Agrobacterium-Mediated Transformation

of Brachypodium distachyon
Fengjuan Chen,1,2 Qi Liu,1,2 John P. Vogel,3,4 and Jiajie Wu1,2,5
1
State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai’an,
Shandong, China
2
College of Agronomy, Shandong Agricultural University, Tai’an, Shandong, China
3
DOE Joint Genome Institute, Walnut Creek, California
4
University of California Berkeley, Berkeley, California
5
Corresponding author: jiajiewu@sdau.edu.cn

Brachypodium distachyon is an excellent model system for the grasses and

has been adopted as a research organism by many laboratories around the
world. It has all of the biological traits required for a model system, including
small stature, short life cycle, small genome, simple growth requirements, and
a close relationship to major crop plants (cereals). In addition, numerous re-
sources have been developed for working with this species, including genome
sequences for many lines, sequenced mutant collections, and a large, freely
available germplasm collection. Fortunately, among grasses B. distachyon is
one of the most easily transformed species, an absolute necessity for a model
system. Agrobacterium-mediated transformation is the preferred method to
transform plants because it usually results in simple insertions of target DNA.
In this article, we describe a method for Agrobacterium-mediated transforma-
tion of the inbred B. distachyon lines Bd21 and Bd21-3. Embryogenic callus
induced from immature embryos is co-cultivated with Agrobacterium tume-
faciens strain AGL1 or Agrobacterium rhizogenes strain 18r12v. Hygromycin
and paromomycin are used as selective agents, with comparable transforma-
tion efficiencies (defined as the percentage of co-cultivated callus that produce
transgenic plants) of 40% to 70%. It takes 20 to 30 weeks to obtain T1 seeds
starting from the initial step of dissecting out immature embryos. This protocol
has been shown to be efficient and facile in several studies that resulted in the
creation of over 22,000 T-DNA mutants.  C 2019 by John Wiley & Sons, Inc.

Keywords: Brachypodium distachyon r immature embryo r genetic transfor-

mation r model plant r grass r Agrobacterium r tissue culture

How to cite this article:

Chen, F., Liu, Q., Vogel, J. P., & Wu, J. (2019).
Agrobacterium-mediated transformation of Brachypodium
distachyon. Current Protocols in Plant Biology, e20088.
doi: 10.1002/cppb.20088

INTRODUCTION
In the last decade, numerous laboratories have used Brachypodium distachyon as a model
system to study a wide variety of topics in plant biology. One of the most important factors
making B. distachyon an attractive system for research is the existence of reliable high-
efficiency transformation methods. Here we describe a highly efficient Agrobacterium-
mediated transformation protocol that uses callus induced from immature embryos as
starting material. The process, which comprises three main parts, is shown in Figure 1.
The time required from dissecting out immature embryos to harvesting T1 seeds is 20

Chen et al.

Current Protocols in Plant Biology e20088 1 of 16

Published in Wiley Online Library (wileyonlinelibrary.com).
doi: 10.1002/cppb.20088

C 2019 John Wiley & Sons, Inc.
 Figure 1 Flow chart for Agrobacterium-mediated transformation of B. distachyon.

to 30 weeks. Hygromycin or paromomycin can be used as selective agents when using

the hygromycin phosphotransferase II (hptII) gene or the neomycin phosphotransferase
(nptII) gene as selectable markers, respectively. Transformation efficiencies, defined as
the percentage of co-cultivated callus that successfully produce transgenic plants, are
typically 40% to 70%. This transformation protocol has been optimized for the inbred
lines Bd21 and Bd21-3. In addition to the transformation protocol, presented in Basic
Protocol 1, techniques and critical considerations for growing B. distachyon plants are
described in Support Protocol 1.

STRATEGIC PLANNING
Because the total time required to get from nontransgenic seed to T1 transgenic seed
is 20 to 26 weeks, careful planning must be used to ensure that required materials,
manpower, and growing space are available when needed. Seeds from the line to be
transformed must be planted 4 to 6 weeks before the main protocol is begun so that
immature seeds at the appropriate stage are available. As shown in Figure 1, starting
from picking immature embryos, the transformation protocol includes three parts and
nine procedures. The purpose and time duration for each procedure are noted in the
figure, above and below the green arrows, respectively.
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AGROBACTERIUM-MEDIATED TRANSFORMATION OF BRACHYPODIUM BASIC
DISTACHYON PROTOCOL 1
The following protocol describes the production of transformed B. distachyon lines via
A. tumefaciens or A. rhizogenes transformation. We use in-house-generated competent
Agrobacterium cells for this procedure; commercial A. tumefaciens AGL1 cells are
available (e.g., Life Science Market, cat. no. CHC00070), although we have not tested
them. To our knowledge, no commercial source of A. rhizogenes 18r12v is available.

