J Appl Genet 51(3), 2010, pp.

243257

Original article

Generation of marker-free Bt transgenic indica rice

and evaluation of its yellow stem borer resistance

S. Kumar, L. Arul, D. Talwar

National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi-110 012, India

Abstract. We report on generation of marker-free (clean DNA) transgenic rice (Oryza sativa), carrying mini-
mal gene-expression-cassettes of the genes of interest, and evaluation of its resistance to yellow stem borer
Scirpophaga incertulas (Lepidoptera: Pyralidae). The transgenic indica rice harbours a translational fusion of
2 different Bacillus thuringiensis (Bt) genes, namely cry1B-1Aa, driven by the green-tissue-specific phosphoenol
pyruvate carboxylase (PEPC) promoter. Mature seed-derived calli of an elite indica rice cultivar Pusa Basmati-1
were co-bombarded with gene-expression-cassettes (clean DNA fragments) of the Bt gene and the marker hpt
gene, to generate marker-free transgenic rice plants. The clean DNA fragments for bombardment were obtained
by restriction digestion and gel extraction. Through biolistic transformation, 67 independent transformants were
generated. Transformation frequency reached 3.3%, and 81% of the transgenic plants were co-transformants. Sta-
ble integration of the Bt gene was confirmed, and the insert copy number was determined by Southern analysis.
Western analysis and ELISA revealed a high level of Bt protein expression in transgenic plants. Progeny analysis
confirmed stable inheritance of the Bt gene according to the Mendelian (3:1) ratio. Insect bioassays revealed com-
plete protection of transgenic plants from yellow stem borer infestation. PCR analysis of T2 progeny plants re-
sulted in the recovery of up to 4% marker-free transgenic rice plants.

Keywords: biolistic transformation, Bacillus thuringiensis, Bt transgenic rice, gene-expression-cassette, marker-free

transgenic, Oryza sativa, Scirpophaga incertulas, yellow stem borer.

Introduction spite screening of more than 30 000 accessions of

Genetic transformation methods have made it pos-
sible to introduce novel genes into plants across
barriers of genomic and sexual incompatibilities
(Lucca et al. 2001; Ho et al. 2006). Transgenic rice
plants carrying desirable traits, such as enhanced
resistance to insect pests, diseases or herbicide tol-
erance, are becoming available (Tu et al. 2000a;
Khanna and Raina 2002; Ho et al. 2006). Stem
borers are considered to be the most destructive
pests of rice, causing estimated losses of 530% of
its total yield (Oerke et al. 1994). Stem borer lar-
vae bore into the plant and damage it while feeding
inside the stem. Dead heart and white head are
common symptoms of stem borer infestation. De-
rice at IRRI, Philippines, no resistance source for
stem borer has been identified (Khan et al. 1991;
Teng and Revilla 1996). Chemical control of stem
borers is difficult, often ineffective, and leads to
environmental pollution (Ho et al. 2006). Genetic
transformation of crop plants with genes from the
soil bacterium Bacillus thuringiensis, i.e. Bt
genes, is an important technological advancement
towards development of environment-friendly in-
sect-pest management strategies (Kumar et al.
2008). Bt proteins (also known as Cry proteins)
are highly toxic to certain groups of insects. How-
ever, as is the case with other insecticides, insects
will eventually develop resistance to Bt toxins
(Ranjekar et al. 2003). Therefore, concerted ef-

Received: January 25, 2009. Accepted: October 27, 2009.

