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A novel Agrobacterium rhizogenes-mediated transformation method of

soybean [Glycine max (L.) Merrill] using primary-node explants from
seedlings

Article in In Vitro Cellular & Developmental Biology - Plant · December 2007

DOI: 10.1007/s11627-007-9050-9

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In Vitro Cell. Dev. Biol.—Plant (2007) 43:536–549
DOI 10.1007/s11627-007-9050-9

BIOTECHNOLOGY

A novel Agrobacterium rhizogenes-mediated transformation

method of soybean [Glycine max (L.) Merrill]
using primary-node explants from seedlings
Paula M. Olhoft & Libby M. Bernal & Leslie B. Grist &
D. Steven Hill & S. Luke Mankin & Yuwei Shen &
Mary Kalogerakis & Hunt Wiley & Effie Toren &
Hee-Sook Song & Helke Hillebrand & Todd Jones

Received: 8 November 2006 / Accepted: 26 April 2007 / Published online: 19 September 2007 / Editor: P. Ozias-Akins
# The Society for In Vitro Biology 2007

Abstract A novel Agrobacterium rhizogenes-mediated of 8 independent events resulted in typical Mendelian inher-
transformation method using a primary-node explant from itance of T-DNA genes. Of seven T0 plants that had two
Dairyland cultivar 93061 was developed for soybean using or more T-DNA fragments, six contained multiple loci
the disarmed Agrobacterium strain SHA17. Transformed segregating in T1 progenies. Further analysis of four lines
plants regenerated from explants inoculated with SHA17 confirmed the presence of PAT, GUS, and/or DsRED2
were fertile and phenotypically normal. In a comparative proteins in transgenic plants that were encoded on the T-
experiment, regeneration frequencies were not significantly DNA into the T2 generation.
different between explants inoculated with A. rhizogenes
strain SHA17 and Agrobacterium tumefaciens strain AGL1; Keyword Agrobacterium rhizogenes . K599 .
however, a 3.5-fold increase in transformation efficiency Soybean transformation .
[(number of Southern or TaqMan-positive independent events/ Primary-node transformation method .
total number of explants inoculated)×100] was found for Soybean [Glycine max (L.) Merrill] . T-DNA integration
explants cocultured with SHA17 compared to AGL1 (6.6
and 1.64%, respectively). Southern analysis of 48 T0 plants
suggested that 37.5, 23, and 39.6% of the T0 plants con-
Introduction
tained 1, 2, and 3 or more T-DNA fragments integrated into
the genome, respectively. Additionally, T1 progeny analysis
Whole plant Agrobacterium-mediated transformation meth-
ods in soybean have been developed using disarmed strains
P. M. Olhoft : L. M. Bernal : L. B. Grist : D. S. Hill :
S. L. Mankin : E. Toren : H.-S. Song : T. Jones (*)
of Agrobacterium tumefaciens, the causative agent for crown
BASF Plant Science L.L.C., gall disease. In order for any of these methods to work,
26 Davis Drive, a reliable regeneration and selection protocol must be
Research Triangle Park, NC 27709-3528, USA coupled with a successful Agrobacterium infection step,
e-mail: todd.jones@basf-corp.com
where the target cells are accessible to Agrobacterium,
Y. Shen : M. Kalogerakis susceptible to Agrobacterium infection, and regenerable
DNA LandMarks Inc., after Agrobacterium infection. Soybean is limited in the
84 Richelieu, type of cells that have the ability to regenerate into healthy,
St-Jean-sur-Richelieu, Quebec J3B 6X3, Canada
fertile plants in vitro. To date, regeneration has only been
H. Wiley observed through somatic embryogenesis or organogenesis
Dairyland Seed Co., Inc., from apical or axillary meristematic cells. Besides direct
7657 S 1050 E, P.O. Box 367, Otterbein, IN 47970, USA regeneration from the apical meristem cells, organogenic
regeneration has been reported in soybean from or near the
H. Hillebrand
BASF Plant Science GmbH, Agricultural Center, axillary meristem cells at or near the cotyledonary node
67114 Limburgerhof, Germany (Kimball and Bingham 1973; Cheng et al. 1980; Hinchee
 AGROBACTERIUM RHIZOGENES-MEDIATED TRANSFORMATION METHOD OF SOYBEAN 537

