全 文 ：Received 1 Jul. 2003 Accepted 25 Nov. 2003
* Author for correspondence: Tel (Fax): +86 (0)27 87199354; E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (5): 610-617
High-efficiency Agrobacterium-mediated Transformation of Soybean
CHEN Shi-Yun*
(Wuhan Institute of Virology, The Chinese Academy of Sciences, Wuhan 430071, China)
Abstract : Soybean (Glycine max (L.) Merrill) is one of the recalcitrant crops to be manipulated in vitro.
To improve Agrobacterium-mediated soybean cotyledonary node transformation efficiency, a new solid-
liquid medium selection system based on using the bar gene as the selectable marker has been developed.
The cotyledonary nodes were first cultured in MS solid medium modified with 2 mg/L 6-BA and 5 mg/L
glufosinate for two weeks, and the surviving explants were transferred into a liquid selection medium with
2 mg/L glufosinate and 0.01 mg/L thidanzuron (TDZ) instead of 6-BA for further selection. The liquid
medium was changed every week until the transgenic shoots were regenerated from the liquid selection
medium. Several factors affecting the transformation efficiency have also been evaluated. GUS assay and
Southern blotting analyses confirmed the integration of the transgenes into the soybean genome. This
transformation system is genotype-independent and efficient, and has less escape due to the tight selection.
Key words: soybean; cotyledonary node; Agrobacterium tumefaciens; transformation; liquid selection
Soybean is one of the important crops around the world.
To increase soybean p roduction as well as to manipulate
seed quality traits such as p rotein and oil, genetic engi-
neering offers a new way to augment traditional soybean
breeding methods. However, genetic modification of soy-
bean has proven to be very difficult. Although two routine
soybean transformation sys tems have been developed,
e.g. Agrobacterium-mediated transformation of cotyledon-
ary nodes from germinated seeds (Hinchee et al., 1988), or
particle bombardment of either embryo axis tissue (McCabe
et al., 1988) or somatic embryos (Stewart et al., 1996). The
major problems of these two transformation systems are
genotype-dependent, low transformation efficiency, labor-
consuming and poor reproducibility.
Since the firs t report of Agrobacterium-mediated soy-
bean transformation of cotyledonary node (Hinchee et al.,
1988), several modifications of this sys tem have been re-
ported (Di et al., 1996; Zhang et al., 1999; Clemente et al.,
2000; Olhoft et al., 2001; Olhoft and Somers , 2001). A ll of
these reports were based on selection of the cotyledonary
node in s olid medium modified with either kanamycin or
glufosinate as the selective agents, and the selection re-
gime resulted in many escaped shoots (Olhoft and Somers,
2001). To develop an efficient geno type-independent
Agrobacterium-mediated soybean transformation system,
one way is to find more efficient selection agents (Olhoft et
al., 2003), the other way is to find better selection methods.
For example, Clemente et al. (2000) reported the efficien t
s elect ion o f t rans gen ic s oybean shoo ts us ing the
herbicide glyphosate, but this selection system faces pro-
prietary limitations.
Liqu id selection has proven to be more efficient than
solid select ion due to the direct contact of the explants
with the medium and the selection agent in the medium. In
this study, a solid/liquid soybean cotyledonary node re-
generation/selection system has been developed. The ex-
plants were first cultured in a so lid BA-containing s elec-
tion medium with 5 mg/L glufosinate for two to four weeks,
and then the surviving explants were transferred into a liq-
uid medium containing thidazuron (TDZ) p lus 2 mg/L
glufosinate for further selection and shoot elongation. In
order to increas e the transformat ion efficiency of th is
sys tem, d ifferent soybean genotypes as well as various
culture and selection conditions were evaluated.
1 Materials and Methods
1.1 Plant transformation
1.1.1 Seed germination Selected soybean seeds stored
in the cold-room were sterilized in 100 mL 15% CloroxÒ
with occasionally s haking . After 15 min, the s eeds were
washed 3-4 times with sterilized distilled water. Ten seeds
were placed onto each 100 mm×15 mm Petri dish contain-
ing about 20 mL seed germination medium (MS basal me-
dium with 1.5 mg/L 6-BA). The seeds were germinated un-
der 24 h light (about 80 mE.m-2.s-1) at 28 ℃ for 3 d.