The basic protocol is divided into three parts (Fig. 1): (i) culture of embryogenic callus,
(ii) transformation of embryogenic callus via A. tumefaciens or A. rhizogenes, and (iii)
regeneration of transgenic plants.
Materials
Brachypodium distachyon plants of inbred line Bd21 (Vogel, Garvin, Leong, &
Hayden, 2006) or Bd21-3 (Vogel & Hill 2008) with immature seeds (Support
Protocol 1) (Bd21 and Bd21-3 seeds are available from the U.S. National Plant
Germplasm System, https://www.ars-grin.gov/npgs/)
Household bleach (5.25% sodium hypochlorite)
Triton X-100
Distilled water, sterile
Callus initiation medium (CIM; see recipe)
Agrobacterium tumefaciens strain AGL1 (Lazo, Stein, & Ludwig, 1991) or A.
rhizogenes strain 18r12v (Collier, Bragg, Hernandez, Vogel, & Thilmony, 2016)
harboring desired binary vector
MG/L medium (see recipe)
Acetosyringone
Synperonic PE/F68
Selection medium (SM; see recipe)
Regeneration medium (RGM; see recipe)
Rooting medium (RTM; see recipe)

50-ml Falcon tube

10-ml glass pipet or Pasteur pipet
Scissors
Laminar-flow hood
9-cm plastic petri dish
Dissecting microscope
Fine forceps (points 0.1 × 0.06 mm, Dumont tweezers, cat. no. 0209-5-PO)
Standard forceps
Long forceps (20 cm)
Parafilm
Spatula
Vortexer
Plant tissue culture incubator, 28°C, without light (environmental chambers
designed to minimize condensation on plates are superior; however, gravity
incubators can be used if necessary)
Micropipet and tips
0.2-µm nylon syringe filters (nylon filters must be used for solutions containing
DMSO)
Spectrophotometer
Grade P4 7-cm circular filter paper (autoclaved and dried at 75°C overnight)
Incubator, 22°C, without light (an inexpensive option is to place a small gravity
incubator in a cold room)
Plant tissue culture incubator, 28°C, 16-hr photoperiod, 65 μmol/m2 /s Chen et al.

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 A D

E
B

Figure 2 Seed selection and dissection of immature embryos for transformation. (A) Seeds
from one spikelet arranged from youngest (from top of spikelet) on the left to oldest (from bottom
of spikelet) on the right. Correspondingly, the sizes of the embryos get larger from left to right.
Counting from left to right, the embryo sizes are: 1–4, too small to dissect; 5, 6, <0.3 mm (ideal
size); 7, 8, 0.3–0.5 mm (acceptable size); 9, 10, 0.5–0.7 mm (too big). (B-E) Illustrations of the
process of dissecting out embryos. Use blunt forceps to hold the seed while using fine forceps to
pierce the seed coat in the top third of the seed (B). Move the fine forceps toward the embryo and
at same time gradually open the forceps to remove the seed coat and allow access to the embryo
(C). The embryo is visible at the tip of the seed (C, D, E). Using one tip of the forceps, remove
the whole embryo. To avoid injury to the embryo, do not pinch it with the forceps. (F) Embryos of
different maturities. The second and third from the left are optimal. Scale bars, 1 mm.

Rooting containers: 25 × 95–mm flat-bottom glass tube (PhytoTech, C935)

covered with tube closure (PhytoTech, C1805) or, when making large numbers
of transgenics, sundae cups made for food service applications [Solo Corp., cat.
nos. SOL-TS5 (cups) and SOL-DL-100 (dome lids); although these are not
guaranteed sterile and cannot be autoclaved, we have never encountered any
problems with contamination in the course of generating >25,000 transgenic
lines]
Culture of embryogenic callus
1. Grow B. distachyon plants following the instructions described in Support
Protocol 1.
2. Cut spikelets carrying immature seeds of suitable stage as shown in Figure 2A
and place in a 50-ml Falcon tube containing a few milliliters of water to avoid
desiccation.
Seeds are at the correct maturity when the caryopsis has grown close to the end of the
palea and has just started filling out. Seeds at the proper maturity are still soft and
pliable. Seeds at the right stage are typically available 7 to 8 weeks after sowing, but the
Chen et al.
exact timing depends on the growth conditions. When learning how to dissect embryos,
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 it is useful to start with more mature seeds to learn where the embryo is located in the
seed.

3. Dump out the spikelets and select seeds of suitable size.

Usually only two to five seeds on each spikelet are of suitable size and should be selected
for dissection of embryos.

4. Using your fingers, remove the lemma from each seed, taking care not to damage
the delicate seed. It is not necessary to remove the palea, but it does not hurt if it
comes off. Place the naked seeds in a sterile 50-ml Falcon tube containing about
10 ml of water to prevent desiccation.
The protocol can be paused at this step by leaving the tube at 4°C or room temperature
for several hours. Pausing overnight is not recommended.

5. Remove the water. Surface sterilize the seeds by soaking in a solution of 10% bleach
(final concentration 0.5% sodium hypochlorite) plus 0.1% Triton X-100, and gently
mix the tube in a shaker or by periodically inverting for 5 min.
A 10-ml glass pipet or Pasteur pipet attached to a vacuum can be used to remove the
liquid in this and the following steps.

6. Remove the bleach solution and rinse the seeds three times with sterile distilled
water.
This step and all those following should be carried out in a laminar-flow hood.

7. After the final rinse, leave about 5 ml water in the tube and transfer the seeds to a
petri dish by swirling and inverting the tube. If necessary, tap the tube or add a small
amount of sterile water to get all the seeds out. Place the lid of the petri dish upside
down on the stage of the dissecting microscope to use as a work surface.
8. Dissect out embryos <0.5 mm in diameter (Fig. 1A) using blunt forceps to hold the
seed and fine forceps to dissect away the overlaying layers to expose the embryo,
using Figure 2 as a guide. The embryo is at the lower end of the seed, close to the
surface (Fig. 2B to E). You can also hold the seed in place with a gloved fingertip,
but take care to keep your finger away from the embryo end to avoid contaminating
the embryo.
Small embryos (<0.3 mm) produce embryogenic callus, while bigger ones (>0.7 mm) pro-
duce soft, disorganized callus. Embryos between 0.3 and 0.7 mm produce intermediate-
quality callus (Fig. 3). Embryos <0.3 mm are clear and can be difficult to see. Larger
embryos are white or yellow. When first learning how to dissect embryos, it is useful to
start with more mature seeds, as shown in Figure 2F.