et al. 1988) and higher nodes (Wright et al. 1987b; Kim unlike other plant species (reviewed by Tempe and Casse-
et al. 1990), or arising indirectly from organogenic callus Delbart 1989), there have been no reports to date of suc-
tissue (Wright et al. 1987a; Sairam et al. 2003). Regeneration cessful plant regeneration from soybean hairy roots (Cho
via somatic embryogenesis in soybean has been reported et al. 2000). Because of natural virulence of K599 to soy-
using a variety of starting explant materials, including bean, we were interested in testing a disarmed K599 strain
embryonic axes, intact zygotic embryos, and excised cot- for transformation improvement in soybean.
yledons (Christianson et al. 1983; Ranch et al. 1985; Lazzeri In our companion paper, we presented evidence of the
et al. 1985). disarming and sequencing of pRi2659 of the A. rhizogenes
Agrobacterium tumefaciens-mediated transformation strain K599 (Mankin et al. 2007). In addition, phenotypic
protocols have been developed for only a few of the regen- data from stably transformed plants of Arabidopsis thaliana,
eration methods established in soybean. The cells com- tomato (Lycopersicon esculentum), soybean (G. max), and
monly targeted include the meristematic cells located at the maize (Zea mays) confirmed that the Ri plasmid was
cotyledonary node (Hinchee et al. 1988; Townsend and completely disarmed and delivery of T-DNA from inde-
Thomas 1994; Zhang et al. 1999; Olhoft et al. 2003; Paz pendent binary vectors into all species tested was successful.
et al. 2005) and freshly germinated meristem cells of soy- In this manuscript, we present a new soybean transformation
bean seeds (Chee et al. 1989; Martinell 2002; Khan 2004). method that targets the axillary meristems at the primary
Other reports of successful transformation protocols include node of 7-day-old seedlings for transformation using SHA17
targeting callus induced from cotyledonary nodes and primary for T-DNA delivery.
nodes (Hong et al. 2007), and cells on the surface of
immature cotyledons (Parrott et al. 1989; Yan et al. 2000;
Ko et al. 2003). Experimental Procedure
Although A. tumefaciens-mediated transformation is the
norm in plant transformation, other Agrobacteria are Seed sterilization and germination. Soybean seeds from
occasionally used. One such Agrobacterium, Agrobacterium cultivar 93061 were used for transformations (Dairyland
rhizogenes, causes hairy root disease in a manner similar to Seed, Otterbein, IN). Soybean seedlings were sterilized in a
crown gall disease caused by A. tumefaciens. Both infect a desiccator with a chlorine gas produced by adding 3.5 mL
wound site and transfer T-DNA from the bacterial cell to 12N HCl into 100 mL bleach. After 24 h, the seeds were
the plant cell (Nilsson and Olsson 1997). Initial interests in removed and immediately used or stored at room temper-
using A. rhizogenes for T-DNA transfer into soybean was, ature until ready for use. Approximately 20 to 40 seeds
in part, to induce plant regeneration from the hairy root were plated onto 100 mL of solid germination medium
growth (Owens and Cress 1985). Further attention in using (GM; Table 1) in PlantCons® (MP Biomedicals, Solon,
A. rhizogenes strains was to use hairy root cultures for OH) with the hilum facing downward (Fig. 1a). To imbibe
propagating obligate soybean root parasites and for testing seeds, warm GM at 50°C was poured around, but not
interactions between soybean and the parasite, especially covering, the seeds. The containers were subsequently placed
the soybean cyst nematode (Heterodera glycines) (Savka under 150 μmol m s−1 cool white lights for approximately
et al. 1990). In a comparison of the virulence of four A. 7 days in an 18/6-h (light/dark) photoperiod at 25°C until
rhizogenes strains on soybean, strain K599 (NCPPB2659) the epicotyl was extended at a minimum of 0.5× the length
induced the greatest number of hairy roots per explant of the cotyledons.
across the broadest range of genotypes (Savka et al. 1990).
K599 is currently the strain of choice in many laboratories Agrobacterium preparation. The disarmed A. tumefaciens
using A. rhizogenes-mediated transformation protocols to strain AGL1 and disarmed A. rhizogenes strain SHA17
induce soybean hairy roots (Mazarei et al. 1998; Narayanan were used for soybean transformations. The binary vectors
et al. 1999; Cho et al. 2000; Cho et al. 2004; Subramanian used for transformations, pBPSEW008 and pRET36, are
et al. 2005). By adding a binary plasmid carrying an extra shown in Fig. 2. Standard Escherichia coli strains, media,
copy of a T-DNA into A. rhizogenes, cotransformation of and growth conditions were used in the construction of the
T-DNAs from both the Ri-plasmid and the binary plasmid deletion plasmids. Standard techniques were used for the
into soybean has been useful in the functional testing of construction of recombinant plasmids and molecular anal-
promoters (Mazarei et al. 1996, 1998), RNAi silencing of yses (Sambrook et al. 1989).
genes (Subramanian et al. 2005), and screening genes that Transformation vectors pBPSEW008 and pRET36 (Fig. 2)
may impart resistance to obligate soybean root parasites (i.e., were constructed using a kanamycin-resistant derivative
soybean cyst nematode) (Narayanan et al. 1999; Cho et al. of the dual origin (VS1 and ColE1) binary vector pSUN1
2000). The hairy root system in soybean (Glycine max) (patent number WO 02/00900) using the following compo-
does have its limitations as a transformation system because, nents. Selective herbicide tolerance was conferred by bar
538 OLHOFT ET AL.

Table 1. Media used in the primary-node transformation method

GMa LCCMb,c CCMb,c LSIMa SIMa SEMd RMb

MS salts – – – – – 1× –
B5 salts 1× 1/10× 1/10× 1× 1× – 1/2×
MS iron stock 1× 1/10× 1/10× 1× 1× 1× 1/2×
B5 vitamins 1× 1× 1× 1× 1× 1× –
MES 3 mM 20 mM 20 mM 3 mM 3 mM 3 mM 3 mM
Sucrose (w/v) 2% 3% 3% 3% 3% 3% 2%
Purified agare 0.8% – 0.5% – 0.8% 0.8% 0.80%
PH 5.8 5.4 5.4 5.6 5.6 5.6 5.6
6-Benzyl-aminopurine – 7.5 μM 7.5 μM – 1 μM – –
Gibberellic acid – 1.44 μM 1.44 μM – – 1.44 μM –
Acetosyringone – 200 μM 200 μM – – – –
L-Cysteine – – 4.4 mM – – – –
Sodium thiosulfate – – 0.5 mM – – – –
Dithiothreitol – – 0.5 mM – – – –
Kinetin – – – – 5 μM – –
Glufosinatef – – – – 0, 5 mg L−1 3 mg L−1 –
Ticarcillin – – – 250 mg L−1 250 mg L−1 250 mg L−1 250 mg L−1
Asparagine – – – – – 0.378 mM –
L-Pyroglutamic acid – – – – – 0.775 mM –
Indole-3-acetic acid – – – – – 0.57 μM –
trans-Zeatin riboside – – – – – 2.85 μM –
Indole-3-butyric acid – – – – – – 0.57 μM
Container PlantCon®g – 15× – 25× 25× PlantCon®
100 mm 100 mm 100 mm
Petri dish Petri dish Petri dish

CCM = cocultivation medium; LSIM = liquid shoot induction medium; MES = 2-morpholinoethanesulfonic acid monohydrate.
a
Sigma® G5893 used; includes B5 salts, MS iron stock, B5 vitamins.
b
Sigma® G5768 used; includes B5 salts, MS iron stock.
c
Sigma® G1019 used; includes B5 vitamins.
d
Sigma® M0404 used; includes MS salts, MS iron stock, B5 vitamins.
e
Difco™ (Becton and Dickinson, Franklin Lakes, NJ) cat #214230.
f
PESTANAL®, glufosinate-ammonium (Riedel-de Haën, Sigma-Aldrich, GMBH).
g
MP Biomedicals