1.1.2 Plasmid and Agrobacterium tumefaciens prepara-
tion A. tumefaciens strain EHA105 harboring the binary
plasmid pCAMBIA3301 (provided by the Center for the
CHEN Shi-Yun: High-efficiency Agrobacterium-mediated Transformation of Soybean 611
Application of Molecular Bio logy to International
Agriculture, Canberra, Australia) was used in this study.
The size o f this plasmid is about 11.3 kb and this binary
vector has the bar selectable marker on the left border and
the gus gene on the right border and both are driven by the
CaMV35S promoter. The A. tumefaciens stock kept at –80
℃ was s treaked on to a s olid LB p late with 100 mg/L
kanamycin, and the plate was incubated at 28 ℃ in the
dark. After 2 d, a single clone was picked from the LB plate
to initiate 2 mL overnight suspension culture. A. tumefaciens
suspension culture was initiated by transferring the 2mL
overnight cu lture to 23 mL LB medium with 100 mg/L
kanamycin, cultured on a shaker for 4-5 h until the OD650
of the cultu re was about 0.6-0.8. After cen trifug ing at
3 000g for 10 min , the pellet was re-suspended in re-sus-
pension medium (B5 basal medium with 1.5 mg/L 6-BA and
200 µmol/L Acetosyringone, pH 5.4). Acetosyringone was
filter-sterilized and added into the medium after autoclaving.
1.1.3 Wounding and co-cultivation of cotyledonary nodes
A # 11 surgical b lade was us ed in the following process.
The hypocotyls of the germinated seeds were first cut about
2 cm below the cotyledon, then the seed coat was removed
and a vertical cut was made between the two cotyledons,
two identical pieces were obtained with portion of the node
region attached. The primary leaf tissue between the junc-
tion region of the cotyledon and hypocotyl was removed
and 6-7 horizontal slices were made in the cotyledonary
node region. The wounded explants were placed in to A.
tumefaciens re-suspension medium.
After all the explants were wounded, the covered plate
was left for 1 h, then blotted dry on a sterilized filter paper,
and placed onto co-cultivation medium (the same as the re-
suspension medium, but with 0.2 mg/L GA3 and 6 g/L agar)
with the wounded side facing the medium with 10 explants
per plate. The plates were placed at 24 ℃ in the dark for 3 d.
1.1.4 Selection and plant regeneration The elongated
hypocotyls from the co-cultivation medium were cut to leave
only 1-2 cm attached to the cotyledons and the exp lants
were briefly washed in the liqu id co-cultivat ion medium,
placed into selection medium with the node region just
above the surface of the medium. The s election medium
was B5 basal medium modified with 2 mg/L 6-BA, 0.2 mg/L
GA 3 , 5 mg/L g lu fos inate, 1 g /L 2-(N-morpholino)
ethanesulfonic acid (MES) and 100 mg/L timentin. The cul-
ture conditions were the same as those for seed germination.
After two weeks selection in the solid selection medium,
4-5 explants that had regenerated shoots were transferred
to 100 mL flasks with 20 mL liquid selection medium. This
medium is composed o f MS basal medium modified with
0.01 mg/L TDZ, 0.2 mg/L GA3, 2 mg/L glufosinate and 100
mg/L timentin. The flasks were put on a shaker at the speed
of 80 r/min under the same temperatu re and light condi-
tions as for s eed germination. The medium was changed
every week, with the dead tissues were removed, briefly
washed with sterilized water and transferred in to the new
selection medium.
1.1.5 Rooting of transgenic shoots The 2-3 cm long
elongated shoots in the liquid selection medium were sepa-
rated from the explants and placed in to rooting medium
(MS basal medium with 100 mg/L timentin). The plants were
transferred to Jiffy pots with soil and were allowed to grow
in a growth chamber at 28 ℃ with 23 h light for 2-3 weeks
and the plants were then moved to the greenhouse for flow-
ering and setting seeds.
1.2 Optimization of the transformation system
1.2.1 Effect of ion chelator and antioxidants One ion
chelator, citric acid, and two antioxidants, L-ascorb ic acid
(vitamin C) and L-cysteine (all purchased from Sigma, St.
Louis , USA) were tested in the co-cultivation medium to
increase transformation efficiency. The three chemicals were
first dissolved in double distilled water, then filter-steril-
ized and added into the autoclaved co-cultivation medium.
The cysteine concentration was 400 mg/L, while both ascor-
bic acid and citric acid were used at 100 mg/L. After co-
cultivation for 3 d and selection for four weeks as described,
the surviving nodes were cut into small pieces and placed
in GUS assay buffer (0.5 mol/L KH2PO4 (pH 7.0), with 100
mg/L X-Gluc).
