In Vitro Cell. Dev. Biol.

ÐPlant 36:456±463, November±December 2000

q 2000 Society for In Vitro Biology
1054-5476/00 $10.0010.00

THE USE OF THE TWO T-DNA BINARY SYSTEM TO DERIVE MARKER-FREE TRANSGENIC SOYBEANS

AIQIU XING1, ZHANGYUAN ZHANG2, SHIRLEY SATO1, PAUL STASWICK3, and TOM CLEMENTE1,3*

1
Center for Biotechnology, University of Nebraska-Lincoln, USA
2
Department of Agronomy, Iowa State University, USA
3
Department of Agronomy, University of Nebraska-Lincoln, USA

(Received 8 March 2000; accepted 20 July 2000; editor J. M. Widholm)

Summary
A binary vector, pPTN133, was assembled that harbored two separate T-DNAs. T-DNA one contained a bar cassette,
while T-DNA two carried a GUS cassette. The plasmid was mobilized into the Agrobacterium tumefaciens strain EHA101.
Mature soybean cotyledonary node explants were inoculated and regenerated on medium amended with glufosinate.
Transgenic soybeans were grown to maturity in the greenhouse. Fifteen primary transformants (T0) representing 10
independent events were characterized. Seven of the 10 independent T0 events co-expressed GUS. Progeny analysis was
conducted by sowing the T1 seeds and monitoring the expression of the GUS gene after 21 d. Individual T1 plants were
subsequently scored for herbicide tolerance by leaf painting a unifoliate leaf with a 100 mg l21 solution of glufosinate and
scoring the leaf 5 d post application. Herbicide-sensitive and GUS-positive individuals were observed in four of the 10
independent events. Southern blot analysis confirmed the absence of the bar gene in the GUS positive/herbicide-sensitive
individuals. These results demonstrate that simultaneous integration of two T-DNAs followed by their independent
segregation in progeny is a viable means to obtain soybeans that lack a selectable marker.

Key words: Agrobacterium tumefaciens; transformation; bar; glufosinate; Glycine max.

Introduction ight et al., 1987; De Block and Debrouwer, 1991); co-transformation

Gene transfer protocols developed for plant species require the
use of visual or selectable markers due to the relatively small
number of cells in which integration of the foreign DNA actually
occurs. With the exception of the plant selectable markers that
provide herbicide tolerance, the marker gene has no agronomic
value. Moreover, once a herbicide-tolerant trait has been introduced
into the germplasm of a plant species, redundancy of the herbicide-
tolerant gene in the genome may be problematic, due to potential
manifestation of homology-dependent gene silencing (Matzke and
Matzke, 1995). Since the germplasm cannot be transformed again
using the same marker, alternative marker systems must be used to
incorporate subsequent transgenes. Given the limited availability of
marker genes and potential problems associated with duplication of
foreign DNA, a transformation system that can produce marker-free
transgenic soybeans is desirable.
Strategies that have been used successfully to derive marker-free
transgenic plants include the use of the bacteriophage P1 Cre/lox
recombination system (Dale and Ow, 1991; Gleave et al., 1999);
mobilization of a selectable marker cassette via transposable
elements (Goldsbrough et al., 1993); co-transformation implement-
ing two Agrobacterium tumefaciens strains (Depicker et al., 1985;
McKn-
*Author to whom correspondence should be addressed: Department of
Agronomy, Plant Transformation Core Research Facility, E324 Beadle
Center, Lincoln, NE 68588-0665, USA, Email tclemente1@unl.edu
utilizing one Agrobacterium tumefaciens strain carrying two binary
vectors (Daley et al., 1998); and an Agrobacterium tumefaciens
strain employing one binary vector carrying two T-DNA elements
(Depicker et al., 1985; Komari et al., 1996). Clearly the most
simplistic approach is the co-transformation strategy and the
subsequent identification of unlinked integration events between
the selectable marker and gene of interest. To determine feasibility
of a co-transformation scheme as a strategy to derive marker-free
transformants for a particular plant species, two critical parameters
must be measured: co-transformation frequency and percentage of
unlinked integration events. The objective of the work reported here
was to evaluate the feasibility of the A. tumefaciens two T-DNA
binary system as a strategy to derive marker-free transgenic
soybean.
Materials and Methods
Assembly of the two T-DNA binary vector. A GUS cassette under the
control of a modified CaMV 35s promoter coupled with the V unit
translational enhancer element from tobacco mosaic virus (Sleat et al., 1987;
Gallie et al., 1989) was subcloned from pBE2113-GUS (Mitsuhara et al.,
1996) as a HindIII/EcoRI fragment into the binary vector pPZP 102
(Hajdukiewicz et al., 1994). The resultant vector is referred to as pPTN132
(Fig. 1). The bar gene (Thompson et al., 1987) was fused to the tobacco etch
virus translational enhancer element (Carrington and Freed, 1990) and the
product fused to the enhanced CaMV 35s (E35s) promoter (Zhang et al.,
1999). This enhanced CaMV 35s bar cassette was subcloned into the binary
vector pPZP 202 (Hajdukiewicz et al., 1994) as a HindIII fragment. The
resultant binary vector is referred to as pPTN130 (Fig. 1). The GUS T-DNA

