Plant Cell Rep (2008) 27:297–305
DOI 10.1007/s00299-007-0463-z
G E N E T I C T R A N S F O R M A T I O N A N D H Y B R I D I Z A T I O N
Improvement of Agrobacterium-mediated transformation
in Hi-II maize (Zea mays) using standard binary vectors
Juan M. Vega Æ Weichang Yu Æ Angela R. Kennon Æ
Xinlu Chen Æ Zhanyuan J. Zhang
Received: 30 July 2007 / Revised: 26 September 2007 / Accepted: 30 September 2007 / Published online: 16 October 2007
Ó Springer-Verlag 2007
Abstract
High-frequency transformation of maize (Zea
transformation frequencies were achieved from a large
mays L.) using standard binary vectors is advantageous for
number of experiments using immature embryos grown in
functional genomics and other genetic engineering studies.
various seasons. The enhanced transformation protocol
Recent advances in Agrobacterium tumefaciens-mediated
established here will be advantageous for maize genetic
transformation of maize have made it possible for the
engineering studies including transformation-based func-
public to transform maize using standard binary vectors
tional genomics.
without a need of the superbinary vector. While maize Hi-
II has been a preferred maize genotype to use in various
Keywords
Agrobacterium tumefaciens Á Binary vector Á
maize transformation efforts, there is still potential and
Hi-II maize Á Zea mays Á Transformation
need in further improving its transformation frequency.
Here we report the enhanced Agrobacterium-mediated
transformation of immature zygotic embryos of maize Hi-
Introduction
II using standard binary vectors. This improved transfor-
mation process employs low-salt media in combined use
Maize (Zea mays) is a major world crop and an important
with antioxidant L-cysteine alone or L-cysteine and dithi-
model monocot plant for studying genetics, genomics, and
othreitol (DTT) during the Agrobacterium infection stage.
molecular biology. Recent advances in maize genome
Three levels of N6 medium salts, 10, 50, and 100%, were
research have generated a wealth of new genetic information
tested. Both 10 and 50% salts were found to enhance the
(http://www.plantgdb.org) (for website use, see Dong et al.
T-DNA transfer in Hi-II. Addition of DTT to the coculti-
2005). These valuable genetic resources will be better utilized
vation medium also improves the T-DNA transformation.
if a high-quality and high-frequency maize transformation
About 12% overall and the highest average of 18%
system is developed. One immediate application of these
resources will be to verify the functions of ESTs through
genetic transformation in a high-throughput manner.
Communicated by P. Ozias-Akins.
Agrobacterium tumefaciens has become a preferred
transgene delivery vehicle in maize transformation in
J. M. Vega
public laboratories. This preference is largely due to the
Departamento de Genetica, Facultad de Biologia,
Universidad Complutense, 28040 Madrid, Spain
advantages that the T-DNA transfer process has over other
gene delivery systems, i.e., the high proportion of simple
W. Yu
insertion events with intact transgenes and stable transgene
Division of Biological Science, College of Art and Science,
expression and inheritance (Dai et al. 2001; Hu et al. 2003;
University of Missouri, Columbia, MO 65211-7400, USA
Shou et al. 2004; Travella et al. 2005). Functional genomic
A. R. Kennon Á X. Chen Á Z. J. Zhang (&)
studies of crop plants such as maize necessitate a cost-
Plant Transformation Core Facility, Division of Plant Sciences,
effective functional testing of a huge number of candidate
College of Agriculture, Food and Natural Resources,
genes or other nucleotide sequences and require high-
University of Missouri, Columbia, MO 65211-7140, USA
e-mail: zhangzh@missouri.edu
throughput transformation systems.