(De Block et al. 1987) and transcriptionally controlled by inoculation, 150 mL of YEP was inoculated in 400-mL
the nopaline synthase promoter (Herrera-Estrella et al. centrifuge bottles with 150 μL working stocks of
1983) and terminator (t-NOS; Depicker et al. 1982). Agrobacterium strains SHA17 or AGL1, respectively. The
Transformation was tracked using either βglucuronidase bottles were shaken overnight at 175 rpm at 28°C until the
(gusINT; Vancanneyt et al. 1990) or DsRed2 (Invitrogen, OD660 was 0.8 to 1.2. The Agrobacterium was spun at
Carlsbad, CA) as reporter genes driven by the parsley 5,600×g for 10 min at 20°C to pellet the cells and
ubiquitin (p-PcUBI4-2; Plesch and Ebneth 2003) and resuspended in liquid cocultivation medium (LCCM; Table 1)
terminated by the nopaline synthase 3′ region (t-NOS; for a final OD660 of 1.5. The Agrobacterium solution was
Depicker et al. 1982). incubated for at least 30 min at room temperature before use.
Agrobacterium glycerol stocks were prepared by streak-
ing Agrobacterium onto solid YEP growth medium Explant preparation and inoculation. Healthy seedlings
(10 g L−1 Bacto-peptone, 5 g L−1 yeast extract, 5 g L−1 with the epicotyl measuring between 1/3 and 1× the length
NaCl, 12 g L−1 agar; pH 7.0) containing 50 mg L−1 of the cotyledon were chosen for experimentation. Explants
kanamycin for antibiotic resistance. After approximately were prepared by first cutting perpendicularly through the
2 days, 50 mL of liquid YEP medium with 50 mg L−1 hypocotyl approximately 1 cm below the cotyledons. After
kanamycin was inoculated with a single colony and shaken discarding the lower hypocotyl and roots, one cotyledon
at 175 rpm at 28°C until an OD660 0.8 to 1.2 was reached. and the adjacent meristematic tissue was subsequently
Working glycerol stocks (15%) for transformation were removed from the aerial portion of the seedling along both
prepared and stored at −80°C. The day before explant primary leaves and the epicotyl tissue above the primary
 AGROBACTERIUM RHIZOGENES-MEDIATED TRANSFORMATION METHOD OF SOYBEAN 539

Figure 1. Steps in the primary

node transformation method in
soybean. (a) Sterile soybean
seeds germinated on GM. (b)
Wounding of the primary node
with a sharp scapel after one
cotyledon, both primary leaves,
and the epicotyl above the pri-
mary node are removed. (c)
Explants on solid cocultivation
plates after inoculation with
Agrobacterium. (d) Shoot
growth at the primary node after
2 weeks on SIM with glufosi-
nate selection. (e) Close-up of a
developing shoot pad at the
primary node. (f) Selection of
glufosinate resistant tissues on
SEM. (g) An elongated,
glufosinate-resistant shoot ready
for removal and transfer to RM.
(h) A rooted shoot growing in
soil. (i) A healthy, fertile trans-
genic soybean plant transformed
with SHA17 growing in the
greenhouse.

node. The primary node was then wounded by carefully wounded explants were placed into a 100×25-mm Petri dish
stabbing 5 to 10 times with a #11 scalpel blade (Feather Safety containing 50 mL liquid Agrobacterium suspension and
Razor, Osaka, Japan) into the epicotyl perpendicular to the cut cocultivated for a minimum of 30 min at room temperature.
surface of the primary node (Fig. 1b). Approximately 50 The explants were then removed from the suspension and
placed on top of a sterile 70-mm Whatman #1 filter paper
(Whatman International, Maidstone, UK) on 15×100-mm
Petri plates containing solid cocultivation medium (Table 1)
with the wounded primary node in direct contact with the
filter paper (Fig. 1c). Stacks of 5 Petri dishes were wrapped
with Parafilm “M” (American National Can, Chicago, IL)
and incubated at 25°C for 5 d in the dark.

Selection and plant regeneration. Excess Agrobacterium

was washed from the explants after cocultivation by
immersing in liquid shoot induction medium (SIM) (Table 1)
containing antibiotics for 5 min. At this time, any axillary
growth at the cotyledonary node was removed to stimulate
Figure 2. Binary plasmids used in soybean transformation. LB, left T-
growth at the primary node. Explants were then transferred
DNA border; RB, right T-DNA border; p-NOS, nopaline synthase into solidified SIM (Table 1) without glufosinate for 7 d
promoter; p-PcUBI4-2, parsley ubiquitin promoter; bar, selectable under 50 to 70 μmol m−2 s−1 cool white lights in an 18/6-h
marker gene for glufosinate resistance; gusINT, βglucuronidase gene; (light/dark) photoperiod at 25°C to allow the explant to
DsRed2, coral reef fluorescent protein gene. Vertical gray bars occur
every 2.5 kb. The EcoRI restriction sites used for Southern analyses
recover from the inoculation procedure and to stimulate
are marked with an asterisk and the HindIII restriction site with a shoot primordial growth at the primary node. After 1 wk,
circle. all shoot growth at the cotyledonary node was removed and
540 OLHOFT ET AL.