456
 RECOVERY OF MARKER-FREE SOYBEANS
Fig. 1. (A), Assembly of the two T-DNA binary vector. (B), Linear representation of the two T-DNA elements in pPTN133.
element harbored in pPTN132 was subcloned as a ScaI fragment into the
unique ScaI site within pPTN130 just outside of its right border region
(Fig. 1). The final two T-DNA binary was called pPTN133 (Fig. 1).
Soybean transformation. The binary vector pPTN133 was mobilized into
Agrobacterium tumefaciens strain EHA101 (Hood et al., 1986) via tri-
parental mating (Ditta et al., 1980). EHA101 transconjugants were selected
on YEP (10 g l21 peptone, 5 g l21 yeast extract, 5 g l21 NaCl) plates
containing 50 mg l21 kanamycin, 25 mg l21 chloramphenicol, 100 mg l21
spectinomycin and 100 mg l21 streptomycin. The integrity of pPTN133 in
EHA101 was confirmed by plasmid rescue.
Soybean transformations were conducted utilizing the Agrobacterium-
mediated transformation of the cotyledonary node explant (Hinchee et al.,
1988; Zhang et al., 1999; Clemente et al., 2000). The protocol implementing
the use of glufosinate as the selective agent was followed as described by
Zhang et al. (1999). The soybean genotype used in this study was A3237
(Asgrow Seed Company, Des Moines, IA). Explants were cultured on shoot
initiation medium for 4 weeks under selection pressure of 4 mg l21
glufosinate. Following the shoot initiation period differentiating nodal
regions were subcultured to shoot elongation medium supplemented with
2 mg l21 glufosinate.
Plant characterization. Putative primary transformants (T0) were
established and grown to maturity in the greenhouse. Co-expression of
GUS was monitored via the histochemical assay (Jefferson et al., 1987).
457
Southern blot (Southern, 1975) analysis was conducted on the primary
transformants to confirm the putative transformants.
Progeny seed derived from the soybean primary transformants were sown
in Metro-Mixw 360 (Scotts Company, Maryville, OH). The T1 individual
progeny plants were monitored histochemically for GUS expression
(Jefferson et al., 1987) 25 d after seeding. The T1 individuals were
subsequently screened for herbicide tolerance by a leaf-painting protocol
(Zhang et al., 1999) for which a 100 mg l21 solution of glufosinate was
applied to the adaxial surface of the unifoliate leaf with a cotton swab.
Herbicide tolerance was monitored 5 d post application. T1 individuals were
placed into four phenotypic categories: herbicide tolerant/GUS positive (HT/
+), herbicide tolerant/GUS negative (HT/2), herbicide sensitive/GUS
positive (HS/+) or herbicide sensitive/GUS negative (HS/2).
The phenotypic categories of a subset of the T1 individuals were
confirmed by Southern blot analysis (Southern, 1975) analysis using the bar
open reading frame (ORF) and the GUS ORF as probes in the hybridizations.
Results
Four soybean transformation experiments with the EHA101/
pPTN133 transconjugant gave transformation efficiencies ranging
458 XING ET AL.