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Plant Cell Rep (2008) 27:297–305
Of the several target maize genotypes, maize hybrid line
States Department of Agriculture-Agriculture Research
Hi-II has been a predominant genotype used for genetic
Service) at the University of Illinois at Urbana-Champaign,
transformation in public laboratories. The primary advantage
USA. They were grown and crossed to generate Hi-IIA ·
of using Hi-II maize is its high-frequency of type II somatic
B (hybrid cross) seeds (F1) under either greenhouse or field
embryogenic callus induction, a type of callus that is friable,
conditions. These seeds were planted and grown in 9.1-l
fast growing, and highly embryogenic (Armstrong and
pots filled with Pro-mix soil in the Sears Plant Growth
Green 1985, 1992; Armstrong 1999). These qualities have
Facility at the University of Missouri-Columbia. The
allowed development of Agrobacterium-mediated T-DNA
greenhouse is located at latitude 38°5708@N and longitude
transfer in Hi-II maize that is either high-throughput (Zhao
92°19048@W. The greenhouse conditions included supple-
et al. 1999, 2002) or adequately efficient (Frame et al. 2002).
mental lighting with high-sodium pressure lights (intensity
Two additional factors have contributed to the increase
600–1,000 lmol m–2 s–1) and a 16:8 h photoperiod from
in transformation frequencies: the superbinary vector and
late May to early November and a 18:6 h photoperiod
the use of antioxidant L-cysteine during the Agrobacterium
during the remaining months. The average temperatures
infection stage. While the superbinary vector has enabled
were set to 22°C during the night and 28°C during the day
very high-frequency transformation of maize in the
throughout the growing seasons. Plants were fertilized once
industrial sector (Ishida et al. 1996; Zhao et al. 1999,
with Osmocot 18-6-12 time-release fertilizer about a
2002), the use of L-cysteine has significantly enhanced Hi-
month after planting or transplanting and watered as
II maize transformation employing standard binary vectors
required. F1 plants were self-pollinated and immature
in public laboratories (Frame et al. 2002). More recently,
embryos (F2) were isolated from kernels 9–13 days after
the use of L-cysteine in combination with modified medium
self-pollination and used for Agrobacterium infection.
salts has improved Agrobacterium-mediated transforma-
Unless otherwise specified, all materials and supplies for
tion of three maize inbred lines (Frame et al. 2006).
maize growth in the greenhouse were purchased from
Several antioxidants have been found to improve
Hummert International, Earth City, IA, USA.
T-DNA transformation of recalcitrant crop species. For
example, dithiothreitol (DTT) coupled with polyvinyl-
polypyrrolidone (PVP) in grape (Vitis vinifera L.) (Perl
Agrobacterium strains and vectors
et al. 1996) or DTT in combination with L-cysteine in
soybean (Olhoft and Somers 2001; Olhoft et al. 2001,
We used Agrobacterium tumefaciens strains LBA4404
2003; Paz et al. 2004) have been shown to enhance T-DNA
(Hoekema et al. 1983) and EHA101 (Hood et al. 1986) to
transfer. However, the potential role of DTT in promoting
harbor the transformation constructs. The first construct
maize transformation remains to be explored.
was a standard binary vector pCAMBIA3301 (CAMBIA,
Use of low-salt media during the Agrobacterium infec-
Australia), which is publicly available. The second stan-
tion stage of transformation represents an additional
dard binary vector, pZY101 (or its derivatives) was used to
strategy to improve T-DNA transfer. Although the precise
subclone a GUS reporter gene or other genes of interest
mechanism of transformation enhancement is not under-
into the multiple cloning sites of the vector. The vector
stood, low salt-medium is now commonly used to improve
pZY101
was
derived
from
pZY101.1
(also
called
T-DNA transformation in a few major crop species, except
PTF101.1) (Frame et al. 2002) by replacing the double
for maize (Fry et al. 1987; Cheng et al. 1997; Zhang et al.
CaMV35S promoter with a single CaMV35S promoter.
1999, 2003; Zeng et al. 2004; Paz et al. 2004).
Figure 1 shows these two vectors. The construct carrying
Here we report the improvement of Agrobacterium-
GUS Intron reporter gene cassette is pZY102 and has been
mediated transformation in maize Hi-II using standard bin-
reported previously (Zeng et al. 2004). All constructs were
ary vectors. We show that the use of low salt media and the
mobilized into Agrobacterium by direct DNA transfer (An
addition of DTT along with L-cysteine in the cocultivation
et al. 1988) and their integrity within the Agrobacterium
medium improved Agrobacterium-mediated stable transfor-
cells was confirmed through restriction digest analysis.
mation frequencies from an average of 5.5 to 12% in Hi-II.