the developing shoots at the primary node were trimmed with SHA17 carrying the binary plasmid, RET36 [available
down to near the base of the growing shoots to induce de through BD Biosciences Clontech, Palo Alto, CA (Clontech
novo shoot growth. The explants were then cultured on 2003)] (Fig. 2). Visualization of the fluorescent proteins in
SIM (Table 1) containing 5 mg L−1 glufosinate for 3 wk for whole in vitro T0 plants was done using a Zeiss SV11
selection of transformed shoots (Fig. 1d, e). stereomicroscope coupled with the mercury vapor short-arc
After a total of 4 wk on SIM, the callus/shoot pad illuminator HBO 100W (Carl Zeiss, Thornwood, NY). The
structure that had formed at the primary node was removed rhodamine filter was used for DsRED2 (563 nm excitation
from the explant by cutting approximately 5 mm below the max and 582 emission max) protein visualization. Images
callus/shoot pad on the epicotyl. All dead shoot material were captured using the Axiocam™ video camera and
was removed at this time and the explant was then placed analyzed using the software Axiovision 3.1 (Carl Zeiss).
with the cut side imbedded into shoot elongation medium Fluorescent protein signals in T1 and T2 plants were also
(SEM; Table 1) containing 3 mg L−1 glufosinate and the captured by scanning tissues on the variable mode imager
base of the shoots in contact with the medium. The explants Typhoon™ 9400 (Amersham Biosciences, Sunnyvale, CA)
were transferred into fresh SEM every 3 wk or when using the fluorescence mode with a 580-nm BP30 according
needed. Shoots elongated after only 1 wk on SEM and to manufacturer specifications for DsRED2 visualization.
continue to elongate for up to 6 mo. (Fig. 1f, g). Healthy Imaging data were processed using ImageQuant™ v.
growing shoots were removed from the explant with a clean 2003.03 (Amersham Biosciences).
excision near the callus/shoot pad when the stem elongated
to >2 cm in length and placed into solid rooting medium ELISA for PAT/bar used for the Evaluation of T1 lines. The
(RM; Table 1) in PlantCon® containers under 150 to QualiPlate™ Kit for LibertyLink® PAT/bar, product AP
175 μmol m−2 s−1 cool white lights in an 18/6-h (light/dark) 013 (EnviroLogix, Portland, ME) was used to detect PAT
photoperiod at 25°C. Emergence of root primordia occurred protein levels in leaf tissues of T1 soybean lines. The high
between 5 and 9 d on RM for the majority of shoots. After sensitivity protocol (0.1%) was applied using a 5-mm-diameter
roots formed, the plantlet was transferred to autoclaved soil leaf punch from a fully expanded soybean leaf, and extrac-
[1:1 (w/w) North Carolina soil/Metro-Mix® 300 (SUNGRO tions were done in a 96-well format. Protein measurements
Horticulture, Bellevue, WA)] and placed in a growth were made using the Tecan Sunrise™ absorbance reader
chamber for approximately 35 d growing under a mix of (Tecan USA, Durham, NC) following the manufacturer’s
incandescent and inflorescence lights between 150 and directions. Absorbance readings were performed at OD450
200 μmol m−2 s−1 in an 18/6-h (light/dark) photoperiod at and analyzed using DeltaSoft PC Microplate Analysis
25°C (Fig. 1h). After flowering began, the plants were Software, v.1.71.2PC 199 (BioMetallics, Princeton, NJ).
moved into a greenhouse under 16/8 h (light/dark) Units reported are absorbance OD450, and values higher
supplemented with 1,000-Watt metal Halide Halogen lamps than 0.5 are recognized as positive for PAT/bar in com-
at 25°C and were kept in the greenhouse for seed mercial lines (i.e., 0.1% Starlink® corn by weight or 1 in
production (Fig. 1i). Leaf tissue was removed from young, 1,000 kernels).
expanding leaves and molecularly checked for transgene
integration and T-DNA copy number using quantitative Southern blot hybridizations. To estimate transgene com-
real-time PCR (TaqMan) analysis as previously described plexity, genomic DNA was isolated from healthy leaf tissue
(Ingham et al. 2001). of soybean plants. Total genomic DNA was isolated using
AutoGen 740 Automatic DNA Isolation System (AutoGen,
GUS histochemical staining in T0 and T1 plants. Regen- Holliston, MA). Eighty-milligrams of silica geldessicated
erated T0 plants and T1 progenies were assayed for GUS leaf tissues were frozen with liquid nitrogen and ground
expression by removing a leaf and placing in GUS histo- using an Autogrinder (AutoGen) for 2 min at 1,000 rpm
chemical stain [80 mM Na2HPO4 (pH 8.0), 8 mM Na2EDTA, three times. The ground tissues were then incubated for 1 h
0.8% (v/v) Triton-X, 1.6% (v/v) dimethyl sulfoxide, 20% at 65°C in AutoGen Plant Lysis Solution (AG00121) with
(v/v) methanol, 0.38 mM K4Fe(CN)6, 1 mM X-glucuro occasional mixing. In the AutoGen 740, DNA was extracted
CHA salt (Calbiochem®, EMD Biosciences, LaJolla, CA)] by adding SDS-N-Lauroyl Sarcosine, potassium acetate,
for 1 day at 37°C (Jefferson et al. 1987; Kosugi et al. 1990). and chloroform; the mixture was shaken at 7 Hz, 9 AMP for
The leaf tissue was then washed in 70% ethanol to remove 300 s and centrifuged twice at 3,500×g for 20 min. After the
excess GUS stain and cleared in 95% ethanol. supernatant was removed, DNA was precipitated using
0.7 vol isopropanol, washed with 70% ethanol, and re-
Fluorescent protein visualization in T0, T1, and T2 plants. suspended in Tris/EDTA buffer (10 mM Tris and 1 mM
Transgenic soybean plants for the DsRed2 fluorescent coral EDTA, pH 8.0) with 0.5 mg L−1 RNase A (Invitrogen). The
reef gene were made by infecting primary-node explants isolated DNA was quantified using Hoechst dye and Packard
 AGROBACTERIUM RHIZOGENES-MEDIATED TRANSFORMATION METHOD OF SOYBEAN 541

