1. Plant Science 180 (2011) 766–774
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Plant Science
journal homepage: www.elsevier.com/locate/plantsci
Improved Agrobacterium-mediated co-transformation and selectable marker
elimination in transgenic rice by using a high copy number pBin19-derived
binary vector
Rajasekaran Sripriya, Manoharan Sangeetha, Chidambaram Parameswari,
Balamani Veluthambi, Karuppannan Veluthambi∗
Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Palkalai Nagar, Madurai 625 021, Tamil Nadu, India
a r t i c l e i n f o
Article history:
Received 6 November 2010
Received in revised form 17 February 2011
Accepted 22 February 2011
Available online 1 March 2011
Keywords:
Co-transformation
Marker elimination
Osmotin
Oryza sativa
a b s t r a c t
A high copy number, selectable marker gene (SMG)-free Agrobacterium binary vector pBin19 nptII was
constructed by deleting the nptII gene from pBin19. The binary vectors with the RK2 and pVS replication
origins exist in 12 and 3 copies, respectively, in Agrobacterium. The tobacco osmotin gene (ap24) was
cloned in pBin19 nptII and the resultant plasmid pBin19 nptII-ap24 was mobilized into the Agrobac-
terium tumefaciens strain C58C1 Rifr
harbouring the single-copy cointegrate vector pGV2260::pSSJ1. The
T-DNA of the cointegrate vector harboured the hph (SMG) and gus genes. Transformation of Oryza sativa
L. var. Pusa Basmati1 with Agrobacterium tumefaciens (pGV2260::pSSJ1, pBin19 nptII-ap24) yielded 14
independent hyg+
/GUS+
transgenic plants. Southern blot analysis with hph and ap24 probes revealed that
12 out of the 14 transgenic plants were co-transformed and harboured hph, gus and ap24 genes. The new
multi-copy binary vector yielded 86% co-transformation efficiency. SMG elimination by genetic separa-
tion of the cointegrate T-DNA with the hph/gus genes and binary vector T-DNA with the ap24 gene was
accomplished in four out of ten primary co-transformants that were forwarded to the T1 generation.
© 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Despite the high commercial adoption rate of genetically mod-
ified crops, many concerns are raised about the biosafety of these
crops [1]. The major concern is the persistence of the selectable
marker genes encoding antibiotic and herbicide resistance. To alle-
viate the risks of the selectable marker gene (SMG) in transgenic
crops, many strategies have been designed for its elimination. These
include the strategy of avoiding the usage of SMG [2,3], excision
of the SMG by site-specific recombination [4,5], repositioning of
the transgene or the marker gene by transposition [6,7], and co-
transformation of two independent T-DNAs, one with the gene of
interest (GOI) and the other with the SMG [8,9].
Co-transformation by Agrobacterium is a simple and clean tech-
nique for SMG elimination and it does not leave behind residual
DNA sequences such as recombination sites and invert repeats in
the SMG-eliminated transgenic plants [10]. Efficient SMG elim-
Abbreviations: ap24, osmotin gene with antifungal activity towards Phytoph-
thora infestans; 2,4-D, 2,4-dichlorophenoxyacetic acid; GOI, gene of interest; gus,
␤-glucuronidase gene; hph, hygromycin phosphotransferase gene; MS, Murashige
and Skoog; nptII, neomycin phosphotransferaseII; SMG, selectable marker gene.
∗ Corresponding author. Tel.: +91 452 2458683; fax: +91 452 2459105.
E-mail address: kveluthambi@rediffmail.com (K. Veluthambi).
ination by co-transformation requires a high co-transformation
efficiency and unlinked integration of an SMG and a GOI. Although
particle bombardment yields a high frequency of co-transformation
[11–13], it is of limited use to obtain SMG-free plants because it fre-
quently results in linked integration of multiple copies of the SMG
and GOI.
Successful co-transformation has been reported using Agrobac-
terium [8,14,15]. In order to achieve high co-transformation
efficiency using Agrobacterium, ‘twin T-DNA’ binary vectors were
constructed in which the same binary vector harboured an SMG
and a GOI in two separate T-DNAs [9,16–18]. In a modified twin
T-DNA strategy, one T-DNA carrying the nptII gene as the posi-
tive SMG and codA as the negative conditional SMG was deployed.
The second T-DNA carried the non-selected gus gene. SMG-free
plants were obtained in the T1 generation by negative selection on
5-flurocytosine-containing medium [19]. By applying a transient
positive selection step followed by negative selection using codA,
SMG-free potato was obtained at a frequency of 6.1% [20].
