Development of a Germline Transformation
System for the Western Corn Rootworm,
Diabrotica virgifera virgifera
Fu-Chyun Chu, Stephanie Gorski, Yasmin J. Cardoza
and Marcé D. Lorenzen
North Carolina State University
Raleigh, North Carolina
[email protected]
The western corn rootworm (WCR) is a major pest of maize and is notorious for
rapidly adapting biochemically, behaviorally and developmentally to a variety of control
methods. Despite much effort, the genetic basis of WCR adaptation remains a mystery.
Transformation-based applications such as transposon tagging and enhancer trapping
have facilitated the genetic dissection of model species such as Drosophila melanogaster.
Following this model, we are developing a genome-wide mutagenesis system for use in
WCR. However, we must first establish a germline transformation system for use in this
beetle. Here we report results from our first sets of experiments. These include: 1) testing
marker genes (EGFP and DsRed); 2) testing heterologous promoters (Tc-alpha-Tubulin
and Dm-heat-shock-70); and 3) testing transposable elements (piggyBac and Minos). Since
our overall goal is to create a jumpstarter mutagenesis system that is analogous to that
used in another coleopteran, the red flour beetle, Tribolium castaneum, we are also using
the CRISPR/Cas9 system to: 1) create a white-eyed mutant strain; and 2) specifically
target transposon insertions to precise genomic locations (i.e. locations analogous to those
known to enable efficient piggyBac remobilization in Tribolium). Establishing transgenic
technologies in WCR is the first step towards bringing a wide-range of transformationbased tools to bear on understanding WCR biology, which can then be extended to other
rootworm species.
260 New DNA-Editing Approaches: Methods, Applications and Policy for Agriculture
Unraveling the Unique Mechanism of EthyleneInduced Abscission in Non-Climacteric
Sweet Cherry, Prunus avium
Benjamin Kilian
Washington State University
Pullman, Washington
[email protected]
Sweet cherry, Prunus avium, is a unique non-climacteric member of Rosaceae that comprises some of the classical climacteric species such as apple, pear and peach. Previous
phenotypic data established that exogenous ethylene treatment has a genotype-dependent
effect on pedicel retention as the fruit nears maturity. This is an intriguing result because
sweet cherry is considered non-climacteric and, therefore, does not display positive reinforcement in ethylene production and elevated respiration. This observation prompted
our inquiry of what could initiate the response to ethylene of certain genotypes of a
non-climacteric fruit in the formation of an abscission zone? Part of the answer at least
can be derived from gene-expression studies. I have addressed this question by measuring
abscission-zone formation indirectly by pedicel fruit retention force (PFRF) and gathering
contemporaneous RNA-Seq profiles to correlate the physiological data. For the experiment, three sweet cherry genotypes were selected which best represented the diversity
of responses to ethylene. ‘Chelan’ does not respond to ethylene application. ‘Skeena’
also does not respond to ethylene; however, it forms an abscission zone naturally. ‘Bing’
is the unique genotype that forms an abscission zone in response to ethylene, lowering
stem-retention force enough to enable mechanical harvesting. The experiments assessed
relative genotypic responses to ethylene in the final phase of fruit maturation prior to
harvest. Surgically defined abscission zone tissues from each genotype were collected and
used to generate quantitative gene-expression data (RNA-Seq). The sequence data have
been de novo assembled into genotype-specific transcriptomes and relative expression for
each gene showed the differences among genotypes. With these studies we have identified
specific genetic elements specifically in ‘Bing’ that are differentially expressed in response
to ethylene. These include transcription factors rapidly responding to the ethylene signal
and their potential target sequences, more delayed cell wall-modifying enzymes, and others
connected to programmed cell death processes. We are currently validating expression
261
of these genes over multiple seasons and plan on performing subsequent experiments to
demonstrate ethylene response. Agricultural labor availability and safety is currently a
large problem in the United States in general, and the Pacific Northwest in particular. One
solution is the facilitation of mechanical harvest, which would lower cost of production
and increase worker safety. Identifying or transferring the required element(s) via classical
breeding or through genetic modification into target species has the potential to facilitate
more efficient harvesting. My PhD work revolves around the approach that we can predict
and/or induce the formation of a pedicel-fruit abscission zone in sweet cherry, a crop
with particularly high labor demand and cost. While aimed primarily at sweet cherry, the
technology could be applied to mechanically harvesting a wide range of crops.
262 New DNA-Editing Approaches: Methods, Applications and Policy for Agriculture
Early Flowering Salvia hispanica (Chia)
Composition
William Serson1, Paul Armstrong2, Timothy Phillips1
and David Hildebrand1
University of Kentucky
Lexington, Kentucky
1
USDA-ARS Grain Marketing and Production Research Center
Manhattan, Kansas
2
[email protected]
A sustainable, affordable source of omega-3 fatty acids has been of high interest to those
interested in human and animal nutrition due to known heart-health benefits. Chia is
an ancient crop that has experienced an agricultural resurgence in recent decades, though
it can only be grown in central latitudes due to its need for short-day flowering. Using
EMS and gamma radiation, researchers at the University of Kentucky have developed
a long-day flowering mutant which can be grown in the heartland of the United States.