FluoroCount™ BF10000 Microplate Fluorometer (Packard labeled gus or bar probe to genomic DNA digested with
Instrument, Meriden, CT). HindIII should result in fragment sizes greater than 3.5 and
DNA fragments used as probes were bar, 804 to 1,303 bp, 1.7 kb, respectively, for each unique T-DNA integration
and gus, 3,273 to 4,158 bp, amplified from pBPSEW008. event. Genomic DNA digested with EcoR1 would release
The primers used for bar gene amplification are BarF: 5′- unique fragments greater than 3.5 kb for gus and a single
ATCTCGGTGACGGGCAG-3′ and BarR: 5′-GACCGCCG 1.48 kb fragment for bar.
GCATGTC-3′. The primers used for gus gene amplification
are GusF: 5′-CGGCAAAGTGTGGGTCAAT-3′ and GusR: Experimental design. Transformation efficiencies (TEs) for
5′-AAGGGTAATGCGAGGTACGGT-3′. PCR reactions the primary-node transformation method were determined
(50 μl) contained 1× manufacturer-supplied PCR buffer, for two Agrobacterium strains, AGL1 and SHA17, both
1.5 mM MgCl2, 50 μM each of dNTP, 10 ng of pBPSEW008 delivering the binary vector, pBPSEW008. The experimental
DNA, two units of Taq DNA polymerase (Qiagen, Valencia, design included seven repetitions over time (1 rep=1 cutting
CA), and 0.4 μM of each PCR primer. The thermal cycler experiment) with two treatments (treatments = inoculation
GeneAmp PCR system 9700 (Applied Biosystems, Foster with either AGL1 or SHA17) per repetition. For each repe-
City, CA) was used and the conditions set at 94°C for 4 min tition, approximately 50 explants were cut four separate
for predenaturation, then 30 cycles of 94°C for 30 s for times and randomly distributed into Petri dishes containing
denaturation, 64°C for 45 s for primer annealing, and 72°C 50 mL of either AGL1 or SHA17 Agrobacterium suspen-
for 2 min for extension. This was followed by 72°C for sion. The primary node transformation protocol was carried
10 min, and a 4°C hold. Both PCR products were separated out through plant establishment in a greenhouse. Because
and cleaned from a 1% agarose gel using Zymoclean™ Gel of the possibility of multiple, transgenic shoots arising from
DNA Recovery Kit (Zymo Research, Orange, CA). Probes a single explant, only one shoot was removed per explant
(approx. 50 ng each) were then labeled with 50 μCi of for GUS and TaqMan analyses. The frequency of shoot
(a-32P)-dCTP (3000 Ci/mmol) (MP Biomedicals) using a regeneration was determined by counting the number of
random labeling system (Rediprime™ II, Amersham, explants with shoot primordia at the primary node after
Piscataway, NJ) according to the manufacturer’s recom- 1 wk on recovery [(number explants with shoot primordia
mendations. The labeled probes were purified on Spin-X® after 1 wk recovery/total number of explants inoculated)×
Centrifuge Tube Filters (Corning Costar, Acton, MA). 100] and after 3 wk on SIM with glufosinate selection
Five micrograms of genomic DNA from transgenic and [(number explants with shoot primordia after 4 wk shoot
nontransgenic controls were digested overnight with EcoRI induction/total number of explants inoculated)×100]. The
and HindIII (New England BioLabs, Beverly, MA) (eight number of GUS+ elongated shoots was determined by pos-
units per microgram DNA) under the conditions recommen- itive GUS-histochemical staining on leaves from elongated
ded by the enzyme manufacturers. Restriction digests were shoots, and the number of positive T0 plants was determined
separated by electrophoresis in 10-cm-long 0.8% agarose by performing TaqMan assays for the bar gene. TEs were
gels and blotted to Hybond N+ nylon membrane (Amersham). calculated by [(number of bar TaqMan+ T0 independent
Southern hybridization was carried out according to Sambrook events in soil/total number of explants inoculated)×100].
et al. (1989). Pair-wise comparisons were made between Agrobacterium
The membranes were prehybridized at 65°C for 2 to 4 h strains, and the significance between the treatments was
and hybridized at 65°C overnight in 30 mL of hybridization determined by calculating the difference between the two
buffer in a Hyb aid MAXI 14 hybridization oven (Thermo population proportions using a t test at a=0.05.
Electron, Waltham, MA). After hybridization, the membranes The effects of eight cytokinin combinations (treatments)
were first washed with 2×SSC, 0.5% SDS at room tem- (Fig. 6a) in the SIM on three measurements of explant
perature for 15 min, 2×SSC, 0.1% SDS at 65°C for 30 min, growth and shoot regeneration were determined by design-
and finally with 0.1×SSC, 0.1% SDS at 65°C for 15 min. ing an experiment with three repetitions over time (1 rep=1
After washing, the membranes were wrapped with plastic cutting experiment) consisting of 14 explants per treatment
wrap and exposed to Hyperfilm™ MP film (Amersham) for 1 (treatment=8 cytokinin combinations) per repetition (repe-
to 4 d depending on the radioactive signal intensity. tition=1 cutting experiment). During explant preparation,
The minimum number of T-DNA integration events and explants were cocultivated in LCCM with A. tumefaciens
loci number were estimated by analyzing the hybridization strain, AGL1, carrying pBPSEW008, cocultivated 5 d in
fragment patterns in the T0 and T1 plants of the Southern the dark, then randomly placed onto one of the eight shoot
blots. The T-DNA sequence from pBPSEW008 has both an induction media and allowed to grow for 4 wk under
EcoR1 and a HindIII restriction site between the bar and normal lighting and temperature conditions. The average
gus genes and a second EcoR1 site between the left border shoot pad width per explant was recorded for each treatment
and the bar gene (Fig. 2). Therefore, hybridizations of 32P- after 4 wk (Fig. 6b) before the explants were transferred
542 OLHOFT ET AL.

onto SEM. After 2 wk on SEM, the average number of no undesirable phenotypes usually associated with the
shoots per explant (Fig. 6c) and the average length of the presence of rol genes from A. rhizogenes developed from
longest shoot per explant were recorded (Fig. 6d). Data regenerated shoots transformed with SHA17.
were evaluated by analysis of variance (SAS, Cary, NC). In
addition, the mean and the least significant difference at a= TEs using SHA17 or AGL1 for gene delivery. Regeneration
0.05 were calculated to determine the differences between and TEs were calculated for the primary node transformation
treatments. method using either SHA17 or AGL1 for delivery of the
binary pBPSEW008 (Fig. 2). Regeneration of a shoot pad
at the tip of the primary node did not significantly differ
between explants inoculated with SHA17 or AGL1 at either
Results time point after inoculation (Table 2). However, a signifi-
cant difference (a=0.05) between the mean TEs of explants
A primary-node transformation method using SHA17 for inoculated with either SHA17 or AGL1 in seven cutting
gene delivery and the bar gene for selection. A plant trans- experiments was found. From these elongated shoots, a
formation method was established that targets the axillary total of 49 (6.6% TE) independent T0 events and 12 (1.64%
meristem cells at the primary node of 7-d-old seedling for TE) independent T0 events developed into mature, healthy
Agrobacterium infection with the disarmed A. rhizogenes plants from explants inoculated with SHA017 and AGL1,
strain, SHA17. The method is illustrated in Fig. 1 and the respectively. The difference between the number of positive
media components in Table 1. When in contact with the elongated shoots and the final TE was a result of the losses
soybean tissue, the growth of the SHA17 strain was fast associated with shoots not rooting or not successfully
and prone to overgrowth, especially during cocultivation. establishing in the soil.
Therefore, smaller, delicate tissues, such as the wounded
primary node on very small or very long epicotyl lengths, Molecular characterization of soybean plants transformed
were more sensitive to cell death than other parts of the with SHA17. Integration of the binary pBPSEW008 into
explant. Optimal regeneration, defined as >80% callus/ the genomic DNA was molecularly confirmed by Southern
shoot pad formation, was obtained from explants prepared hybridizations for 48 T0 plants transformed with SHA17. A
from healthy seedlings with the epicotyl measuring between Southern blot of genomic DNA from six of the 48 inde-
1/3 and 1× the length of the cotyledon (data not shown). In pendent T0 events digested with HindIII and probed with
addition, regeneration of shoots at the primary node was bar is shown in Fig. 3. In this panel, four of the five positive
necessary by the removal of the meristem cells at the cot- events contained one to two hybridization signals, and one
yledonary node. In this protocol, healthy, fertile plants with had a more complex pattern with six fragments. Overall,

