The following student awards were presented at the 2006 In Vitro Biology Meeting, Minneapolis, Minnesota. Information related to the available specific student awards can be found on the SIVB website (www.sivb.net) or by contacting the SIVB Business Office at (919) 420-7940, sivb@sivb.org, or Dr. Pamela Weathers, Chair, Student Affairs and Awards Committee, at (508) 831-5196, email: weathers@wpi.edu.

2006 John S. Song Award

Characterization of Populus tremuloides COMT, 4CL1 and 4CL2 Gene Promoters to Identify Regulatory Elements.
The phenylpropanoid pathway supplies a wide range of secondary compounds that play important roles in plant growth and development. Two major classes of phenylpropanoid products are lignin and flavonoids. Lignin serves as a structural component of cell walls and plays a major role in plant defense. Flavonoids form a large class of phenolic compounds associated with stress-induced responses, signal transduction, symbiotic interactions and reproductive-related functions. The diverse functions of the phenylpropanoid products make this pathway a target for genetic engineering. Our study aims at identifying cis regulatory elements that are important for spatiotemporal regulation of phenylpropanoid gene expression. Promoters of three aspen genes, encoding caffeic acid O-methyl transferase (COMT), and 4-Coumarate: CoA ligase 1 (4CL1) and 2 (4CL2), are targeted for this research. 4CL activates the hydroxycinnamates to their corresponding high-energy CoA-esters, with the two isoforms, 4CL1 and 4CL2, differentially involved in lignin and flavonoid biosynthesis, respectively. COMT, on the other hand, catalyzes the O-methylation of 5-hydroxyconiferaldehyde specifically for syringyl (S) lignin biosynthesis. We have generated independent lines of transgenic aspen, harboring serial promoter deletions fused to a GUS reporter gene for studies aimed at identifying the putative regulatory elements. Histochemical analysis of the transgenic aspen have revealed various tissue-specific GUS expression patterns. COMT and 4CLI promoters show xylem and phloem specific expression patterns, while 4CL2 promoter display an epidermis-specific expression. The 4CL1 promoter is strongly induced by nitrogen stress treatment.

Edward Anino, Biotechnology Research Centre, Michigan Technological University, 1400 Townsend Dr., Houghton, MI 49931. In Vitro Cellular and Developmental Biology, 42:40-A, 2006


2006 Honor B. Fell Award, 2006 Hope E. Hopps Award, and 2006 Cellular Toxicology Award

Determination of the Effects of Ecdysteroids and JH on Nodulation Responses
Insects have a highly developed innate immune system, including humoral and cellular components. The cellular immune responses refer to hemocyte-mediated processes such as phagocytosis, nodulation, and encapsulation. Nodulation, which is considered the predominant defense reaction to infection in insects, is a complex process influenced by various endogenous factors. However, the precise mechanisms underlying nodulation remain largely unknown. In the present study, we examined the influence of the insect hormones 20-hydroxyecdysone (20E) and juvenile hormone (JH) on the laminarin-induced nodulation reaction in larvae of the flesh fly Neobellieria bullata. Treating third-instar larvae of N. bullata with 20E prior to laminarin injection enhanced the nodulation response in a dose-dependent manner. The ecdysone agonists RH2485, RH5849 and RH0345 similarly enhanced the nodulation reaction, although they were less active than 20E. In contrast to ecdysone stimulation, supplying larvae with JH or the juvenile hormone analogs (JHA), fenoxycarb and pyriproxyfen, significantly impaired their ability to form nodules in response to laminarin. These findings demonstrate for the first time that 20E and JH play an important regulatory role in the nodulation process.

Vanessa Franssens, Laboratory of Developmental Physiology, Genomics and Proteomics, Department of Animal Physiology and Neurobiology, Zoological Institute, K.U. Leuven, Naamsestraat 59, B-3000 Leuven, Belgium. In Vitro Cellular and Developmental Biology, 42:6-A, 2006.


