1. Spring 2015 Student-Initiated SPUR Proposal
Principal Investigator: Peggy G. Lemaux
Student: Max Benjamin, Microbial Biology Major
Supply and Expense Amount Requested: $2000
Project Title: Agrobacterium-mediated Transformation of Setaria viridis as a Model for
Assessing Genetic Engineering Strategies in Sorghum bicolor
Abstract:
Setaria viridis is a C4-photosynthesizing plant that is closely related to several major
feed, fuel, and bioenergy crops. Its rapid life cycle, simple growth requirements, and
well-documented genome make it an excellent model system for studying genetic
engineering strategies in valuable cereal crops, such as sorghum and corn. Sorghum is an
important cereal crop due to its current use in the U.S. as a bioenergy crop and its
widespread cultivation and consumption in food-limited regions throughout the world.
Sorghum serves as a valuable source of nutrition, and is often a staple food, in regions
with unfavorable growth conditions due to its strong drought and flood tolerance.
Unfortunately, sorghum is less digestible than other cereal crops, such as maize, rice, and
wheat, especially after cooking. The decreased bioavailability of starch and protein in
sorghum grains, compared to those nutrients in grains of other cereal crops, reduces its
overall nutritional value as a food. Manipulation of the NADPH-thioredoxin reductase
(NTR)/thioredoxin (TRX) system in sorghum was found to increase seed size and
digestibility in a limited number of engineered lines, providing a potential strategy to
increase sorghum’s nutritional value. Cells in immature sorghum embryos can be
transformed to increase TRX expression via inoculation with Agrobacterium tumefaciens,
a bacterium that is able to transfer genes to plant cells. This method of Agrobacterium-
mediated transformation can also be used to transform embryogenic Setaria viridis callus
tissue. Because Setaria can be transformed more rapidly than sorghum, this model system
can be used to assess the outcome of a number of additional approaches to modifying
TRX expression in sorghum. By increasing the TRX h expression in the Setaria, using an
endosperm-specific promoter, we hope to demonstrate increased seed size in the grain,
and therefore, provide proof-of-concept for increased grain yield via enhanced seed size
in sorghum.
Objectives:
The goal of my project is to increase the starch content in seeds of the A10.1 variety of
Setaria through Agrobacterium-mediated transformation, using previously developed and
newly constructed plasmids. Successful transformation will result in the expression of the
barley thioredoxin h gene (BTrxh) in the endosperm of setaria seeds.
Introduction:
The process of C4 photosynthesis occurs in plants like maize and sorghum and is
much more efficient than C3 photosynthesis, especially in hot environments. Many C4-
photosynthesizing plants have been extensively characterized, including both maize and
sorghum, which have readily available and well-annotated genomic sequence data.

2. Unfortunately, their inefficient transformation systems, large stature, and long generation
times prevent high-throughput genetic studies from being performed on these plants. This
calls for the use of a C4-photosynthesizing model system, which could revolutionize
research on these crops, much as Arabidopsis has done for other crops. In the past,
model genetic systems have been restricted by the availability of their genomic
sequences. However, advances in sequencing technology have reduced the role of
sequence availability as a limiting factor, instead allowing for factors such as generation
time, size, self-fertility, and ease of transformation to dictate an organism’s utility as a
model system.
The recent publication of the Setaria viridis genome (Bennetzen et al. 2012), along
with advancements in the development of stable Setaria viridis transformation protocols
(Brutnell et al. 2010), have allowed Setaria viridis to progress as the most promising
model system for C4-photosynthesizing plants. Setaria grows and flowers quickly, self-
crosses readily, and is small enough to grow in a lab chamber environment (growing only
to 6-8 inches tall). Its capacity for C4-photosynthesis and close evolutionary relationship
to sorghum makes it particularly useful as a system for examining the effects of
transgenes in sorghum compared to other widely used models, such as Brachypodium
distachyon, a C3 model grass, and A. thaliana, a C3 model dicot plant.
Setaria is capable of producing totipotent embryogenic callus tissue from dry seed
when supplemented with nutrients on agarose media. Setaria callus tissue grows
particularly fast, taking only 2-3 months to produce transformable callus tissue from each
seed. Callus tissue can then be split into multiple pieces prior to transformation,
increasing the total possible number of independent transformation events. Transformed
plants, capable of producing transgenic T0 seed, can be obtained in 3-4 months, allowing
for expedited prediction of the effects of various transgenes – in our case on seed size and
digestibility. The use of Setaria as a model for genetic engineering approaches
translatable to Sorghum bicolor would allow for accelerated research and development
(via higher-throughput genetic screens) on future transgene efforts in sorghum.
Sorghum is a food staple in resource-limited regions all over the world. In fact, more
than 80% of the total land dedicated to sorghum production resides in developing
countries. Sorghum’s ability to grow in severe conditions, like flood- and drought-prone
regions, has caused it to become a valuable source of human nutrition in many
impoverished regions where common cereal crops, such as maize, cannot grow.
Unfortunately, protein digestibility is markedly lower in sorghum grain than in other
common cereal grains, rice, corn, wheat, especially after cooking, which is the primary
method of preparation of sorghum foods for human consumption. The reduced
bioavailability of protein in sorghum grains reduces its overall nutritional value as a food
source. In vitro manipulation of the NADPH-thioredoxin reductase (NTR)/thioredoxin
(TRX) system in sorghum has been found to increase protein content and bioavailability,
increasing digestibility and, in turn, providing a potential strategy for increasing
sorghum’s current limited nutritional value.
Thioredoxin, a protein found in the cytosol, mitochondria and chloroplasts of plant
cells, is an important part of the NADPH-thioredoxin reductase/thioredoxin (NTS)
system. This system affects protein and carbohydrate availability in the seeds of plants.
NTS modulates plant nutrient metabolism and availability through reductive processes,
by specifically targeting the redox state of proteins involved in these processes. TRX is a