Table 2. Regeneration and transformation efficiencies for explants inoculated with either AGL1 or SHA17

Repetition Agrobacterium strain n GUS+ elongated shoots TaqMan bar+ plants TE %a Regen.% (1)b Regen.% (2)c

1 SHA17 97 4 3 3.1 91.9 89.7

AGL1 66 0 0 0.0 95.5 92.4
2 SHA17 100 10 10 10.0 89.0 87.0
AGL1 95 6 5 5.3 95.2 92.6
3 SHA17 98 6 2 2.0 84.5 79.6
AGL1 110 4 4 3.6 89.1 88.2
4 SHA17 120 14 14 11.7 94.4 94.2
AGL1 88 0 0 0.0 96.8 96.6
5 SHA17 92 7 6 6.5 98.0 96.7
AGL1 24 0 0 0.0 100.0 100.0
6 SHA17 115 14 11 9.6 94.8 92.2
AGL1 115 3 3 2.6 93.9 92.2
7 SHA17 99 3 3 3.0 92.2 89.9
AGL1 149 0 0 0.0 93.0 86.6

Details of explants inoculated (n), GUS+ shoots, TaqMan bar+ plants, TE, and regeneration efficiencies (Regen. %) are shown for seven
experimental repetitions.
a
[(number of bar TaqMan+ T0 independent events in soil/total number of explants {n} inoculated)×100].
b
[(number explants with shoot primordia after 1 week recovery/total number of explants {n} inoculated)×100].
c
[(number explants with shoot primordia after 4 weeks shoot induction/total number of explants {n} inoculated)×100].
 AGROBACTERIUM RHIZOGENES-MEDIATED TRANSFORMATION METHOD OF SOYBEAN 543

Table 3. Estimation of T-DNA integration complexity in 48 T0 plants

transformed with SHA17 based on Southern hybridization patterns for
the bar gene using genomic DNA digested with HindIII

Hybridization Number Percent (%)

fragments (bar) of T0 plants of T0 plants

1 18 37.5
2 11 23.0
3 2 4.2
>3 17 35.4

LMB25, with a more complex T-DNA integration pattern in

the T0 plant, had at least four independent loci, each
segregating normally in the T1 plants (Fig. 4). Of the seven
lines with putative multiple T-DNA insertions, six had two
or more T-DNA loci independently segregating from one
another in the T1 progenies (Table 4). We did not observe
any loss of T-DNA fragments between generations in these
eight lines.

Expression of transgenes delivered into soybean using

SHA17. The functionality of transgenes delivered by SHA17
into soybean was addressed by assaying for the presence
of GUS, PAT, and DsRED2 proteins in T0 and T1. GUS
expression was typically detected in the leaf tissue of T0
Figure 3. Southern hybridization showing the integration of the T- plants in varying degrees of intensity from event to event
DNA region from BPSEW008 into the soybean genome of five (Fig. 5a, b). In addition, it was not uncommon to find plants
independent transformation events. The genomic DNA was digested that were negative for GUS expression but positive for the
with HindIII and probed with 32P-labeled bar. Lane M, lambda
HindIII marker; lane NC, nontransformed control plant; lanes R1 and
gus gene. In these cases, the presence of the protein may
R4, R0 plants negative for bar gene; lanes R2, R3, R5, R6, and R7, R0 have been below visual detection in the tissues sampled or
plants positive for bar gene. not expressed at all due to possible silencing of the gene.
GUS expression was commonly seen segregating in the T1
progenies (Fig. 5c), which complements the conclusions
greater than half of the plants (60.5%) had one to two gathered from the Southern hybridization experiments
hybridizing fragments, suggesting that a T-DNA fragment presented above that the T-DNAs are segregating normally
delivered using SHA17 was able to undergo simple, low- in the T1 generation.
copy integration events into the soybean genome (Table 3). To test the efficacy of the coral reef florescent proteins in
Of the complex T-DNA insertion events that had greater our transformation method, glufosinate-resistant T0 plants
than three hybridization fragments, many of the predicted were regenerated from soybean explants inoculated with
T-DNA structures based on integration models, i.e., head- SHA17 carrying the binary pRET36 (Fig. 2). The DsRED2
to-head, tail-to-tail, and concatamers, were found; however, protein was seen as red fluorescence in the developing
there was no preponderance of one type over another (data tissues of glufosinate-resistant plantlets in vitro (data not
not shown). shown). The shoots that were positive for red fluorescence
Transmission of the T-DNA into the T1 progenies was in vitro remained positive in the mature leaf tissues of T0
evaluated for eight independent transformation events whose plants. Seeds from one positive T0 plant for red fluorescence,
Southern blots of T0 genomic DNA digested with HindIII LMB92, which was also TaqMan-positive for the bar gene,
and probed with 32P-labeled bar revealed hybridization were harvested and planted, and leaves from 10 T1 plants
fragments ranging from one to greater than three. Although were scanned for fluorescence using the Typhoon™ image
only 10 plants were characterized per event, all eight T1 scanner. Of the 10 plants screened, five were positive for the
lines showed transmission of the T-DNA into the T1 prog- DsRED2 protein. Seeds were once again harvested from
enies in an expected Mendelian fashion. For example, two positive T1 plants, LMB927 and LMB92-10, and leaves
PO287 had one hybridization fragment using both bar and from T2 plants from each T1 line were scanned for expres-
gus probes in the T0 and in eight of the 10 T1 plants, and sion of the DsRED2 protein (Fig. 5d, e). The red fluo-
544 OLHOFT ET AL.

