. Hamamouch, C. Li, T. J. Baum, M. G. Mitchum, and E. L. Davis

P-3002 Phloem Specific Gene Expression in Vegetative Tissues of Transgenic Citrus Plants M. Dutt, University of Florida/IFAS, A. Govindarajulu, M. K. Jaromin, R. H. Brlansky and J. W. Grosser

P-3003 RNA Interference (RNAi) Induced Resistance for Plant Parasitic Nematodes Jiarui Li, Kansas State University, Kerri Neugebauer, Timothy C. Todd, Tom R. Oakley, and Harold N. Trick

P-3004 Towards Development of a Sterile Tobacco Hybrid for Pharmaceutical Production J. Hollis Rice, The University of Tennessee, Richard Mundell, Reginald J. Millwood, Orlando Chambers, H. M. Davies, and C. Neal Stewart, Jr.

P-3005 The Effects of Light vs. Darkness on the Accumulation of Storage Reserves in Developing Soybean Somatic Embryos S. A. Sparace, Clemson University, Y. He, and T. E. Young

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P-3006 RNAi Silencing of the Plant Pathogen Phytophthora capsici Rio A. Stamler, New Mexico State University, Natalie P. Goldberg, Soum Sanogo, and Jennifer J. Randall

P-3007 Development of a Super Spud Through Repeated Cycles of Marker-free Transformation. A. Stivison, J. R. Simplot Company, S. Ringold, T. Carroll, and C. Rommens

P-3026 The Molecular Events During the Induction of Shoot Regeneration in Plant Tissue Culture Moshe Reuveni, ARO, Volcani Center, Dalia Evenor, and Mike Timko

P-3027 Expression of an Arabidopsis Vacuolar H+-Pyrophosphatase Gene (AVP1) in Peanut Enhances Salt- and Drought-tolerance and Improves Yield in the Field Qiang Gu, Texas Tech University, Hua Qin, Yizheng Zhang, Li Sun, Roberto Gaxiola, Paxton Payton, and Hong Zhang

CELLULAR AND MOLECULAR PATHOLOGY P-3008 Cell Line Selection for Sugar Beet Resistance to Cercospora beticola Sacc O. V. Kryliuk, State Enterprise Research Farm “Shevchenkivske,” and V. M. Venger EMBRYOGENESIS/MICROPROPAGATION/REGENERATION P-3009 Somatic Embryogenesis Induction in Zygotic Embryos of Asparagus racemosus Willd. - A Medicinal Plant J. Chaudhary, Deemed University, and P. K. Dantu P-3010 Comparative Initial In Vitro Stabilization and Stage II Shoot Multiplication of Sea Oats (Uniola paniculata L.)

Genotypes Established from Seedlings J. J. Sadler, University of Florida, M. E. Kane, and N. L. Philman P-3011 Triploid Citrus Regeneration via Embryo Rescue X. Shen, University of Florida, K. J. Song, H. S. Dhaliwal, M. Holt, M. Wendell and F. G. Gmitter, Jr. P-3012 Micropropagation of Veratrum californicum Through Tri-scale Culture Y. P. Sun, Clemson University, J. Naylor-Adelberg, S. A. White, and J. W. Adelberg P-3028 Induction and Multiplication of Embryogenic Callus of Coffea arabica L. Elite Genotypes

Carlos H. S. Carvalho, Embrapa Coffee, Ana Carolina R. S. Paiva, Elizane Q. Silva, Aline A. Custódio, Jaqueline Pala, and Vanessa R. Paulino

GENE TRANSFER TO PLANTS P-3013 Antiviral Lectins as Components of Plant Based Microbicides for HIV Prevention M. Sabalza, Universitat de Lleida, C. van Dolleweerd, T. Capell, B. O’Keefe, J. Ma, and P. Christou P-3014 A Transgenic Rice Seed Based Platform for the Production of Next Generation Multi-component

Microbicides Against HIV E. Vamvaka, Universitat de Lleida, M. Sabalza, B. O’Keefe, J. Ma, T. Capell, and P. Christou

IN VITRO TOOLS TECHNIQUES AND OPTIMIZATION P-3015 Induction of Tetraploidy in Diploid Wild Peanut (Arachis paraguariensis) O. O. Aina, University of Florida, M. Gallo, and K. H. Quesenberry P-3016 Macronutrient Optimization for Micropropagation and Acclimatization Using Turmeric Curcuma longa L as a

Model Plant S. M. Halloran, Clemson University, and J. Adelberg P-3017 Optimization of Micropropagation Protocols: The Genotype Effect J. Jasinski, University of Florida, M. E. Kane, N. Philman, and T. Johnson

MONOCOT TRANSFORMATION P-3018 Tetracycline-inducible Expression of the ODP2 Gene in Maize Produces Callus from Leaves

K. Lowe, Pioneer Hi-Bred International, Inc., G. Hoerster, X. Sun, C. Scelonge, Z. Bao, L. Newman, J. Zheng, B. Shen, W. Bruce, and W. Gordon-Kamm

P-3019 Transformation of Explants from Either Mature Seed or Germinating Seedlings in Maize Aided by the ODP2 and WUS2 Genes N. Wang, Pioneer Hi-Bred International, Inc., E. Wu, K. Lowe, C. Scelonge, L. Wang, B. Lenderts, G. Hoerster, C. Sweeney, L. Ryan, W. Hua, P. Shen, J. Chow-Yiu, Z.-Y. Zhao, and W. Gordon-Kamm

PLANT TRANSFORMATION P-3020 Medicago truncatula Insertion Mutants Using the Tnt1 Retrotransposon H. K. Lee, The Samuel Roberts Noble Foundation, J. Yarce, M. Tadege, J. Wen, J. Gallaway, C. Elles, X. Cheng, P.

Ratet, and K. S. Mysore P-3021 Characterization of Nonhost Resistance Mechanisms in Rice Using RNA Interference-mediated Gene

Silencing H. K. Lee, Samuel Roberts Noble Foundation S. H. Lee, H. Hisano, Z. Wang, and K.S. Mysore

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P-3022 Use of the ODP2 Gene Alone or in Combination with WUS2 Improves Transformation Frequencies in Maize Inbreds

E. Wu, Pioneer Hi-Bred International, Inc., N. Wang, K. Lowe, C. Scelonge, L. Wang, B. Lenderts, G. Hoerster, L. Ryan, H. Wei, P. Shen, J. Chow-Yiu, Z.-Y. Zhao, and W. Gordon-Kamm

P-3029 Agrobacterium-mediated Transformation of Mature Citrus M. Marutini-Hert, USDA-ARS-U.S. Horticultural Research Laboratory, T. E. Mirkov, T. J. Evens, and R. P. Niedz

STRESS RESISTANCE P-3023 The Comparison of P5CS Gene Expressions of Two Soybean Varieties Under Salt Stress

Ö.Çelik, Istanbul Kultur University, and Ç. Atak P-3024 Identification of Rice Genes Induced by Abiotic Stress by Proteomics and qRT-PCR

S. Dashevskaya, Universitat de Lleida, R. Horn, D. Zimmermann, I. Chudobova, S. Schillberg, T. Capell, and P. Christou

SILENT ABSTRACT P-3025 A Series of Modular Vectors for Cloning of Target Genes and Regulatory Elements for Providing Stable and

Effective Expression of Heterological Genes in Plants A. O. Vyacheslavova, Institute of the General Genetics Russian Academy of Sciences, I. N. Berdichevets, H. R. Shimshilashvili, E. A. Romanov, O. Mustafaev, and I. V. Goldenkova-Pavlova

SYMPOSIUM ABSTRACTS SINGLE MOLECULE SEQUENCING TECHNOLOGY: THE APPLICATIONS AND IMPLICATIONS FOR BIOLOGICAL RESEARCH J-2 Dealing with the Threads of Life: A Singular View of the Genome. D. C. SCHWARTZ, S. Zhou, S. Goldstein, K. Potomousis, M. Place, B. Teague, and K. Kounovsky-Shafer. Laboratory for Molecular and Computational Genomics, Departments of Genetics and Chemistry, University of Wisconsin-Madison, Madison WI 53706. Email: dcschwartz @wisc.edu Technological advances broaden our understanding of genomes, and new approaches employing single molecule analytes offer unique advantages for the discovery and characterization of genomic alterations complementing discernment of single nucleotide polymorphisms (SNPs) and copy number variants (CNVs). CNVs commonly represent genomic events, such as amplifications and deletions usually found by DNA hybridization. Although such measures of genomic alteration are relatively comprehensive and have characterized small populations, CNVs effectively flag broad classes of genomic alterations, but do not readily discern genomic structural alterations embodied as translocations, gene-fusion events, amplifications, insertions, or rearrangements--both large and small scale (sub-genic). Furthermore, next-generation sequencing also offers limited structural insights, due to short read lengths, which attenuate genome coverage and discernment of complex events. The Optical Mapping System, the first genomics platform to utilize single molecule analytes enables the construction of genome-wide physical maps (consensus maps) from ensembles of ordered, single-DNA molecule restriction maps developed from genomic sources, obviating clone libraries, PCR, and hybridization. Comparisons of optical consensus maps against a reference map reveals structural alterations as “differences,” in the form of novel restriction sites (missing or extra cuts; MCs or ECs), or indels (insertions or deletions), which are statistically assessed, in part, based on the number of single-DNA molecule optical maps collectively represented by

the consensus map. Since high-resolution restriction maps intrinsically reveal genome structure, elusive differences such as indels are discoverable and physically characterized. These advantages are further advanced by the de novo construction of optical maps that guide the sequence assembly of complex genomes, examples of which include the rat, maize, mouse, rice and medicago genomes. CURRENT TOPICS IN SYSTEMS BIOLOGY PS-10 Fish Nets and Ecotoxicology: Network Methods for Exploring Perturbations of the Hypothalamic Pituitary Gonadal Axis. L. D. BURGOON. 109 T.W. Alexander Drive, National Center for Environmental Assessment, US Environmental Protection Agency, RTP, NC 27711. Email: burgoon.lyle@epa.gov Alternative species, including ecologically relevant species, are a significant part of our Next Generation (NexGen) Risk Assessment paradigm. NexGen consists of three tiers, where high throughput and computational screening occurs in the Tier 1, medium throughput assays and alternative species are used in Tier 2 on active compounds from Tier 1, and more traditional toxicology and risk assessment data are used in Tier 3. The alternative species models in Tier 2 tend to be cheaper and higher throughput in vivo models than typical mammalian models, and have the benefits of fully functioning drug clearance and metabolism mechanisms compared to most high throughput in vitro models used in Tier 1. Recently, EPA and the US Army Corps of Engineers completed a large study in fathead minnow exploring the transcriptomic response of the ovary in response to perturbation of several specific parts of the hypothalamic pituitary gonadal axis using numerous specific inhibitors. This talk will describe how networks can be used to dissect the similarities and differences between vehicle and treated animals, as well as between different HPG-targets. This work is critical to NexGen as it lays the groundwork for creating a better understanding of what endocrine modes of action may be present in this alternative species model, and allow us to generate more informed comparisons between the fathead minnow and more traditional model data.