TABLE 1 5.4 kb (Fig. 3A). Hybridizing pPTN133 digested with SstI with the
bar ORF resulted in a hybridizing signal at 9.0 kb (Fig. 3B),
SUMMARY OF SOYBEAN TRANSFORMATION EXPERIMENTS
representing the remaining bar T-DNA region along with the aadA,
Experiment na I.E.b Frequency (%) ori, rep, bom and sta regions of the binary vector (Hajdukiewicz et
al., 1994). In contrast, genomic DNA from plants transformed with
257 125 4 3.2
258 195 2 1.0 this construct will likely produce hybridizing fragments of variable
259 200 1 0.5 size because SstI cuts only once in each of the two T-DNAs.
260 225 3 1.3 A total of 10 T1 individuals derived from event 257-5A were
a
characterized by Southern blot analysis which included five HS/+
Total number of explants put into culture. individuals, three HT/+ individuals, and two HT/2 individuals
b
Total number of independent events established in the greenhouse.
along with the original T0 parent (Fig. 3). The 257-5A T0 appears to
contain four GUS hybridizing fragments presumably due to multiple
TABLE 2
integration events in this line (Fig. 3A). Three of these signals range
PROGENY ANALYSIS ON SOYBEAN TRANSFORMATION EVENTS in size from approximately 5.0 kb to 7.0 kb, while the last GUS
DERIVED FROM pPTN133 hybridizing signal in the 257-5A T0 parent resides at approximately
12.0 kb (Fig. 3A). All of the HS/+ individuals characterized from
T0a T1b
event 257-5A harbored the approximate 12.0 kb GUS hybridizing
Event GUS HT/+ HS/2 HS/+ HT/2 signal. The two HT/2 individuals from event 257-5A were identical
257-1B + 10 2 0 4 to the parental genotype for GUS hybridizing bands [individual #5
257-2A 2 3 16 0 0 (T15) and #19 (T119)], while the three HT/+ individuals derived
257-4B + 9 1 1 5 from 257-5A were either identical to the parental genotype
257-5A + 5 0 6 8
[individual #4 (T14) and #15 (T115)] or missing the approximate
258-2A 2 0 13 0 5
258-5B1 + 5 6 0 1 12.0 kb GUS hybridizing signal [individual #6 (T16)]. The
258-5C1 + 5 1 0 0 membrane in Fig. 3A was stripped and re-probed with the bar
259-4A2 + 8 3 1 0 ORF (Fig. 3B). The result demonstrated only herbicide-tolerant T1
259-4C2 + 7 3 0 2 individuals, along with the T0 parent possessing a single hybridizing
260-1B3 2 0 3 13 0
260-1C3 + 2 1 4 0 signal at approximately 5.0 kb (Fig. 3B). The five HS/+ T1
260-2A4 + 11 4 0 0 individuals were devoid of a bar hybridizing signal.
260-3A4 + 6 1 0 1 Southern blot analyses on T1 individuals derived from event 257-
260-5A5 2 0 4 0 2 4B and 260-1B are shown in Fig. 4. From event 257-4B two HT/2
260-5B5 2 0 4 0 10
T1 individuals, one HS/+ individual and one HT/+ individual were
a characterized. From event 260-1B four HS/+ T1 individuals were
Refers to GUS expression in T0 generation (+) positive or (2) negative.
b
Refers to the total number of individuals within the respective characterized. In Fig. 4A a GUS hybridizing signal was observed at
phenotypic category. approximately 8.0 kb for the one HS/+ [individual #13 (T113)] and
one HT/+ [individual #16 (T116)] T1 plants derived from event 257-
4B. A GUS hybridizing signal was observed at approximately
from 0.5% to 3.2% on a Southern blot positive T0 plant in soil per 19.0 kb in the four HS/+ T1 individuals derived from event 260-1B.
explant basis (Table 1). Progeny from 15 primary transformants, The parental T0 plant of 257-4B possessed only the approximate
representing 10 independent events, were characterized (Table 2). 8.0 kb GUS fragment (Fig. 4A). The membrane in Fig. 4A was
In five cases, two plants were recovered from the same explant, stripped and re-probed with the bar ORF. The data shown in Fig.
these T0 plants were assumed to be clones derived from the same 4B demonstrates only the 257-4B HT T1 individuals and the 257-
integration event. The putative clones were 258-5C and 5B, 259-4C 4B T0 with a bar hybridizing fragment at approximately 3.0 kb
and 4A, 260-1B and 1C, 260-2A and 3A and 260-5A and 5B (Table (Fig. 4B).
2). Progeny analysis was not conducted on event 257-1A (Fig. 2) Two HT/2 T1 individuals from event 260-5B and one HT/2 T1
which was a clone of 257-1B (Table 2). A total of 7 of the 10 T0 individual from event 258-5B were characterized by Southern blot
independent events co-expressed GUS (Table 2). A Southern blot analysis (Fig. 5). Included in the Southern blot analyses in Fig. 5
analysis probed with the bar cassette, which will hybridize to both are T1 individual numbers 4 (T14), 5 (T15) and 6 (T16) derived from