9. Carefully transfer each embryo onto CIM using one tip of the forceps, taking care
not to push the embryo into the medium (Fig. 1A). To minimize losses due to
contamination, place no more than 10 embryos on a plate. After finishing each plate,
check the placement of each embryo under the microscope and adjust any that are
below the surface of the medium.
Surface tension is enough to hold the embryo on the tip of the forceps. Do not clamp the
embryo in the forceps or you will crush it. It does not matter if the scutellum side is facing
up or down. However, it is easier to judge the size of an embryo when placed scutellum
side down. A skilled person can dissect out 30 embryos in 20 min.

10. Seal the petri dish with Parafilm and incubate at 28°C in the dark for 3 to 4 weeks
(Fig. 1B and C).
After 7 to 10 days, yellow callus with organized structures will begin to form. Shoots
may sprout from some embryos and can be ignored. The surface of high-quality callus Chen et al.

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 A C

B D

Figure 3 Calluses induced from immature embryos of different sizes. (A) Callus induced from
embryos of optimal size. (B–D) Calluses induced from larger embryos. Note that these calluses
are dominated by soft, watery undefined callus. Also note the shoots emerging from the older
embryos. Scale bars, 1 mm.

will be dominated by yellow organized structures that may be interspersed with amor-
phous callus. Bd21-3 callus is yellower than Bd21 callus. The interior of the callus
and the area touching the medium are watery and amorphous, and are not useful for
transformation.
The time required before subculturing varies between plant lines. Usually we incubate
3 weeks for Bd21 and 4 weeks for Bd21-3. Longer incubation time will decrease callus
quality. Avoid incubating the callus longer than 4 weeks before subculturing. Check the
plates twice a week and, if contamination is noted, move uncontaminated embryos to
fresh CIM.

11. Subculture embryogenic callus under a microscope. Discard amorphous parts and
only keep the yellow organized parts. Split these embryogenic regions into small
pieces (2 mm) and transfer onto fresh CIM. Seal the petri dish with Parafilm and
incubate at 28°C in the dark for 2 weeks (Fig. 1D).
It is important to select only the embryogenic callus for transfer. The embryogenic parts
are compact, structured and yellow in color. They feel firm when pinched with forceps.
To obtain a high quality of embryogenic callus, this step and the following subculturing
step should be done with a microscope placed in a laminar-flow hood. One callus can
usually be broken into three to five pieces. Transfer 25 to 30 pieces onto one fresh plate
and leave space for the callus to grow.

12. After 2 weeks, subculture the callus again and incubate at 28°C in the dark for
Chen et al.
1 week (Fig. 1E).
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 As with the first subculture, select only the embryogenic callus to transfer. Break the
callus into small pieces (usually three to five pieces) using forceps. The callus is usually
more organized than in the first subculture, and skilled workers may not need the aid of a
microscope. After this subculture, each embryo should have produced 10 to 20 calluses.

Transformation of embryogenic callus

13. Two days before the day of transformation, heavily streak out the desired Agrobac-
terium strain from a single colony or glycerol stock onto solid MG/L medium con-
taining the appropriate antibiotics, and incubate the plate at 28°C for 2 days. One
plate is usually enough to transform at least eight plates of callus (200 calluses).
For AGL1, use carbenicillin (final concentration 100 mg/liter) to select for the helper
plasmid. The second antibiotic, used to select for the plasmid containing the T-DNA,
depends on the binary vector. Commonly used antibiotics are kanamycin or spectinomycin
at a final concentration of 50 mg/liter.
Agrobacterium rhizogenes strain 18r12v works with similar efficiency to AGL1 in this
protocol.

14. Scrape the Agrobacterium layer from the MG/L plate using a small sterile spatula
or 1-ml pipet tip and transfer to a sterile 50-ml Falcon tube containing 10 ml liquid
CIM. Vortex the tube or shake vigorously by hand until the bacteria are completely
suspended and then adjust the OD600 to 0.6 using spectrophotometer by adding
additional CIM.
The OD600 value of 0.6 is not critical. Add an excess of bacteria to the medium first,
vortex, and check the OD600 , and then dilute the suspension as necessary. How much
volume to prepare depends on the amount and volume of calluses being transformed.
Usually we prepare 20 ml of suspension for every 100 calluses.

15. Add acetosyringone stock solution to the Agrobacterium suspension to a final con-
centration of 200 µM, and add Synperonic PE/F68 stock solution to a final concen-
tration of 0.1%.
16. Remove the calluses, without intentionally breaking them into pieces, from the plates
and place them into sterile 50-ml Falcon tubes in preparation for Agrobacterium co-
cultivation. To allow efficient mixing in the next step, do not fill tubes more than
three-quarters full.
It is not necessary to examine the callus with a microscope at this stage, but any callus
that is entirely white, watery, and soft should be discarded.

17. Optional: Test the quality of the callus by transferring a few pieces to RGM prior to
transformation. Simply place a few calluses onto petri plates containing RGM (with-
out any selective agents) as described in step 23; researchers new to the procedure
may find it particularly informative to note the appearance of the callus pieces so
they can better judge callus quality by appearance. Immediately use the remainder
of the calluses for transformation (next step).
Experienced researchers can omit this step; however, it can be invaluable when trou-
bleshooting problems.