Correspondence: S. Kumar, Division of Crop Improvement, Indian Grassland and Fodder Research Institute, Jhansi-284 003,
India; e-mail: sureshkumar3_in@yahoo.co.uk
244 S. Kumar et al.
forts must be made to delay insect resistance de-
velopment against Bt toxins. Many strategies to
manage insect resistance to Bt toxins have been
proposed (Cohen et al. 2000; Shelton et al. 2002).
There is widespread agreement that the
high-dose/refuge strategy is the most promising
and practical approach to prolong the effective-
ness of Bt toxin (Frutos et al. 1999; Cohen et al.
2000; Ranjekar et al. 2003). Another important
recommendation is to employ 2 or more insecti-
cidal genes with different binding sites and/or dif-
ferent modes of action (Frutos et al. 1999;
Gatehouse 2008). Currently, several protocols for
transformation of indica rice by
Agrobacterium-mediated methods (Hiei et al.
1994; Rashid et al. 1996; Cheng et al. 1998;
Khanna and Raina 1999) and biolistic particle de-
livery (Christou and Ford 1995; Ghareyazie et al.
1997; Khanna and Raina 2002) are available.
A major advantage of biolistic transformation is
that a wide variety of tissues can be used as targets
for gene delivery. Moreover, this method can be
used for co-transformation with multiple genes
(Hadi et al. 1996; Chen et al. 1998; Romano et al.
2003; Maqbool et al. 2001). When combined with
the clean DNA transformation method, it pro-
duces transgenic plants with 1-2 transgene copies,
resulting in stable expression of the transgene over
several generations (Fu et al. 2000; Loc et al.
2002).
Direct DNA transfer, as well as
Agrobacterium-mediated transformation using
multicopy binary vectors, may lead to the integra-
tion of vector backbone sequences into the host
genome (Knonov et al. 1997; Afolabi et al. 2004;
Altpeter et al. 2005; Lange et al. 2006; Lee and
Gelvin 2008). The vector backbone integration
may exert undesirable effects in cis as well as pro-
motes transgene rearrangements (Artelt et al.
1991). As a rule, all the vector backbone se-
quences were removed before microinjecting for-
eign DNA into animal eggs or embryos (Palmiter
and Brinster 1986), while whole-plasmid bom-
bardment is a common practice for plant transfor-
mation. In case of microprojectile-mediated direct
DNA transfer, the vector backbone sequence of
the linear or super-coiled plasmid gets integrated
into the host plant genome along with the
transgene(s). Even after Agrobacterium-mediated
transformation, initially thought to generate only
clean transgenic events, up to 33% transformants
have T-DNA exceeding the left border repeat
(Wenck et al. 1997).
In Agrobacterium-mediated transformation the
use of whole plasmid is obligatory, but in biolistic
transformation the vector backbone serves no spe-
cific purpose. Fu et al. (2000) investigated
transgene integration and expression patterns in
rice, following microprojectile-mediated direct
DNA transformation using minimal transgene cas-
settes (promoter open reading frame termina-
tor). They observed simple integration events
with low-copy number and low frequency of
transgene re-arrangements in transgenic plants.
Higher levels of transgene product accumulation
were observed in transgenic rice plants derived
from minimal gene cassette transformations (Loc
et al. 2002). Several plant species, including rice
(Breitler et al. 2002; Xia et al. 2006; Zhao et al.
2007a), potato (Romano et al. 2003), wheat (Yao
et al. 2006; Shi et al. 2007), maize (Huang et al.
2004), and grapevine (Vidal et al. 2006), have al-
ready been successfully transformed by using
minimal gene cassettes via particle bombardment.
Co-bombardment with 2 gene-cassette fragments
allows integration of transgenes at various loci.
Such integrations facilitate separation of a marker
gene from the gene of interest in the segregating
progenies (Zhao et al. 2007a; Darbani et al. 2007).
Tissue-specific expression of Bt genes is an-
other strategy to manage resistance development
in insects and to address certain biosafety con-
cerns as well. The light-inducible phospho-
enolpyruvate carboxylase (PEPC) promoter has
been used for this purpose in transgenic plants
(Koziel et al. 1993; Ghareyazie et al. 1997; Hagh
et al. 2009). Enhanced stem borer resistance was
observed by Ghareyazie et al. (1997) in indica rice
cultivar Tarom Molaii transformed with cry1Ab
under the regulatory control of the PEPC pro-
moter. The gene was expressed in leaf blades but
not in dehulled mature grains, and provided effec-
tive resistance to first-instar larvae of stem borers.
Datta et al. (1998) used the cry1Ab gene driven by
green-tissue-specific and/or pith-specific promot-
ers for microprojectile bombardment of rice. A
high level of Cry1Ab protein expression in green
tissues (leaves and stem) resulted in enhanced re-
sistance to yellow stem borer (YSB), Scirpophaga
incertulas (Lepidoptera: Pyralidae), and mini-
mized the expression of the toxin in seeds and
other tissues.
A selectable marker gene for antibiotic or her-