Medium formulations
Materials and methods
The types of media and composition of maize cultures used
Plant materials
at various stages were derived from Zhao et al. (1999) and
are listed in Table 1. Critical modifications in our estab-
Maize genotypes Hi-II A and Hi-II B were acquired from
lished standard protocol included the use of 50% N6 basal
the Maize Genetic Cooperation Stock Center (United
salt mixture and full-strength vitamins (Chu et al. 1975) in
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299
S
C
M
pCAMBIA3301
5’
LB
T35S
bar
P35S
P35S
Int
GUS
Tnos
B
R
3’
S
C
M
pZY101
5’
LB
Tvsp
bar
TEV
P3 S
5
B
R
3’
probe
Fig. 1 Diagram of the two binary transformation vectors used in the
selecting transformants; Int-GUS intron-containing b-glucuronidase
study. Shown are only T-DNA regions of standard binary vectors
(GUS) gene; Tnos nopaline synthase gene terminator; Tvsp soybean
pCAMBIA3301 (top) and pZY101 (bottom). LB and RB, T-DNA left
seed storage protein gene terminator; TEV tobacco etch virus
and right borders, respectively; P35S and T35S, CaMV35S promoter
translational enhancer; MCS multiple cloning sites
and terminator, respectively; bar bialaphos resistance gene for
both inoculation and cocultivation media and the addition
our earlier experiments which tested the impact of three
of L-cysteine and DTT to the cocultivation medium. Basal
levels of N6 salt mixture.
salt medium and sucrose were autoclaved while additives
including vitamins, plant growth regulators, antioxidants,
selective agent and antibiotics were filter-sterilized into the
Transformation protocol
medium. Minor modifications included the use of 8 g l–1
washed agar (Cat: A8678, Sigma-Aldrich, USA) to replace
Described below is our established standard protocol,
gelrite in the cocultivation medium. This cocultivation
which is modified from the previous ones (Zhao et al. 1999;
medium was overlaid with a piece of sterile filter paper in
Frame et al. 2002). The variations of this protocol used for
Table 1 Medium formulation
Medium compositions
Unit per liter
Concentrations
A
B
C
D1
D2
E
F
N6 salta
g
2.0
2.0
4.0
4.0
4.0
–
–
MS salt
g
–
–
–
–
–
4.3
2.9
Sucrose
g
68.5
30
30
30
30
60
30
Glucose
g
36.0
–
–
–
–
–
–
L-proline
g
0.7
0.7
0.7
0.7
0.7
–
–
MES
g
0.5
0.5
0.5
0.5
0.5
–
–
2,4-D
mg
1.5
1.5
1.5
1.5
1.5
–
–
pH
5.2
5.8
5.8
5.8
5.8
5.6
5.6
Agar (washed)
g
–
8.0
–
–
–
–
–
Gelrite
g
–
–
3.0
3.0
3.0
3.0
3.0
N6 vitamins, 1,000·
ml
1.0
1.0
1.0
1.0
1.0
–
–
MS vitamins, 1,000·
ml
–
–
–
–
–
1.0
1.0
Glycine
mg
–
–
–
–
–
2.0
2.0
Silver nitrate
mg
0.85
0.85
0.85
–
–
–
–
L-cysteine
g
–
0.4
–
–
–
–
–
DTTa
g
–
0.15
–
–
–
–
–
Acetosyringone
lM
–
100
–
–
–
–
–
Cefotaxime
g
–
–
0.25
0.25
0.25
0.25
–
Bialaphos
mg
–
–
–
1.5
3.0
3.0
–
The N6 and MS salts are pre-mixed basal salts mixtures without vitamins, respectively. Treatments (salt levels) of N6 salt mixture in inoculation
(A) and cocultivation (B) media were 10, 50, and 100%, while remaining media and their compositions for all treatments were identical. Salt and
vitamin compositions were the same as described for MS (Murashige and Skoog 1962) and N6 (Chu et al. 1975). Sigma-Aldrich Inc., USA was
supplier of all reagents except for MES (Fisher Scientific, USA), DTT (Invitrogen, USA), and bialaphos (Shinyo Sangyo Co. Ltd, Japan)
MES, 2-(4-morpholino)-ethane sulfonic acid; 2,4-D, 2,4-dichlorophenoxyacetic acid; acetosyringone, 30,50-dimethoxy-40-hydroxyacetophenone
a Only 50% salts and 0.15 g l–1 (1 mM) DTT are listed as our established, standard protocol
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testing the effect of different treatment conditions are also
a side-by-side comparison between no and addition of
described in detail in the Results section.