18. Add Agrobacterium suspension to the 50-ml tube containing the calluses, making
sure the calluses are completely covered. Cap the tube and invert gently by hand for
30 sec, then let it stand still for 5 min.
19. Carefully discard the Agrobacterium suspension and transfer all the calluses into
an empty petri dish. The Agrobacterium suspension can be removed by careful
decanting using the cap to prevent the calluses from falling out. Alternatively, the
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 suspension can be removed using a 10- to 50-ml pipet, taking care not to fragment
or remove calluses.
20. Tilt the petri dish and remove the remaining liquid using a 1-ml micropipet.
It is critical to remove excess Agrobacterium suspension from the callus. After all visible
liquid has been removed, push the pipet tip underneath the calluses to remove the last
bits of Agrobacterium suspension.

21. For each 20 ml of calluses, place a single piece of dry, sterile filter paper into an
empty petri dish. Transfer up to 20 ml of calluses onto each filter paper using forceps
and distribute the calluses evenly around the filter paper (Fig. 1F).
Do not intentionally break the calluses into smaller pieces.

22. Seal the petri dish with Parafilm and incubate at 22°C in the dark for 3 days
(Fig. 1F).
23. Transfer the callus pieces onto SM and incubate at 28°C in the dark for 1 week
(Fig. 1G and H). Place about 20 to 25 calluses on each plate.
After 3 days on the filter paper, the callus will appear somewhat dry and shrunken.
Calluses usually break into smaller pieces due to handling, so there will be more callus
pieces placed onto SM than initially placed into co-cultivation. Very small pieces can be
placed together on the SM and treated as one callus in subsequent steps.
SM contains 300 mg/liter Timentin to kill Agrobacterium and the appropriate selective
agent to kill untransformed plant tissue. Several selective agents have been tested in this
protocol. Hygromycin (40 mg/liter) works best and is most commonly used. Comparable
efficiency is obtained using paromomycin (200 to 400 mg/liter) selection. BASTA selection
is much less efficient because it does not kill untransformed callus as efficiently.

24. Subculture the callus and transfer healthy parts to a new plate of SM. Incubate at
28°C in the dark for 2 weeks (Fig. 1I and J).
The appearance of calluses on different selective agents varies considerably. Hygromycin
provides very strong selection and kills nontransgenic callus. Thus, the sectors of healthy
callus growing from brown/black dying callus are very easy to pick. By contrast, BASTA
is a weak selective agent, and nontransgenic callus typically does not die but just grows
more slowly. Thus, transgenic callus will appear as sectors of rapidly growing callus
that are difficult to distinguish from untransformed callus that may or may not appear
brownish. When selecting callus, err on the side of selecting more callus rather than less.
Any untransformed callus transferred will be selected against over time.
If several small pieces are selected from one callus, keep them together as one callus line.
Keeping callus lines separate ensures that transformants are independent. A convenient
way to demarcate callus lines is to gently score the surface of the Phytagel around the
callus fragments. Typically, 16 to 20 callus lines can be placed on one plate, but this will
vary depending on callus growth rate and selection efficiency.

Regeneration of transgenic B. distachyon

25. Select the healthy parts of the calluses as described in step 22 and place them
on RGM, taking care to keep all pieces from each callus line together to ensure
independence of the transgenics. Typically, 8 to 10 lines fit on one plate. Wrap with
Parafilm and incubate at 28°C in the light for 2 to 3 weeks (Fig. 1K).
Standard light conditions are cool-white fluorescent lighting at a level of 65 μEm/m2 /s
with a 16 h light/8 h dark cycle. Relatively low light intensity is used to avoid excessive
heating and condensation in the plates, and long days are used to promote flowering after
plantlets are transferred to soil. However, we have not rigorously tested other light levels
and daylengths. Timentin (300 mg/liter) and plant selective agent should be included in
Chen et al. the regeneration medium. It is normal for most of the callus to turn black and die, leaving

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 only interspersed regions of healthy callus. In 1 to 2 weeks, shoots will emerge from small
green dots of callus (Fig. 1L). Albino shoots and shoots that gradually die are probably
untransformed escapees.

26. When the shoots are large enough to handle, 1 to 2 cm tall, transfer a small piece
of callus containing the shoot onto RTM in a container that is tall enough to allow
vertical growth (Fig. 1M). The small piece of callus is included to avoid damaging
the shoot. Incubate in the light at 28°C for 2 to 3 weeks.
If space permits, place only one line in each container, because they often root at different
rates.
Timentin (150 mg/liter) should be included in RTM to avoid growth of Agrobacterium.
When using BASTA as the selective agent, it should be added to the RTM to reduce
escapes. When using hygromycin it is not necessary to add it to the RTM because the
rate of escapes is extremely low.

27. Examine the shoots periodically (Fig. 1N) and when they are 2 to 5 cm tall and have
developed one to three healthy roots (1 to 2 cm long), they can be transferred to soil.
Carefully remove the medium and callus adhering to the plantlets using forceps or
by dipping the roots in water. Callus and medium that cannot be easily removed
should be left attached to the plantlets to avoid damaging them. Grow the plants
under conditions that promote flowering: 16- to 20-hr daylength, 24°C during the
day and 18°C at night, cool-white fluorescent lighting at a levels of 600 μEm/m2 /s.
Cover the plantlets with a clear plastic dome (Fig. 1O) to prevent desiccation during
the first 3 to 5 days, and then gradually remove the cover.
Most shoots will form roots within 4 weeks. However, shoots that do not form roots should
still be transplanted to soil because many will form roots once in soil.
If the daylength after transplanting is <16 hr, flowering can be promoted by vernalizing
the plantlets at 4°C for 2 to 3 weeks immediately before transplanting. It is acceptable
for the light intensity to be low (e.g., a pair of 40-watt fluorescent tubes 30 cm above the
plants) during vernalization. If plants are placed into a greenhouse after transplanting,
shade the plastic dome or place the transplants in a shady place without a dome to avoid
excessive heat buildup.
See Support Protocol 1 for instructions on how to grow B. distachyon.