bicide resistance is generally incorporated into the
transgenic plants for efficient and successful se-
lection of transgenics. However, the selectable
markers have potential pitfalls (Bai et al. 2008),
including public concerns raised because of their
unnecessary presence in transgenic plants
 Marker-free Bt transgenic indica rice
(Ebinuma et al. 2001; Miki and McHugh 2004;
Afolabi 2007). To obtain marker-free transgenic
plants, several systems (Ac transposable element,
homologous recombination, site-specific recom-
bination) for deleting selectable markers have
been developed (Goldsbrough et al. 1993; Zubko
et al. 2000; Sreekala et al. 2005; Bai et al. 2008).
Most of these systems require extra genetic ma-
nipulation efforts (in addition to being time- and
labour-intensive) for deletion of the selectable
marker from transgenic plants. Recently, the
Cre/LoxP-based auto-excision system, controlled
by tissue-specific promoter, has been used for de-
velopment of marker-free transgenic plants (Bai
et al. 2008). Earlier on, Mlynarova et al. (2006)
and Luo et al. (2007) also used the
Cre/LoxP-based system for deletion of the
transgene to avoid pollen- and seed-mediated
transgene flow. This approach, again, requires ad-
ditional genetic engineering and shows a lower
frequency of marker excision along with partial
auto-excision events. To improve auto-excision
frequency and to avoid chimeras, a strong tis-
sue-specific promoter needs to be employed (Bai
et al. 2008).
The present study was undertaken to develop
marker-free (clean DNA) Bt transgenic indica
rice plants by using biolistic transformation. We
attempted to overexpress 2 dissimilar Bt genes
(namely cry1B and cry1Aa), driven by a tis-
sue-specific promoter (rather than a constitutive
promoter), with a view to delay the development
of resistance in insects against this insecticidal
system. As a result, marker-free transgenic rice
plants were obtained, based on the co-transforma-
tion and segregation approach.
Materials and methods
Plant material
Mature and healthy seeds of Pusa Basmati-1 (an
indica rice cultivar highly responsive to regenera-
tion and transformation protocols) were collected
from a pure-line seed maintenance plot at the In-
dian Agricultural Research Institute, New Delhi.
Next, they were dehusked manually and used for
callus induction. The dehusked seeds were sur-
face-sterilized and plated on MS medium
(Murashige and Skoog 1962) for callus induction
on 90-mm Petri plates. The medium was solidified
by using 3 g L1 Phytagel (Sigma, USA). The
plates were incubated in the dark at 252oC for
5 days. Calli induced from the scutellar region of
245
mature seeds were used as explants for
microprojectile bombardments. The details of me-
dia used for tissue culture and transformation are
given in Table 1. The tissue culture and regenera-
tion protocol of Khanna et al. (1997) was adopted
with minor modifications.
Vector construction and
gene-expression-cassette
The green-tissue-specific PEPC promoter (driv-
ing the expression of phosphoenolpyruvate
carboxylase gene in maize) was obtained from
Novartis Agribusiness Biotechnology Research
Inc., USA. The synthetic PEPC promoter (re-
ceived in pCIB8759) was digested with
HindIII-BamHI to take out a 2.3-kb fragment of
the promoter. The promoter fragment was cloned
into pGEM-7Zf (+) (Promega, Madison, USA,
Accession No. X65310) at HindIII-BamHI sites,
to construct the pSKPEPC-7Z vector. A
codon-optimized, synthetic, translational fusion
cry1B-1Aa gene (obtained in pC1300-Ubi-
1B-1Aa) was received from Dr. R Frutos, CIRAD,
France (constructed following Bohorova et al.
2001) and used for construction of the Bt
gene-expression-cassette. Translational fusion of
cry1B-1Aa, along with the nos terminator (4.2 kb),
was excised by using BamHI. The fragment was
cloned into pSKPEPC-7Z at the BamHI site, to
construct the pSKPBA-7Z vector (Figure 1A).
GUSPlus and hygromycin-resistance gene-cas-
settes were excised from pCAMBIA- 1305.1
(GenBank accession no. AF354045). The
hygromycin-resistance gene (hpt) is driven by a 2x
CaMV35S promoter and terminates by CaMV35S
polyA. The gene-cassette (2.0 kb) is flanked by
HindIII and SacII sites (Figure 1B). The GUSPlus
in pCAMBIA-1305.1, originally isolated from
Staphylococcus sp., was codon-optimized for ex-
pression in plants (http://www.cambia.org/
daisy/cambialabs/3850.html). The gene is driven
by the CaMV35S promoter, contains an intron
from the castor bean catalase gene, and is termi-
nated by nos polyA. The gene-cassette (2.9 kb) is
flanked by SphI sites (Figure 1C). Two or three
gene-expression-cassettes (clean DNA fragments)
were used for co-bombardments: one carried the
gene of interest (Bt gene), while others carried
marker genes (hpt and GUSPlus).
Clean DNA preparation
Gene-cassette fragments were isolated by using
restriction digestion of the source plasmid DNA,
followed by agarose gel electrophoresis and gel
extraction of the fragment of interest (Kumar et al.
246 S. Kumar et al.