0.15 g l–1 (1 mM) DTT in the cocultivation medium using
the most advanced public procedure for maize Hi-II (Frame
et al. 2002) as a basic protocol except that 50% N6 salt
Agrobacterium culture initiation
mixture was used in both treatments. In this test, a 4-h pre-
inoculation
induction
of
Agrobacterium
cells
was
Agrobacterium tumefaciens strain LBA4404 (harboring
employed and cocultivation lasted 3 days at 20°C. In all
construct pCAMBIA3301) or EHA101 (harboring pZY101
cases, the cocultivation medium B plate was wrapped with
derivatives) was streaked out from a –80°C glycerol stock
parafilm and placed in darkness.
onto an AB minimal medium (Chilton et al. 1974) plate
containing appropriate antibiotics. LBA4404 (carrying
Culture selection and maturation
pCAMBIA3301) required 100 mg l–1 streptomycin and
50 mg l–1 kanamycin; whereas EHA101 (carrying pZY101
After cocultivation, embryos were then transferred to the
derivatives) required 25 mg l–1 chloramphenical, 50 mg l–1
medium C plate for 5–8 days without herbicide selection
kanamycin, 100 mg l–1 each of spectinomycin and strepto-
(the resting stage). Embryos were then subcultured on
mycin. The plate was incubated at 28°C for 3 days until
medium D1 containing 1.5 mg l–1 bialaphos for 2 weeks
single colonies developed. This master plate was used on a
for initial herbicide selection. Subsequent cultures used
weekly basis for up to a month. A single colony was streaked
3 mg l–1 bialaphos in the medium D2; embryogenic, bi-
out onto YEP (5 g l–1 yeast extract, 10 g l–1 peptone, 5 g l–1
alaphos-resistant calluses were transferred to fresh medium
NaCl, and pH 7.0) containing the same antibiotics as the AB
D2 biweekly until somatic embryos turned opaque. During
plate. The YEP plate was then incubated at 20°C for
the subculture on medium D2 the growing embryogenic
2–3 days until bacterial colonies developed fully.
calluses (over 1 cm in size) were divided into smaller
pieces and only highly-embryogenic calluses (i.e., dry,
friable, and fast growing tissues) were maintained. All
Inoculation and cocultivation
divided calluses from the same embryo that later developed
into fertile transgenic plants were treated as one event. The
Agrobacterium colonies were taken from the YEP plate,
total culture period on the medium D2 lasted about
suspended in 5 ml of A medium (in a 15 ml tube) with cell
3–4 months. Maturation took place when embryos showing
density of OD550 = 0.3–0.4. The culture tube was then
opaque color were transferred to the medium E for
shaken horizontally on a platform at 160 rpm at room
3–4 weeks. All of the above culture plates were wrapped
temperature (23°C) for 4 h, i.e., pre-incubation (Frame
with parafilm and maintained in darkness at 28°C, except
et al. 2002) before being used for embryo infection. Before
for maturation plates, which were wrapped with venting
embryo isolation, ears were sterilized in 50% commercial
tape and placed in darkness at 25°C.
bleach containing 5.25% sodium hypochlorite for 20 min
and washed three times with sterile water. These ears were
either freshly harvested or stored in 4°C refrigerator for up
Regeneration, acclimatization and greenhouse care
to 2 days (ears with husk were wrapped in a plastic bag).