T-DNAs, on a set of T0 plants derived from the pPTN133 two T- event 257-5A (Fig. 3). The data for the GUS hybridization are
DNA binary vector is shown in Fig. 2. shown in Fig. 5A. As expected, the 257-5A individuals were
We identified four independent events in which T1 individuals identical to the result presented in Fig. 3. A single GUS hybridizing
were recovered which fit into the HS/+ phenotypic category (257- fragment was observed at approximately 8.0 kb for both HT/2 T1
5A, 257-4B, 259-4A and 260-1B) (Table 2). Southern blot analyses individuals [individual #1 (T11) and #2 (T12)] from event 260-5B
hybridized with the GUS and bar ORFs of T1 individuals derived and a single GUS hybridizing fragment at approximately 5.0 kb for
from event 257-5A are shown in Fig. 3. Total genomic DNA was one HT/2 T1 individual from event 258-5B [individual #2 (T12)].
digested with SstI which cuts twice in pPTN133 (Fig. 1B), dropping The membrane in Fig. 5A was stripped and re-probed with the
out an approximate 5.4 kb fragment harboring the promoter region, bar ORF (Fig. 5B). The data for the 257-5A T1 individuals was
GUS ORF, left border (LB) region of the GUS T-DNA and right identical to that observed in the previous hybridization analysis
border (RB) region of the bar T-DNA. Hence, hybridization with the shown in Fig. 3B. The banding patterns observed for the HT/2 T1
GUS ORF to the SstI digest of pPTN133 resulted in a signal at individuals derived from both 260-5B and 258-5B events in Fig. 5B
 RECOVERY OF MARKER-FREE SOYBEANS
Fig. 2. Southern blot analysis on putative primary transformants (T0) of soybean. Probe used in the hybridization was the bar cassette
(see Fig. 1B). T0 T-DNA lane refers to a primary transformant which was transformed with a binary vector carrying a single T-DNA
element. ck lane is wild-type soybean DNA.
were identical to that observed when the GUS ORF was used as a
probe (Fig. 5A). This result is suggestive that in these T1 plants both
GUS and bar T-DNA regions integrated at linked positions within
the genome, however, the GUS gene is not expressed.
Discussion
A reliable and efficient strategy to generate marker-free
transgenic soybeans is needed and would be extremely useful.
Published reports on soybean transformation protocols include the
use of different marker genes for herbicide tolerance, bar (Zhang et
al., 1999) and CP4 (Clemente et al., 2000); the antibiotic resistance
genes hpt (Stewart et al., 1996; SantareÂm and Finer, 1999; Maughan
et al., 1999) and npt II (Hinchee et al., 1988; Di et al., 1996), or the
visual gene GUS (McCabe et al., 1988). For soybean, as with other
plant species, there is a limited number of marker genes available
for use in transformation protocols. Moreover, the perceived risks
that have been associated with some selectable markers (Malik and
Saroha, 1999) may delay public acceptance of genetically enhanced
soybeans.
A number of strategies have been successful for obtaining
marker-free transgenic plants. The use of the maize Ac/Ds
transposon system to derive marker-free tomatoes was described
by Goldsbrough et al. (1993). This approach is clearly a viable
option to recover marker-free plants, but information on frequency
of unlinked transpositions will be needed in order to assess the
efficiency of this strategy for soybean. Moreover, amplification of
the Ds delineated region may arise within the primary transformant
(Yoder and Goldsbrough, 1994) resulting in progeny with high copy
number of the Ds delineated regions (Goldsbrough et al., 1993).
This may increase the probability of homology-dependent gene
silencing (Matzke and Matzke, 1995). Ebinuma et al. (1997)
designed a transformation vector coupling the maize Ac system with
the Agrobacterium ipt gene involved in cytokinin synthesis that can
be employed as a visual marker gene for transformation. This
system, referred to as multi-auto-transformation (MAT), to derive
459
marker-free plants was reduced to practice in tobacco and hybrid
aspen. The frequency of obtaining marker-free plants with the MAT
system was rather low, 0.032%, and required prolonged time in
culture to identify the marker-free plantlets (Ebinuma et al., 1997).
The MAT system is clearly advantageous for vegetative propagated
plant species and species with prolonged life cycles, but
implementation with soybean may require significant labor inputs,
and thus not be cost effective.