Table 1. Tissue culture media used for rice transformation

Medium
Callus induction medium
Selection medium
Pre-regeneration medium
Regeneration medium
Rooting medium
Hardening medium
Potting medium
The media were solidified (wherever necessary) with Phytagel (3.0 g L1) and autoclaved at 121oC for 18 min
Composition (mg L1)
MS + 2,4-D (2.0)
MS medium + hygromycin (50)
MS + NAA (2.5) + kinetin (0.1) + 2,4-D (0.5)
MS + BAP (2.0) + NAA (0.5) + Kinetin (0.5)
1/2 MS with full concentration of iron
liquid 1/10 MS medium without sucrose
Agropeat
Application
callus induction from seeds
selection of transformed cells/calli
pre-regeneration treatment of selected
embryogenic calli
shoot organogenesis from treated embryogenic
calli
rooting from regenerated shoots
hardening of regenerated plants
transplantation of regenerated plants in pots

Figure 1. Linear map of gene-expression-cassettes. (A) Translational fusion cry1B-1Aa gene-cassette in pSKPBA-7Z.
Horizontal line indicates the position of the probe used for Southern hybridization. (B) Hygromycin-resistance (hpt)
gene-cassette in pCAMBIA1305.1. (C) GUS gene-cassette. Restriction enzymes (red) flanking the gene-cassette were
used for isolation of the gene-cassettes for clean DNA transformation.

2006). Restriction enzymes, flanking the
gene-cassettes (Figure 1), were used for restriction
digestion of the plasmid DNA. The desired
gene-cassette fragment DNA was eluted from
agarose gel (1%) by using a QIAEX II gel extrac-
tion kit (QIAGEN GmbH, Germany).
Genetic transformation
From the seeds germinated on MS medium, 2530
five-day-old scutellar calli were excised and
closely arranged within a circular area 2 cm across
at the centre of a 90-mm plate containing freshly
prepared MS medium. Gold particles (0.6 mm;
Bio-Rad Laboratories, USA) were coated with
clean DNA fragments by using the procedure de-
scribed by Klein et al. (1988) with minor modifi-
cations. The clean DNA fragments for the gene of
interest and marker gene (2.5 mg, in a molar ratio
of 4:1 of Bt gene:hpt or 4:1:1 of Bt gene:hpt:
GUSPlus) were mixed and used for precipitation
on the gold particles. A clean DNA fragment for
the GUSPlus gene was used in a parallel experi-
ment to monitor the gene delivery process. The
biolistic apparatus (He-driven PDS-1000/BioRad)
was used for bombardment of the calli at a fixed
distance (9 cm) from the microcarrier launch as-
sembly. The calli were bombarded once under
vacuum (25 mm Hg) by using 900 psi rupture
discs. The bombardment process was carried out
under aseptic conditions, and the protocol of
Khanna et al. (1997) was adopted with minor mod-
ifications. A total of 1440 calli, arranged on
52 plates, were bombarded in a set of experiments.
Bombarded calli were incubated on the same me-
dium in the dark for 24 h, and then transferred to a
selection medium containing hygromycin
(50 mg L1). One-third of each callus was sub-
merged in the selection medium while transfer-
ring. The plates were incubated in the dark for
 Marker-free Bt transgenic indica rice 247

21 days. The small secondary calli that appeared
from the selected calli (Figure 2B) were trans-
ferred to the freshly prepared selection medium
and subjected to second and third rounds of selec-
tion, each of 21 days.
ened by growing in liquid 1/10 MS medium without
sugar for 14 days. The hardened plants were then
transplanted into pots filled with Agropeat and
transferred to a phytotron (National Phytotron Fa-
cility, I.A.R.I., New Delhi) for their growth till

Figure 2. (A) Transient GUS gene expression in bombarded calli 24 h after bombardment (microscopic view), indicating
proper DNA coating on gold particles and normal delivery of the gene-cassettes into the calli. (B) Secondary calli
(marked with arrows) emerging from calli selected on hygromycin-containing medium. (C) Shoot organogenesis from
hygromycin-resistant calli. (D) Putative transgenic plants grown in a phytotron.