3. small, soluble disulfide-containing protein that reduces disulfide bonds. The reducing
activity of TRX has been shown to affect the mobilization of carbohydrates and proteins
within the seed. More specifically, increased NTR/TRX activity has been shown to result
in increased carbohydrate and protein mobilization during seed germination through the
modulation of expression of degradative enzymes. By manipulating TRX levels in the
seed through genetic engineering, we hope to increase mobilization of protein and
possibly carbohydrates in transgenic grain, which will ultimately result in improved
nutritional value of the sorghum grain.
The increase in TRX h expression will be accomplished through Agrobacterium-
mediated transformation of the Setaria viridis variety, A10.1, according to a modified
transformation protocol, published by Brutnell et al. Modifications in the Lemaux
laboratory were influenced by addendums to the published protocol by Joyce van Eck at
Cornell and Arcadia Biosciences in Davis CA. After selection, regeneration, rooting, and
planting of putatively transformed plantlets, DNA will be collected from leaf tissue and
used to determine using PCR whether the plants were successfully transformed. All
putatively transformed Setaria plants, which successfully grow on antibiotic selection,
will be tested by PCR and Southern blot, and confirmed transgenic progeny will be
assayed for Trx h expression via Western blot. Size of transformed seeds will be
determined using a seed assay program, available through a former SPUR student in
graduate school at UC Davis. Digestibility of grain will be determined through standard
methods established in the Buchanan laboratory (Wong et al. 2009). The immediate goal
of this project will be to manipulate TRX levels in Setaria, improving grain yields by
increasing seed size. Setaria will serve as a rapid model for studying the potential effects
of TRX modulation in sorghum on seed size and in vitro protein digestibility.
Materials:
Plant Materials
Seeds from Setaria viridis cultivar A10.1 were obtained from J. Van Eck, Cornell
University. Seeds are kept at -80°C for 3 days to improve germination prior to initial
germination on callus-induction media (CIM). Callus is grown and regularly subcultured
on CIM until adequate transformation target tissue has been obtained.
Other Materials
LBA4404 Agrobacterium tumefaciens strain, various cell and tissue culture reagents
for inoculation and media for in vitro culture, pPZP201 pgkaff-ss-trx-pmi (pTrx-HptII)
constructs, Phusion polymerase and other PCR, western blot, and pepsin digestion
materials.
Methods:
For transformation of A10.1 with the pTrx-HptII construct, a modified transformation
protocol, based initially on Brutnell et al., will be used. Bleach-sterilized seeds will be
plated onto callus induction media and will be subcultured in 3 phases: initial growth
phase, followed by separation/isolation of optimal embryogenic callus tissue in transition
to secondary growth phase, which is followed by further separation/splitting of optimal
tissue in transition to pre-transformation phase (Benjamin, unpublished). Embryogenic
callus tissue that is 2-3 mm in diameter will be inoculated with Agrobacterium