1.2.2 Effects of carbohydrates Three different carbon
sources: sucrose, glucose and maltose, were tested for the
selection efficiency in the selection medium. After co-culti-
vation for 3 d, the explants were put into selection medium
with either 30 g/L sucrose or 30 g/L glucose or 60 g/L mal-
tose and four weeks later, the surviving explants were sub-
jected to GUS assay buffer as described before.
1.3 Confirmation of transformed events
1.3.1 GUS assay When elongated shoots were about 2-
3 cm long, a small leaf piece was excised from each p lant
and was placed into GUS assay buffer as described in 1.2.1
and incubated at 37 ℃ overnight. Chlorophyll was extracted
with 70% ethanol at 65 ℃.
1.3.2 Herbicide resistance test Leaf tissues from puta-
tive transgenic soybean plants and from wild type s oy-
bean plan t from the greenhouse were sterilized in 15%
CloroxÒ for 15 min, washed in sterilized distilled water three
times, cut into 1 cm× 1 cm leaf disks and placed on MS
basal medium with 2 mg/L 6-BA and 5 mg/L glufosinate.
1.3.3 S outhern blotting analysis Part ially expanded
Acta Botanica Sinica 植物学报 Vol.46 No.5 2004612
trifoliate leaf tissues were collected from the T0 transgenic
plants in the greenhous e and were immediately frozen in
liquid nitrogen. The tissues were ground into fine powder
in liquid n itrogen. Genomic DNA was is olated us ing a
nucleon PhytoPure plan t DNA ext raction kit (#RPN8511,
Amers ham Life Science, Eng land) fo llowing the
manufacturer’s protocol.
About 10 mg genomic DNA was digested using either
Hind Ⅲ or EcoRⅠ at 37 ℃ overnight. Bo th enzymes are
single cutters of the transformation construct. The digested
DNA samples and the control plasmid DNA (also digested
with the enzymes ) were fractioned in 0.8% agarose gel
overn ight , and then b lo tted on to a nylon membrane
(Hybond-N+, Amersham Pharmacia Biotech Limited ,
England) following the manufacture’s protocol. The DNA
materials were then fixed on to the membrane by a UV
crosslinker. The membrane was placed into pre-hybridiza-
tion buffer at 65 ℃ for 1-2 h. Probes were prepared by
XhoⅠ digestion from pCAMBIA3301 (resu lting in about
500 bp fragment of the bar gene), or by NheⅠ and NcoⅠ
double digestion from the same plasmid (about 2 kb frag-
ment of the gus gene). The probes were purified after en-
zyme restrict ion using a QIAEXÒⅡ gel extract ion kit
(QIAGEN Inc., Valencia, USA). The probes were labeled
with 32P and were incubated at 37 ℃ for 1-2 h before puri-
fied with a Bio-Rad resin, denatured at 100 ℃ for 10 min and
added to the pre-hybridization buffer. Hybridization was
carried out overn ight at 65 ℃. The membrane was then
washed with low stringency buffer (5× SSC, 0.5% SDS) at
65 ℃ for 10-15 min and washed twice in high stringency
buffer (0.1×SSC, 0.5% SDS) at 65 ℃ for 15 min. The mem-
brane was placed onto a Kodak X-ray film, placed at -80 ℃
for 12-24 h until the film was developed.
2 Results and Discussion
2.1 Establishment of cotyledonary node transformation
system
Soybean seeds were first germinated on seed medium
for 3 d (Fig.1A), the cotyledonary node explants were ex-
cised and wounded as shown in Fig.1B. It was important
that the primary leaf tissue be completely removed from the
node area to avoid any regeneration from the primary leaf
nodes. After incubation for 1 h in an A. tumefaciens
suspension, the cotyledonary nodes were co-cultivated for
3 d on co-cultivation medium with the wounded side facing
the medium.
Following co-cultivation, the explants were exposed to
glufosinate selection, as shown in Fig.1C, most explants
regenerated shoots after two weeks in selection medium
containing 5 mg/L glufosinate. At this stage, the explants
with green shoot-buds can be transferred into liquid shoot
selection/elongation medium modified with 0.01 mg/L TDZ
and 1 mg/L glufosinate for further selection (Fig.1D).
Shoot elongation is one of the major problems with this
system under glufosinate selection. Some of the node re-
gions were GUS positive, bu t cou ld no t regenerate into
plants. To efficiently select the transformed shoots from
the non-transformed ones, explants were cultured in liquid
select ion as well as in liquid shoot elongation medium.