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ANIMAL POSTER ABSTRACTS IN VITRO ANIMAL CELL SCIENCES POSTERS A-3000 Insect-derived Antifreeze Peptides for Cryopreservation of Cells and Tissues: A Cryomicroscopy Evaluation. D. O. Halwani1, K. G. M. Brockbank1,2,3, J. G. Duman4, and L. H. CAMPBELL1. 1Cell & Tissue Systems, Inc., North Charleston, SC; 2 Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA; 3Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC; and 4Department of Biological Sciences, University of Notre Dame, Notre Dame, IN. Email: dhalwani@celltissuesystems.com, lcampbell@celltissuesystems.com Expanding cryopreservation methods to include a wider range of cell types such as those sensitive to freezing is needed for maintaining the viability of cell-based regenerative medicine products. Conventional crypreservation protocols which include commonly used cryoprotectants such as dimethly sulfoxide (DMSO) are limited by the damaging effect of ice formation during freezing. The aim of this study was to examine recombinant insect-derived antifreeze peptides (AFPs) from the larvae of a beetle, Dendroides canadensis, which survives sub-zero arctic temperatures. Different combinations of AFPs in concentrations ranging from 0-3mg/ml in 1M DMSO were evaluated by cryopmicroscopy to determine their effect on supercooling temperature and ice crystal growth patterns. Solutions containing combinations of AFPs in general demonstrated significant supercooling point depressing activity (~8º difference) along with alteration of ice crystal morphology when compared to single AFPs or DMSO-only solutions (P<0.05). It was also noted that this AFP activity was not necessarily concentration-dependent; dilutions as low as 1µg/ml were sufficient to trigger this effect. Low and effective AFP concentrations are advantageous because they minimize concerns regarding cell cytotoxicity and manufacturing cost. These findings support the potential of incorporating AFPs in solutions used to cryopreserve cells and tissues, while reducing the damaging effect of ice crystals. Preliminary studies with the most effective AFP/DMSO combinations have promoted cell viability over DMSO alone during cryopreservation. Supported by NIH Grant Number 1 R43GM088900-01. A-3001 Organ-cultured Human Colon Tissue Growth Optimized for Stroma: Isolated Mesenchymal Cells Respond to Calcium and Lanthanide Metals with Increased Proliferation and Differentiation. M. K. DAME, M. Naik, I. Veerapaneni, and J. Varani. University of Michigan Medical School, Department of Pathology, Med. Sci. 1, 1301 Catherine St. , Ann Arbor, MI 48109-5602. Email: mdame@med.umich.edu We have recently demonstrated that human colon tissue can be maintained in organ culture. The present study is part of our effort to optimize culture conditions. When the medium was optimized for the epithelial component (0.15mM Ca2+) the tissue rapidly became necrotic. When optimized for stromal cell function (1.5 mM Ca2+), whole tissue histological structure and biochemical function were preserved. Immunostained tissue

revealed Ki67 expression in the proliferating cells at the base of glandular crypts. E-cadherin staining indicated progressively more differentiated cells towards the crypt surface and lumen. Organized around the crypts in the lamina propria were stromal cells with strong staining for smooth muscle actin (SMA). Mesenchymal cells were isolated from explanted colon tissue. The cells exhibited F-actin fibers, but were desmin negative (suggesting the origin of these cells was lamina propria rather than muscularis). Isolated cells were examined under conditions that have been shown to modulate whole tissue function in organ culture. We found that increasing Ca2+ to 4.5mM induced stromal cell proliferation and promoted differentiation as indicated by increased SMA expression. Lanthanoid metal ions (known to mimic effects of Ca2+) promoted growth and differentiation at low micromolar concentrations. The level of growth induction and SMA expression was dependent on their initial growth potential. Cells isolated from aged individuals with low growth potential did not respond to Ca2+ or to lanthanoid metal ions. Based on these findings, we believe that the health of the stromal tissue surrounding the colonic crypts is critical to the preservation of mucosal structure/function. Lanthanoid-stimulated changes in the stroma may lead to improved epithelial function. A-3002 Development and Characterization of a Pituitary Derived Cell Line from Adult Atlantic Salmon, Salmo salar. LUCY E.J. LEE1, Nguyen T. K. Vo1, Mike S. Mikhaeil1, Una Adamcic-Bistrivoda1, Heather E. Braid2, and Robert H. Hanner2. 1Department of Biology, Wilfrid Laurier University, Waterloo, ON N2L 3C5, CANADA and 2Biodiversity Institute of Ontario, University of Guelph, Guelph, ON N1G 2W1, CANADA. Email: llee@wlu.ca Atlantic salmon, Salmo salar, are highly valued fish for both fisheries and aquaculture. Growth and reproduction in this species have been widely studied and factors regulating their life cycle including somatic growth, sexual maturation and reproduction, spawning, smoltification have been extensively investigated. However, the molecular mechanisms controlling expression of the hormones involved in these processes are largely unknown. Pituitary cell cultures could be valuable for elucidating these mechanisms, and a cell line derived from this teleost‘s pituitary could make significant impacts in understanding growth and hormonal regulation as well as endocrine disruption by environmental contaminants. Here we report on the development and histochemical characterization of a continuous cell line derived from adult Atlantic salmon pituitary dubbed ASP309. The cells display a fibroblastic morphology and have been grown for almost 2 years in L15 media supplemented with 10% FBS at 18-20°C. DNA barcoding confirmed the cells as derived from Salmo salar. A-3004 Cryopreservation of Cells with Recombinant Insect-derived Anti-freeze Proteins. L. H. CAMPBELL1, L. Freeman1, H. Kershaw1, D. Halwani1, J. G. Duman2, and K. G. M. Brockbank1. 1Cell & Tissue Systems, Inc., North Charleston, SC 29406 and 2Dept. of Biological Sciences, University of Notre

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Dame, Notre Dame, IN 46556. Email: lcampbell@ celltissuesystems.com Long term preservation is crucial as an enabling technology for regenerative medicine products that contain living cells. However, while cryopreservation works well for most cells, some cells are more sensitive and hard to cryopreserve by freezing. Vitreous cryopreservation is an alternative cryopreservation strategy that stabilizes the tissue as a glass, no ice crystallization. The high concentrations of cryoprotectants required for vitrification, however, can be cytotoxic to cells. Avoidance of high concentration cryoprotectant formulations by reduction of cryoprotectant concentrations while reducing or inhibiting ice formation may be possible by mimicking the strategy employed by certain insects to survive sub-zero temperatures. These insects combine increases in cryoprotectant content (glycerol) with the onset of cold environmental conditions with production of antifreeze peptides (AFPs) to survive temperatures as low as -80ºC. In this study, several AFPs from Dendroides canadensis have been evaluated for their ability to improve the viability of a smooth muscle cell line (A10), that has been difficult to cryopreserve while growing on tissue culture plastic substrates, using conventional cryoprotectants such as dimethylsulfoxide (DMSO). A series of concentrations of the AFPs alone and in combination was evaluated using a 96-well plate format in the presence DMSO or glycerol. In this plate format, the best viability of the A10 cells in DMSO alone is between 25-40%. The addition of AFPs either alone or in combination improved cell viability over cells cryopreserved in DMSO alone in a concentration dependent manner. The best combination was several concentrations of AFPs 1, 2, 4 and 6 combined in equal amounts with viability that measured ~20-30% above the DMSO treated group. There are some indications that varying the concentrations of the AFPs in differing combinations may have even more benefits. In contrast, neither glycerol alone or in combination with AFPs have demonstrated any benefits. Future experiments will include evaluating more identified AFPs and defining optimal concentrations and combinations. A-3005 Generation and Analysis of a New Immortalized Human Corneal Epithelium Cell Line. N. Yamamoto1, K. Hirano2, M. Sumitomo1, H. Yamashita1, M. Nakamura3, K. Hara3, A. Tanikawa2, M. Horiguchi2, K. Taniguchi1 and H. KOJIMA4. 1Laboratory of Molecular Biology and Histochemistry, Joint Research Laboratory, Fujita Health University, Aichi, JAPAN; 2Department of Ophthalmology, School of Medicine, Fujita Health University, Aichi, JAPAN; 3Hoyu Co., Ltd., Aichi, JAPAN; and 4Japanese Center for the Validation of Alternative Methods, Division of Pharmacology, Biological Safety Research Center, National Institute of Health Sciences, Tokyo, JAPAN, Email: h-kojima@nihs.go.jp We previously reported that p75NTR (CD271)-positive cells are observed among the tissue stem/progenitor cells in the crystalline lens and retina, allowing immunological purification of tissue stem/progenitor cells. We observed and separated p75NTR-positive stem/progenitor cells from the human corneal limbus epithelium, and the cultured them using serum-free

medium (In Vitro Cell Dev Biol-Animal, 2010). However, during culture, it proved difficult to maintain the properties of corneal epithelium for more than 5 passages. Furthermore, past efforts to create transgenic corneal epithelium frequently involved the use of defective viral vectors. We transduced an immortalizing gene into corneal epithelium stem/progenitor cells from the corneal limbus without using a defective viral vector, and analyzed the resulting novel immortalized human corneal epithelium cell line (iHCE-NY). We used normal human corneal epithelium cells (HCE), obtained during research on normal human cornea tissue, from an American eye bank. For transduction, we used a GFP vector that contains the SV40 Large T Antigen gene (SV40), purchased from JCRB Gene Bank. We performed transgenesis using a modified electroporation protocol. We cloned the resulting novel immortalized human corneal epithelium cell line, iHCE-NY, and analyzed its gene expression and proteomics. In addition, we used another immortalized corneal epithelial cell line, HCE-T (RCB2280, Riken BRC), as a standard for comparison. We successfully constructed iHCE-NY without using a defective viral vector. On analysis of gene expression, the markers of corneal epithelium barrier function were detected; these included E-cadherin, ZO-1 and Occludin. Proteomics of HCE, HCE-T and iHCE-NY revealed very small changes following SV40 transgenesis. However, enzymes associated with vitamin A were detected within the corneal epithelium tissue, but were barely detectable in any of the cultured cells. We found it was possible to generate immortalized cells of the corneal epithelium without using a defective viral vector. We found that the protein expression of iHCE-NY and HCE-T was relatively similar to that of HCE. In future, we will examine the capacity of iHCE-NY to undergo differentiation. A-3006 Rational Design of Engineered Botulinum Neurotoxin B-insensitive VAMP-2 Mutants. ADAM SWARTZ, Kaylie Tuznik, and Patrick McNutt. USAMRICD, 3100 Ricketts Point Rd, Gunpowder, MD, 21010. Email: adam.m.swartz@us.army. mil Botulinum neurotoxins (BoNTs) are the most toxic substances known to man, with estimated human LD50s as low as 0.5 ng/kg. The botulinum neurotoxin serotype B light chain (LC/B) is a zinc-dependent endopeptidase capable of selectively cleaving the v-SNARE protein VAMP-2 between residues Q76 and F77. This results in the inhibition of neurotransmitter exocytosis at the neuromuscular junction, leading to flaccid paralysis. Exogenous expression of a functional, cleavage-resistant mutant of SNAP-25 has been shown to rescue synaptic transmission from BoNT/A intoxication in vitro and in vivo. In this study, eight human VAMP-2 mutants, with amino acid substitutions at one or both residues flanking the scissile bond, were evaluated for resistance to LC/B-mediated proteolysis in crude mammalian cell extracts and purified bacterially expressed protein elutions. Despite the reported high specificity of LC/B, substitution of Q76 with acyclic residues (i.e., alanine, serine, and threonine) or F77 with aromatic residues (i.e., histidine and tyrosine) did not result in any appreciable change in proteolytic sensitivity; however, substitution of F77 with acyclic residues (i.e., alanine, serine, and threonine) appeared to confer complete resistance to 1 uM LC/B over a 24-hour exposure. In