One disadvantage of the twin T-DNA approach is the high
frequency of ‘linked co-delivery’ of T-DNA along with the adja-
cent intervening non-T-DNA sequences [16,21]. High frequency of
unlinked integration of the T-DNAs with the SMG and the GOI was
achieved in the conventional co-transformation system in which
the GOI and the SMG were placed on two separate plasmids in a
single Agrobacterium strain [14,22–24]. By separating the SMG and
0168-9452/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.plantsci.2011.02.010

2. R. Sripriya et al. / Plant Science 180 (2011) 766–774 767
1 kb
> 2.1 kb
LB RB
PstI
P35S ap24 35S3’
HindIII EcoRIEcoRI EcoRI HindIII
KpnI
LB hphint-gus P35Stml3’nos3’P35S RB
>2.0 kb1.0 kb
hph probe
A
B
KpnIEcoRI
Fig. 1. Linear maps of the T-DNA regions. (A) The T-DNA region of the cointegrate vector pGV2260::pSSJ1 [22] which harbours P35S-hph and P35S-int-gus genes. The right
border junction fragment (>2.0 kb, the distance between the EcoR1 site and RB) is marked as a broken arrow. The hph probe is marked in a bold line. P35S, Cauliflower mosaic
virus (CaMV) 35S promoter; int-gus, ␤-glucuronidase gene with the catalase intron; nos3 , nopaline synthase gene polyadenylation signal; LB, left T-DNA border; RB, right
T-DNA border; tml3 , tumor morphology large gene polyadenylation signal. (B) The T-DNA region of pBin19 nptII-ap24, which harbours the CaMV 35S promoter-driven
tobacco ap24 gene. The left border junction fragment (>2.1 kb, the distance between the PstI site and the LB) is marked as a broken arrow. The ap24 probe is marked in a bold
line. RB, right T-DNA border; LB, left T-DNA border; ap24, tobacco osmotin gene; P35S, CaMV 35S promoter; 35S3 , CaMV 35S polyadenylation signal. Scale (1 kb) is marked.
the GOI on two plasmids, linked integrations of two T-DNAs along
with the intervening non-T-DNA sequences is avoided. Besides, it
is also feasible to alter the ratio of the T-DNA with SMG and the
T-DNA with the GOI. Co-transformation using the cointegrate vec-
tor (one copy/cell) with the SMG and a multi-copy binary vector
with pVS replicon (3 copies/cell) [25] with the GOI yielded 20%
co-transformation efficiency in rice [24].
In the present work, we constructed an SMG-free binary vec-
tor pBin19 nptII with the RK2 replicon [26] to clone the GOI
(ap24). The high copy number (10–15 copies) of RK2 plasmids in
Agrobacterium [14] was expected to improve the co-transformation
efficiency. The SMG-free, multi-copy binary vector pBin19 nptII-
ap24 was used in combination with the single-copy cointegrate
vector pGV2260::pSSJ1 [22] to transform rice. We report here a
higher co-transformation frequency of 86%. SMG elimination was
achieved through genetic separation in four out of ten primary
co-transformants that were forwarded to the T1 generation.
2. Materials and methods
2.1. Agrobacterium strains and plasmids
The binary vector pBin19 [26] has the RK2 replication origin and
has nptII as the SMG. It is a broad host-range binary vector which
is reported to exist in 10–15 copies per Agrobacterium cell [14].
The following subcloning steps were performed to delete the pnos-
nptII from pBin19. A 3.7-kb BglII fragment from pBin19, containing
the nptII gene (SMG) and the T-DNA borders, was cloned in the
BglII site of pIC19H ClaI [27] to yield the plasmid pRP3. The nptII
gene from pRP3 was deleted by digesting the plasmid with ClaI
and SacII, treated with Klenow polymerase to destroy ClaI and SacII
sites and self-ligated to yield pRP4. The SMG-free T-DNA, obtained
as a 1.8-kb BglII fragment from pRP4, was ligated with the BglII-
digested 7.97-kb pBin19 backbone devoid of the T-DNA. This clone
(named as pBin19 nptII) has an SMG-free T-DNA with a multiple
cloning site between the right (RB) and left (LB) T-DNA borders. The
tobacco osmotin gene (ap24) [28] was obtained as a 1.6-kb EcoRV
fragment from pKVD2 (comprising the CaMV 35S promoter-driven
tobacco ap24 gene) and cloned in the SmaI site of pBin19 nptII
to obtain pBin19 nptII-ap24. The T-DNA portion of pBin19 nptII-
ap24 is shown in Fig. 1B.
The binary vector pBin19 nptII-ap24 was mobilized by tri-
parental mating into the Agrobacterium tumefaciens strain C58C1
Rifr harbouring the cointegrate vector pGV2260::pSSJ1 (Fig. 1A)
which carries the hph and gus genes in its T-DNA [22]. The transcon-
jugants were selected on AB minimal medium with 10 mg/l
rifampicin, 300 mg/l streptomycin, 100 mg/l carbenicillin, 100 mg/l
kanamycin and 100 mg/l spectinomycin. The presence of both plas-
mids in Agrobacterium was confirmed by Southern blot analysis.