Immature zygotic embryos, around 1.5 mm (1.4–1.8 mm)
The maturation stage ended when embryos turned ivory in
in size, were isolated from maize kernels and placed in
color; embryos were subsequently transferred to medium F
1 ml of the medium A (about 100 embryos in a 1.5 ml
plates (wrapped with venting tape) for regeneration using a
Eppendorf tube), followed by 3–4 washes with the same
18:6 h photoperiod with light intensity of 100–150 lmol m–2
medium. Then 1 ml of Agrobacterium suspension was
s–1 at 24°C. Shortly thereafter, both shoots and roots
added to the 1.5 ml tube, and inoculation was carried out
developed from each mature embryo. Each small plantlet
for 10 min before embryos were transferred onto the
was subsequently transferred to a 150 · 25 mm tube con-
medium B plate, with embryo scutellum facing up. To test
taining the medium F for further development. Plantlets with
the effect of the salt concentrations in media A and B on
fully grown shoots and roots were then transferred to Deep
stable transformation, three levels of the N6 basal salt
Traditional Inserts (3100 Â 3100; Hummert International) con-
2
2
mixture, i.e., 10, 50, and 100%, were examined. In this test,
taining Pro-mix soil and allowed to acclimatize for
0.4 g l–1 cysteine was added to the cocultivation medium
2–3 weeks in growth chamber conditions (a 18:6 h photo-
for all three levels of salt mixture treatments and no pre-
period and mixed cool white florescence and high-sodium
inoculation
induction
of
Agrobacterium
cells
was
pressure lights with light intensity of 300 lmol m–2 s–1 at
employed. The cocultivation lasted 3 days at 25°C. To test
24°C) before being moved to 9-l pots in greenhouse con-
the impact of DTT on stable transformation, we conducted
ditions. We carried 2–5 clones per event to the greenhouse
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301
to secure the fertility. Subsequent greenhouse plant care
each ear (block) to minimize the variation between the
followed the procedures described in the Plant materials
experimental units (embryos). Analysis of variance was
section.
conducted using the SAS GLM program (Der and Everitt
2001) and means were separated by Duncan’s multiple
range test at a = 0.05. Progeny segregation was analyzed
Analysis of transgenes
using the v2-test for Goodness of fit. Transformation fre-
quency (%) was calculated based on stable transformation
The histochemical b-glucuronidase (GUS) assay (Jefferson
and was defined as the number of independent fertile
et al. 1987) was used to evaluate early events of T-DNA
transgenic maize events per explant, excluding contami-
transfer to maize cells located in the embryo scutellum area
nated explants when treatment effects were compared, or
at the end of the resting stage. The number of GUS-positive
including contaminated explants when large scale stable
sectors and their distribution in the scutellum region were
transformation experiments were conducted. Each inde-
scored. Thus, the status of T-DNA transfer was estimated
pendent event was defined as a bialaphos-resistant plant
by percent areas infected on scutellum as indicated by GUS
derived from a single inoculated embryo that also trans-
staining. Six infection ranges were used to estimate the
mitted the transgene to progeny. Random samples of some
magnitude of infection: 0, \25, 25–50, 50–75, [75, and
of these bialaphos-resistant plants were then tested for
100% of scutellum areas showing GUS staining. The
confirmation of different transgene integration events by
infection status was also evaluated using the ‘‘infection
Southern blot analysis and for transgene transmission by
frequency (%)’’ that is defined as the number of embryos
progeny segregation analysis.
infected over the total number of embryos cocultivated.
We used a leaf-painting assay (Zhang et al. 1999) for
preliminary confirmation of T-DNA insertion and progeny
Results
segregation to verify the functional expression of the bar
gene. Leaf-painting was conducted 2–3 times on each
Impact of low-salt media during the cocultivation
putative transgenic event and progeny plant using 0.5%
on transformation
Liberty1 (Aventis CropScience, USA). Southern blot
analysis (Southern 1975) was used to confirm the integra-
The binary vector pCAMBIA3301 (Fig. 1) harbored by
tion and stable inheritance of transgene inserts. For
Agrobacterium strain LBA4404 was used to test the effect
Southern blot analysis, genomic DNA was extracted from
of three different salt strengths of N6 basal medium, 10, 50,
Hi-II maize leaf tissues following a modified CTAB-based
and 100% on enhancing stable transformation. In these
protocol (Dellaporta et al. 1983). Thirty lg of DNA were
experiments, no pre-inoculation induction of Agrobacte-
digested with SwaI restriction enzyme that cut only once
rium cells was employed and cocultivation lasted 3 days at
within the T-DNA region. The digested genomic DNA was
25°C. Our results demonstrate that as the salt concentration
fractionated on a 1.0% agarose gel prior to transferring to
decreased in the inoculation (A) and cocultivation (B)
Zeta-Probe1 GT nylon membrane (Bio-Rad, CA, USA).
media, the transformation frequency increased to as high as
DNA was fixed to the membrane by UV cross-linking.