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comparison, a 24-hour treatment of homogeneous glutamatergic neuron cultures derived from mouse embryonic stem-cells (mESNs) with 12pM BoNT/B cleaved 50% of endogenous VAMP-2 (McNutt et al., 2011). We are currently conducting in silico modeling to evaluate the changes caused by these mutations and their effects on the interaction between mutant VAMP-2 and LC/B. Additionally, the ability of the F77T mutant to support neurotransmission and resist intoxication by BoNT/B holotoxin is being assessed in situ in transgenic mESNs by using functional neurotransmitter release assays and molecular characterization of VAMP-2 cleavage. Determination of a functional LC/B-insensitive mutant will be applied to many efforts: a more thorough characterization of the proteolytic mechanism of LC/B; as a negative control for BoNT/B cell-based assays; and as a component of a broad-spectrum gene therapy approach to expedite recovery from intoxication. Disclaimer: This research was supported by the Defense Threat Reduction Agency – Joint Science and Technology Office, Medical S&T Division [grant numbers 3.10037_07_RC_B and CBM.THRTOX.01.10.RC.021]. The views expressed in this article are those of the authors and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government. SILENT ABSTRACT A-3003 Prostaglandins Alter Protein Phosphorylation Levels in the Insect Cell Line BCIRL-HzAM1. David Stanley1, CYNTHIA L. GOODMAN1, and Qisheng Song2. 1USDA-ARS-BCIRL, 1503 S. Providence Rd., Columbia, MO 65203-3535 and 2Division of Plant Sciences-Entomology, University of Missouri, Columbia, MO 65211. Email: cindy.goodman@ars.usda.gov We are exploring the roles prostaglandins play in the physiology of insect cells. We have identified a large number of proteins that are up-/down-regulated by specific prostaglandins, such as PGA1, PGA2, and PGE1, including those that as act in signal transduction, protein structure/degradation, cellular protection and metabolism/energetics. We are now determining the extent to which prostaglandins influence phosphorylation of specific proteins to identify pathways responsible for transducing prostaglandin signals. We incubated the cell line, BCIRL-HzAM1, with 15 µM PGA2, PGE1 or PGF2α for 20 min and subjected the resulting cell homogenates to 2D electrophoresis. The gels were stained with the phosphoprotein-specific stain ProQ Diamond, as well as with Coomassie Blue G250. Changes in phosphorylation were determined using Delta2D software. PGA2 influenced phosphorylation of 16 proteins, decreasing 9 and increasing phosphorylation of 7. PGF2α, decreased phosphorylation of 6 proteins and increased 10. PGE1 decreased phosphorylation in 22 proteins and increased 8. Three proteins had decreased phosphorylation after treatments with PGA2, PGE1 and PGF2: #270 (37%, 45% and 59%, respectively), #477 (50%, 51%, 62%, respectively), and #660 (46%, 72%, 30%, respectively). Protein #482 stands out from the rest with phosphorylation reduced by 83% after treatment with PGF2α. Protein #242 is the only protein that had increased levels of phosphorylation after all PG treatments (2.5 to 2.7 fold increase). Proteins #706 and #4356 similarly increased phosphorylation after exposure to PGF2α. Phosphoproteins were excised from the gels and prepared for MS/MS (MALDI

TOF/TOF) analysis. We will use bioinformatic analysis to determine the protein identities and report the results in this presentation. PLANT POSTER ABSTRACTS BIOTECHNOLOGY P-3000 Preliminary Safety Assessment of Two Genetically Engineered Corn Lines Expressing Carotenoid and Other Vitamin Biosynthetic Genes. G. ARJÓ1, T. Capell2, C.Piñol1, and P. Christou2,3. 1 Departament de Medicina, Universitat de Lleida, Av. Alcalde Rovira Roure, 80, Lleida, 25198, SPAIN; 2Departament de Producció Vegetal i Ciència Forestal, Universitat de Lleida, Av. Alcalde Rovira Roure, 191, Lleida, 25198, SPAIN; and 3Institució Catalana de Recerca i Estudis Avançats (ICREA), Passeig Lluís Companys, 23, 08010 Barcelona, SPAIN. Email: gemma.arjo@medicina.udl.cat Safety assessment is an integral component of the approval process for genetically engineered (GE) crops, prior to wide cultivation and market introduction. My experiments focus on a preliminary assessment of a multivitamin corn line accumulating high levels of β-carotene (as a vitamin A source), ascorbate and folate, and a second line high in ketocarotenoids as well as carotenoids. Bioinformatics analysis to compare the expressed recombinant protein sequences to known allergens was performed to assess any allergenic potential. Preliminary short (acute toxicity) and long-term feeding trials were performed in vivo. Mice fed with diets containing the GE corn lines (individually) and control diets (their conventional maize counterpart and standard mice diet) were carried out to study potential toxicity. Based on a comparative approach, statistical methods were used to analyze data for food consumption, body weight, hematological and biochemical blood parameters, organ weight and histopathology. Potential allergenicity in vitro tests have been planned to assess the resistance of the recombinant proteins to human simulated fluid digestions and also to determine their heat stability. Our preliminary short-term study indicates that the two GE corn lines exhibited no toxicity and no potential for allergenicity as determined by bioinformatics analysis. P-3001 Evaluation of a Cyst Nematode Parasitism Gene in Arabidopsis thaliana. J. M. CHIERA1, N. Hamamouch1, C. Li1, T. J. Baum2, M. G. Mitchum3, and E. L. Davis1. 1North Carolina State University, Department of Plant Pathology, Box 7903, 840 Method Road Unit 4, Raleigh, NC 27607; 2Iowa State University, Department of Plant Pathology, 351 Bessey Hall, Ames, IA 50011; and 3University of Missouri, Division of Plant Sciences, 371H Life Sciences Center, Columbia, MO 65211. Email: jmchiera@ncsu.edu Heterodera shachtii (Beet Cyst Nematode; BCN) is an obligate sedentary endoparasite that has a wide host range including Arabidopsis thaliana. The nematode migrates through root tissue until it locates the proper root vascular cell and then uses a protrusible stylet to perforate cell walls and secrete proteins into the plant cell. The secreted nematode proteins initiate the transformation of the recipient plant cell into an elaborate

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feeding site and to maintain the syncytium through the remainder of the nematode life-cycle. After initiating a feeding site, the nematode forms a feeding tube that acts as a molecular sieve, preventing transmission of large molecules (>40 kDa) to the nematode, however smaller molecules, like small interfering RNA (siRNA) can be transferred to the nematode. A number of genes encoding secreted parasitism proteins have been identified in the closely related soybean cyst nematode (H. glycines; SCN), however their role in parasitism remain largely unknown. The obligatory parasitic nature of the nematode prohibits a simple method of evaluating the effect of parasitism genes in the infection process. One way to partially evaluate the effect of a parasitism gene is to express each of them in the host plant. Additionally, the passage of siRNA from host to nematode provides a way to knock down genes within the nematode. In an attempt to elucidate the role a parasitism gene plays in parasitism, Arabidopsis plants were genetically transformed to constitutively express nematode parasitism gene transcripts that form double-stranded RNA in order to target genes within the nematode. Nematode feeding studies using the transgenic RNAi producing Arabidopsis plants and the nematode H. schachtii were conducted to test whether the selected nematode genes have any effect on nematode infection. P-3002 Phloem Specific Gene Expression in Vegetative Tissues of Transgenic Citrus Plants. M. DUTT, A. Govindarajulu, M. K. Jaromin, R. H. Brlansky, and J. W. Grosser. Citrus Research and Education Center, University of Florida/IFAS, 700 Experiment Station Road, Lake Alfred, FL 33850. Email: manjul@ufl.edu Epicotyl segments of Mexican lime (Citrus aurantifolia Swingle) were transformed with A. tumefaciens EHA105 harboring a binary vector containing one of four heterologous phloem specific promoters (Arabidopsis sucrose-H+ symporter AtSUC2, rice sucrose synthase l, Agrobacterium rhizogenes rolC and rice Tungro Bacilliform virus) fused to the gus gene. Plants with the gene under control of the 35S promoter were used as control. Histochemical analysis of transgenic plants demonstrated tissue specific promoter activity. Fluorometric GUS activity ranging from 10 to 260 pmol MU/mg/min was observed after analysis of transgenic plants. Overall, GUS activity was highest in vascular tissues of leaves and petiole cells and lower in root cells. These results were confirmed by Northern blotting of selected single copy transgenic lines. Relative quantitation of promoter activity was evaluated using qRT-PCR. In vascular tissues, expression levels of the rice Tungro Bacilliform virus promoter were higher than 35S. Among the four promoters evaluated, plants containing the Rice Tungro Bacilliform virus promoter had the highest GUS expression levels followed by the Agrobacterium rhizogenes rolC promoter, Arabidopsis sucrose-H+

symporter AtSUC2 promoter and rice sucrose synthase l promoter. Use of strong phloem specific promoters can be potentially useful in genetic engineering of citrus to express desired genes whose products are preferentially needed in vascular organs. P-3003 RNA Interference (RNAi) Induced Resistance for Plant Parasitic Nematodes. JIARUI LI, Kerri Neugebauer, Timothy C. Todd,