28. Plants usually flower 3 to 6 weeks after transplantation (Fig. 1P), and T1 seeds
will be ready to harvest when they are completely dry, 6 to 12 weeks after
transplantation.
T0 plants usually produce 50 to 150 T1 seeds.

GROWING BRACHYPODIUM DISTACHYON SUPPORT

PROTOCOL 1
In this support protocol, we describe the procedure for growing B. distachyon plants
to produce immature embryos in Basic Protocol 1 or to propagate transgenic seeds.
This species can be grown a number of ways as long as attention is paid to these key
parameters: soil, daylength, light intensity, watering, and vernalization. Optimization of
these parameters is described in the Critical Parameters section.
Materials
Potting soil (see recipe in Reagents and Solutions for a list of suitable commercial
and custom-made potting soils)
Slow-release fertilizer: e.g., Scotts Osmocote Plus 15-9-12 (cat. no. 903246)
B. distachyon inbred lines of Bd21 (Vogel et al., 2006) or Bd21-3 (Vogel & Hill,
2008)—seeds can be ordered from the USDA National Plant Germplasm
System, https://www.ars-grin.gov/ Chen et al.

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 5- to 20-cm-diameter pots
Labels and markers
Stakes
Growth chamber or greenhouse
1. Prepare pots by filling them with soil and pressing gently so that the soil level is
below the pot rim (to allow space for watering). Add slow-release fertilizer [1.2 ml
(0.25 teaspoon) for a 5-cm (2-inch) pot or 5 ml (1 teaspoon) for a 15-cm (6-inch)
pot] and lightly work into the top of the soil with your finger. When preparing many
small pots, add an appropriate amount of fertilizer to a large quantify of soil and
mix thoroughly before filling the pots.
Various pot sizes can be used. The minimum size for single plants is 5 cm in diameter.
Inserts for greenhouse flats can also be used. Plants grow better and are easier to keep
evenly moist in larger pots of 8 cm or greater diameter. If experiments allow, growing
multiple plants in a single large pot is another option. About 5 to 10 plants can be
grown in a single 15-cm-diameter pot; this is the approach we use to grow plants for
embryos.

2. Water pots until water comes out the drainage holes.

3. Plant B. distachyon seeds 1 cm deep and cover with soil.
4. Incubate planted pots at 4°C for 1 week to ensure uniform germination. This strati-
fication step is especially important for fresh seed (<6 months after harvest).
5. Optional: Incubate the planted pots at 4°C for another 1 to 2 weeks for vernalization
(2 to 3 weeks total incubation time). Do this when growing plants in a greenhouse
in winter or when using a growth chamber with short day length.
If large numbers of lines need to be stratified/vernalized, seeds can be placed on moist
paper towels and planted after the cold treatment. We use small plastic bags for this
purpose. Simply place the seeds and a wet paper towel in the bag and fold it over. If using
ziplock bags, leave them unzipped to facilitate air exchange.
Bd21-3 requires a slightly longer period of vernalization than Bd21.

6. Move plants into a growth chamber or greenhouse.

Our standard growth chamber conditions are 28°C day 18°C night, 16 h light/8 h dark
light cycle, light intensity of 600 μEm/m2 /s (cool-white fluorescent plus incandescent).
Ideal greenhouse conditions are 28°C day and 18°C night, supplemental light to extend
daylength to 16 h, and no shading.

7. Depending on the growth conditions, it may be necessary to stake the plants after
flowering to avoid tangling.
8. After the seed heads have turned brown, stop watering.
Do not stop watering if the seeds are still green.

9. Harvest seeds after they are all dry. Place in paper envelopes.
Paper envelopes are preferred because they allow seeds to continue drying. If using
plastic bags, leave them open for at least a month after harvest. Seeds in envelopes
can be stored at room temperature for several years. For longer storage or to maintain
maximum viability, seeds should be stored at 4°C with <10% humidity.

