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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.org) 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.
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