4. tumefaciens bearing the binary vector containing pTrx-HptII. Agrobacterium will be pre-
induced with acetosyringone and synperonic. Inoculated callus tissue will be placed on
co-cultivation medium and, after co-cultivation, tissue will be transferred to callus
induction medium containing cefotaxime to kill off excess Agrobacterium. Callus pieces
will then be moved to callus selection medium containing hygromycin, which will give
transformed calli the ability to continue to grow while untransformed calli stagnate and
die, given the inability of plant cells to survive in the presence of hygromycin without the
introduced hygromycin phosphotransferase gene (hptII). Tissue will be subcultured and
transferred to fresh selection medium for 2-3 weeks, and then subcultured and transferred
to selective regeneration media with different plant hormones for 1-2 weeks until small
green shoots form. The tissue with actively growing, green, opaque shoots will be
transferred to non-selective regeneration medium for 1 week, and the resulting plantlets
will then be divided (if necessary) and transferred to rooting selection medium for 1-2
weeks before finally being planted in soil.
Full sized plants will flower, producing seed, within 1-3 weeks of being planted in
soil (depending on initial size of plants transferred from rooting selection). DNA will be
collected from the leaves of putative transformants, which have grown successfully in
soil. This DNA will be analyzed via PCR to determine whether the transferred genes
from the Trx-hpt construct exist in DNA from putative transformants. Heads of each
plant will be bagged to prevent crossing between different transgenic lines. Grain from
confirmed transformants will be germinated and tested for expression of the thioredoxin
protein, using western blots to determine the levels of TRX expression relative to plants
from the null segregant control grain. Time permitting screening of T3 grain from T2
generation plants will be used to identify homozygous plants and seed size determined. In
addition, if possible, pepsin digestions will be conducted on grain from homozygous
plants to determine whether the increased TRX levels in the grain of the transformants
correlate with increased digestibility of Setaria grain.
Timeline:
December: Continue previous pipeline of tissue culture from fall semester and
perform two transformations in parallel, one with a DsRed-HptII construct and another
with the desired Trx-HptII construct.
January: Identify transformation-ready callus tissue and perform another
transformation with Trx-HptII Agrobacterium. Transfer newly transformed callus to
CIM selective. Continue tissue culture of previous transformations by transferring from
CIM selective to regenerative selection. Germinate new wild-type seeds on CIM.
February: Continue transferring tissue from CIM selective to regenerative selection,
and from regenerative selection to rooting selection respectively.
March: Identify new transformation-ready callus tissue and perform another
transformation with Trx-HptII. Transfer remaining putative transformants from
regenerative selection to rooting selection. Plant rooting transformants in soil, extract
DNA from leaves, and perform analysis of DNA via PCR.
April: Transfer corresponding tissue to regenerative selection rooting selection
respectively. Perform Western and Southern blot analyses on confirmed transformants.
Bag flowering heads of adult plants in growth chamber. Harvest T2 seed from T1 plants,
screen DsRed-HptII plants for fluorescence (after seed coat removal). Plant T2 seeds

5. from initial DsRed-HptII and Trx-HptII transformants.
May: Transfer plants from second Trx-HptII transformation from rooting selection to
soil. Bag flowering heads of adult plants. Extract DNA from leaves, and perform
analysis of DNA via PCR. Perform Western and Southern blot analyses on confirmed
transformants. Harvest T2 seed of confirmed transformants. Utilize grain from
homozygous lines to perform western blots and determine levels of TRX expression.
Conduct pepsin digestions on grain from homozygous and companion null segregant
lines to assess differences in digestibility
Expected Results:
Using the modified Lemaux laboratory transformation protocol and recently
constructed pTrx-HptII vector, I expect to obtain transformed plants that can be used for
further analysis to determine effects of overexpression of TRX in grain endosperm on
grain digestibility, with phenotypic and molecular results from homozygotes that are
translatable to sorghum. As TRX has been previously shown to improve endosperm
starch content in seeds of a number of cereals in vitro, I expect that increased TRX
expression in the grain of transformed A10.1 Setaria will result in greater size of the
grain.
References:
Bennetzen et al. 2012. Reference genome sequence of the model plant Setaria.
Nature Biotech 30: 555-561.
Brutnell et al. 2010. Setaria viridis: A Model for C4 Photosynthesis. The Plant
Cell 22: 2537-2544.
Buchanan, BB. (2002). Thioredoxin: a photosynthetic regulatory protein finds
application in food improvement. Journal of the Science of Food and
Agriculture, 84.
Cho, Myeong-Je et al. “Overexpression of Thioredoxin h Leads to Enhanced
Activity of Starch Debranching Enzyme (pullulanase) in Barley
Grain.”Proceedings of the National Academy of Sciences of the United States
of America 96.25 (1999): 14641–14646. Print.
Howe, Arlene, Shirley Sato, Ismail Dweikat, Mike Fromm, and Tom Clemente.
"Rapid and Reproducible Agrobacterium-mediated Transformation of
Sorghum." Plant Cell Reports 25.8 (2006): 784-91.
Mauro-Herrera, Margarita et al. “Genetic Control and Comparative Genomic
Analysis of Flowering Time in Setaria (Poaceae).” G3:
Genes|Genomes|Genetics 3.2 (2013): 283–295. PMC. Web. 5 Dec. 2014.
Overexpression of thioredoxin h leads to enhanced activity of starch debranching
enzyme (pullulanase) in barley grain. PNAS, 96(25).
Sebastian J, Wong MK, Tang E, Dinneny JR (2014) Methods to Promote
Germination of Dormant Setaria viridis Seeds. PLoS ONE 9(4): e95109.
doi:10.1371/journal.pone.0095109.
Shahpiri, Azar, Birte Svensson, and Christine Finnie. “The NADPH-Dependent
Thioredoxin Reductase/Thioredoxin System in Germinating Barley Seeds:
Gene Expression, Protein Profiles, and Interactions Between Isoforms of

6. Thioredoxin h and Thioredoxin Reductase.” Plant Physiology 146.2 (2008):
789–799. PMC. Web. 5 Dec. 2014.
"Sorghum and Millets in Human Nutrition." FAO Food and Nutrition Series 27th
ser. (1995). FAO: FAO Home. Food and Agriculture Organization of the
United Nations, 1995.
Wong et al. 2009 Digestibility of protein and starch from sorghum (Sorghum
bicolor) is linked to biochemical and structural features of grain endosperm. J
Cereal Science 49:72-83.