10. Wait at least 3 weeks after harvest before sowing seeds.

Fresh seeds germinate poorly. This can be overcome somewhat by stratification, but even
with stratification, very fresh seed will have low and erratic germination.
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REAGENTS AND SOLUTIONS
Acetosyringone stock solution (200 mM, 1000×)
Dissolve 0.392 g acetosyringone (Sigma, D134406) in 10 ml DMSO
Filter sterilize with a 0.2-µm nylon syringe filter
Aliquot and store at −20°C. Can be stored for at least 6 months.
BASTA (DL-phosphinothricin) stock solution (60 mg/ml, 1000×)
Dissolve 1.2 g DL-phosphinothricin (Phytotechnology, P679) in 20 ml
double-distilled water
Filter sterilize with a 0.2-µm syringe filter
Aliquot and store at −20°C. Can be stored for at least 6 months.
Callus initiation medium (CIM), per liter
4.43 g Linsmaier and Skoog (LS) basal medium (Phytotechnology, L689)
30 g sucrose (Phytotechnology, S829)
1.0 ml 0.6 mg/ml CuSO4 stock solution (see recipe)
0.5 ml 5 mg/ml 2,4-D stock solution (see recipe)
Adjust pH to 5.8 with 0.1 N KOH and sterilize by autoclaving
For solid medium only: Add 2.5 g Phytagel (Sigma, P-8169) when using
hygromycin selection or 5 g Phyto Agar (Research Products International Corp.,
A20300) for paromomycin selection before autoclaving (paromomycin
precipitates in the presence of Phytagel)
Autoclave for 45 min
After the medium has cooled to 65°C, pour into storage bottles (liquid medium) or
9-cm petri plates (solid medium)
Store at 4°C for <2 weeks
Note that Phytagel is difficult to melt after it solidifies, so it is easiest to pour plates
after autoclaving rather than melting previously autoclaved medium.
For all solid media, add the Phytagel or agar to dry bottles to avoid clumping.

Carbenicillin stock solution (100 mg/ml, 1000×)

Dissolve 10.0 g carbenicillin (Phytotechnology, C346) in 100 ml double-distilled
water
Filter sterilize with a 0.2-µm syringe filter
Aliquot and store at −20°C. Can be stored for at least 6 months.
CuSO4 stock solution (0.6 mg/ml, 1000×)
Dissolve 0.6 g CuSO4 ·5H2 O in 1 liter double-distilled water
Store at −20°C. Can be stored indefinitely.
2,4-D stock solution (5 mg/ml)
Dissolve 50 mg 2,4-D (Sigma, D7299) in 10 ml of 95% ethanol
Aliquot and store at −20°C. Can be stored for at least 6 months.
Hygromycin stock solution (40 mg/ml, 1000×)
Dissolve 4.0 g hygromycin (Gold Biotechnology, H-270-10) in 100 ml
double-distilled water
Filter sterilize with a 0.2-µm syringe filter
Aliquot and store at −20°C. Can be stored for at least 6 months.
Kanamycin stock solution (50 mg/ml, 1000×)
Dissolve 5 g kanamycin in 100 ml double-distilled water Chen et al.

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Current Protocols in Plant Biology
 Filter sterilize with a 0.2-µm syringe filter
Aliquot and store at −20°C. Can be stored for at least 6 months.
Kinetin stock solution (0.2 mg/ml, 1000×)
Dissolve 2.0 mg kinetin (Phytotechnology, K750) in 10 ml of dimethyl sulfoxide
(DMSO). Filter sterilize with a 0.2-µm nylon syringe filter. Aliquot and store at
−20°C. Can be stored for at least 6 months.
MG/L medium, per liter
5 g tryptone
2.5 g yeast extract
5 g NaCl
5 g mannitol
0.1 g MgSO4
0.25 g K2 HPO4
1.2 g glutamic acid
Adjust pH to 7.2 with 0.1 N NaOH
Add 15 g agar
Autoclave for 30 min
After the medium has cooled to 65°C:
Add 1 ml 100 mg/ml carbenicillin stock solution (see recipe) and a second
antibiotic depending on the plasmid to be transformed
Pour into 9-cm petri dishes
Store at 4°C for < 2 weeks.
Paromomycin stock solution (200 mg/ml, 500×)
Dissolve 20 g paromomycin (Phytotechnology, P710) in 100 ml double-distilled
water
Filter sterilize with a 0.2-µm syringe filter
Aliquot and store at −20°C. Can be stored for at least 6 months.
Potting soils (use one)
Commercial potting soils:
Sungro professional growers mix, also called Sunshine #1 (Sun Gro Horticulture,
USA and Canada)
Pindstrup Peat Moss (0-10 mm) (Pindstrup, Denmark).
Custom soil mixes:

Ingredient Formula 1 Formula 2

Sandy loam 1 part
Clay loam 1 part
Sand 2 parts
Peat moss 3 parts 1 part
Vermiculite, medium grade (#3 or 2–5 mm) 3 parts 1 part

The sandy loam or clay loam can be moistened and autoclaved on a liquid cycle for
30 min, if desired.
Regeneration medium (RGM), per liter
4.43 g Linsmaier and Skoog (LS) basal medium (Phytotechnology, L689)
30 g maltose (Phytotechnology, M588)
Chen et al.
Adjust pH to 5.8 with 0.1 N KOH
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Current Protocols in Plant Biology
 Add 2.5 g Phytagel (Sigma, P-8169) for hygromycin selection or 5 g Phyto Agar
(Research Products International Corp., A20300) for paromomycin selection
(Phytagel precipitates paromomycin).
Autoclave for 45 min
After the medium has cooled to 65°C:
Add 1.0 ml 0.2 mg/ml kinetin stock solution (see recipe)
Add 2.0 ml 150 mg/ml Timentin stock solution (see recipe)
Add the appropriate selective agent: 1.0 ml 40 mg/ml hygromycin stock solution
(see recipe) for vectors with hptII or 2 ml 200 mg/ml paromomycin stock
solution (see recipe) for vectors containing nptII.
Pour medium into 9-cm petri dishes (1.6 cm in height)
Store at 4°C for < 2 weeks.
Rooting medium (RTM), per liter
4.42 g Murashige and Skoog (MS) basal medium with vitamins (Phytotechnology
M519)
30 g sucrose (Phytotechnology, S829)
Adjust pH to 5.7 with 0.1 N KOH
Add 2.5 g Phytagel (Sigma, P-8169) for hygromycin selection or 5 g Phyto Agar
(Research Products International Corp., A20300) for paromomycin selection
Autoclave for 45 min
After the medium has cooled to 65°C:
Add 1.0 ml 150 mg/ml Timentin stock solution (see recipe)
Optional: Add 1.0 ml 40 mg/ml hygromycin stock solution (see recipe) or 2 ml
200 mg/ml paromomycin stock solution (see recipe), depending on the
selectable marker gene used
Pour medium into sterile glass tubes or tissue culture boxes
Store at 4°C for < 2 weeks.
Selection medium (SM), per liter
To 1 liter of 65°C CIM, add:
2.0 ml 150 mg/ml Timentin stock solution (see recipe)
Appropriate selective agent: 1.0 ml 40 mg/ml hygromycin stock solution (see
recipe) for vectors with hptII or 2 ml 200 mg/ml paromomycin stock solution
(see recipe) for vectors containing nptII
Pour medium into 9-cm petri dishes
Store at 4°C for <2 weeks.
Note that B. distachyon is resistant to kanamycin, which is commonly used as the selective
agent when using nptII as the selectable marker in dicot transformations.