The bacteriophage P1 Cre/lox recombination system has been
exploited as a tactic to recover marker-free tobacco plants (Dale
and Ow, 1991; Gleave et al., 1999). This system has the advantage
of allowing breeders to utilize the herbicide tolerance marker gene
for efficient introgression of transgenes into their breeding program
and subsequently remove the herbicide-tolerant marker at a desired
generation. However, this approach requires either sexual crossing
or re-transformation to introduce the Cre recombinase expression
for the site-specific excision of the lox flanked region. Hence, a
transformation event that possesses the desired phenotype and elite
agronomic traits may be compromised if the re-transformation
approach is employed to introduce cre due to potential somalclonal
variation (Larkin and Scowcroft, 1981). If sexual crossing is used to
introduce cre, then additional backcrossing will be required which
will add time and labor to the breeding program.
The use of a co-transformation approach to derive marker-free
rice (Komari et al., 1996) and rape seed (Daley et al., 1998) has
been reported. The frequency of recovery of marker-free plants was
over 25% of the initial primary transformants regenerated for the
respected crop plants rice and rape seed. We have extended this
strategy for use in obtaining marker-free soybeans. Our results
demonstrate a co-expression frequency of 70% (7 out of 10
independent events, Table 2) in primary transformants in using this
strategy. The co-expression frequency of the non-selected transgene
cassette within the primary transformants was essentially the same
if compared to that observed in previous reports of soybean
transformation utilizing the Agrobacterium-mediated transformation
coupled with the cotyledonary-node explant (Zhang et al., 1999;
460
Fig. 3. Characterization of progeny derived from soybean transformation event 257-5A: (A) GUS probe; (B) bar probe. Lane
designations refer to the respective T1 individual phenotype. T0 lane is parent. ck lane represents wild-type soybean DNA. Lanes 25 pg
and 50 pg are pPTN133 plasmid control lanes.
Clemente et al., 2000). However, GUS expression in the T0
generation was not predictive of GUS expression at the T1
generation in this study (Table 2). The same observation was
reported by Clemente et al. (2000). In two cases, 257-2A and 260-
1B, the primary transformants were histochemically GUS negative,
yet GUS positive T1 plants were identified (Table 2).
We assumed that all T0 plants derived from the same explant
were clones. For example, events 259-4A and 4C (Fig. 2, Table 2)
displayed identical hybridization patterns in Southern blot analysis
and arose from the same explant. This is highly suggestive that
these two T0 plants were derived from the same integration event.
Although the segregation ratio at the T1 generation was different
between 259-4A and 4C, this may have been due to the low number
of T1 plants characterized. Events 260-1B and 1C were also derived
from the same explant, yet 260-1B was GUS negative at the T0
generation and 260-1C was GUS positive at the T0 generation
(Table 2). The segregation ratios at the T1 generation were different
between these two events. Event 260-1C generated progeny in all
XING ET AL.
the phenotypic categories except HT/2, while HT/+ or HT/2 T1
plants were not observed in progeny derived from 260-1B (Table 2).
The most likely explanation for this observation is that 260-1B at
the T0 generation was a chimera.
If each of the two T-DNA elements were integrated at two
unlinked loci and were inherited as simple Mendelian traits the
expected segregation ratio at the T1 generation would be 9:3:3:1 for
HT/+:HT/2:HS/+:HS/2 phenotypes, respectively. The number of
progeny evaluated for each event in this study is not sufficient for
meaningful statistical analysis of the ascertained ratios. Moreover,
the recovery of putative chimeral T0 plants (i.e. events 257-2A and
260-1B, Table 2) will confound the interpretation of the analysis at
the T1 generation. Thus, better estimates on inheritance of the
foreign alleles can be made in subsequent generations.
The overall transformation frequency using the two T-DNA
binary ranged from 0.5 to 3.2% (Table 1). These numbers are
similar to that observed when employing a binary vector carrying a
single T-DNA element in the same soybean transformation protocol
 RECOVERY OF MARKER-FREE SOYBEANS 461