The hygromycin-selected calli were trans-
ferred to a pre-regeneration medium and incu-
bated in the dark for 14 days. The calli were next
transferred to a regeneration medium (Alam et al.
1998) and incubated at 25 2oC under 16-h
photoperiod (using Philips F40/CW fluorescent
tubes, 110130 mM/m2/s PAR). When the shoots
reached 45 cm in height, they were transferred to
the rooting medium (Ghareyazie et al. 1997) in
culture tubes for root organogenesis. The plants
with well-developed roots and shoots were hard-
maturity. Thus the Bt transgenic rice plants and
their progenies were grown under controlled con-
ditions: initially in the laboratory and later in the
phytotron.
PCR screening of transgenic plants
Genomic DNA from putative transgenic and un-
transformed control plants for PCR screening was
isolated by using a simplified protocol standard-
ized in our laboratory. A small piece (1 cm long) of
248 S. Kumar et al.
young leaf tissues was ground in 200 L of AP1
buffer (QIAGEN) with a disposable plastic pestle,
specially used for grinding soft tissues in micro-
centrifuge tubes. A few grains of acid-washed,
sterilized sand were added to help grinding of the
tissues. The ground sample was incubated at 65oC
for 15 min. To this, 65 L of AP2 buffer
(QIAGEN) was added and incubated on ice for
5 min. The content was centrifuged and 0.6 vol-
umes of isopropanol were mixed with the
supernatant. After centrifugation the pellet was
washed with 70% ethanol, air-dried, and dissolved
in 30 L of sterilized double-distilled water.
Gene-specific primers (cry1B: forward 5-CGA
GAT CAT CAA CGC TGT GTC C-3, reverse
5-CAG CCT GAG ACA CGT CCA TCA AC-3;
hpt: forward 5-ACA CAG CCA TCG GTC
CAG-3, reverse 5-CGA CCT GAT GCA GCT
CTC-3) were used for PCR amplification of a part
of the transgene(s) to screen the putative transgen-
ic plants. These primers target to amplify 524-bp
and 897-bp DNA sequences from the coding re-
gions of cry1B and hpt, respectively. The
PCR-amplified products were subjected to
agarose gel (1.2%) electrophoresis and visualized
under UV light.
T1 and T2 progenies of Bt transgenic plants
were analysed using the gene-specific primers for
PCR amplification. The available T1 progeny
(1520 plants) of selected T0 plants was analysed
to determine the segregation ratio, and T2 proge-
nies were analysed to select marker-free Bt trans-
genic rice plants.
RT-PCR and Southern analysis
Transcription of the integrated Bt gene was tested
by analysing reverse transcription of mRNA. To-
tal RNA was isolated from 100 mg of young leaf
tissues by using RNeasy Plant Mini Kit
(QIAGEN). Reverse transcriptase-PCR was car-
ried out using 50 g of total RNA and OneStep
RT-PCR Kit (QIAGEN), following the procedure
suggested by the manufacturer.
For Southern blot analysis, genomic DNA was
isolated from young leaf tissues of plants by using
DNeasy Plant Mini Kit (QIAGEN) and quantified
by running a standard DNA marker
(l DNA/HindIII digest) along with the test DNA
samples. About 15 g of genomic DNA (equiva-
lent to 100 copies of the rice genome) of transgen-
ic and untransformed control plants was digested
with SacII, which cuts the Bt gene insert once at
the 5 end of the coding region (Figure 1A). The
plasmid used for isolation of the Bt gene clean
DNA fragment was also digested with SacII, to be
used as a positive control. Digested samples were
electrophoresed in a 0.8% agarose gel and trans-
ferred to a positively charged nylon membrane
(Amersam) for Southern blot. DNA was
crosslinked to the membrane by UV exposure for
90 s. Radio-labelled probes were prepared by us-
ing HexaLabel Plus DNA labelling kit (MBI
Fermentas, Lithuania). PCR-amplified internal re-
gion of the cry1B gene was used as a template for
generation of gene-specific radiolabelled (with
a-32P dCTP) DNA probes.
ELISA and western analysis
Total soluble protein was extracted from leaves of
transgenic and untransformed rice plants. Direct
antigen-coated ELISA (DAC-ELISA) and west-
ern blotting were used for detection of Bt proteins
in transgenic plants. Soluble protein was estimated
according to Bradford (1976). The extracted pro-
tein (200 mL sample) was coated with sodium car-
bonate coating buffer in wells of ELISA plate.
Polyclonal rabbit antibody specific for the Bt pro-
tein, alkaline phosphatase-conjugated goat
anti-rabbit IgG as the secondary antibody, and
p-nitrophenyl phosphate (PNPP) as substrate for
alkaline phosphatase were used for DAC-ELISA.
Various dilutions of the purified Bt protein were
used as positive controls, and the protein extracted
from an untransformed plant as negative controls.
For western analysis, protein was extracted
with sample buffer, following the procedure of
Khanna and Raina (2002). The samples were
electrophoresed on 10% SDS-PAGE and trans-
ferred to PVDF membrane (Amersham). Using
the polyclonal antibodies, alkaline phosphatase-
conjugated IgG, and NBT/BCIP as a substrate, the
Bt protein was detected in western blotting. West-
ern analysis was performed for the T0 and T2
plants showing Bt titres of more than 0.2%.
Insect bioassays
YSB moths were collected from unprotected
paddy fields of the Rice Research Station,
Kapurthala (Punjab), a hot-spot area of rice stem
borers. Moths were collected and reared as de-
scribed by Khanna and Raina (2002). The freshly
hatched neonate larvae were used in the cut-stem
or whole-plant bioassays. Insect bioassays were
conducted at vegetative and reproductive stages of
plants, following the procedure adopted by
Khanna and Raina (2002). Selected PCR- and
 Marker-free Bt transgenic indica rice
Southern-positive T0 plants (showing normal mor-
phology and growth) and their progenies were
evaluated for YSB resistance. Non-transgenic re-
generated plants and untransformed control plants
were also included in the cut-stem bioassay. Mor-
tality and growth rate (in terms of the increase in
body weight) of larvae were observed in the vege-
tative stage cut-stem bioassay, whereas the ap-
pearance of typical symptoms (dead heart and
white head) of YSB infestation was recorded dur-
ing the reproductive stage whole-plant bioassay.
Selected T0 plants and their T2 progenies, showing
high Bt titres (>0.20 and >0.30%, respectively),
were evaluated by the whole-plant bioassay. Indi-
vidual tillers/plants of T0 and T2 generations were
evaluated in 3-replication assays. Cut stems were
evaluated after 4 days of infestation, while whole
plants were examined 25 days after infestation.
Results and discussion
Vector construction and
gene-expression-cassette
Cloning of the 2.3-kb synthetic PEPC promoter in
the 3.0-kb pGEM-7Zf (+) plasmid resulted in
5.3-kb pSKPEPC-7Z constructs. The cloning of
the 4.2-kb translational fusion cry1B-1Aa gene
fragment at the BamHI site resulted in a 6.5-kb Bt
gene-expression-cassette with the PEPC promoter
and nos terminator in the 9.5-kb pSKPBA-7Z
plasmid vector.
Clean DNA preparation
Restriction digestion of pSKPBA-7Z with MluI
and SmaI restriction enzymes resulted in the sepa-
ration of a 6.5-kb clean DNA fragment of the Bt
gene-expression-cassette. This yielded 63.5% of
DNA containing the gene of interest (Bt gene).
The hygromycin-resistance (hpt) gene-cassette
was excised from pCAMBIA-1305.1. The flank-
ing HindIII and SacII sites separated a 2.7-kb frag-
ment of hygromycin-resistance gene-cassette.
This yielded 44.5% of the DNA containing the
fragment of interest (hpt gene-cassette). Due to the
limitation of restriction sites, 265 bp upstream
from the promoter and 399 bp downstream from
the terminator were included in the gene-cassette