Synperonic PE/F68 (10%, 100×)

Dissolve 1 g Synperonic PE/F68 (Sigma, 81112) in 10 ml double-distilled water
Filter sterilize with a 0.2-µm syringe filter
Aliquot and store at −20°C. Can be stored for at least 6 months.
Timentin stock solution (150 mg/ml, 500×)
Dissolve 15 g Timentin (Gold Biotechology, T-104-100) in 100 ml double-distilled
water
Filter sterilize with a 0.2-µm syringe filter
Aliquot and store at −20°C. Can be stored for at least 6 months.
Triton X-100 (10% (v/v), 100×)
Add 10 ml Triton X-100 (Sigma, T8787) to 90 ml double-distilled water
Mix and store at room temperature. Can be stored for at least 6 months. Chen et al.

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Current Protocols in Plant Biology
 COMMENTARY
Background Information
Brachypodium distachyon has many desir-
able features that make it an excellent model
organism for functional genomics research in
monocots, which include cereal crops, for-
age grasses, and biomass crops (Draper et al.,
2001). Owing to the intensive and thoughtful
development of this grass as a model organism,
many resources have been created for work in
B. distachyon in the past decade (Scholthof,
Irigoyen, Catalan, & Mandadi, 2018; Vogel,
2016; https://jgi.doe.gov/our-science/science-
programs/plant-genomics/brachypodium/).
With these public resources, B. distachyon is
widely used in fundamental research for un-
derstanding diverse grass traits such as their
cell walls, roots, vernalization, and biotic and
abiotic stresses (Scholthof et al., 2018).
Since Draper et al. (2001) reported that
a polyploid accession of B. distachyon (now
classified as a separate species, Brachypodium
hybridum) was amenable to microprojectile
bombardment–mediated transformation, sev-
eral improved protocols have been developed.
As in other plant species, the bombardment-
mediated transformation of B. distachyon is
characterized by a complex integration pat-
tern (Christiansen, Andersen, Didion, Folling,
& Nielsen, 2005). Efficient Agrobacterium
tumefaciens– mediated transformation was
established in several laboratories (Alves
et al., 2009; Bragg, Anderton, Nieu, & Vogel,
2015; Pacurar, Thordal-Christensen, Nielsen,
& Lenk, 2008; Vogel & Hill, 2008; Vogel et al.,
2006). These studies proved that B. distachyon
is much more amenable to genetic transfor-
mation than are small grains, including wheat
and barley. Embryogenic callus was used as
material for transformation in the above pro-
cedures. To produce embryogenic callus, it is
more efficient to use small immature embryos
than whole seeds (immature or mature) (Vogel
et al., 2006).
Hygromycin is the most widely used se-
lective agent. BASTA selection has also been
used for B. distachyon transformation in few
studies, and the transformation efficiency was
5.5% for bombardment-mediated transfor-
mation of BDR018 (Christiansen et al., 2005).
A much higher transformation rate, 55%,
for the same accession was achieved us-
ing Agrobacterium-mediated transformation
(Pacurar et al., 2008). However, the latter
study co-cultivated embryos without subcul-
turing, which is much more labor intensive
Chen et al. and not directly comparable to other methods.
14 of 16
A direct comparison of hygromycin and
BASTA as selective agents showed that hy-
gromycin was much more efficient (average
transformation efficiency 23% to 56%) than
BASTA (average transformation efficiency 3%
to 8%) (Bragg et al., 2012). Paromomycin
has also been proved effective for selecting B.
distachyon transformants, and transformation
efficiencies similar to those for hygromycin
selection have been obtained (Collier et al.,
2016). The promoter driving the selectable
marker also has a large impact on transforma-
tion efficiency, and the maize ubiquitin pro-
moter and CaMV 35S promoter with a 3 in-
tron both work well (Alves et al., 2009; Bragg
et al., 2012; Pacurar et al., 2008).
Agrobacterium tumefaciens AGL1, a super-
virulent strain, has been used for the majority
of B. distachyon transformations (Alves et al.,
2009; Bragg et al., 2012; Hsia et al., 2017;
Vogel & Hill, 2008). However, a recent study
found that A. rhizogenes strain 18r12v can also
transform B. distachyon with efficiency com-
parable to or slightly higher than that of AGL1
(Collier et al., 2016).
The protocol described here has been suc-
cessfully employed to create over 22,000 T-
DNA mutants (Bragg et al., 2012; Hsia et al.,
2017) and has been used for studying individ-
ual genes, including the functional validation
of the flowering-time gene FT (Qin et al., 2017;
Wu et al., 2013) and wheat male sterile gene
Ms2 (Ni et al., 2017), among others.
Critical Parameters and
Troubleshooting
The Agrobacterium strain, selectable mar-
ker, promoter driving the selectable marker,
and quality of callus are the major factors in-
fluencing transformation efficiency. For initial
transformation we suggest using a control vec-
tor expressing a reporter gene such as GFP
or GUS. This allows investigators to identify
transgenic callus early in the process, which
facilitates troubleshooting. We also encourage
researchers to routinely test the quality of their
callus by placing a small sample on RGM
without selective agent prior to co-cultivation