Tom R. Oakley, and Harold N. Trick. Department of Plant Pathology, Kansas State University, Manhattan, KS 66506. Email: hnt@ksu.edu (corresponding author), jli2@ksu.edu Plant parasitic nematodes are the primary biotic factors limiting crop production, resulting in over $100 billion losses annually. In effort to find an alternative method of control, we evaluated the use of RNAi strategy for controlling plant parasitic nematodes. RNA interference (RNAi) has emerged as an important tool in improving the quality, productivity or reducing yield loss due to biotic and abiotic stresses of food crops. We made RNAi constructs of more than 25 different genes, transformed into soybean or wheat plants. Transgenic plants were evaluated by PCR, RT-PCR, Southern blot, siRNA Northern blot and small RNA sequencing. Bioassays performed on transgenic plants expressing RNAi of nematode genes resulted in up to 85% reduction for nematodes, which is approaching conventional resistance. Furthermore, we amplified several genes from different nematode populations, and observed very high similarity of gene sequences between different nematode populations, suggesting that knockout of these genes should provide broad resistance against nematodes. Our research results indicated that RNAi strategy is effective for controlling plant parasitic nematodes. P-3004 Towards Development of a Sterile Tobacco Hybrid for Pharmaceutical Production. J. HOLLIS RICE1, Richard Mundell2, Reginald J. Millwood1, Orlando Chambers2, H. M. Davies3, and C. Neal Stewart, Jr.1. 1Plant Sciences, The University of Tennessee, Knoxville, TN 37996; 2Kentucky Tobacco Research & Development Center, University of Kentucky, Lexington, KY 40546; and 3Plant & Soil Sciences, University of Kentucky, Lexington, KY 40546. Email: jrice1@utk.edu Plant-made-pharmaceuticals can potentially lower production costs of many medicines. Uncontrolled transgene flow from cultivated fields into other Nicotiana populations may pose environmental and human health risks. Therefore, biocontainment strategies, including host sterility, are needed to prevent potential negative consequences from transgene flow. Based on preliminary data, it is hypothesized that Nicotiana tabacum cv. TN90 crossed with N. glauca produces a functionally sterile hybrid population. To study the sterility of the hybrid population, transgenic hybrid events were generated by transformation of both parental lines with a vector carrying the green fluorescent protein (GFP) for visually monitoring gene flow. Homozygous parental lines were selected in the T2 generation and crossed. A field experiment was conducted to examine the gene flow from the transgenic Nicotiana hybrid to non-transgenic tobacco. Male sterile N. tabacum cv. MSTN90 plants were used as pollen recipient plants and placed at varying distances around a central plot containing transgenic Nicotiana hybrids and fertile population of N. tabacum cv. SN2108, a positive control for pollen flow. Seed pods from transgenic Nicotiana hybrids and recipient MSTN90 plants were separately collected, germinated, and screened for GFP expression. The capability of pollination within transgenic Nicotiana hybrid population and outcrossing with recipient MSTN90 plants was

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analyzed. Results indicate that the Nicotiana hybrid has a low capacity for cross and self- pollination and outcrossing to non-transgenic tobacco. Although the plant is not completely sterile, this reduced fertility of the Nicotiana hybrid may have potential to be a transgene containment strategy. We aim to repeat this field experiment with pollen-specific fluorescent proteins allowing for real time detection of gene flow. P-3005 The Effects of Light vs. Darkness on the Accumulation of Storage Reserves in Developing Soybean Somatic Embryos. S. A. SPARACE, Y. He, and T. E. Young. Biological Sciences Dept., 132 Long Hall, Clemson University, Clemson, SC 29634. Email: smsprc@clemson.edu Soybean (Glycine max L.) somatic embryos are being used as a model for investigating the regulation of storage reserve accumulation in this important crop. An important question that remains to be resolved is the role of light in the accumulation of the main storage materials. To address this question, we have measured the growth of embryos over the course of two weeks in Finer and Nagasawa soybean histodifferentiation and maturation medium, including the relative amounts of starch, protein, lipid and soluble sugars accumulated in light-grown vs. dark-grown embryos. Overall, dark-grown embryos accumulated 31% less fresh weight and 57% less chlorophyll (mg/gFW) than light-grown embryos. Similarly, on a % fresh weight basis, dark-grown embryos accumulated 12% and 33% less starch and soluble sugars, and 20% more protein than light-grown embryos. There was no effect of light vs. darkness on lipid content, which remained at about 0.6% of embryo fresh weight. These observations suggest that light promotes the growth of embryos and the accumulation of chlorophyll and protein, but not carbohydrate or lipid. However, it is not known if the effects of light are mediated directly via modulation of the photosynthetic activities of the embryos or indirectly through modification of gene expression. Further work is necessary to clarify this issue. This research was supported by project no. 8233 from the United Soybean Board. P-3006 RNAi Silencing of the Plant Pathogen Phytophthora capsici. RIO A. STAMLER1, Natalie P. Goldberg2, Soum Sanogo1, and Jennifer J. Randall1. 1Department of Entomology, Plant Pathology and Weed Science, New Mexico State University, 3BE Skeen Hall, Las Cruces, NM 88003 and 2Department of Extension Plant Sciences, New Mexico State University, MSC 3A Skeen Hall, Las Cruces, NM 88003. Email: rstamler@nmsu.edu, jrandall@nmsu.edu Phytophthora capsici is a soilborne oomycete plant pathogen that attacks a broad range of plant species including chile pepper (Capsicum annum). P. capsici infects all parts of the pepper plant including root, stem, leaves, and fruit. Severe root infection results in wilting and death of the plant. Management schemes that reduce soil water saturation are the most effective in controlling root infection by P. capsici. We are currently exploring RNA interference (RNAi) gene silencing as a viable option for controlling P. capsici. Two genes from P. capsici were isolated by PCR for silencing. The first gene is cdc14, which is necessary

for zoospore production, and the second gene is enolase that is utilized in glycolysis. These genes were cloned in various vectors to evaluate dsRNA production and silencing efficacy using both in-vitro and in-vivo techniques. Silencing of in-vitro produced dsRNA was initially evaluated by electroporation into zoospores. The frequency of silencing by electroporation was low, however, the silenced cdc14 lines had on average a 59% reduced zoospore production. We are currently evaluating a second in-vitro method for silencing using hyphal uptake of dsRNA. Using fluorescent microscopy and a fluorescein labeled dsRNA probe we visualized dsRNA uptake by P.capsici and its movement through the hyphal network. We are also evaluating in-planta production of P. capsici cdc14 and enolase dsRNA using the binary vector pFGC5941 in Agrobacterium tumefaciens. Preliminary data indicates that dsRNA produced in-planta is capable of silencing P. capsici during infection. Zoospore production was reduced by approximately 46% and 35% in response to enolase and cdc14 dsRNA respectively as compared to controls. P-3007 Development of a Super Spud Through Repeated Cycles of Marker-free Transformation. A. STIVISON, S. Ringold, T. Carroll, and C. Rommens. J. R. Simplot Company, Plant Sciences, 5369 Irving Street, Boise, ID. Email: artesia.stivison@simplot.com The potato industry is faced with numerous issues related to disease susceptibility, tuber quality, and, most importantly, acrylamide formation. A new approach to genetic engineering (GE) was developed to overcome many of these problems while addressing negative public perception issues that are associated with conventional GE methods. Using marker-free and all-native DNA transformation, we introduced a first series of traits into five potato varieties. The ten resulting “intragenic” or InnateTM events displayed black spot bruise tolerance and reduced cold sweetening, and also had an acrylamide-forming potential that was four-fold lower than that of untransformed controls. A petition for deregulation of the events will be submitted to the USDA in 2011. Efforts have been initiated for the marker-free stacking of three additional traits: further enhanced cold sweetening, late blight resistance, and resistance to potato virus Y. Frequencies for backbone-free transformation were about 5% for simple plant-derived transfer (P-) DNAs, 1.5% for more complex P-DNAs containing inverted repeats, and 0.5% for P-DNAs containing a late blight resistance gene. These commercially-feasible frequencies were achieved by using optimized tissue-culture media and super-virulent Agrobacterium strains. P-3026 The Molecular Events During the Induction of Shoot Regeneration in Plant Tissue Culture. MOSHE REUVENI1, Dalia Evenor1, and Mike Timko2. 1ARO, Volcani Center, Department of Ornamental Horticuture, ISRAEL and 2Department of Biology, University of Virginia. Email: vhmoshe@agri.gov.il The regeneration of shoots in vitro and the subsequent formation of whole plants from various plant tissues is an invaluable tool in modern agriculture and crucial for the genetic transformation of

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plants. However, many crop species are recalcitrant to regeneration, which causes a major obstacle for transgenic improvement of these crop species. A methodical understanding of the molecular events that occur during the process of shoot induction in tissue culture will provide the foundation for increased ability to regenerate recalcitrant species or screen for related species that can be regenerated. To this aim, we are studying the molecular events during the early stages of the process of shoot induction using tobacco as a model system. We exploit conditions that we characterized in tobacco plant leaf segments which display dramatic differences in their ability to induce and regenerate shoots. We also have T2 tobacco plants transformed with ARR5::GUS as a reporter gene for cytokinin presence, T2 plants with IAA4/5::GUS as a reporter for auxin. Using these transgenic plants and microarray technology we are studying the molecular events that occur in tobacco tissue during induction of shoot regeneration. Our findings show that the regulation of specific NAC family genes is modified as well as others and it seems to be related to the process of shoot induction. P-3027 Expression of an Arabidopsis Vacuolar H+-Pyrophosphatase Gene (AVP1) in Peanut Enhances Salt- and Drought-tolerance and Improves Yield in the Field. QIANG GU1, Hua Qin1, 2, Yizheng Zhang2, Li Sun1, Roberto Gaxiola3, Paxton Payton4, and Hong Zhang1. 1Department of Biological Sciences, Texas Tech University, Lubbock, TX, 79409; 2College of Life Sciences, Sichuan University, Chengdu, Sichuan Province, CHINA; 3School of Life Sciences, Arizona State University, Tempe, AZ; and 4USDA Cropping Systems Research Laboratory, Lubbock, TX. Email: hong.zhang@ttu.edu Peanut (Arachis hypogaea) is an important oil crop and widely grown in the Southeast and Southwest regions of US. Environmental stresses such as salinity and drought in these areas can cause yield loss in peanut. To improve crop productivity or to maintain high yield in crop production, it's necessary to develop drought- and salt-tolerant peanut. The Arabidopsis H+- pyrophosphatase gene AVP1 is a proton pump on the vacuolar membrane, which facilitates sequestering of ions and sugars into the vacuole, reducing water potential and stimulating the root development through auxin transportation. Over-expression of the AVP1 gene has been reported to enhance the salt- and drought- tolerance in various plants, such as rice, tomato and cotton. Using the same approach, over-expression of Valencia peanuts were created by using the agrobacterium-mediated transformation. PCR analysis was used to confirm that about half of the 62 regenerated T1 plants were transgenic and carried the AVP1 gene. The expression of transgene was verified by using RT-PCR and Northern blot analyses. Under high salt condition, the AVP1-expressing peanuts showed more vigorous growth than wild-type plants. Under dry-land conditions in the field, AVP1-expressing peanuts also displayed improved tolerance to drought and increased the yield by at least 15%. This research proves that AVP1 can be used to improve crop's performance under drought and salt conditions, which is applicable to other types of crops in saline and semi-arid areas of the world.