Compared with the s olid s elect ion procedure, the liquid
system readily produced elongated shoots, but some of
these shoots were escapes when they were analyzed for
GUS expression , even though these shoots survived the
selection with 2 mg/L glufosinate. However, there was a
possibility that some of the shoots were transformed, but
for some reason the gus gene was not expressed. Xing e t
al. (2000) also reported that they obtained some GUS nega-
tive T0 plants that were resis tant to the herb icide when
using the leaf-painting assay.
2.2 Confirmation of transformed events
To confirm if the recovered tis sues were transformed,
different tissues at different selection stages were analyzed
for GUS expression. As shown in Fig.2A, some of the coty-
ledonary node regions were GUS positive after four weeks
of selection, and some shoot primordia began to develop
GUS positive s hoot-buds . After they were transferred to
shoot elongation medium, some of the small shoots elon-
gated and a s mall piece of leaf tis sue cou ld be used to
confirm by GUS expression if they were transformed or es-
capes (Fig.2B). Also, leaf tissue from the plants in the green-
house was used to confirm whether the bar gene was ex-
pressed o r no t by placing leaf tis sue on medium with 5
mg/L glufosinate. Leaves from a transformed plant formed
callus on medium with 5 mg/L glufosinate while the wild
type leaves did not. Alternatively, a leaf-callus assay method
was used to test the herbicide resistance as shown in Fig.
2C. The induced callus could be subcultured on the same
medium. This leaf callus assay was simple and reliable, and
might be us ed to eliminate the escapes in the select ion
process as well as to test expression of a number of differ-
ent selectable marker genes (Wang and Waterhouse, 1997).
Other indirect methods such as the chlorophenol red assay
can als o be used to confirm the bar gene expres sion in
plant tissues (Trieu and Harrison, 1996; Wright et al., 1996).
When the plants began to set seeds in the greenhouse,
immature pods were also analyzed for GUS expression (Fig.
2D).
To confirm that the transgenes were integrated into the

Acta Botanica Sinica 植物学报 Vol.46 No.5 2004614
soybean genome, Southern blotting analysis was carried
out with s oybean genomic DNA cut with Hind Ⅲ, which
cu t un iquely in the T-region. As s hown in Fig.3, all the
tested transgenic plants were carrying one to two copies of
the transgene. Re-probe of the membrane with the bar gene
also showed the same result (data not shown).
highest. This result also showed that Dwight is a genotype
amenable to A. tumefaciens-mediated cotyledonary node
transformation.
Many experiments have also been done before to try to
identify soybean genotypes that are more susceptible to A.
tumefaciens infection (Delzer et al., 1990; Mauro et al., 1995).
Most of the work used wild type A. tumefaciens st rains,
resulting in crown gall format ion in vivo o r in vitro. The
result s were not consistent, as the infection not on ly de-
pends on soybean genotypes, but also on A. tumefaciens
strains. In addition, the use of additional virulence genes
has been reported to increas e A. tumefaciens-mediated
transformation (Liu et al., 1992; Park et al., 2000). Ke et al.
(2001) reported that A. tumefaciens strains with a point mu-
tation in the vir G gene-increased transient GUS expression
of soybean tissues, but no t ransgenic plants were recov-
ered since most of the GUS expression was in non-regener-
able regions. From Table 1, we can also conclude that the
soybean genotypes tested are susceptible to A. tumefaciens
infection, but most of the transformed tiss ues canno t re-
generate into plants. This regeneration problem was thought
to be the limitation o f increasing s oybean transformation
efficiency (Donaldson and Simmonds, 2000).
2.4 Optimization of transformation conditions
Various factors that could affect the transformation effi-
ciency were investigated using the genotype Dwigh t in-
cluding the effect of co-cultivation time on transformation
efficiency. As shown in Tab le 2, 4 d co-cu ltivation pro-
duced fewer blue sectors than that of 3 d. Transformation
efficiency can also be influenced by culture temperature
(de Block et al., 1995; Dillen et al., 1997), but there was no
big difference observed of culture temperatures of 24 ℃ or
28 ℃ for soybean transformation, as summarized in Table
3.
de Block et al. (1995) reported that glufosinate selection
is influenced by different carbon sources with many es-
capes when sucrose was used, while few escapes occurred
Fig.3. Southern blotting analysis of transgenic soybean lines.