fragment (Figure 1B). GUSPlus gene-cassette was
also excised from pCAMBIA-1305.1. In this case,
the flanking SphI sites generated a 3.2-kb
gene-cassette fragment. Again, due to the limita-
tion of restriction sites, the inclusion of 220 bp up-
249
stream from the promoter and 129 bp downstream
from the terminator was inevitable (Figure 1C).
Here the DNA yield for the fragment of interest
was only 37.5% of the whole pCAMBIA-1305.1
plasmid.
Genetic transformation of rice
Calli induced from the scutellar region of mature
seeds were bombarded with 23 gene-expres-
sion-cassettes (clean DNA fragments) by using a
biolistic system. Clean DNA fragments were pre-
cipitated on 0.6-m gold particles in a molar ratio of
4:1 or 4:1:1 for the gene of interest (Bt gene) and
the marker genes (hpt and GUSPlus). A similar
bombardment strategy was adopted earlier by
Zhao et al. (2007a). One of the major advantages
of particle bombardment-mediated transformation
has been its ability to transform plants simulta-
neously with several (35) different constructs
(Maqbool and Christou 1999; Maqbool et al.
2001; Loc et al. 2002). The higher molar ratio
(4:1) of the Bt gene used for co-bombardments re-
sulted in a maximum (81%) of the transgenic
plants harbouring both the transgenes. The use of a
higher ratio of the Bt gene to increase the probabil-
ity of co-transformation was as suggested by Tang
et al. (1999). Fu et al. (2000) used a molar ratio of
3:1 for the gene of interest and plant-selectable
marker. Similar co-bombardment strategy was
adopted by Loc et al. (2002), where they used a
molar ratio of 3:3:1 for gene-cassettes of 2 genes
of interest and a selectable marker gene. With a
similar bombardment strategy (molar ratio 3:1),
Zhao et al. (2007a) reported co-transformation fre-
quency of 5060%. We could monitor the gene de-
livery process and functional activity of the
gene-cassette by mixing GUSPlus gene-cassette in
parallel preparation for co-bombardments. When
assayed for transient GUS gene expression,
well-scattered GUS foci were observed on the
bombarded calli (Table 2; Figure 2A). This indi-
cated proper precipitation of the clean DNA frag-
ments on gold particles and their evenly spread
delivery into the calli.
When the bombarded calli were plated on the
selection medium containing hygromycin, many
of the calli were found proliferating in the first
round of selection. The number of proliferating
calli was lesser in the subsequent rounds of selec-
tion. Even after 3 rounds of selection, all the
non-transgenic calli escaping hygromycin selec-
tion could not be eliminated because more than
13% of the regenerated plants were found to be
negative for the hpt gene. A more extensive selec-
tion of calli on hygromycin-containing medium
250
Table 2. Rice calli bombarded with different gene-cassettes, T0 plants regenerated, and their molecular response
Gene-cassettes used (molar No. of calli
ratio) bombarded*
Bt gene + hpt (4:1) 1440 (52)
Bt gene + hpt + GUS (4:1:1) 259 (10) all GUS positive
N/A = not applicable. *Value in parentheses indicates the number of plates bombarded
has not been recommended, particularly for
Basmati rice, since it seriously affects callus re-
generation (Khanna and Raina 2002). We used
hygromycin only in the callus induction medium
for selection of transformed cells. Shoot regenera-
tion from hygromycin-resistant embryogenic calli
was observed after 67 weeks of incubation on the
regeneration medium (Figure 2C). A total of
67 putative transformants were regenerated from
calli selected on hygromycin-containing medium,
as a result of independent transformation events
(Table 2; Figure 2D). The observed transforma-
tion frequency (as revealed by PCR analysis) for
the gene of interest was 3.3%, while 4.4% for the
selectable marker gene (hpt). The use of
hygromycin in the callus induction, regeneration
and rooting medium (Table 1) may help to reduce
the number of non-transgenic plants surviving on
hygromycin selection, but the observed transfor-
mation frequency was very low. A similar trans-
formation frequency has been reported when
using hygromycin-B in callus induction medium
(Khanna and Raina 2002).
Transgenic analysis
Regenerated T0 transgenic plants were screened
by using PCR detection of integrated transgenes.
The amplification products of expected sizes
(524 bp for cry1B and 897 bp for hpt) were de-
tected. Among 67 regenerated putative transgenic
plants, 47 were found PCR-positive for the Bt
gene, while the hpt gene was detected in 58 plants,
including those positive for the Bt gene. Transfor-
mation frequency (no. of independent
transformants / no. of embryonic-calli bombarded
100) for the Bt gene was 3.3%. About 13% of the
regenerated plants (negative for the hpt gene) were
regenerated from the calli that escaped
hygromycin selection, as revealed by PCR analy-
sis. On the other hand, 19% of the transformants
were PCR-positive for only the hpt gene. About
81% of the transformants (47 out of 58) were
PCR-positive for the Bt gene as well as hpt. The
higher molar ratio (4:1) of Bt and hpt improved
co-transformation frequency. Co-transformation
S. Kumar et al.
GUS assay No. of plants No. of No. of
response regenerated PCR-positive Southern-positive
plants plants
hpt gene Bt gene
N/A 67 58 47 47
(independent events)
N/A N/A N/A N/A
frequency of 5060% (Zhao et al. 2007a) and 71%
(Loc et al. 2002) has been earlier reported by using
a similar co-transformation strategy.
Stable integration and copy number of the
transgene was confirmed by Southern hybridiza-
tion, using gene-specific radiolabelled probes.
Copy number analysis for the integrated Bt gene in
T0 plants revealed that 2-5 copies of the transgene
were integrated (Figure 3A). Variation in the in-
tensity of bands was also observed. In most of the
cases, 2 copies (a relatively simple pattern) of
transgene integration were observed. However, in
a few cases (e.g. plant T0-38), 35 copies of the
transgene (complex pattern of transgene integra-
tion) was detected. Simple to complex patterns of
transgene integration have been reported when us-
ing the whole-plasmid biolistic method of trans-
formation (Datta et al. 1998; Khanna and Raina
2002). Fu et al. (2000) and Loc et al. (2002) re-
ported a simple pattern of transgene integration
when using a similar approach for biolistic-medi-
ated transformation of rice. However, Zhao et al.
(2007b) reported integration of multiple copies
and complex segregation behaviours of gene-cas-
settes in rice.
PCR analysis of T1 progenies indicated that
transgenes were integrated at one or more loci, as
evidenced by segregation ratios (Table 3). In many
cases, 2 copies of the Bt gene were found inte-
grated at one locus as tandem repeat (showing seg-
regation ratio of 3:1), but in a few cases (e.g. T0-7),
they were integrated at 2 different loci. In plant
T0-38, the segregation ratio and sites of integration
of the Bt gene could not be determined due to
smaller size (19) of the T1 progeny of the plant
(Table 3), and probably because they were inte-
grated at more than 2 loci. Yao et al. (2006) also
reported Mendelian segregation ratio of 3:1 for
gene-cassettes for the gene of interest and marker
gene in wheat. Southern analysis of the T1 progeny
confirmed integration of the Bt gene at 2 different
loci in plant T0-7 (Figure 3B). In other plants, band
size remained unchanged even in T2 progenies,
confirming that the gene was integrated at a single
locus (Figure 3C). Thus a stable inheritance of the
 Marker-free Bt transgenic indica rice 251