CELLULAR AND MOLECULAR PATHOLOGY P-3008 Cell Line Selection for Sugar Beet Resistance to Cercospora beticola Sacc. O. V. KYRYLIUK1 and V. M. Venger2. 13 Gagarina St., State Enterprise Research Farm “Shevchenkivske”, Village Denykhivka, Tetiiv District, Kyiv Region, 09832 UKRAINE and 27 Staryy Bulvar St., Zhytomyr National University of Agriculture and Ecology, Zhytomyr, 10008 UKRAINE. Email: info@shevchenkivske.com.ua Cercospora leaf spot disease is the most widespread beet disease of the central regions of Ukraine. Annually the disease involves 90-95% beet shoots until the end of vegetation, while the number of affected plants fluctuates from 30 to 62% with extent of the disease development from 22 to 54%. The purpose of research is to develop the methods for evaluation of sugar beet resistance to Cercospora leaf spot disease in vitro culture and in field conditions, and also to create the initial material of sugar beet resistant to Cercospora leaf spot on the basis of evaluation methods improvement to be used in selection process. The researches have been held during 2006-2010 in Laboratory of viral diseases and immunity of the Institute of Bioenergy Crops and Sugar Beet of Ukrainian Academy of Agrarian Sciences, Veselopodilska Pilot Selection Station of the Institute for Sugar Beet and State Enterprise Research Farm “Shevchenkivske” of the Institute for Sugar Beet. Formation of infectious background of Cercospora leaf spot disease agent has been conducted using artificial inoculation of sugar beet plants by isolates culture of the fungus C. beticola according to the method developed by Syumka. As a result of research a collection of 29 isolates of Cercospora leaf spot disease agent selected from the different soil-climatic areas of Ukraine and Russia has been formed. It has been revealed that examined isolates of C. beticola show considerable changeability of cultural characteristics and cercosporin content, which does not depend on geographical origin of isolates. Application of luminescent microscopy method has allowed defining the C. beticola cultures, isolated from tissues of sugar beet leaves affected by Cercospora. The method of infectious background formation of the Cercospora leaf spot disease agent using isolates culture of the fungus C. beticola has been improved, that has allowed investigating their virulence and influence on the indices of sugar beets resistance. There has been a method for in vitro culture developed using selective medium and cultural filtrate for acceleration of sugar beet selection for resistance to Cercospora leaf spot. EMBRYOGENESIS/MICROPROPAGATION/ REGENERATION P-3009 Somatic Embryogenesis Induction in Zygotic Embryos of Asparagus racemosus Willd. - A Medicinal Plant. J. CHAUDHARY and P. K. Dantu. Plant Biotechnology Lab, Department of Botany, Dayalbagh Educational Institute (Deemed University), Dayalbagh, Agra (Uttar Pradesh), INDIA. Email: juhi.chaudhary@gmail.com Asparagus racemosus (Shatavar in Sanskrit) of Asparagaceae is rich in the sapogenins Shatavarin I and IV. It is medicinally important for its oestrogenic properties as antioxidant, anti-tumour, and anticancer activities. Of the 150 species distributed

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throughout tropical Asia, Africa and Australia, 17 species are reported from India. Being a rasayana (a rejuvenating herb), its restorative action is beneficial in women's complaints. The interest in plant-derived oestrogens has increased tremendously making A. racemosus particularly important. The plant is propagated through seeds and roots but due to poor seed germination results in very limited number of propagules. Somatic embryogenesis is a powerful tool for clonal propagation that has opened avenues for deployment of superior clonally replicated planting stock of medicinal plants. Zygotic embryos were inoculated in Murashige & Skoog (1962) media with 2, 4-Dichlorophenoxyacetic Acid and Kinetin for callus induction. Two types of callus were formed: embryogenic and nonembryogenic. Compact and nodular callus found to be embryogenic was subsequently subcultured in the same medium. During subculture proembryogenic masses were observed though the globular and bipolar embryos developed in 2, 4-Dichlorophenoxyacetic Acid with kinetin medium remained weak and small. Proper conversion of the globular embryos into bipolar embryos was achieved by transforming the proembryogenic mass to Murashige & Skoog medium supplemented with Naphthalene Acetic Acid, Kinetin and Ancymidol. Secondary somatic embryos were formed when cultures were left in the same medium. The somatic embryos could be germinated by supplementing the above medium with 800mg/l Glutamine and 500mg/l Casein Hydrolysate. P-3010 Comparative Initial In Vitro Stabilization and Stage II Shoot Multiplication of Sea Oats (Uniola paniculata L.) Genotypes Established from Seedlings. J. J. SADLER, M. E. Kane, and N. L. Philman. Environmental Horticulture Department, P.O. Box 110675, University of Florida, Gainesville, FL 32611-0675. Email: jjsadler@ufl.edu Coastal dunes serve as a natural barrier against the destructive forces of hurricanes and storms but are continually being eroded. Dune stabilization is usually accomplished by planting native dune species, such as sea oats (Uniola paniculata L.). Sea oats are commercially propagated by seed; however, frequent hurricane damage to donor populations has significantly limited seed availability. A complete sea oats micropropagation protocol was developed to complement seed propagation. To maintain genetic diversity, the protocol must be applicable to production of numerous sea oats genotypes. The influence of genotype on time for culture stabilization and sustained shoot multiplication of sea oats established from in vitro germinated seedlings from ten sea oats Florida populations was evaluated. Surface sterilized seeds were germinated on ½ strength Murashige & Skoog (MS) mineral salts, 87.6 mM sucrose, 56 mM myo-inositol, 1.2 µM thiamine-HCL, solidified with 0.8% TC agar. After 8 weeks, 48 seedlings from each population were transferred to a multiplication medium consisting of MS mineral salts, 87.6 mM sucrose, 0.56 mM myo-inositol,1.2 µM thiamine-HCL supplemented with 2.2 µM BA and solidified with 0.7% TC agar and then subcultured to fresh multiplication medium at 4-week culture intervals. Genotypes from all populations initially exhibited low shoot multiplication rates (1 – 1.4 fold) during the first culture cycle. Progressive differences in shoot multiplication rates (2.0 – 18 fold) and decreased rooting were observed by the end of the fourth

multiplication subculture cycle. Culture stabilization for shoot multiplication was achieved by the fourth subculture. However, these responses were highly dependent on genotype and population. P-3011 Triploid Citrus Regeneration via Embryo Rescue. X. SHEN, K. J. Song, H. S. Dhaliwal, M. Holt, M. Wendell, and F. G. Gmitter, Jr. Citrus Research and Education Center (CREC), University of Florida, 700 Experiment Station Road, Lake Alfred, FL 33850. Email: xis300@ufl.edu Citrus is an important fruit crop all over the world. Seediness is a major hindrance for consumers’ preference for fresh fruit and regarded as unacceptable in the international market; therefore one of the primary goals of citrus breeding is seedlessness. Triploid citrus (2n=3x=27) can be seedless even with cross pollination, thus have a great commercial potential. An extensive study on triploid breeding was conducted with a goal toward selection of seedless citrus cultivars. Interploid hybridization between diploid female (2n) and tetraploid male (4n) parents is used to regenerate triploids. Twenty-seven monoembryonic diploids, primarily mandarin-derived hybrids (C. reticulata) and pummelo (C. maxima Merr.), were selected as seed parents. Thirteen tetraploids (autotetraploids or obtained via somatic hybridization from elite diploids) were used as pollen parents. Eighty-two interploid crosses were made in 2008. To avoid embryo abortion which is common in interploid crosses due to failure in normal endosperm development, embryo rescue was conducted to recover triploid hybrids. Immature embryos at different developmental stages were excised and cultured on MS basal medium supplemented with 0.5 g/l malt extract, 25 mg/l adenine and 50 g/l sucrose. Embryo germination occurred within 2 months. Both seed and pollen parents had significant effects on germination. Germination percentage ranging from 0 to 100% was obtained from 82 crosses. Embryo developmental stages also affected embryo survival and germination. A total of 2782 individual triploid hybrids were generated. Triploid nature was confirmed by chromosome counting. These generated triploids were top-grafted on to Carrizo (C. sinensis Osb. X Poncirus trifoliata L.) rootstocks and planted in the field for future evaluation. P-3012 Micropropagation of Veratrum californicum Through Tri-scale Culture. Y. P. SUN, J. Naylor-Adelberg, S. A. White, and J. W. Adelberg. Clemson University, Department of Horticulture, E143 Poole Ag. Center, Clemson SC, 29634. Email: youpins @clemson.edu Veratrum californicum Durand (corn lily), is native to alpine regions in the western USA. Corn lily produces steroid alkaloids such as cyclopamine and jervine, which have been reported to have anti-tumor activity. The development of micropropagation techniques is necessary to ensure a sustainable supply of field harvestable corn lily to meet potential therapeutic demand and understand its propagation rate. To meet this objective, we obtained bulbs from the field and sterilized them. The sterilized bulbs were dissected and cultured on either MS + 0.05 µm Thidiazuron (TDZ) or 0.16 µm 6-benzylaminopurine (BA) for 2

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weeks. Triscales from the TDZ treatment were cultured on MS + TDZ (0.05, 0.5, or 5 µm), and triscales from the BA treatment were subcultured on BA (0.16, 1.6, or 16 µm) for 1 month. The triscales were then subcultured on the same fresh media for an additional month. The highest BA and TDZ treatment concentration resulted in increased callus and bulb formation (<0.0001). Callus or bulblets formed on about 90% of the triscales grown on 16 µm BA or 5 µm TDZ media. The optimal concentration of BA and TDZ was 6.2 µm and 6.0 µm for callus induction and 6.1 µm and 2.8 µm for bulblet formation, respectively. As plant growth regulator (PGR) concentration increased, the percent of explants with shoots increased, but no difference for shoot inductions by PGRs was observed. An average of 48% of explant regenerated shoots at an optimal concentration of 3.4 µm BA or TDZ. Explants on TDZ media regenerated more shoots (0.58 ± 0.1) than those on BA media (0.07 ± 0.1) at all concentrations. However, shoots regenerated on BA were morphologically normal, while shoots regenerated on TDZ appeared hyper-hydric. Corn lily clones varied greatly with regard to shoot regeneration. Shoot development was enhanced across clones in medium with 1% charcoal. Work continues to develop stable multiplication of good quality shoots. P-3028 Induction and Multiplication of Embryogenic Callus of Coffea arabica L. Elite Genotypes. CARLOS H. S. CARVALHO1, Ana Carolina R. S. Paiva2, Elizane Q. Silva2, Aline A. Custódio2, Jaqueline Pala2, and Vanessa R. Paulino2. 1Embrapa Coffee, PqEB s/n°, Brasília, DF, BRAZIL, CEP 70770-901 and 2Procafé Foundation, Varginha, MG, BRAZIL. Email: carlos.carvalho@embrapa.br Somatic embryogenesis is the method of choice for industrial propagation of heterozygous coffee plants. This technique applied to genetic improvement of arabica coffee allows for the selection of mother plants in only 10 years. This work reports the optimization of a protocol for induction and proliferation of embryogenic callus of 17 elite Coffea arabica L. plants with leaf miner and rust resistance selected in Brazil. This protocol has now been used for large scale propagation of coffee plants. It was found that embryogenic callus induction was much higher when the leaves were collected during fruit development, from January to June (81.50%) than from July to December (9.35 %) and that the 2nd and 3rd pairs produced more embryogenic callus than the 1st pair. The results also indicated that explant subculture is not necessary during the second phase of callus induction (four to five months). The percentage of explants with embryogenic callus depended on the genotype and also on the general state of the plant. In general, healthy plants with high vigor produced the higher percentage of explants with embryogenic callus, 60 to 80%. In average, 34% of the explants of the 17 genotypes plated in a two year assay produced embryogenic callus. Callus proliferation was much faster in liquid than in gelled medium with an increase of callus mass of 6.7 times in liquid medium and only 3.0 times in the gelled. Callus growth rate in liquid medium ranged from 53.3 mg to 1051.1 mg, with an overall average of 328.7 mg, for every 15 days. Callus growth was highly genotype-dependent, but varied among