While the agronomics continue to be hashed out, we are interested in observing seed
composition. If EMS and gamma radiation did not mutagenize genes relevant to seed
composition, then we would anticipate no significant changes in seed composition. Five
lines with agronomic potential were evaluated for fiber, oil, protein, and fatty-acid content
and compared to the parent line, ‘Pinta’. A Dunnett’s test confirmed that there were 3
varieties with significantly higher fiber, one with higher protein, and one with higher 16:0
levels. Some of these changes could be due to mutations of genes that are responsible for
helping determine seed composition. However, further trials will be needed to investigate
genotype by environment interactions.
263
Effect of Different Gums on the Functional
Properties of Soy-Based Nile Tilapia Feed
P. Singha and K. Muthukumarappan
South Dakota State University,
Brookings, South Dakota
[email protected]
Plant-origin polymeric binding agents, cellulosic gum, and starch are often used to improve
the cohesiveness of extruded aquafeed. Exopolysaccharide gum such as pullulan and
xanthan secreted by microorganisms has unique properties that have been used in food
and pharmaceutical products for decades. Isocaloric ingredient blends were formulated
for Nile tilapia using a constant level of defatted soybean meal (DFSBM) as a fishmeal
replacer and graded levels of five different binders (guar, wheat gluten, xanthan, CMC,
and pullulan). Extrusion trials were performed using a single-screw extruder at two levels
of screw speed (100 and 150 rpm). To acquire more distinct results for the effect of utilized
binders, the extrusion-barrel temperatures, blend-moisture content, die diameter, and
screw-compression ratio were kept constant at 100–120–140ºC, 20% db, 3 mm, and
3:1 respectively. A fishmeal-based diet was used as the control diet. Physical properties
of the extruded products including expansion ratio, durability, water stability, water
solubility, and densities were quantified. It is hypothesized that exopolysaccharide binder
can provide similar binding properties in extruded aquafeed formula when compared to
conventional binders.
264 New DNA-Editing Approaches: Methods, Applications and Policy for Agriculture
Evaluation of Carrot (Daucus carota L.) for
Traits Related to Early Seedling Establishment
and Canopy Growth at Different
Planting Densities
Sarah Turner and Philipp W. Simon
USDA-ARS, University of Wisconsin
Madison, Wisconsin
[email protected]
Carrot production is limited by erratic germination, poor seedling growth, and delayed
canopy establishment, all of which make weed control a major challenge. Plants with early
germination, quick seedling growth, and competitive growth response are one viable option
for improving weed management. Preliminary field trials have demonstrated that carrot
genotypes have variable germination rates and responses to planting density, ranging from
no response to an increase in canopy growth as planting density increases. This project
aims to elucidate competitive growth response in diverse carrot-breeding stocks. Four
genotypes with small canopy size and four with large canopy size were planted at different
densities (30, 60, and 90 plants per meter) using a randomized complete-block design
with three replications. Emergence, canopy height, and canopy width were monitored
throughout the growing season and postharvest measurements of fresh leaf weight, root
weight, and dry leaf weight were taken. Plant height was significantly affected by planting
density, genotype, location, and genotype by location interaction. In general, increasing
planting density promoted top growth at the expense of root weight, but this response
varied by genotype. Current progress will be reported.
265
Targeted Mutation and Precise Genome Editing
in Plants with CRISPR/Cas9 System
Kabin Xie, Bastian Minkenberg, Xiangling Shen, Qin Wang
and Yinong Yang
The Pennsylvania State University
University Park, Pennsylvania
[email protected]
The bacterial cluster regularly interspaced short palindromic repeats (CRISPR)-associated
nuclease (Cas) system has recently emerged as an efficient and versatile tool for genome
editing. In this study, we have demonstrated targeted mutation and precise genome editing
with CRISPR-Cas9 system in both monocot and dicot plants. The engineered gRNAs
were shown to direct the Cas9 nuclease for precise cleavage at the desired genomic sites
and introduce specific mutations (insertion or deletion) at a high efficiency by errorprone non-homologous end-joining repairing. In addition, new strategies and tools are
being developed for simultaneous mutation of multiple genes, site-directed mutagenesis,
site-specific gene integration and precise deletion of chromosomal fragment in plants. To
assess potential off-target effects and increase the specificity of CRISPR-Cas9 system, we
have performed genome-wide prediction of highly specific gRNA spacer sequences and
targetable transcription units in eight model plants and major crops. A bioinformatic
database and web tool have been developed to help design highly specific gRNAs and assess
their off-target potential. With improved bioinformatic prediction and new experimental
strategies, CRISPR-Cas9-mediated genome editing is rapidly becoming a powerful tool
for plant functional genomics and genetic improvement of agricultural crops.
266 New DNA-Editing Approaches: Methods, Applications and Policy for Agriculture