Figure 3. (A) Southern blot of T0 plants. PC = positive control, pSKPBA-7Z (9.5 kb) restricted with SacII and probed
with the coding region of cry1B; NC = negative control, untransformed rice plant. (B) Southern blot of plant T0-7 and its
randomly selected T1 progeny: genomic DNA restricted with SacII and probed with the coding region of cry1B. (C)
Southern blot of plant T0-64 and its randomly selected T2 progeny: genomic DNA restricted with SacII and probed with
the coding region of cry1B.

Table 3. Inheritance of the Bt gene in the T1 progeny of Bt transgenic

rice plants
T0 plant No. of T1 plants No. of Bt-positive Segregation P value*
analysed plants ratio
T0-4 15 12 3:1 0.200
T0-7 20 19 15:1 0.053
T0-11 18 14 3:1 0.074
T0-38 19 19 ?
T0-43 16 13 3:1 0.333
T0-64 20 14 3:1 0.266
Available T1 progeny plants were analysed by PCR using cry1B specific primers. Segrega-
tion ratio in plant T0-38 could not be determined due to the smaller size of the population
available for the analysis; *c2 test (no significant difference between the observed and the
expected ratios was recorded).

Bt gene was observed through the T2 generation. plants were found to carry only the Bt gene (Ta-
PCR analysis of segregating T1 and T2 progenies ble 4). Thus, using co-transformation approach
clearly indicated independent segregation of the (Komari et al. 1996; Vain et al. 2003; Afolabi et al.
Bt gene and hpt, as a few T1 and many T2 progeny 2005; Zhao et al. 2007a), we generated marker-
252
Table 4. PCR analysis of T1 and T2 progenies for
screening of marker-free Bt transgenic rice plants
(Bt+ hpt = positive for the Bt gene but negative for the
hpt gene).
T0 plant No. of Bt+ hpt plants*
T1 plants T2 plants
T0-4 1 (15) 3 (181)
T0-7 2 (20) 8 (304)
T0-11 2 (18) 7 (214)
T0-38 1 (19) 7 (289)
T0-43 1 (16) 5 (203)
T0-64 2 (20) 9 (224)
Available T1 and T2 progeny plants were analysed by PCR using
cry1B and hpt specific primers; *Value in parentheses indicates the
total number of plants analysed
free transgenic plants (up to 4%) in the T2 genera-
tion without additional genetic manipulation for
removal of the selectable marker gene, as required
in the Cre/loxP-based approach (Mlynarova et al.
2006; Luo et al. 2007; Bai et al. 2008). Zhao et al.
(2007a) also reported a similar frequency for gen-
eration of selectable-marker-free transgenic plants
by using minimal gene-cassette, co-transforma-
tion, and segregation approach.
Expression of the Bt gene
Transcription of the integrated genes was detected
by RT-PCR. The cry1B-specific primers in
RT-PCR produced a 524-bp band in South-
ern-positive T0 plants (Figure 4A) as well as in
most of the Bt-positive T2 progenies. Only
2 Southern-positive T0 plants (T0-47 and 59) were
detected negative for RT-PCR. Absence of the
amplification product in these T0 plants might be
due to a lack of transcription or very low level of
transcription of the Bt gene, which could not be
detected by RT-PCR. A similar observation has
been reported earlier by Khanna and Raina (2002).
A wide variation (0.030.35% of total soluble
proteins) in Bt titre was observed in South-
ern-positive T0 and PCR-positive T2 plants. Many
of the progenies had high (0.10.3%) Bt titres.
Western analysis of selected T0 and T2 plants con-
firmed the presence of the expected high-molecu-
lar-weight Bt protein (Figure 4B). Additional
bands of lower molecular weights were also ob-
served, probably because of degradation of the Bt
protein. This observation is in agreement with the
earlier reports (Maqbool and Christou 1999;
Khanna and Raina 2002; Ho et al. 2006).
Transgene copy number was not correlated with
Bt titre because a high-copy-number plant (e.g.
T0-38 plant) did not show higher Bt titre or vice
S. Kumar et al.
versa. Similar observations have been reported by
Maqool and Christou (1999) and Khanna and
Raina (2002).
Insect bioassays
The cut-stem bioassay demonstrated significantly
higher (38% to 83%) mortality of YSB larvae in
transgenic plants than in control plants (up to 14%,
Table 5). The larvae recovered from cut stems of
control plants were well-developed (3rd instar),
while the larvae recovered from those of transgen-
ic plants were dead or sluggish, weak, and under-
developed (Figure 5A). During the whole-plant
bioassay, observations were made on appearance
of typical symptoms (dead heart and white head)
of YSB infestation. Up to 4 white heads per plant
(with 57 tillers) were recorded in control plants
(Table 6, Figure 5B), while no dead heart or white
head was observed on transgenic plants (Fig-
ure 5D) with Bt titres of more than 0.28%.
Well-developed larvae were recovered from con-
trol plants. One or 2 white heads were observed on
T0-47, T0-49, T0-56, and T0-62 (Table 6). A few
underdeveloped surviving larvae were recovered
from these plants, showing a low level of resis-
tance. Three white heads were observed on tis-
sue-cultured non-transgenic plants.
In the present study, the level of YSB resis-
tance in Bt transgenic plants was estimated on the
basis of appearance of the typical symptoms of
YSB infestation in the whole-plant bioassay. Al-
though dead hearts and white heads were observed
on a number of T2-segregating progenies in the
whole-plant bioassay, no symptom of YSB infes-
tation was observed on T2 plants showing high
Bt titres (Table 7). Dead hearts and white heads
with considerable yield loss were observed on un-
transformed control plants. The whole-plant
bioassay is suggested to be a more appropriate
method of assessment of transgenic plants for in-
sect resistance (Tu et al. 2000b; Khanna and Raina
2002). A few T2 transgenic plants showed a re-
duced level of resistance, indicating a lower level
of Bt gene expression (data not shown). Similar
findings have been reported earlier (Nayak et al.
1997; Ghareyazie et al. 1997; Datta et al. 1998;
Khanna and Raina 2002). A lower level of Bt gene
expression has been reported in the leaf bases
and/or developing panicles of maize (Mellon and
Rissler 1998) and rice plants (Alinia et al. 2000),
where the Bt gene was placed under the control of
the PEPC promoter. In spite of the varying level of
Bt gene expression, a high level of Bt protein accu-
mulation and YSB resistance was observed in the
 Marker-free Bt transgenic indica rice 253

Figure 4. (A) RT-PCR amplification product of mRNA from T0 Bt transgenic plants amplified with cry1B-specific
primers. PC = positive control, plasmid pSKPBA-7Z DNA amplified with cry1B-specific primers; NC = negative
control, untransformed rice plant; M = 100-bp ladder. (B) Western analysis of T2 Bt transgenic plants showing high
(0.3%) Bt titres. PC = positive control: Bt protein expressed and purified from E. coli; NC = negative control,
untransformed plant.