callus lineage within the same genotype suggesting that callus selection can be used to increase multiplication rate. GENE TRANSFER TO PLANTS P-3013 Antiviral Lectins as Components of Plant Based Microbicides for HIV Prevention. M. SABALZA1, C. van Dolleweerd2, T. Capell1, B. O’Keefe3, J. Ma2, and P. Christou1, 4. 1Departament de Produccio Vegetal I Ciencia Forestal (PVCF), Universitat de Lleida, Av. Alcalde Rovira Roure, 177, Lleida, E-25198, SPAIN; 2 Centre For Infection, Division of Cellular and Molecular Medicine (CMM), St. George’s University of London, London SW17 0RE, UNITED KINGDOM; 3Molecular Targets Laboratory, National Cancer Institute at Frederick (NCI), Frederick, MD 21702; and 4Institució Catalana de Recerca I Estudis Avancats (ICREA). Email: msabalza@pvcf.udl.cat Protein based microbicide components against HIV are promising alternatives to the current generation of small molecule drugs. An effective microbicide will need to consist of a cocktail of anti-HIV molecules to prevent the rapid evolution of HIV resistance and to provide sufficient cross-clade protection. It is unlikely that such drugs will be broadly available because of their high production and distribution costs in conventional production systems. Their production in plants (molecular pharming) could provide a low-cost platform for the manufacture of protein microbicide candidates. We put forward a cost-effective plant based system for the simultaneous production of two of the most promising microbicide candidates under evaluation, griffithsin and cyanovirin-N. In preliminary experiments simultaneous agroinfiltration of N. benthamiana leaves with griffithsin and cyanovirin-N followed by ELISA and western blot analyses demonstrated that both proteins were expressed and functional. Stable transformation experiments were performed utilizing endosperm-specific maize expression vectors. Putative transgenic plants are currently being screened. The short term objective of this work is to identify a number of lead events co-expressing both molecules at high levels and obtain T2 homozygous lines. Subsequent analysis will focus on determining the HIV-binding efficacy of the two plant-produced molecules. Additional experiments co-expressing griffithsin, cyanovirin-N and highly potent anti-HIV neutralizing antibodies constitute additional elements in our strategy to generate a plant-based combination microbicide against HIV. P-3014 A Transgenic Rice Seed Based Platform for the Production of Next Generation Multi-component Microbicides Against HIV. E. VAMVAKA1, M. Sabalza1, B. O’Keefe2, J. Ma3, T. Capell1, and P. Christou1,4. 1 Departament de Produccio Vegetal I Ciencia Forestal (PVCF), Universitat de Lleida, Av. Alcalde Rovira Roure, 191, Lleida, E-25198, SPAIN; 2 Molecular Targets Laboratory, National Cancer Institute at Frederick (NCI), Frederick, MD 21702, Molecular Targets Development Program, National Cancer Institute at Frederick (NCI), Frederick, MD 21702; 3 The Hotung Molecular Immunity Unit, Division of Clinical Sciences, St. George’s University of London, London SW17 0RE, UNITED KINGDOM; and 4 Institucio Catalana de Recerca I Estudis Avancats (ICREA) Barcelona, SPAIN. Email: evangelia.vamvaka@pvcf.udl.cat

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Rice has a number of distinct advantages as a plant-based production platform for molecular pharming applications. Some of these include a well-developed gene transfer system, inexpensive greenhouse- or field-based large scale production, dispensing with fermentors required for microbial or cell-culture based systems, and its ability to carry out essential post-translational modifications. Rice, like other seed-based production systems, offers high stability of proteins for many years at ambient temperatures. With these advantages as a backdrop we have been exploring rice seeds as a production vehicle for the production of next generation multi-component prophylactic microbicides, such as the human monoclonal antibody 4E10 in concert with the anti-HIV proteins Cyanovirin-N and Griffithsin. We report preliminary experiments and a strategy aiming towards the creation of transgenic plants accumulating these three molecules simultaneously in rice seeds. Subsequent experiments will focus on the identification and biochemical characterization of a lead event for further in depth studies to assess the feasibility of simultaneous production of microbicide multi-component candidate ingredients in a single plant. IN VITRO TOOLS, TECHNIQUES, AND OPTIMIZATION P-3015 Induction of Tetraploidy in Diploid Wild Peanut (Arachis paraguariensis). O. O. AINA, M. Gallo, and K. H. Quesenberry. University of Florida, Agronomy Department, 304 Newell Hall, Gainesville, FL 32611-0300. Email: ainab@ufl.edu The diploid wild Arachis species are important sources of novel genes for improving the tetraploid cultivated peanut (Arachis hypogaea L.). In an attempt to overcome the ploidy barriers that exist in gene transfer between the wild species and cultivated peanut, this study investigated the capacity of the antimitotic agent colchicine for in vitro induction of tetraploidy in wild peanut Arachis paraguariensis Chodat & Hassl. The experiment was laid out in a split plot with 4 (colchicine concentration) x 5 (treatment duration) factorial main plot and 3 explant-types as the subplot. Quarter-seed, callus and shoot tip explants were immersed in aqueous solutions of colchicine (0.05%, 0.1%, 0.2% and 0.5%) dissolved in 1% dimethyl sulfoxide for 4, 8 , 16, 20 and 24 hours. Controls were held in sterile, distilled water for similar durations. The treated explants were then regenerated on semi-solid MS callus induction medium supplemented with 4.4 g-

1 thidiazuron (TDZ) and 2.2 g-l 6-( γ,γ-dimethylallylamino) purine (2iP). Plantlets were allowed to form roots on MS basal medium with no growth regulators before ex-vitro acclimatization. The ploidy levels of plantlets were determined via flow cytometry after two months in culture. The best results in which 39% and 43% of the explant produced tetraploid plants were 0.5% colchicine for 4 h and 8 h respectively. The flow cytometric analysis of induced tetraploids derived from quarter-seeds revealed that they were true-to-type with absence of chimerism but the plants derived from colchicine-treated callus were mixoploids. Besides, treating explants with high concentrations of colchicine for 24 h proved to be very lethal. Overall, the findings from this study should contribute towards the enhancement of gene introgression from wild Arachis into the cultivated peanut.

P-3016 Macronutrient Optimization for Micropropagation and Acclimatization Using Turmeric Curcuma longa L as a Model Plant. S. M. HALLORAN and J. Adelberg. Clemson University, E-143 Poole Agricultural Center, Department of Horticulture, Clemson, SC 29631. Email: shallor@clemson.edu Murashige and Skoog 1962 optimized regeneration of tobacco callus on agar-solidified media, and this media formulation is still widely used. However, many MS media formulations may not be optimal for specific plant systems, and OFAT (one-factor-at-a-time) approaches to media design cannot get at true optimal conditions for plant growth. Simultaneous optimization for multiple responses is possible in tissue culture. Successful media formulation requires proper selection of media factors and responses. This macronutrient experiment altered the most massive components of media: media volume (25-45 ml), plant density (3-9 divisions), sucrose concentration (1.5-6% m/v), macronutrient concentration (20-100 mM [NO3-

]=[NH4+]+[K+]), and [NH4+]:[K+] ratio (0 to 0.5). Both laboratory propagation and greenhouse acclimatization were optimized. Vessels with the fewest plants, most media volume, and 4% sucrose had the highest multiplication ratio. Those massive plantlets from the laboratory gained the most fresh and dry mass in the greenhouse. The largest, fast-growing plantlets came from vessels with fewest plants per vessel, most medium, greatest sucrose concentration, 75 mM macronutrients, and 0.12:0.38 proportion [NH4+]:[K+]. Producing the most plantlets per vessel had different optima than multiplication ratio via the plants per vessel term. By increasing plants per vessel from 3 buds to 9, we more than doubled the number of new plants (10 to 25) while slightly increasing multiplication ratio (4.29X to 4.33X). Plantlets transferred to greenhouse were found to be deficient in phosphorus and magnesium when plantlet dry mass nutrient level estimates were compared with published literature for field-grown turmeric. The response surface methodology used in this experiment produced massive plantlets that multiplied quickly, survived well in the greenhouse; while the space identified clearly indicated less than optimal conditions for several of the meso-nutrients (P, Mg) analyzed herein. P-3017 Optimization of Micropropagation Protocols: The Genotype Effect. J. JASINSKI, M. E. Kane, N. Philman, and T. Johnson. University of Florida, Environmental Horticulture, P.O. Box 110675, Gainesville, FL, 32611-0675. Email: beetlebo@ufl.edu Uniola paniculata L. (Poaceae; sea oats), an ecologically important dune species in the southeastern United States, plays an integral role in coastal dune stabilization and dune building. Sea oats is nursery propagated using field collected seed. U. paniculata is not a prolific seed producer and seed donor sites in Florida have been diminished due to recent hurricane activity. Micropropagation has the potential to supplement seed propagation of a wide range of sea oats genotypes. A micropropagation protocol was originally optimized using a single sea oats genotype utilizing the cytokinin N6-benzyladenine (2.2 μM) for Stage II multiplication. Further studies indicated that in vitro multiplication, rooting, and ex vitro acclimatization varied widely among genotypes when this protocol was used. To

13

assess genotypic responses, 43 genotypes from 13 Florida populations were micropropagated using this “optimized” protocol. Explants were cultured on Stage II medium supplemented with 2.2 μM N6-benzyladenine for four weeks and subsequently cultured on Stage III rooting medium containing 10 µM naphthaleneacetic acid, then acclimatized in the greenhouse after six weeks. Stage II shoot multiplication and Stage III percent rooting varied significantly between the selected genotypes. Ex vitro survival rates ranged from 0-100% after six weeks with 23 genotypes having a survival rate of 60% or less. These results highlight the challenges associated with developing micropropagation protocols suitable for a wide range of plant genotypes. MONOCOT TRANSFORMATION P-3018 Tetracycline-inducible Expression of the ODP2 Gene in Maize Produces Callus from Leaves. K. LOWE1, G. Hoerster1, X. Sun1, C. Scelonge1, Z. Bao1, L. Newman1, J. Zheng3, B. Shen1, W. Bruce2, and W. Gordon-Kamm1. 1Pioneer Hi-Bred International, Inc. 7300 NW 62nd Ave, Johnston, IA, 50131; 2BASF Plant Sciences, 26 Davis Dr, Research Triangle Park, Durham, NC 27709; and 3Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268. Email: william.gordon-kamm@pioneer.com, keith.lowe@pioneer.com