2.2. Copy number analysis of the binary plasmids with RK2 and
pVS replicons
Southern blot analysis using [␣-32P]dCTP-labelled probes [29]
was used to determine the copy numbers of the binary plasmids
with the pVS replicon (pCAMBIA3301) [25] and the RK2 replicon
(pGA472) [30] relative to that of the single-copy cointegrate vec-
tor pGV2260::pSSJ1. The probes simultaneously hybridized to the
single-copy cointegrate plasmid and the multicopy binary plas-
mid in the same Agrobacterium strain. The autoradiograms and the
Zeta Probe nylon membranes were matched and positions corre-
sponding to bands in the autoradiogram were marked on the nylon
membrane. Rectangular strips of similar dimensions, correspond-
ing to the bands were cut and the radioactivity was estimated by
liquid scintillation counting. The relative copy number of the binary
plasmid was estimated by dividing the radioactivity (cpm) in the
binary vector fragment by the radioactivity in the fragment corre-
sponding to the cointegrate vector. A normalization procedure [29]
was used to calculate cpm/kb which was used in the copy number
estimation of pGA472 with the RK2 replicon.
2.3. Plant material and transformation
Scutellum-derived callus was generated from the mature
seeds of the indica rice variety Pusa Basmati1. Callus induction
and Agrobacterium-mediated transformation were performed as
described earlier [31]. The calli were initiated from dehusked
and surface-sterilized mature seeds on a callus-induction medium
[Murashige and Skoog (MS) salts [32], B5 vitamins, 100 mg/l
proline, 2.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 300 mg/l
casein hydrolysate, 30 g/l sucrose and 2.25 g/l Phytagel, pH 5.8]. The
21-day-old scutellum-derived calli were excised and preincubated

3. 768 R. Sripriya et al. / Plant Science 180 (2011) 766–774
on fresh callus induction medium for 4 days. The preincubated
calli were infected with 1.0 OD (A600) Agrobacterium culture. The
infected calli were co-cultivated on the co-cultivation medium
(MS salts, B5 vitamins, 2.5 mg/l 2,4-D, 300 mg/l casein hydrolysate,
30 g/l sucrose, 10 g/l glucose, 3 g/l Phytagel and 100 ␮M acetosy-
ringone, pH 5.6) [33]. After 3 days of co-cultivation, the calli were
washed with liquid callus-induction medium supplemented with
250 mg/l cefotaxime and 150 mg/l timentin. The co-cultivated
calli were placed on the selection medium (callus-induction
medium supplemented with 50 mg/l hygromycin, 250 mg/l cefo-
taxime and 150 mg/l timentin). Selection, regeneration and
establishment of transgenic plants were done as described ear-
lier [31]. ␤-Glucuronidase (GUS) staining [34] of portions of
leaves and roots was done before transferring the plants to a
greenhouse.
2.4. Southern blot analysis
Total DNA was extracted from control and transgenic plants
[35]. DNA was estimated in a DNA Fluorometer using the Hoechst
dye 33258. About 2.5 ␮g of DNA was digested with a restric-
tion enzyme and electrophoresed in a 0.8% agarose gel. DNA was
transferred to the Zeta-Probe nylon membrane (Bio-Rad Labora-
tories, Hercules, CA). Southern blot analysis was performed with
probes labelled with [␣-32P]dCTP (BRIT, Hyderabad, India) using
the MegaprimeTM DNA labelling system (GE Healthcare UK Limited,
Little Chalfont, UK). Prehybridization, hybridization and washes
were done as reported earlier [33].
2.5. Segregation analysis
The T0 plants were transferred to the greenhouse, selfed and
seeds were collected. The seeds were dehusked, surface-sterilized
and germinated on half-strength MS basal medium in the dark at
25 ◦C. The sprouted seedlings were acclimatized in a greenhouse
and were analysed by GUS histochemical staining. The GUS+ and
GUS− seedlings were scored and the data were validated by 2 test.
3. Results
3.1. Copy number analyses of the binary plasmids pCAMBIA3301
with the pVS replicon and pGA472 with the RK2 replicon
The A. tumefaciens strain C58C1 (pGV2260::pSSJ1, pCAM-
BIA3301, pGA472) was used for copy number estimation of the
binary vectors. pCAMBIA3301 has the pVS replicon [25]. Restric-
tion digestion of pCAMBIA3301 with KpnI yielded a 9.7 kb fragment
comprising the complete intron-gus sequence of 2.1 kb. Digestion of
the cointegrate vector pGV2260::pSSJ1 with KpnI, yielded a 4.8 kb
fragment with the complete intron-gus sequence of 2.1 kb (Fig. 1A).
The intron-gus probe of 2.1 kb will hybridize with equal efficien-
cies to the binary vector fragment of 9.7 kb and the cointegrate
vector fragment of 4.8 kb. The relative differences in the radioac-
tivity of these two bands will reveal the relative copy numbers of
the two vectors (Fig. 2A). The radioactivity in the nylon membrane
corresponding to the positions of these two bands was measured
by liquid scintillation counting. The radioactivity was taken as a
measure of the amount of probe DNA present on the membrane
after blotting. The entire length of the same probe hybridized to
the 4.8 kb and 9.7 kb fragments. The relative copy number was
calculated by dividing the counts obtained for the 9.7 kb band of
the binary plasmid (pCAMBIA3301) by the counts obtained for the
4.8 kb band of the cointegrate plasmid (pGV2260::pSSJ1). The coin-
tegrate plasmid, a derivative of the Ti plasmid, is inferred to exist
as a single copy. Counts were obtained from DNA extracted from
four individual Agrobacterium colonies. The results presented in
Fig. 2. Southern blot analysis for copy number estimation of the binary plas-
mids pCAMBIA3301and pGA472 in the Agrobacterium tumefaciens strain C58C1
(pGV2260::pSSJ1, pCAMBIA3301, pGA472). (A) Copy number estimation of the
binary plasmid pCAMBIA3301 with the pVS origin of replication. Total DNA (1 ␮g)
from four individual colonies of the same strain of Agrobacterium tumefaciencs was
digested with KpnI and loaded in each lane. A 2.1-kb gus-intron fragment labelled
with [␣-32
P]dCTP was used as probe. (B) Copy number estimation of the binary
plasmid pGA472 with the RK2 origin of replication. Total DNA (1 ␮g) from individ-
ual colonies of the same A. tumefaciens strain was digested with both SmaI + SalI
and loaded in each lane. The plasmid pVK102 + Sal13b was digested with SmaI + SalI,
labelled with [␣-32
P]dCTP and used as the probe.