with Agrobacterium. This will localize prob-
lems to the callus induction or transformation
steps. With high-quality callus, nearly all cal-
luses will regenerate shoots on RGM with-
out selective agent. If a high efficiency is ob-
served for this test, but at the same time low
efficiency is obtained from Agrobacterium-
mediated transformation, then troubleshooting
Current Protocols in Plant Biology
should be focused on transformation is-
sues such as Agrobacterium strain, selectable
marker, and promoter.
To obtain high-efficiency transformations,
the most critical factor is the quality of the
embryogenic callus. It is essential that very
small embryos be used and that only highly
embryogenic callus be selected at each sub-
culture. Inexperienced investigators can test
their skill at identifying embryogenic callus
by placing callus with different appearances
on RGM without selective agent. With high-
quality callus, nearly all calluses will produce
shoots on RGM. Incubation of callus for too
long or additional rounds of subculturing will
decrease the transformation efficiency.
After co-cultivation with Agrobacterium,
it is critical to remove all visible liquid;
however, overdesiccation of the callus sig-
nificantly reduces transformation efficiency.
Overdesiccated callus appears very dry and
does not fully recover on selection medium.
At step 19 of Basic Protocol 1, leaving the
petri dish (with callus on filter paper) uncov-
ered in the laminar-flow hood for more than
10 min may cause overdesiccation.
In growing B. distachyon, soil and water-
ing have to be carefully controlled because the
plants are very sensitive to root rot. In addition,
we have observed phytotoxicity of some com-
mercial soil mixes (Vogel & Bragg, 2009). The
symptoms include slow growth and yellowing
or browning leaves. The latter symptoms can
be easily confused for a foliar disease. Any-
time plants show signs of poor health, includ-
ing foliar lesions, the first thing to check is the
health of the roots. Gently knock the plant out
of its pot, taking care not to disturb the root
ball. There should be an extensive network
of white roots. If the roots are tan, brown,
or black, change the watering regimen, soil,
or both. In our experience, however, once a
root system is compromised the plant rarely
recovers (Vogel & Bragg, 2009). Peat-based
potting mixes are typically suitable. In our ex-
perience, products made from forest products,
such as ground bark, sometimes suffer from
batch-to-batch variation in performance. You
can also make your own mix to have more con-
trol over its composition (see recipe for potting
soil in Reagents and Solutions). When grow-
ing B. distachyon for the first time, or if you
are having problems, it is useful to try sev-
eral soil mixes to find the best one for your
conditions. In addition, overwatering should
be avoided, as plants left in standing water
or in saturated soil for a long time will grow
poorly.
Current Protocols in Plant Biology
Light is another key factor. Both intensity
and duration of light affect plant health and
flowering. As a grass, B. distachyon needs
more light than Arabidopsis, but much less
than crops such as rice, wheat, and all C4
grasses. We grow plants under fluorescent
plus incandescent lights at an intensity of
600 μEm/m2 /s. Light levels as low as 150
μEm/m2 /s can be used, but the plants will be
much taller, will flower more slowly, and may
be more susceptible to disease. Daylength and
vernalization greatly influence flowering time.
Lines Bd21 and Bd21-3 will flower without
vernalization when grown in growth cham-
bers with 16 to 20 hr of light per day. In
greenhouses, we have noted that extending
daylength with supplemental lighting may not
work as well, possibly due to the low light
intensity. In these cases, flowering can be
promoted by vernalization of 2 to 3 weeks.
Excessive vernalization will cause the plants
to flower extremely rapidly and produce few
seed. If larger quantities of seed are required,
you can grow plants under short-day condi-
tions, 8 to 12 hr daylength, for a few weeks and
then vernalize them for 3 weeks before moving
them back to a growth chamber or greenhouse
with long days. Note that these conditions are
for lines Bd21 and Bd21-3. For most other
lines, daylength cannot overcome the need for
vernalization, and most have longer vernaliza-
tion requirements (Tyler et al., 2014).
Time Considerations
From dissecting immature embryos to har-
vesting T1 seeds, the whole process takes 20
to 30 weeks. Since it takes 7 to 8 weeks for
plants to produce immature embryos suitable
for transformation, it is useful to plant a few
pots of plants every 1 to 2 weeks to ensure that
a ready supply of embryos is always available
when needed.
Acknowledgements
Development of this protocol was sup-
ported by the National Key Research and De-
velopment Program of China (2016YFD0101
004) and the China Research and Develop-
ment Initiative on Genetically Modified Plants
(2016ZX08009003). The work conducted by
the US DOE Joint Genome Institute is sup-
ported by the Office of Science of the US De-
partment of Energy under contract no. DE-
AC02-05CH1123.
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Current Protocols in Plant Biology