Traditional maize transformation methods are highly dependent on using standard tissue culture manipulations to obtain a desired culture response. Over 10 years ago we initiated a project to see if we could take a genetic approach to stimulating cell divisions rather than the traditional media approach. Earlier we have shown that introduction of expression cassettes containing viral replication proteins, maize cell cycle genes and Lec1 had a stimulatory effect on cell division. Continued screening lead us to two genes, Zm-ODP2 (a Babyboom ortholog) and Zm-WUS2 (a Wuschel ortholog) that greatly stimulated cell divisions and worked together synergistically. Expression of these genes was sufficient to induce a culture response in normally unresponsive tissues such as leaves, stems and older immature embryos. In developing this transformation strategy, we were interested in tightly controlling expression of these two genes, ideally not having them express after they were no longer needed. With this in mind we began investigating methods to control expression of our stimulatory gene cassettes. By modifying the tet repressor gene we were able to demonstrate tight control over gene expression. By placing Zm-ODP2 under the control of the tet repressor we were able to using Doxycycline to induce embryo formation from maize leaves. P-3019 Transformation of Explants from Either Mature Seed or Germinating Seedlings in Maize Aided by the ODP2 and WUS2 Genes. N. WANG, E. Wu, K. Lowe, C. Scelonge, L. Wang, B. Lenderts, G. Hoerster, C. Sweeney, L. Ryan, W. Hua, P. Shen, J. Chow-Yiu, Z.-Y. Zhao, and W. Gordon-Kamm. Pioneer Hi-Bred International, Inc. 7300 NW 62nd Ave, Johnston, IA. Email: william.gordon-kamm@pioneer.com, ning.wang@ pioneer.com

For maize and a number of other crops, immature embryos have been the primary transformation target for almost 20 years. Using Pioneer proprietary inbreds, Agrobacterium-mediated delivery of T-DNA was evaluated and protocols specific to this explant were developed. When transformed with constitutively-expressed CFP + moPAT, no transgenic sectors grew from these explants. When a strongly-expressed ODP2 plus a weakly-expressed WUS2 were included in the T-DNA, growing transgenic sectors were readily observed and vigorously growing callus events were obtained. In the inbreds PHN46 and a PHI-Flint, the frequency of recovering transgenic callus was 12 and 25%, respectively. Before regeneration, the callus was transferred to dry filter papers for a few days, activating RAB17::CRE::pinII whose encoded product efficiently mediated excision of CRE, WUS2 and ODP2 (leaving behind the transgenes of interest). Analysis of regenerated plants showed that approximately 60% of the events were single copy for the T-DNA, and of these approximately 90% showed evidence of complete excision. Healthy, fertile plants were readily produced and T1 and T2 progeny showed Mendelian inheritance of the transgenes left behind after excision. Leaf bases of Pioneer inbreds were also readily transformed using Agrobacterium, and when WUS2 & ODP2 were included, regenerable callus grew out of these leaf pieces. Healthy, fertile plants were readily produced and T1 and T2 progeny showed Mendelian inheritance of the transgenes left behind after excision. PLANT TRANSFORMATION P-3020 Medicago truncatula Insertion Mutants Using the Tnt1 Retrotransposon. H. K. LEE1, J. Yarce1, M. Tadege2, J. Wen1, J. Gallaway1, C. Elles1, X. Cheng1, P. Ratet3, and K. S. Mysore1. 1Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, OK 73401; 2Department of Plant and Soil Science, Oklahoma State University, Stillwater, OK 74078; and 3Institute des Sciences du Vegetal, CNRS, 91198 Gif sur Yvette Cedex, FRANCE. Email: hklee@noble.org Retrotransposons are retrovirus-like elements which encode proteins required for their own replication and transposition. Retrotransposons can be activated by tissue culture and preferentially insert in gene-rich regions of the genome. The absence of excision during transposition makes retrotransposons ideal for saturation mutagenesis with stable tags. We are using tobacco retrotransposon Tnt1 to mutagenize and tag the whole genome of a model legume, Medicago truncatula. We show that Tnt1 is very active and transpose into, on average, 25 different locations during M. truncatula tissue culture. Mutations induced by Tnt1 insertion are stable during seed to seed generation. We have generated over 20,000 independent Tnt1-containing lines encompassing approximately 500,000 insertion events. Over 25,000 Tnt1 flanking sequence tags (FSTs) have been recovered. We have pooled genomic DNA from 18,000 lines for customized reverse-genetic screening, and the frequency of insert identification in this pool for average-sized-gene is approximately 82% percent. The range and diversity of mutant phenotypes obtained to date suggest that M. truncatula offers a great opportunity to dissect symbiotic and developmental pathways for comprehensive understanding of legume biology.

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P-3021 Characterization of Nonhost Resistance Mechanisms in Rice Using RNA Interference-mediated Gene Silencing. H. K. LEE, S. H. Lee, H. Hisano, Z. Wang, and K.S. Mysore. Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, OK 73401. Email: hklee@noble.org Rice (Oryza sativa) is one of the world’s most important food crops and serves as a model organism for the grass family. However, diseases are among the most significant limiting factors that affect rice production worldwide. To understand the mechanisms of nonhost disease resistance against fungal and bacterial pathogens in rice, RNA interference (RNAi)-mediated gene silencing was used in japonica rice cultivars, Nipponbare, whose genome has been sequenced, and Kusabue containing the rice blast resistance gene Pi-k. Previously, we identified several genes that play a role in nonhost disease resistance and basal resistance in Arabidopsis. To further characterize the function of these genes in rice, we searched their homologs in the rice genome (cv. Nipponbare) and RNAi constructs were developed to transform into both Nipponbare and Kusabue. We will examine how silencing these genes in rice affect basal and nonhost resistance to bacterial and fungal pathogens and the preliminary data will be presented. P-3022 Use of the ODP2 Gene Alone or in Combination with WUS2 Improves Transformation Frequencies in Maize Inbreds. E. WU, N. Wang, K. Lowe, C. Scelonge, L. Wang, B. Lenderts, G. Hoerster, L. Ryan, H. Wei, P. Shen, J. Chow-Yiu, Z.-Y. Zhao, and W. Gordon-Kamm. Pioneer Hi-Bred International, Inc., 7300 NW 62nd Ave, Johnston, IA. Email: william.gordon-kamm@ pioneer.com, emily.wu@pioneer.com As part of our continuing effort to find maize genes that enhance transformation, we evaluated ZmODP2 (BBM), either alone or in conjunction with ZmWUS2 for their ability to increase transformation frequencies after Agrobacterium-mediated transformation of maize immature embryos. For the Pioneer inbreds PH581, PHN46 and PHP38, transformation with constitutively expressed CFP and moPAT resulted in frequencies of 1% or lower. When a strongly-expressed ODP2 gene was added, transformation frequencies were 20%, 32% and 12%, respectively. Further addition of a weakly-expressed WUS2 gene resulted in frequencies of 20%, 38% and 50%, respectively. For another Pioneer inbred PHH5G, no events were recovered in either the control treatment or with ODP2 alone. However, when both ODP2 and WUS2 were included in the T-DNA, callus events were recovered at approximately a 45% frequency, with no chemical selection. Before regeneration, calli were placed onto dry filter paper for a few days to activate Rab17::CRE::pinII, resulting in excision of CRE, WUS2 and ODP2. PCR analysis for the components within the T-DNA revealed that on average 50% of the recovered events were single-copy for the T-DNA and of these, roughly 90% showed complete excision of the loxP-flanked genes. For all the genotypes above, fertile plants were readily regenerated and the non-excised transgenes were inherited in typical Mendelian fashion.

P-3029 Agrobacterium-mediated Transformation of Mature Citrus. M. MARUTINI-HERT1, T. E. Mirkov2, T. J. Evens1, and R. P. Niedz1. 1USDA-ARS-U.S. Horticultural Research Laboratory, 2001 South Rock Road, Ft. Pierce, FL 34945-3030 and 2Department of Plant Pathology and Microbiology, Texas AgriLife Research, Weslaco, TX 78596. Email: randall.niedz@ ars.usda.gov Four populations of adult phase trees were maintained in the greenhouse and included Valencia sweet orange, Ruby Red grapefruit, US-942 citrange rootstock, and Etrog Arizona 861-S1 citron. To identify and characterize factors important in shoot regeneration, several experiments were conducted. The first experiment was a 4-factor response surface design that included AgNO3 (0-5 mg/L), GA (0-5 µM), plant growth regulators (10 µM ZR or 15 µM BA+10 µM NAA), and basal medium (MS or WPM). The most important factor for shoot regeneration was 10 µM ZR. The second experiment examined the effects of ‘time in the dark’ on shoot regeneration. Internode explants were cultured in the dark (0 to 8 weeks) and then moved to the light where shoot regeneration was recorded 30 days later. The results for each citrus type showed an absolute requirement to achieve shoot regeneration of a dark period of at least two weeks. The third experiment involved Agrobacterium-mediated transformation of internode explants from the four citrus types using the beta-glucuronidase reporter gene driven by the d35S promoter. In vitro shoots were micrografted, moved to the greenhouse, and tested for GUS expression. Transformed plants were obtained for each of the four types and included sweet orange (1 plant), grapefruit (1 plant), US-942 (8 plants), and Etrog citron (8 plants). Current efforts are directed toward characterizing this system for routine transgenic plant production. STRESS RESISTANCE P-3023 The Comparison of P5CS Gene Expressions of Two Soybean Varieties Under Salt Stress. Ö. ÇELİK and Ç. Atak. Istanbul Kultur University, Faculty of Science and Letters, Department of Molecular Biology and Genetics, 34156, Atakoy, Istanbul, TURKEY. Email: ocelik@iku.edu.tr In this study, we aimed to determine the expression levels of P5CS gene in salt-sensitive and salt-tolerant two soybean varieties. We subjected two different soybean genotypes (Ataem-7 and Üstün-1) to three different concentrations of NaCl for 7 days. We evaluated the ∆-1-pyrroline-5-carboxylate synthetase (P5CS) gene expression under 50, 100 and 150 mM NaCl stress. Lipid peroxidation levels of Üstün-1 and Ataem-7 were 0.20 and 0.30 µmol/g FW increased at 150 mM NaCl respectively with respect to control (p<0.05). In Ataem-7 variety, 1.39 fold more increment was observed in proline accumulation compared to Üstün-1 variety (p<0.05). At 150 mM NaCl concentration, P5CS gene expression level was 8.7 fold increased at Üstün-1 variety when compared to Ataem-7. The interesting point is the differences between high proline content and low expression level of P5CS in sensitive cultivar Ataem-7 at 150 mM NaCl. This finding indicated that the proline accumulation can not be a