Table 1 show that the relative copy number of pCAMBIA3301 in
A. tumefaciens is three [2.94 ± 0.13].
pGA472 is a binary vector based on the RK2 repli-
con. pBin19 nptII-ap24, the binary vector used in our
co-transformation experiments, also has the RK2 replicon.
Agrobacterium DNA was digested with SalI + SmaI and the Southern
blot was probed with [␣-32P]dCTP-labelled pVK102-Sal13b plas-
mid [36] in which a 3.5-kb virG fragment (Sal13B) of an octopine
type Ti plasmid was cloned. pVK102-Sal13b will hybridize to a

4. R. Sripriya et al. / Plant Science 180 (2011) 766–774 769
Table 1
Estimation of the copy number of pCAMBIA3301 in Agrobacterium tumefaciens
(pGV2260::pSSJ1, pCAMBIA3301, pGA472).
Colony
no.
cpm in 4.8 kb
cointegrate plasmid
fragment (CI)
cpm in 9.8 kb
pGAMBIA3301
fragment (CAMBIA)
Relative copy
number
(CAMBIA/CIa
)
1 693 1927 2.78
2 643 1937 3.01
3 613 1779 2.9
4 663 2043 3.08
Mean copy number of pCAMBIA3301 = 2.94 ± 0.13.
a
Cointegrate Ti plasmid is taken as 1 copy per Agrobacterium cell; cpm, counts
per minute.
3.5 kb SalI fragment of the cointegrate plasmid (comprising virG)
and a 0.8-kb SmaI fragment of the binary plasmid (comprising
the tetracycline resistance [Tcr] gene) of A. tumefaciens C58C1
(pGV2260::pSSJ1, pCAMBIA3301, pGA472). The probe will have
equimolar amounts of virG and Tcr genes. The complete lengths
of the 3.5 kb virG sequence and 0.8 kb Tcr sequence are the same
between the probe (pVK102-Sal13b) and the Agrobacterium DNA
transferred to the nylon membrane. Therefore, the relative differ-
ences in the radioactivity of the 3.5-kb virG band of the cointegrate
vector and 0.8-kb Tcr band of the binary vector will provide an
estimate of the copy number of the RK2-based binary vector
pGA472 (Fig. 2B).
As the lengths of probes hybridizing to different fragments
from the binary vector pGA472 (0.8 kb) and the cointegrate vector
pGV2260::pSSJ1 (3.5 kb) are different, the normalization procedure
described by Froissard et al. [29] was used. The cpm of the 3.5-kb
and 0.8-kb bands was normalized by dividing them by the length
of the probes hybridizing to them, to obtain cpm per kb length
of the probe (Table 2). The normalized cpm of the 0.8-kb band of
pGA472 was divided by the normalized cpm of the 3.5-kb band
of pGV2260::pSSJ1 in four independent Agrobacterium colonies.
The relative copy number of pGA472 was determined as 12
(11.8 ± 0.26) with respect to the cointegrate vector pGV2260::pSSJ1
(Table 2). It is inferred that pBin19 nptII, with the RK2 repli-
con, will also have a copy number of 12 in Agrobacterium. Three
other fragments of 6.0-, 3.0- and 0.5-kb which are common
between pGA472 in Agrobacterium and pVK102-Sal13b which was
used as the probe, displayed hybridization (Fig. 2B). These are
not relevant for copy number estimation and not discussed any
further.
3.2. Co-transformation of rice with pGV2260::pSSJ1 and the
SMG-free binary plasmid pBin19 nptII-ap24
The A. tumefaciens strain C58C1 Rifr (pGV2260::pSSJ1,
pBin19 nptII-ap24) was used to transform the scutellum-derived
calli of Pusa Basmati1. pGV2260::pSSJ1 is a single-copy cointegrate
vector with the hph (SMG) and gus genes. pBin19 nptII-
ap24 with the RK2 replicon is a 12 copy SMG-free binary
vector (Table 2). In three transformation experiments, 15
hygromycin-resistant (hygr) plants were regenerated. All
15 plants were GUS+. Southern blot analysis of the GUS+
plants was performed with the hph and ap24 probes to study
co-transformation.