Figure 4. Southern hybridiza-

tions of two T1 soybean lines
confirming the inheritance of the
T-DNA from the T0 plant. Ge-
nomic DNA from leaves of 10
T1 plants for each line, LMB25
and PO287, were digested with
HindIII and probed with 32P-
labeled bar. Lane M, lambda
HindIII marker; lanes 1–10, T1
progeny from line LMB25;
lanes 11–20, T1 progeny from
line PO287.

rescence in T2 plants LMB92-10-1, LMB92-10-2, and Optimization of SIM in the primary-node transformation
LMB92-7-2 was intense compared to the nontransformed method. The type and concentration of a hormone in a
control and LMB92-7-1 plants. Those plants with the DsRED2 growth medium has a substantial impact on the growth of
expression were confirmed positive for the DsRed2 and bar the explant not only while physically in contact with the
genes, and those without expression lacked the genes. growth medium but also long after it is removed. Therefore,
PAT/bar ELISA assays were used to detect the presence combinations of various concentrations of two cytokinins,
of the protein product of the bar gene, PAT, in T1 plants BAP and kinetin (Fig. 6a), in the SIM were tested to see
from four different transgenic events. The PAT protein was how these hormone combinations affect the size of the
detected in all T1 plants that contained at least one bar callus/shoot pads, number of shoots, and the length of
hybridization fragment (data not shown). In Table 5, PAT/bar elongating shoots per explant (Fig. 6b–d). Explants exposed
ELISA data from two of these T1 lines are shown along to BAP at levels tested developed significantly larger shoot
with the respective GUS-staining and Southern hybridiza- pads than in the two treatments with only 5 μM kinetin or
tion data from HindIII digested genomic DNA and probed no hormones, treatments A and H, respectively (Fig. 6b).
with either bar or gus. In all lines where the bar gene was The addition of BAP at levels between 2.5 and 7.5 μM did,
detected by Southern hybridization, a positive result for however, have an inhibitory effect on the elongation of the
PAT/bar ELISA was found for all plants, confirming the developing shoots when compared to explants cultured in
presence of PAT protein. In addition, the GUS protein was treatments A, B, and H (Fig. 6d). Explants that were cultured
also detected in all T1 plants that had a positive hybridiza- with only 5 μM kinetin produced the longest shoot per
tion signal for the gus gene, except plant PO287-4. explant of all treatments tested. Kinetin (5 μM) alone had no
influence on explant shoot pad width or shoot elongation
(Fig. 6b, c). The combination of kinetin with 1 μM BAP
resulted in explant developing medium-size shoot pad widths
Table 4. Transmission of both linked and unlinked T-DNA loci into
the T1 progenies of eight independent events was confirmed using and the greatest number of elongating shoots without a
Southern analysis negative effect in shoot elongation (no significant difference
between the no-hormone control). Therefore, of the hormone
Event T0 hybridization Minimum # of
concentrations tested, 5 μM kinetin with 1 μM BAP showed
fragments of bar gene (#)1 segregating loci
the best combination of shoot pad width, elongated shoots per
PO287 1 1 explant, and longest shoot per explant for this primary node
PO021-2 2 2 transformation method.
LMB37 2 2
PO261 2 2
PO094 >3 1
Discussion
PO101 >3 2
PO090 >3 2
LMB25 >3 4 We demonstrated that the disarmed A. rhizogenes strain
SHA17 is capable of T-DNA delivery into soybean and
The eight events chosen represented the range in the number of bar results in healthy, fertile soybean plants that express the
fragments found in the T0 Southern analysis. To determine the
minimum number of loci, total genomic DNA of T1 plants was
integrated transgenes into two generations. This finding
digested with either HindIII or EcoR1 and probed with either complements the companion paper that reported for the first
a 32 P-labeled gus or bar probe. time the successful disarming of an A. rhizogenes strain and
 AGROBACTERIUM RHIZOGENES-MEDIATED TRANSFORMATION METHOD OF SOYBEAN 545

Figure 5. Expression of genes

carried on the T-DNA in trans-
genic plants from the T0, T1, and
T2 generations. Examples of
strong GUS expression (a) and
weak GUS (b) expression in
leaves of two different T0 plants.
GUS expression was observed
in the T1 segregating population
for the transgene (c). Leaves
transgenic for the DsRed2 gene
are shown under fluorescence
(d) or white light (e). 1, non-
transformed control leaf; 2,
LMB92-10-1; 3, LMB92-10-2;
4, LMB92-7-1; 5, LMB92-7-2.

the transformation of three plant species in regenerated T0 geous for shoot induction and regeneration (presumably
plants of maize (Z. mays), tomato (L. esculentum), soybean due to the storage of nutrients and hormones in the coty-
(G. max), and in T1 progenies of A. thaliana. In addition, edon) and, as a result, was included as part of the explant
the normal phenotype of the regenerated soybean plants for the first 33 d in the transformation protocol.
without hairy root formation is in accordance with the We optimized shoot regeneration from primary-node
sequence data of the Ri-plasmid in that the endogenous explants by testing BAP and kinetin at various concentrations
T-DNA region containing the genes responsible for hairy during shoot induction. Our results are similar to those of
root formation, the rol genes, was removed in entirety and Kim et al. (1990) in that, although BAP was required for
no second T-DNAs were present. the development of large shoot pads, higher levels were
To produce soybean transformants, a transformation pro- found inhibitory to shoot elongation. In our study, explant
tocol was developed that targets the primary node’s axillary exposed to only 5 μM kinetin developed small shoot pads,
meristem cells for transformation and regeneration. This is but elongated shoots rapidly grew into plantlets. By combin-
the first report of a successful transformation method based ing 5 μM kinetin with 1 μM BAP, the explants developed
on using the primary-node explant. The meristem cells medium-sized shoot pads with long, sturdy elongating
targeted for transformation are different from those targeted transgenic shoots within 4 wk of transfer to elongation
in other transformation methods involving organogenesis, medium. In Kim et al. (1990), the addition of proline and
which are the cells located at the cotyledonary node of increased micronutrients was found essential for shoot
seedlings (Hinchee et al. 1988; Townsend and Thomas regeneration, whereas only friable callus was induced when
1994), the freshly germinated meristem cells of soybean these compounds were omitted from SIM. We had no prob-
seeds (Chee et al. 1989; Martinell 2002; Khan 2004), and lems with shoot formation at the primary node with the
callus tissue (Hong et al. 2007). In Kim et al. (1990), the media presented in Table 1, and therefore, these compounds
attachment of the cotyledon to the explant was advanta- were not added to the media.
546 OLHOFT ET AL.