2006 Wilton R. Earle Award and 2006 SIVB Travel Award

Quantitative Multiplex Real-time PCR as a Screening Tool for Estimating Transgene Copy Number in Transgenic Citrus.
Quantitative real-time PCR (RT-PCR) was adapted to estimate transgene copy number in transgenic citrus plants. This task is normally achieved by Southern analysis, a procedure that requires relatively large amount of plant material and is both costly and labor-intensive. ‘Hamlin’ sweet orange (Citrus sinensis (L.) Osbeck) transgenic plants were generated using protoplast/GFP transformation system. These transgenic materials represented a range of copy number. The standard curve is the key element for the quantitative assay: since it is based on the standard DNA used, the choice and the preparation of this DNA is extremely important. One of the proposed methods to prepare the standard DNA consists of mixing plant DNA with a plasmid carrying the transgene (Ingham et al., 2001). This procedure, however, introduces several sources of error that cannot be controlled: previous absolute quantification of both plant and plasmid DNA is necessary, together with precise knowledge of the nuclear genome size of the plant species to be assayed. Unfortunately for most plants, only approximate estimates are available. All these problems were by-passed by simply taking the DNA of one transgenic line and using its dilutions to construct the standard curve. From the standard DNA stock solution, accurate four-fold serial dilutions were prepared and utilized to obtain the standard curves necessary for relative quantification of an endogenous gene and transgene. Estimated copy number in the plants using RT-PCR was correlated with the actual copy number found with Southern blot analysis. The results indicated that there was a significant correlation between the two methods. Thus, RT-PCR can provide an efficient means of estimating copy number in transgenic citrus.

Quantitative real-time PCR assay for determining transgene copy number in transformed plants. Biotechniques 31, 132-134, 136-140.

Ahmad Al-Sayed Omar, University of Florida, IFAS, CREC, 700 Experiment Station Road, Lake Alfred, Fl 33850. In Vitro Cellular and Developmental Biology, 42: 22-A, 2006.


2006 SIVB Travel Award

Production of Biologically Active Human Secretory Leukocyte Protease Inhibitor (SLPI) in Transgenic Tobacco and Rice Biomass.
The largest component of the HIV treatment is drug costs. For a typical HIV patient in 2004, the treatment cost ranged from 8,500-$13,600/patient/year. Currently, T-20 (fuzeon) is the most expensive anti-AIDS drug in the market, at $20,000/patient/year. Regardless of the costs, none of these drugs are naturally human-based product, and therefore patients in certain cases develop allergenic reactions. Human Secretory Leukocyte Protease Inhibitor (SLPI) is an anti-HIV protein naturally produced in the human body in regions with high levels (saliva) and/or individuals with high levels of this protein have a notably reduced acceptance to HIV transmission. At present, SLPI is sold for $215/100 microgram, translating into $2.15 M/gram. This extreme high cost has prohibited conducting large-scale human trials, and certainly has prohibited the use of this protein in a drug regimen. The main reasons for such high costs is the fact that production of its non-glycosylated cationic protein form in E. coli requires very extensive denaturation and renaturation to refold this disulfide-rich protein in its biologically active form. A plant-specific vector containing SLPI gene, polyhistidine tag, ß-glucuronidase (gus) and bar herbicide resistance selectable marker genes was used to develop transgenic plants. Molecular and histochemical GUS analyses of T1 rice and T2 tobacco plants confirmed presence and expression of the transgenes. Production of the protein had no apparent deleterious effects on plant growth and development. Work is in progress to test the biological activity of plant-produced SLPI. Should it be biologically active, a series of clinical trials will be performed with collaboration of Mayo Clinic HIV Research Center. The plant-produced human SLPI protein in transgenic plants could be marketed as low as $5000/kilogram in the future.

Hesham Farouk Oraby, Department of Crop and Soil Sciences, Michigan State University, East Lansing, MI 48824. In Vitro Cellular and Developmental Biology, 42:22-A, 2006.