Total plant DNA (2.5 ␮g) from 15 GUS+ T0 plants was digested
with EcoRI and the blot was hybridized to the [␣-32P]dCTP-labelled
hph probe. A junction fragment of the cointegrate vector T-DNA
that hybridizes to the hph probe is expected to be longer than
2.0 kb (Fig. 1A). All 15 GUS+ plants displayed hybridization of
junction fragments (Fig. 3A and B). Plants 2 and 3 harboured junc-
tion fragments of same sizes and were inferred as siblings (two
plants which regenerated from one transformed callus). Among
Fig. 3. Southern blot analysis of transgenic rice plants co-transformed with the SMG
(hph/gus) in the cointegrate vector pGV2260::pSSJ1 and the GOI (tobacco ap24) in
the binary vector pBin19 nptII-ap24 using the hph probe. Plant DNA (2.5 ␮g) was
digested with EcoRI and loaded in each lane. The blot was hybridized to the [␣-
32
P]dCTP-labelled hph probe. (A) Analysis of T0 rice plants CoTosm1–9. Lanes 1–9,
T0 plants CoTosm1–9. (B) Analysis of the T0 rice plants CoTosm10–15. Lanes 10–15,
T0 plants CoTosm10–15; lane C, DNA from the control untransformed plant.
the 14 independent transformants, nine plants (CoTosm1, 4, 5,
7, 8, 9, 10, 11 and 14) had single copies of the cointegrate vec-
tor T-DNA, whilst three plants (CoTosm3, 6, and 15) harboured
two T-DNA copies (Fig. 3). As reported earlier [24], the cointe-
grate vector favoured the integration of one or two copies of the
T-DNA.
Co-transformation of the T0 plants with the binary plasmid
pBin19 nptII-ap24 was analysed with the ap24 probe. Total
DNA (2.5 ␮g) from the GUS+ plants was digested with PstI
and the blot was hybridized to the [␣-32P]dCTP-labelled ap24
probe. Junction fragments longer than 2.1 kb were expected
to hybridize to the ap24 probe (Fig. 1B). Twelve out of 14
independent transgenic plants harboured junction fragments of
the binary vector T-DNA (Fig. 4A and B). The plants 9 and
11 were not co-transformed. The co-transformation efficiency
was 86% (12 out of 14). Hence, a GOI placed in a high copy
number SMG-free binary plasmid (pBin19 nptII-ap24) yielded

5. 770 R. Sripriya et al. / Plant Science 180 (2011) 766–774
Table 2
Estimation of the copy number of pGA472 in Agrobacterium tumefaciens (pGV2260::pSSJ1, pCAMBIA3301, pGA72).
Colony
no.
3.5 kb
(Ti)
cpm
0.8 kb (Bi)
cpm
Ti/3.5
(cpm/kb)
Bi/0.8
(cpm/kb)
Relative copy no Bi
(cpm/kb)/Ti
(cpm/kb)
1 811 2192 232 2740 11.8
2 785 2143 224 2679 12
3 894 2318 255 2898 11.4
4 812 2213 232 2766 11.9
Mean copy number of pGA472 = 11.8 ± 0.26; cpm, counts per minute; Ti, cointegrate vector; Bi, binary vector.
high co-transformation efficiency. The plants CoTosm5, 6, 8
and 10 harboured single copies of the binary vector T-DNA.
The plants CoTosm1, 3, 4, 7, 12, 13, 14 and 15 harboured
two or more T-DNA copies. Junction fragments of same sizes
hybridized to the ap24 probe in plants CoTosm2 and 3 confirming
that both plants regenerated from the same transformed cal-
lus.
Fig. 4. Southern blot analysis of transgenic rice plants co-transformed with the
SMG (hph/gus) in the cointegrate vector pGV2260::pSSJ1 and the GOI (tobacco ap24)
in the binary vector pBin19 nptII-ap24 using the ap24 probe. Plant DNA (2.5 ␮g)
was digested with PstI and loaded in each lane. The blot was hybridized to the [␣-
32
P]dCTP-labelled ap24 probe. (A) Analysis of T0 rice plants CoTosm1–9. Lanes 1–9,
T0 plants CoTosm1–9. (B) Analysis of the T0 rice plants CoTosm10–15. Lanes 10–15,
T0 plants CoTosm10–13; lane C, DNA from the control untransformed plant.
3.3. Segregation of the SMG (hph/gus) and the GOI (ap24)
T-DNAs in the T1 generation
Twelve co-transformed rice plants (CoTosm1, 3, 4, 5, 6, 7, 8,
10, 12, 13, 14 and 15) were selfed, grown to maturity in a green-
house and seeds were collected. The plants CoTosm12 and 15 did
not set seeds. The seeds of the ten co-transformed rice plants were
germinated and the T1 plants from each line were subjected to seg-
regation analysis based on GUS histochemical staining (Table 3).