Fig. 4. Characterization of progeny derived from soybean transformation events 257-4B and 260-1B: (A) GUS probe; (B) bar probe.
Lane designations refer to the respective T1 individual phenotype. T0 257-4B is parental DNA from that event. ck lane represents wild-
type soybean DNA. Lanes 25 pg and 50 pg are pPTN133 plasmid control lanes.

(Zhang et al., 1999). More importantly the frequency of deriving
marker-free T1 transgenic soybeans was 40% (4 out of 10
independent events; Table 2). Characterized marker-free T1
individuals suggested they possess simple inserts with copy number
of the gene of interest (GUS) of one to two copies per genome (Figs
3±5).
Delivering marker-free genetically enhanced soybean lines to
breeding programs is more desirable if the transformation protocol
employs non-agronomically valuable marker genes or if the value-
added marker gene is already present within soybean germplasm.
Other published protocols for deriving marker-free plants are
potentially applicable to soybean. However, due to the lack of
information in soybean regarding the frequency of unlinked
transposition events with heterologous transposon systems, and
the requirement of re-transformation to induce marker gene removal
with the Cre/lox system, the co-transformation of two separate
462 XING ET AL.
T-DNAs within one Agrobacterium strain appears to be a practical
alternative to obtain marker-free transgenic soybean.
Acknowledgments
This research was supported by funds from the Nebraska
Research Initiative and the University of Nebraska's Center for
Biotechnology. The soybean biotechnology program at UNL's Plant
Transformation Core Research Facility is also supported by funds
from the Nebraska Soybean Board and the North Central Soybean
Research Program. Gratitude is extended to R. A. Zimmerman for
keeping things in perspective.
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