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result of increased stress-inducible expression of delta-pyrroline-5-carboxylate synthetase gene. P-3024 Identification of Rice Genes Induced by Abiotic Stress by Proteomics and qRT-PCR. S. DASHEVSKAYA1, R. Horn2, D. Zimmermann2, I. Chudobova2, S. Schillberg2, T. Capell1, and P. Christou1,3. 1Departament de Producció Vegetal i Ciència Forestal, Universitat de Lleida, Av. Alcalde Rovira Roure, 191, Lleida, 25198, SPAIN; 2Fraunhofer IME Forckenbeckstraße 6, 52074 Aachen, GERMANY; and 3Institucio Catalana de Recerca i Estudis Avancats (ICREA), Passeig Lluís Companys, 23, 08010 Barcelona, SPAIN. Email: svetlana@pvcf.udl.cat Drought and salinity are the major abiotic stresses constraining crop productivity. Limited water supplies, shrinking arable land and increases in world population particularly in areas subject to chronic and long-term drought and other forms of abiotic stresses are additional factors that threaten food security. One component of a broader strategy to combat food shortages is to create crops with enhanced tolerance to abiotic stresses. Identifying new genes that play potential roles in stress adaptation through contemporary “omics” technologies facilitates the analysis of gene expression and elucidation of mechanisms involved in stress tolerance or adaptation. In turn, results from such studies might identify candidate genes useful in generating plants tolerant to such stresses. Rice, one of the most important food crops in the world, is very sensitive to drought stress because of its limited adaptation to water-deficit conditions. Salinity stress also affects considerably rice growth and productivity. In order to identify candidate genes involved in stress adaptation, we subjected rice plants to moderate-long term drought or salinity stress followed by a period of recovery during which the stress stimulus was withdrawn. A time course analysis was carried out on vegetative tissues of plants subjected to stress. This analysis allowed us to identify a number of proteins which were over expressed under salinity stress. Good correlations were established between mRNA levels as measured by qRT-PCR and protein levels for group 3 late-embryogenesis abundant (OsLEA3) protein, a mitochondrial import inner membrane translocase and a putative fumarylacetoacetate hydrolase. Under salinity stress accumulation of mRNA of the three genes was increased further when ABA was applied immediately prior to

the onset of the stress. These and similar studies might facilitate the use of such candidate genes to generate stress-tolerant rice plants. PLANT SILENT ABSTRACTS P-3025 A Series of Modular Vectors for Cloning of Target Genes and Regulatory Elements for Providing Stable and Effective Expression of Heterological Genes in Plants. A. O. VYACHESLAVOVA, I. N. Berdichevets, H. R. Shimshilashvili, E. A. Romanov, O. Mustafaev, and I. V. Goldenkova-Pavlova. Institute of the General Genetics Russian Academy of Sciences, Gubkin's Street, 3, Moscow, RUSSIA 119991. Email: alisavo@gmail.com, irengold@vigg.ru Transgenic plants are widely used objects of fundamental and applied researches. The success in these directions, in many respects, depends on efficiency of an expression of the transferred gene (transgene) in plants. Efficiency of the transgene expression is caused by a series of factors: codon composition of transgene; regulatory elements and others. It will be observed, that expression vectors, which used for transformation of plants, plays an important role in creation of transgenic plants. Currently there is a whole spectrum of plant expression vectors but, however, they have both advantages and disadvantages. So, creation of new plant vector systems for providing stable and effective expression of transgenes in plants is essential. Based on the analysis of the literature and databases of plant genomes we designed a series of modular vectors in which most part of the factors, providing stable and effective expression of transgenes in plants, was considered, e.g.: a context of initiating ATG codon of a target gene; the sequences coding stabilizing amino acids in the second position after the initiating codon and many other things. Besides, the modular structure of the designed vectors allows to easily replace the regulatory elements regulating an expression of a transgene in plants, such as promotors, sequences of leader peptides and others regulatory elements. Designed plant vectors have been successfully approved by transient expressions of some heterological genes and regulatory elements. These plant vectors are offered both for the analysis of new regulatory elements, and for providing effective expression of transgenes in plants.

INDEX

Adamcic-Bistrivoda, Una A-3002 Adelberg, J. P-3016 Adelberg, J. W. P-3012 Aina, O. O. P-3015 Arjó, G. P-3000 Atak, Ç. P-3023 Bao, Z. P-3018 Baum, T. J. P-3001 Berdichevets, I. N. P-3025 Braid, Heather E. A-3002 Brlansky, R. H. P-3002 Brockbank, K. G. M. A-3000 Brockbank, K. G. M. A-3004

Bruce, W. P-3018 Burgoon, L. D. PS-10 Campbell, L. H. A-3000 Campbell, L. H. A-3004 Capell, T. P-3000 Capell, T. P-3013 Capell, T. P-3014 Capell, T. P-3024 Carroll, T. P-3007 Carvalho, Carlos H. S. P-3028 Çelik, Ö. P-3023 Chambers, Orlando P-3004 Chaudhary, J. P-3009

Cheng, X. P-3020 Chiera, J. M. P-3001 Chow-Yiu, J. P-3019 Chow-Yiu, J. P-3022 Christou, P. P-3000 Christou, P. P-3013 Christou, P. P-3014 Christou, P. P-3024 Chudobova, I. P-3024 Custódio, Aline A. P-3028 Dame, M. K. A-3001 Dantu, P. K. P-3009 Dashevskaya, S. P-3024

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Davies, H. M. P-3004 Davis, E. L. P-3001 Dhaliwal, H. S. P-3011 Duman, J. G. A-3000 Duman, J. G. A-3004 Dutt, M. P-3002 Elles, C. P-3020 Evenor, Dalia P-3026 Evens T. J. P-3029 Freeman, L. A-3004 Gallaway, J. P-3020 Gallo, M. P-3015 Gaxiola, Roberto P-3027 Gmitter, Jr., F. G. P-3011 Goldberg, Natalie P. P-3006 Goldenkova-Pavlova, I. V. P-3025 Goldstein, S. J-2 Goodman, Cynthia L. A-3003 Gordon-Kamm, W. P-3018 Gordon-Kamm, W. P-3019 Gordon-Kamm, W. P-3022 Govinarajulu, A. P-3002 Grosser, J. W. P-3002 Gu, Qiang P-3027 Halloran, S. M. P-3016 Halwani, D. O. A-3000 Halwani, D. O. A-3004 Hamamouch, N. P-3001 Hanner, Robert H. A-3002 Hara, K. A-3005 He, Y. P-3005 Hirano, K. A-3005 Hisano, H. P-3021 Hoerster, G. P-3018 Hoerster, G. P-3019 Hoerster, G. P-3022 Holt, M. P-3011 Horiguchi, M. A-3005 Horn, R. P-3024 Hua, W. P-3019 Hua, Wei P-3022 Jaromin, M. K. P-3002 Jasinski, J. P-3017 Johnson, T. P-3017 Kane, M. E. P-3010 Kane, M. E. P-3017 Kershaw, H. A-3004 Kojima, H. A-3005 Kounovsky-Shafer, K. J-2 Kyryliuk, O. V. P-3008 Lee, H. K. P-3020 Lee, H. K. P-3021 Lee, Lucy E. J. A-3002 Lee, S. H. P-3021 Lenderts, B. P-3019 Lenderts, B. P-3022 Li, C. P-3001 Li, Jianrui P-3003 Lowe, K. P-3018 Lowe, K. P-3019 Lowe, K. P-3022

Ma, J. P-3013 Ma, J. P-3014 Marutini-Hert, M. P-3029 McNutt, Patrick A-3006 Mikhaeil, Mike S. A-3002 Millwood, Reginald J. P-3004 Mirkov, T. E. P-3029 Mitchum, M. G. P-3001 Mundell, Richard P-3004 Mustafaev, O. P-3025 Mysore, K. S. P-3020 Mysore, K. S. P-3021 Naik, M. A-3001 Nakamura, M. A-3005 Naylor-Adelberg, J. P-3012 Neugebauer, Kerri P-3003 Newman, L. P-3018 Niedz, R. P. P-3029 Oakley, Tom R. P-3003 O'Keefe, B. P-3013 O'Keefe, B. P-3014 Paiva, Ana Carolina R. S. P-3028 Pala, Jaqueline P-3028 Paulino, Vanessa R. P-3028 Payton, Paxton P-3027 Philman, N. P-3017 Philman, N. L. P-3010 Piñol, C. P-3000 Place, M. J-2 Potomousis, K. J-2 Qin, Hua P-3027 Quesenberry, K. H. P-3015 Randall, Jennifer J. P-3006 Ratet, P. P-3020 Reuveni, Moshe P-3026 Rice, J. Hollis P-3004 Ringold, S. P-3007 Romanov, E. A. P-3025 Rommens, C. P-3007 Ryan, L. P-3019 Ryan, L. P-3022 Sabalza, M. P-3013 Salbalza, M. P-3014 Sadler, J. J. P-3010 Sanogo, Soum P-3006 Scelonge, C. P-3018 Scelonge, C. P-3019 Scelonge, C. P-3022 Schillberg, S. P-3024 Schwartz, D. C. J-2 Shen, B. P-3018 Shen, P. P-3019 Shen, P. P-3022 Shen, X. P-3011 Shimshilashvili, H. R. P-3025 Silva, Elizane Q. P-3028 Song, K. J. P-3011 Song, Qisheng A-3003 Sparace, S. A. P-3005 Stamler, Rio A. P-3006 Stanley, D. A-3003

Stewart, Jr., C. Neal P-3004 Stivison, A. P-3007 Sumitomo, M. A-3005 Sun, Li P-3027 Sun, X. P-3018 Sun, Y. P. P-3012 Swartz, Adam A-3006 Sweeney, C. P-3019 Tadege, M. P-3020 Taniguchi, K. A-3005 Tanikawa, A. A-3005 Teague, B. J-2 Timko, Mike P-3026 Todd, Timothy C. P-3003 Trick, Harold N. P-3003 Tuznik, Kaylie A-3006 Vamvaka, E. P-3014 van Dolleweerd, C. P-3013 Varani, J. A-3001 Veerapaneni, I. A-3001 Venger, V. M. P-3008 Vo, Nguyen T. K. A-3002 Vyacheslavova, A. O. P-3025 Wang, L. P-3019 Wang, L. P-3022 Wang, N. P-3019 Wang, N. P-3022 Wang, Z. P-3021 Wei, H. P-3022 Wen, J. P-3020 Wendell, M. P-3011 White, S. A. P-3012 Wu, E. P-3019 Wu, E. P-3022 Yamamoto, N. A-3005 Yamashita, H. A-3005 Yarce, J. P-3020 Young, T. E. P-3005 Zhang, Hong P-3027 Zhang, Yizheng P-3027 Zhao, Z.-Y. P-3019 Zhao, Z.-Y. P-3022 Zheng, J. P-3018 Zhou, S. J-2 Zimmerman, D. P-3024