The hph gene was not used for segregation analysis since SMG−
and ap24+ plants will be lost in such an analysis. All co-transformed
plants which had single copies of the cointegrate vector T-DNA seg-
regated in the typical 3:1 ratio characteristic of integration at single
loci. The plant CoTosm14 was an exception. Although it had a sin-
gle cointegrate T-DNA copy (Fig. 3B), all 28 T1 plants displayed GUS
staining with variable intensities (Table 3). Even though the plants
CoTosm3 and 6 had two copies and the plant CoTosm13 had three
copies of the cointegrate vector T-DNA, the multiple copies were
linked and consequently segregated as single loci in the T1 genera-
tion. A single-locus integration of the SMG is advantageous because
it can be easily segregated out from the GOI in the T1 generation. The
GUS−, SMG-free plants were further analysed by Southern blotting
with hph and ap24 probes to identify the SMG-eliminated plants
which harboured the GOI (ap24). Unlinked integration of the SMG
and the GOI will result in their segregation and yield SMG-free
plants with the GOI.
Out of the 13 T1 plants of the line CoTosm1, three were GUS−
(Table 3). Southern blot analysis of the three GUS− plants with the
ap24 probe showed the presence of two copies of the ap24 gene
(GOI) in two T1 plants (Fig. 5A). It is interesting to note that in spite
of a complex integration of the ap24 gene in CoTosm1, SMG-free,
ap24+ plants could be recovered because of the single-copy status of
the SMG. Among the 34 T1 plants of the line CoTosm3, 25 T1 plants
were GUS+ and nine were GUS− (Table 3). Southern blot analysis
with the ap24 probe showed the presence of two copies of the ap24
gene in seven out of eight GUS− plants that were analysed (Fig. 5B).
The presence of two ap24 junction fragments of 6.6 and 2.2 kb in all
GUS−, ap24+ T1 plants indicated that both these integration events
were linked in one ap24 locus. Two other integration events in the
T0 plant (Fig. 4A) corresponding to the junction fragments of 8.0-kb
and 5.5-kb appear to be linked to the hph locus.
In the co-transformed line CoTosm4, out of 40 T1 plants analysed
by GUS histochemical staining, 31 plants were GUS+ and nine were
GUS− (Table 3). Southern blot analysis of nine GUS− plants with the
ap24 probe revealed that eight plants had two copies of the ap24
gene (Fig. 6A). Two copies of the ap24 gene were linked to each
other but were unlinked to the single SMG (hph/gus) locus. Among
the 56 T1 plants analysed in the line CoTosm13, eight plants were
GUS− (Table 3). PCR analysis of the eight GUS− plants with the ap24
primers showed that four plants were ap24+ (data not shown). The
four ap24+ plants and two ap24− plants were analysed by Southern
blotting with the ap24 probe. All four ap24+ plants harboured two
copies of the ap24 gene (Fig. 6B). The two copies of the ap24 gene
were linked to each other and unlinked to the three copies of the
hph gene.

6. R. Sripriya et al. / Plant Science 180 (2011) 766–774 771
Table 3
Segregation analysis of ten co-transformed rice plants obtained by transformation with Agrobacterium tumefaciens C58C1 Rifr
(pGV2260::pSSJ1, pBin19 nptII-ap24) to
determine the number of GUS+
/GUS−
loci. The gus gene is linked to the hph gene (SMG) in T-DNA of the cointegrate plasmid pGV2260::pSSJ1.
T0 lines T-DNAcopynumber No. of T1 plants analysed No. T1 plants Segregation ratio GUS+
/GUS− 2
value hph/gus loci
hph ap24 GUS+
GUS−
CoTosm1 1 5 13 10 3 3:1 0.533 1
CoTosm3 2 4 34 25 9 3:1 0.039 1
CoTosm4 1 2 40 31 9 3:1 0.233 1
CoTosm5 1 1 40 26 14 3:1 1.9 1
CoTosm6 2 1 40 35 5 3:1 3.7 1
CoTosm7 1 3 40 28 12 3:1 0.433 1
CoTosm8 1 1 20 14 6 3:1 0.2 1
CoTosm10 1 1 40 32 8 3:1 0.53 1
CoTosm13 3 3 56 48 8 3:1 3.42 1
CoTosm14 1 2 28 28 – 4:0 0 NE
NE, the locus number could not be estimated due to lack of segregation.
All GUS− plants which were identified to harbour the ap24 gene
by Southern blotting (Figs. 5 and 6) were subjected to Southern blot
analysis with the hph probe to confirm the elimination of the SMG.
The T1 plants 1-1 and 1-2 of CoTosm1 (Fig. 7A), 4-1, 4-2, 4-3, 4-4,
4-6, 4-7, 4-8 and 4-9 of CoTosm4 (Fig. 7A), 3-1, 3-2, 3-3, 3-4, 3-5,
3-7 and 3-8 of CoTosm3 (Fig. 7B) and 13-1, 13-4, 13-5 and 13-6 of
CoTosm13 (Fig. 7C) did not display hybridization to the hph gene.
In the co-transformed lines CoTosm5, 6, 7, 8 and 10, the GOI was
inferred as linked to the SMG since none of the GUS− plants showed
Fig. 5. Southern blot analysis to check the presence of GOI (tobacco ap24) in the
GUS−
T1 plants of the co-transformed lines CoTosm1 and 3. Plant DNA (2.5 ␮g)
digested with PstI was loaded in each lane. The blot was hybridized to the [␣-
32
P]dCTP-labelled ap24 probe. (A) Analysis of the T1 plants of the line CoTosm1.