17.5% at 10% salt concentration (Table 2). This was three
Hybridization and washing conditions for Southern blot
times higher than the 5.5% transformation frequency pre-
analysis followed the Zeta-Probe1 GT manufacturer’s
viously reported by Frame et al. (2002). Since 10% salt in
instructions. The bar ORF from vector pZY101 was used
some cases decreased the quality of the embryogenic
to generate a 32P-labeled probe. The probe was prepared by
response, 50% was used for further experiments.
random primer synthesis incorporating 32P-dATP utilizing
the Prime-it1 II kit (Stratagene, USA).
For progeny analysis, ten random sets of progeny plants
Impact of addition of DTT in cocultivation medium
were analyzed first using the leaf-painting assay to examine
on T-DNA transfer
segregation patterns. One random set of progeny was fur-
ther analyzed by Southern blot analysis to verify transgene
Previous studies showed enhanced transformation of Hi-II
inheritance.
by employing L-cysteine during cocultivation (Frame et al.
2002). To determine if the addition of DTT in the cocul-
tivation medium could further enhance transformation, we
Experimental design and data analysis
conducted side-by-side experiments to compare this mod-
ification with the Frame et al. (2002) method. Since 50%
Randomized complete block design (RCBD) was applied
salts was proven to be superior to 100% salts in inoculation
to all experiments in which all treatments were assigned to
(A) and cocultivation (B) media for the T-DNA transfer in
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Table 2 Impact of medium salt concentrations on stable transfor-
80
mation of Hi-II maize
0

<
7
. 5 scutellum
70
Medium salt
Number of
Number of
Transformation
0

<
2
. 5 scutellum

strength (%)
embryos
transgenic events
frequency (%)
60
recovered
50
10
200
35
17.5a
40
50
200
8
4.0b
100
200
0
0.0c
30
Data were from three independent experiments using immature
Infection frequency (%)
20
embryos from Hi-IIA · B:F1 maize plants. L-cysteine at 0.4 g l–1
(3.3 mM) as a sole antioxidant was used in combination with three
10
different salt concentrations in the cocultivation medium (B) to test
the effect of medium salt levels on stable transformation. Transfor-
0
L-c s
y e
t
e
n
i
L-c s
y e
t
e
n
i
D
+
T
T
mation frequencies (%) followed by the same letter are not
statistically different from one another as detected by Duncan’s
o
i
t
n
A
a
d
i
x
s
t
n
multiple range test at a = 0.05 level
Fig. 2 Impact of addition of dithiothreitol (DTT) to the cocultivation
medium on early events of T-DNA transfer. Results were evaluated
our preliminary experiments, we used 50% salt media
based on GUS assays of infected embryos right after resting stage.
across the two treatments contrasting L-cysteine alone and
The status of T-DNA transfer was estimated by percent areas infected
L-cysteine + DTT in cocultivation medium. The Agrobac-
on scutellum as indicated by GUS staining. Six infection ranges were
terium strain EHA101 carrying the binary vector pZY102
used for estimation: 0, \25, 25–50, 50–75, [75, and 100% of
scutellum areas. Data were from two independent experiments with a
(PTF102) was used to transform the embryos after a 4-h
total of 64 embryos per treatment. Each error bar stands for a
pre-incubation (see also Materials and methods section)
standard error of a mean for each treatment
and the cocultivation lasted 3 days at 20°C.