Table 5. Expression of the bar

and gus genes were detected in Event T1 GUS PAT/bar ELISA Southern # gus Southern # bar
segregating T1 progenies plant expression absorbance (OD450) fragments fragments
transformed using SHA17
LMB25 1 + 2.876 5 6
LMB25 2 + 2.973 4 4
LMB25 3 + 2.83 3 4
LMB25 4 + 2.822 2 2
LMB25 5 + 2.975 4 6
LMB25 6 + 0.792 1 1
LMB25 7 + 2.862 5 6
LMB25 8 + 2.588 3 3
LMB25 9 + 2.658 5 6
LMB25 10 + 2.893 5 6

PO287 1 + 1.784 1 1
For each T1 plant, GUS histo- PO287 2 + 1.388 1 1
chemical staining for visuali- PO287 3 − 0 0 0
zation of the GUS protein and
PO287 4 − 2.125 1 1
a BAR ELISA assay for pres-
PO287 5 + 2.138 1 1
ence of the BAR protein were
performed on leaf tissue and PO287 6 − 0 0 0
compared to the approximate PO287 7 + 2.125 1 1
number of gus and bar frag- PO287 8 + 1.192 1 1
ments detected by Southern PO287 9 + 1.245 1 1
hybridization using HindIII PO287 10 + 1.562 1 1
digested genomic DNA.

The average TE of 6.6% obtained using the primary et al. 2004). The reason for this increase could be due to the
node transformation method with glufosinate selection and differences in explant tissue, growth media, Agrobacterium
the SHA17 strain for mobilization of the T-DNA is higher strain, environment, or interactions between these factors.
than those efficiencies reported using the A. tumefaciens- The increase in TE found for explants inoculated with A.
mediated cotyledonary node transformation method with rhizogenes strain SHA17 compared to those inoculated with
glufosinate selection; 2.1% (Olhoft and Somers 2001), 3.8% A. tumefaciens strain AGL1 indicates that SHA17 does play
(Paz et al. 2005), 4.0% (Paz et al. 2004), and 5.4% (Zeng a role (6.6 and 1.64%, respectively). This difference in

Figure 6. Influence of cytoki-

a b
nins in the SIM on shoot pad
growth of primary-node
Treatment Kinetin BAP Shoot Pad Width after 4w on SIM
explants inoculated with A. (µM) (µM)
tumefaciens strain, AGL1. Eight A 5 0
Average width (mm)

10
different hormone combinations B 5 1 a ab ab
c abc bc
of BAP and kinetin (a) were 8
C 5 2.5
added to the SIM and the aver- D 5 5 6
d
age shoot pad width at the d
E 5 7.5 4
primary node on explants was
measured after 4 weeks on SIM
F 5 10 2

(b). The explants were then G 0 7.5 0

transferred to SEM, and after H 0 0 A B C D E F G H
2 weeks, the average number of BAP/Kinetin Treatments
shoots per explant (c) and the
average length of the longest
c d
Elongated Shoots per Explant Longest Shoot per Explant
shoot per explant (d) were
recorded. Bars topped with dif-
Average Length (mm)
Average # of Shoots

15
ferent letters show significant 10
differences among treatments 8 a a
ab
acd abc
10
according to least significant bcd cd b
6 cd d b
difference (a=0.05).
4 5
2
c c c c c
0 0
A B C D E F G H A B C D E F G H

BAP/Kinetin Treatments BAP/Kinetin Treatments

 AGROBACTERIUM RHIZOGENES-MEDIATED TRANSFORMATION METHOD OF SOYBEAN 547

virulence between different Agrobacterium strains is well (Nishizawa et al. 2006). The protein accumulated to levels
known and is also influenced by different laboratory strains, in the seeds that the gene product could be visualized as a
soybean target tissue, and cultivars, to name a few variables faint pink color under white light. Wenck et al. (2003)
(Owens and Cress 1985; Byrne et al. 1987; Savka et al. reported green fluorescence from soybean explants trans-
1990; Torisky et al. 1997). The higher TEs of explants formed with two other fluorescent proteins isolated from
inoculated with SHA17 may be partially attributed to the nonbioluminescent species of reef coral, ZsGREEN1 and
fact that soybean is a natural host for A. rhizogenes infection AmCYAN1. In this study, we showed that the DsRED2
(Savka et al. 1990). Further comparative experiments will protein was successfully visualized in the soybean tissues
need to be initiated to ascertain whether the TEs seen using into the T2 generation. The DsRED2 florescent protein may
SHA17 remain as significant when other commonly used A. prove to be a valuable tool for early transgene detection
tumefaciens strains for soybean transformation are tested without tissue destruction.
(Zhang et al. 1999; Ko et al. 2003; Olhoft et al. 2003; Zeng This is the first report of (1) an A. rhizogenes transfor-
et al. 2004; Paz et al. 2005). mation method in soybean using a disarmed strain and (2) a
The Southern hybridization data indicated that the new transformation method based on targeting the primary-node
explant source and the chromosomal background and the explant. The normal phenotypes of transformed plants, the
Ri-plasmid of the A. rhizogenes strain did not significantly simple T-DNA integration patterns and Mendelian inheri-
change integration patterns of the mobilized T-DNA com- tance of the T-DNA, and expression of genes on the T-DNA
pared to other published reports in soybean despite the all support the functionality of the disarmed A. rhizogenes
different mechanisms of T-strand protection (Hodges et al. strain SHA17. The application of the SHA17 strain to other
2004). We found that 37.5% of T0 plants had a single plant species, especially those recalcitrant to transfor-
hybridization fragment when probed with the bar gene. mation, may provide the additional virulence needed that
This is similar to a study by Olhoft et al. (2004) that found is not being met with the disarmed A. tumefaciens strains
31.5% of 270 T0 transgenic plants obtained from A. currently available.
tumefaciens inoculated cotyledonary-node explants had a
single hpt-hybridizing fragment. Moreover, each primary Acknowledgements The authors are grateful for all the technical
support Drs. Michael Kock, Stephan Krieger, and Klaus Daeschner
transformant had an average of two unlinked loci in both have given us for this project.
studies. This propensity for the Agrobacterium to deliver
the T-DNA fragments in less complex patterns, i.e., infre-
quent recovery of plants with large concatamers of T-DNA
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