Lanes 1–3; DNA from GUS−
T1 plants1-1 to 1-3; lanes 4 and 5, GUS+
T1 plants as
positive control; T0, DNA from the CoTosm1 T0 plant; lane C, DNA from the untrans-
formed control plant. (B) Analysis of the GUS−
T1 plants of the line CoTosm3; lane
C, DNA from the untransformed control plant; lane T0, DNA from the CoTosm3 T0
plant; lanes 1–8, GUS−
T1 plants 3-1 to 3-8.
hybridization to the ap24 probe or displayed amplification with the
ap24 gene primers (data not shown). Hence, SMG elimination was
not feasible in the lines CoTosm5, 6, 7, 8 and 10. Segregation of the
GOI and SMG was accomplished in four (CoTosm1, 3, 4 and 13) out
of ten primary transformants that were taken up for marker elimi-
nation through segregation in the T1 generation. CoTosm14, which
harboured a single copy of the cointegrate T-DNA and expected to
yield GUS+ and GUS− plants in a 3:1 ratio, unexpectedly did not
yield any GUS− plants among 28 T1 plants. It is not clear whether
this plant carried additional truncated T-DNAs with the gus
gene.
Fig. 6. Southern blot analysis to check the presence of GOI (tobacco ap24) in the
GUS−
T1 plants of the co-transformed lines CoTosm4 and 13. Plant DNA (2.5 ␮g)
digested with PstI was loaded in each lane. The blot was hybridized to the [␣-
32
P]dCTP-labelled ap24 probe. (A) Analysis of the T1 plants of the line CoTosm4.
Lanes 1–9, DNA from GUS−
T1 plants 4-1 to 4-9; lanes 10 and 11, GUS+
T1 plants. C,
DNA from the untransformed control plant. (B) Analysis of the T1 plants of the line
CoTosm13. Lanes 1–6, DNA from GUS−
T1 plants 13-1 to 13-6. Lane T0, DNA from
the CoTosm13 T0 plant; lane C, DNA from the untransformed control plant.

7. 772 R. Sripriya et al. / Plant Science 180 (2011) 766–774
Fig. 7. Southern blot analysis to confirm the elimination of the SMG (hph) in the
T1 plants of the lines CoTosm1, 3, 4 and 13. Plant DNA (2.5 ␮g) was digested with
EcoRI and loaded in each lane. The blot was hybridized to the [␣-32
P]dCTP-labelled
hph probe. (A) Analysis of the T1 plants of the lines CoTosm1 and 4. Lane 1, DNA
from GUS+
T1 plant of the line CoTosm4, as a positive control; lanes 2 and 3, DNA
from GUS−
, ap24+
T1 plants 1-1 and 1-2 of the line CoTosm1; lanes 4–11, DNA from
GUS−
, ap24+
T1 plants 4-1, 4-2, 4-3, 4-4, 4-6, 4-7, 4-8 and 4-9 of the line CoTosm4.
(B) Analysis of the T1 plants of the line CoTosm3. Lanes 1–7, DNA from GUS−
, ap24+
T1 plants 3-1, 3-2, 3-3, 3-4, 3-5, 3-7 and 3-8 of the line CoTosm3; lane C, DNA from
the untransformed control plant; T0, DNA from the CoTosm3 T0 plant. (C) Analysis
of the T1 plants of the line CoTosm13. Lanes 1–6, DNA from GUS−
plants 13-1, 13-2,
13-3, 13-4, 13-5 and 13-6; lane C, DNA from the untransformed control plant; T0,
DNA from the CoTosm13 T0 plant as a positive control.
4. Discussion
A high co-transformation frequency and segregation of the T-
DNAs with the GOI and SMG in the T1 generation are prerequisites
for the generation of SMG-free transgenic plants. Although co-
transformation has been achieved by particle-bombardment, the
efficiency of SMG elimination is very low due to complex and linked
integrations [37–40]. Agrobacterium-mediated co-transformation
with simple integration patterns is more suitable for SMG elim-
ination. Co-transformation frequency of two T-DNAs from two
different Agrobacterium strains (mixed strain method) is equal to
the product of the probability of two independent transformation
events, leading to low co-transformation frequencies [8]. Compar-
ative co-transformation studies using two Agrobacterium strains
with independent binary vectors and one Agrobacterium strain with
two compatible binary vectors revealed that co-transformation
of two T-DNAs from a single strain of Agrobacterium is more
efficient [9,18,41]. A single strain of Agrobacterium with two com-
patible binary plasmids yielded a 50% co-transformation efficiency
and a 50% segregation frequency [14]. It was concluded that in
order to produce an SMG-free transgenic plant it would be nec-
essary to generate and screen four times more transgenic plants in
comparison to a normal transformation experiment wherein SMG
elimination is not intended [14]. Hence, further improvement of
co-transformation frequency is needed for effective generation of
SMG-free transgenic plants.