We first conducted preliminary experiments to examine
improved infection conditions (50% salts and L-cyste-
the effect of adding DTT to the cocultivation medium on
ine + DTT) with a large number of immature embryos
T-DNA transfer by using a GUS assay at an early culture
from various growing seasons. Table 3 lists the transfor-
stage, i.e., right after the resting stage. The percent infected
mation results obtained from the experiments using
area as indicated by GUS-positive sectors was estimated in
Agrobacterium strain EHA101 carrying 12 different con-
scutellum areas, since only scutellum areas are responsible
structs (containing different genes of interest) derived from
for somatic embryo formation and T-DNA transfer to this
pZY101 (PTF101.1). The overall transformation frequency
area is more relevant. Data from two independent experi-
across these constructs was 12.2%, and the highest average
ments with a total of 64 embryos per treatment indicated
frequency was 18%. The overall recovery was consistent,
that addition of DTT plus L-cysteine was superior to
although embryo vigor influenced transformation fre-
L-cysteine alone. The former treatment led to 63.7% of
quency (data not shown). The time frame of this entire
embryos exhibiting GUS staining on scutellum areas as
transformation process from embryo isolation to harvesting
compared with 17.2% of embryos in the latter treatment
transgenic T1 seeds was 7–9 months.
(L-cysteine alone) (Fig. 2). Furthermore, 12.5% of embryos
in DTT plus L-cysteine treatment exhibited large (25–75%)
scutellum areas infected as compared with no scutellum
Confirmation of transgene inheritance and integration
displaying this infection range in L-cysteine alone.
patterns
We then conducted four independent, side-by-side stable
transformation experiments comparing the two treatments
To test the transgene inheritance and integration patterns,
(L-cysteine alone and L-cysteine + DTT) using the same
we randomly chose ten transgenic Hi-II maize events (T0)
experimental conditions. The L-cysteine alone and L-cys-
expressing the HcPro construct (carrying a virus helper
teine + DTT treatments included a total of 192 and 190
component for gene silencing study). Progeny plants of
embryos, respectively. Our results showed that addition of
these events were first screened by herbicide leaf-painting
DTT in cocultivation medium increased the transformation
to verify transgene segregation. Because all T0 lines were
frequency fourfold over that using L-cysteine alone. This
crossed with wild type maize plants, the expected segre-
enhanced transformation was also statistically significant
gation ratio was 1:1 (resistant versus susceptible). The
according to Duncan’s multiple range test at a = 0.05 level.
results showed that six out of ten events followed the
To further confirm the reproducibility of our improved
expected
1:1
segregation
ratio
for
a
single
locus
transformation process, we then conducted multiple inde-
(P ‡ 0.059) and four events deviated from this ratio
pendent stable transformation experiments using the
(P £ 0.018) (Table 4). The deviation in these four events,
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Plant Cell Rep (2008) 27:297–305
303
Table 3 Stable transformation of Hi-II maize using the improved
protocol
Binary
Number of
Number of
Transformation
vectors
embryos
transgenic events
frequency (%)
recovered
WY76
746
85
11.4
WY86
377
65
17.2
HcPro
1,013
84
8.3
MT101
275
37
13.5
MT102
672
70
10.4
MT103
273
29
10.6
HK191
378
68
18.0
HK245
380
58
15.3
MIPT
476
84
17.6
MOY
530
40
7.5
MYFP
536
51
9.5
MMRP
469
79
16.8
Total
6,125
750
12.2
Data were from at least three independent experiments for each
construct using immature embryos from Hi-IIA · B: F1 maize plants
grown in various seasons. The transformation frequency was calcu-
lated by the number of independent fertile transgenic events over the
total number of embryos to start with (see also Materials and methods
for event definition)
which was always a deficiency of resistant offspring, could
be attributable to the small number of progeny plants
Fig. 3 Southern blot analysis of transgenic maize events and a set of
representative progeny. a Analysis of transgene integration of ten
examined or bar gene silencing. To examine the transgene
putative events derived from vector HcPro. b Analysis of progeny
integration patterns, we analyzed the genomic DNA sam-
plants 1–15 from event JB13–12b, showing segregation of transgene;
ples of ten randomly selected maize events derived from
two distinct bands, one at 5 kb and the other at 1.4 kb are indicated. A
construct HcPro using Southern blot analysis with the bar
k/HindIII ladder was used as molecular marker and sizes are labeled
as kb; WT wild-type control, 1X and 5X 1X and 5 X genome
equivalent copy number controls using 24 and 120 pg of vector
Table 4 Progeny segregation analysis of primary transgenic Hi-II
HcPro, respectively. A total of 30 lg DNA per sample was digested
maize events
with SwaI, which cuts once within the T-DNA region of vector
HcPro. Membranes were probed with bar ORF
Events
Total
Progeny
Segregation:
v2-value
P-value
progeny
R:S ratio
R
S
gene ORF as a probe. We used restriction enzyme SwaI,
which cuts once within the T-DNA region of the HcPro.