Soybean genomic DNA was diges ted with Hind Ⅲ and p robed
with the gus gene. 1, pCAMBIA3301 plasmid DNA diges ted
with Hind Ⅲ; 2, Wild type soybean control; 3-9, seven indepen-
dent transgenic soybean lines.
2.3 Genotype evaluation
To determine if these results were the same as those
found using the cotyledonary node transformation system,
six soybean genotypes were us ed for A. tumefaciens
transformation. After four weeks s election with 5 mg/L
glufosinate, the explan ts were cu t into small pieces and
assayed for GUS expression. As shown in Table 1, Jack had
no GUS pos itive expression, while Dwigh t, Peking and
PI227328 were the highest with almost the same infection
rate. Thorne and P9341 had a lower response than the three
Table 1 Evaluation of six soybean genotypes for Agrobacterium
tumefaciens transformation
Genotype Total cot nodes GUS+ nodes Percentage(%)
Dwight 80 5 6.2
Peking 88 6 6.8
PI227328 88 6 6.8
Thorne 72 2 2.8
P9341 111 2 1.8
Jack 77 0 0
Table 2 Effect of co-cultivation time on transformation effi-
ciency
Co-cultivation time (d) Total cot nodes GUS + nodes
3 98 4
4 96 1
Table 3 Effect of culture temperature on transformation effi-
ciency
Temperature Total cot Nodes with GUS+
(ºC) nodes shoots nodes
24 50 30 3
28 50 42 4
CHEN Shi-Yun: High-efficiency Agrobacterium-mediated Transformation of Soybean 615
on glucose-based selection medium with glufosinate as the
selective agent for transformation of Brassica napus. When
comparing the effect of three carbon sources in the selec-
tion medium on shoot regeneration and transformation, glu-
cose had a negative effect on shoot regeneration, but did
not decrease the transformation efficiency relative to that
found with sucrose and maltose (Table 4). Three genotypes
were chosen to test the efficiency of the cotyledonary node
transformation system as des cribed in Materials and
Methods. The transformation efficiency ranged from 1.8%
(Dwight) to 0.5% (PI227328), the results are summarized in
Table 5. These results are similar to those of Table 1, where
Dwight had higher expression than that of Thorne. However,
due to the difficulty of rooting, the small transgenic shoots,
some of the transgenic shots were not recovered. To solve
this problem, several plant rooting growth regulators (IAA,
IBA and NAA) at different concentrations were tested and
the most effective one was found to be MS basal medium
with IBA at a concentration of 2 mg/L. Most of the shoots
can be rooted in this medium in one week, while roo ting
medium containing IAA and/or NAA produced only callus.
There have been reports of using either antioxidants or
chelators to increase A. tumefaciens-mediated transforma-
tion efficiency on rice and sugarcane (Enriquez et al., 1999).
The role o f thes e compounds was thought to be ant i-
necrotic, thus minimizing the oxidative process after wound-
ing to provide better environment for the interaction of A.
tumefaciens with the plant cells (Olhoft et al., 2001; Olhoft
and Somers, 2001). The effects of one chelator (citric acid)
and two ant ioxidan ts (cys teine and ascorbic acid) in the
co-cultivation medium on transformation was tested. While
citric acid and ascorbic acid have negative effects on trans-
formation as compared with the control, cysteine did have
a positive effect on trans format ion efficiency (Table 6).
The mechanism for this is not clear, but cysteine may pre-
vent the browning of the soybean tissues after wounding,
as the tissue browning was reduced and the explants were
more yellowish in colo r than control. However, more ex-
periments need to be done to confirm this result.
In summary, a high-efficiency soybean transformation
system was developed. Soybean cotyledonar nodes were
first under glufosinate selection in solid selection medium
for two weeks, and transferred into liquid selection medium
for further selection and shoot regeneration. The transfor-
mation efficiency was ranged from 0.5% to 1.8%, depend-
ing on differen t genotypes tested. Although the transfor-
mation efficiency was comparable to the procedure devel-
oped by Zhang e t al . (1999), the time needed to get
transgen ic shoots was only 3-4 weeks compared with at
least 5-6 weeks using the solid selection alone. In addition,
due to the tight selection pressure using this liquid selec-
tion system, there were less escapes of the shoots, and the
elongation of the transgenic shoots was stimulated by the
addition of TDZ in the liquid medium . T-DNA integration
was confirmed by Sou thern blo tting hybridization. The
majority of the transgenic soybean plants were found to
contain one or two copies of the transgenes.
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