Co-transformation using two T-DNAs (twin T-DNAs) in one
binary vector was used to improve co-transformation frequency
[9]. A two T-DNA binary vector was developed, in which the first
T-DNA was delimited by the A. tumefaciens borders and the second
T-DNA was delimited by the A. rhizogenes borders [16]. This sys-
tem yielded 90% co-transformation of two T-DNAs. In a twin T-DNA
binary vector system used for co-transformation of soybean, a co-
transformation frequency of 70% was achieved [42]. The large size
of the twin T-DNA binary vectors, the complexity in the construc-
tion of the vectors and the high frequency of ‘linked co-delivery’
of the GOI and the SMG along with the intervening non-T-DNA
sequences are the major limitations of this method.
The molar ratio of the T-DNA with the SMG and the T-DNA
with the GOI influences the co-transformation frequency. In parti-
cle bombardment mediated gene delivery, higher gene ratios of the
GOI (non-selected gene) over the SMG enhanced co-transformation
efficiency [43]. In a twin T-DNA vector, placing the GOI in a shorter
T-DNA and the SMG in a T-DNA twice longer than the former
increased co-transformation of the unselected GOI [21]. By placing
the GOI between the regular T-DNA borders and repositioning the
SMG in the vector back bone, efficiency of SMG elimination could be
increased [44]. Agrobacterium-mediated co-transformation involv-
ing two T-DNAs on different plasmids offers an advantage of
altering the ratio of the SMG T-DNA to that of the GOI T-DNA.
Arabidopsis roots were co-transformed with two strains of Agrobac-
terium, one carrying a cointegrate vector (one copy per cell) and
the other carrying a binary vector (2–3 copies per cell) [45]. Co-
transformation frequency was 47% when initial selection was based
on the low copy cointegrate vector, and the co-transformation fre-
quency decreased to 21% when the selection was based on the high
copy number binary vector. In the single-strain method involving
a cointegrate vector and a binary vector, the co-transformation
efficiency of the non-selected T-DNA (carrying nptII) was high
(56–74%) when the non-selected GOI was placed in a multi-copy
binary plasmid [22]. The initial selection was based on the hph gene
in a single-copy cointegrate vector.
Using a single-copy cointegrate vector and a binary vec-
tor with pVS replicon (3 copies/Agrobacterium cell, Table 1) in
one Agrobacterium strain, a co-transformation frequency of 20%
was observed and SMG elimination was achieved in 10% of
the primary transformants [24]. In the present study, a broad
host-range SMG-free binary vector pBin19 nptII was constructed
and used for co-transformation experiments. By placing the GOI
(ap24) in the T-DNA of pBin19 nptII with the RK2 replicon (12
copies/Agrobacterium cell, Table 2) and the SMG in a single-copy
cointegrate vector, an improved co-transformation efficiency of
86% was achieved. Daley et al. [14] proposed a copy number of
10–14 for RK2 replicon-based plasmids in Agrobacterium. Haj-
dukiewicz et al. [25] state that plasmids with pVS replicon exist
as three copies per Agrobacterium cell. However, in a recent report,
Oltmanns et al. [46] have reported that binary plasmids with RK2
and pVS replicons maintain a copy number of seven to 10 per cell.
We made a quantitative estimate of the binary plasmid copy num-
ber by Southern blotting and liquid scintillation counting in four
independent Agrobacterium colonies. Our results show that the
copy number of RK2 and pVS replicon-based binary plasmids is
three and 12, respectively, with respect to the cointegrate Ti plas-
mid, which is inferred to have one copy per Agrobacterium cell.

8. R. Sripriya et al. / Plant Science 180 (2011) 766–774 773
pBin19 nptII is a relatively small SMG-free binary vector with a
convenient multiple cloning site to clone a GOI.
In comparison to our earlier report in which we used a
pCAMBIA-based SMG-free binary plasmid (3 copies/Agrobacterium
cell) with the GOI [24], the use of an SMG-free pBin19 nptII (12
copies/Agrobacterium cell) harbouring the GOI in the present work
increased co-transformation frequency from 20% to 86%. When ten
of the co-transformed plants were forwarded to the T1 generation,
SMG elimination was achieved in four co-transformed plants in
which SMG and GOI were unlinked.
In an independent study of successive co-transformation of
transgenic rice harbouring SMG-free chitinase (chi11) gene [24]
with A. tumefaciens C58C1 (pG2260::pSSJ1, pBin19 nptII-ap24),
aimed at stacking marker-free chi11 transgenic plants with
the ap24 gene, we found that 12 of the 18 transgenic plants
(67%) were co-transformed [47]. Thus, in the single strain-based
Agrobacterium-mediated co-transformation, placing the GOI in an
RK2-based multi-copy binary vector significantly improved the co-
transformation efficiency.
Acknowledgements
We thank Dr. Leo S. Melchers, MOGEN International NV,
Netherlands, for ap24. We thank Dr. Stanton B. Gelvin, Purdue
University, USA for pGA472 and pVK102-Sal13b plasmids. RS is
thankful to the Department of Biotechnology (DBT) and University
Grants Commission (UGC), Government of India for her Research
Fellowships. DBT, Govt. of India is thanked for research funding.
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