JB13-1
18
2
16
1:1
10.89
0.001
Our results confirmed that at least nine of the ten putative
JB13-2
16
7
9
1:1
0.25
0.617
events carrying the transgene were independent, as evi-
JB13-3
15
8
7
1:1
0.67
0.796
denced by different banding patterns (Fig. 3a). Plants
JB13-4
18
12
6
1:1
2.00
0.157
JB13-9a-7 and JB13-9a-11 are apparently from the same
JB13-7
18
13
5
1:1
3.56
0.059
event, as expected (bearing the same core code JB13-9a),
JB13-8
18
4
14
1:1
5.56
0.018
because of the same banding pattern. Furthermore, five of
JB13-9
16
5
11
1:1
2.25
0.134
nine events were simple insertions, with 1–2 transgene loci
JB13-10
18
2
16
1:1
10.89
0.001
per genome; only one event exhibited a complex integra-
JB13-12
18
9
9
1:1
0.00
1.000
tion pattern (with seven transgene loci). It is possible that
JB13-14
14
2
12
1:1
7.14
0.007
event JB13-1a may still carry a transgene but failed to
Progeny R and S columns indicate the number of progeny plants
exhibit insertion signal due to an insufficient amount of
showing herbicide resistance and susceptible, respectively. Resistant
genomic DNA sample loaded, as is the case with the single
progeny plants were scored as R and susceptible ones as S. v2-test was
copy control using vector HcPro that is undetectable on the
used to test Goodness of Fit of observed progeny plants against the-
Southern blot. Moreover, we analyzed genomic DNA
oretical number of plants showing R and S, respectively. A single
degree of freedom was used to obtain P-values
samples from a random set of progeny (event JB13-12b
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304
Plant Cell Rep (2008) 27:297–305
showing 1:1 segregation) using Southern blot analysis with
the above observations including the correlations between
the bar probe. The result indicates that the transgene was
the embryo sizes and vigor and their transformation
indeed transmitted to the progeny plants; thus the presence
frequencies.
of a Southern band corroborated the observed herbicide
resistance (Table 4, Fig. 3b).
Acknowledgments
We thank Regina Wamsley (from Zhanyuan J.
Zhang’s laboratory) for her excellent technical assistance; CAMBIA
(Australia) for providing pCAMBIA3301; Aventis CropScience
(Research Triangle Park, NC, USA) for herbicide Liberty1, and
Discussion
James A. Birchler and Seth D. Findley (University of Missouri-
Columbia) for a critical review of this manuscript. University of
We have improved Agrobacterium-mediated gene delivery
Missouri-Columbia Life Science Mission Enhancement program
in maize Hi-II using standard binary vectors. These trans-
supported Angela R. Kennon and, in part, Xinlu Chen (from
Zhanyuan J. Zhang’s lab). All transformation experiments were
formation results are representative because the immature
conducted in the Plant Transformation Core Facility at the University
embryos we used were from various growing seasons
of Missouri-Columbia.
including winters when embryos exhibit relatively lower
embryogenic responses (anecdotal observations). Southern
blot and progeny segregation analyses confirmed stable
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Document Outline

• Improvement of Agrobacterium-mediated transformation in Hi-II maize (Zea mays) using standard binary vectors
  • Abstract
  • Introduction
  • Materials and methods
    • Plant materials
    • Agrobacterium strains and vectors
    • Medium formulations
    • Transformation protocol
      • Agrobacterium culture initiation
      • Inoculation and cocultivation
      • Culture selection and maturation
      • Regeneration, acclimatization and greenhouse care
    • Analysis of transgenes
    • Experimental design and data analysis
  • Results
    • Impact of low-salt media during the cocultivationon transformation
    • Impact of addition of DTT in cocultivation mediumon T-DNA transfer
    • Confirmation of transgene inheritance and integration patterns
  • Discussion
  • Acknowledgments
  • References

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