Table 5. Cut-stem insect bioassay of selected Southern-positive T0 plants

Plant no. Bt titre (%) No. of larvae (mean SE) Mortality a Average weight
alive dead unrecovered (%) (mg)
T0-4 0.33 1.00 0.33 3.33 0.33 0.66 0.33 76.90 1.12 0.35 0.029
T0-7 0.35 0.66 0.58 3.33 0.33 1.00 0.66 83.46 1.72 0.37 0.033
T0-11 0.28 1.33 0.67 3.00 0.58 0.33 0.33 69.28 1.19 0.38 0.030
T0-21 0.20 2.00 0.58 2.66 0.67 0.33 0.33 57.08 1.23 0.40 0.034
T0-32 0.28 1.66 0.67 3.66 0.67 1.00 0.66 64.38 0.66 0.37 0.022
T0-38 0.31 1.33 0.33 3.66 0.33 0.00 0.00 73.34 1.45 0.34 0.026
T0-43 0.30 1.33 0.33 3.33 0.67 0.33 0.33 71.46 0.57 0.37 0.021
T0-51 0.10 2.66 0.88 1.66 0.58 0.66 0.33 38.42 1.18 0.44 0.025
T0-56 0.21 2.00 0.58 3.00 0.58 0.00 0.00 60.00 0.96 0.39 0.023
T0-62 0.27 1.66 0.33 3.00 0.67 0.33 0.33 64.38 1.06 0.36 0.024
T0-64 0.31 1.00 0.67 3.33 0.67 0.66 0.33 76.90 1.12 0.35 0.022
UTCb 0.00 4.00 0.33 0.66 0.33 0.33 0.33 14.16 0.67 0.65 0.033
NTCb 0.00 4.33 0.33 0.66 0.33 0.00 0.00 13.22 0.66 0.66 0.031
The cut-stem bioassay was carried out at the vegetative stage of transgenic plants, using neonate larvae (5 larvae/cut-stem). Three cut-stem
pieces (as replicates) were taken from each plant. Average body weight of surviving larvae indicates their growth rate; a Mortality (%) = (dead /
total larvae) 100%; b UTC = untransformed control; NTC = non-transgenic control.

transgenic plants with simple integration and sta-
ble inheritance of the transgenes.
Co-transformation has been reported to be an
efficient and simple strategy for generation of
marker-free transgenic plants. Sufficient data have
been generated to believe that co-transformation
followed by rounds of segregation creates
marker-free plants (Huang et al. 2004; Park et al.
2004; Xia et al. 2006; Zhao et al. 2007a). Results
of the present study on utility of the clean DNA
transformation of rice with genes conferring
agronomically important trait are consistent with
earlier reports of Loc et al. (2002) and Zhao et al.
(2007a). Although comparative evaluation was
not made in the present study, it has been reported
earlier (Fu et al. 2000; Loc et al. 2002; Vidal et al.
2006) that bombardments with clean DNA frag-
ments and whole plasmids give rise to transgenic
plants with similar efficacy. Moreover, this
method provides additional advantages of simple
254 S. Kumar et al.

Figure 5. (A) Yellow stem borer larvae recovered from untransformed control (L = live) and transgenic (D = dead) plants
in the cut-stem assay; (B) Untransformed control plant with white heads in the whole-plant bioassay; (C) White heads
(arrow marked) on the control plant; (D) T0 transgenic plants without any symptom of infestation in the whole-plant
bioassay.

Table 6. Whole-plant bioassay of yellow stem borer (YSB) resistance of selected T0 plants
Plant no. Bt titre (%) No. of No. of white Damagea (%) No. of larvae Average larva weight SE (mg)
tillers/plant heads recovered
T0-4 0.33 6 0 0 0 0
T0-7 0.35 8 0 0 0 0
T0-11 0.28 5 0 0 0 0
T0-38 0.31 7 0 0 0 0
T0-43 0.30 6 0 0 0 0
T0-56 0.21 5 1 20 2 0.32 0.023
T0-62 0.27 5 1 20 1 0.23 0.000
T0-64 0.31 7 0 0 0 0
UTCb 0.00 5 4 80 16 0.72 0.056
NTCb 0.00 5 3 60 13 0.73 0.051
The whole-plant bioassay was carried out at the early panicle initiation stage, using neonate YSB larvae (25 larvae/plant). Tillers with white
heads were dissected and surviving larvae were recorded; a Damage (%) indicates loss of panicle on tiller basis; b UTC = untransformed control;
NTC = non-transgenic control

integration (low copy number) of transgenes and
recovery of marker-free transgenic plants from the
segregating progenies.
Acknowledgements. We are grateful to Dr. R
Frutos, CIRAD, France, for providing the
translational fusion cry1B-1Aa gene. Thanks are also
due to Novartis Agribusiness Biotechnology Re-
search Inc., North Carolina, USA (for the PEPC pro-
moter) and to CAMBIA, Australia (for
pCAMIA1305.1). We thank Dr. SK Raina, Dr. NK
Singh, Dr. KR Koundal, N.R.C. on Plant
Biotechnology, and Dr. KV Prabhu, National
Phytotron Facility, New Delhi, for the support and
facilities provided during this study. Scientific and
technical supports provided during insect bioassays
by Dr. VS Singh, Division of Entomology, I.A.R.I.,
and Mr. Upendra Kumar, N.R.C.P.B., are gratefully
acknowledged. We give our sincere thanks to the
staff of the Regional Rice Research Station,
Kapurthala, Punjab, for the help and assistance pro-
vided during collection of YSB moths. Financial
 Marker-free Bt transgenic indica rice 255

Table 7. Whole-plant bioassay of yellow stem borer (YSB) resistance of selected T2 progenies
Line Bt titre (%) No. of tillers/plant Symptom of YSB infestation No. of damaged No. of normal
dead heart white head tillers/plant panicles/plant
T2-4-3-7 0.34 6 0 0 0 5
T2-4-3-12 0.33 8 0 0 0 7
T2-4-3-16 0.33 7 0 0 0 7
T2-7-2-4 0.35 6 0 0 0 6
T2-7-2-10 0.34 6 0 0 0 6
T2-7-2-13 0.34 8 0 0 0 7
T2-38-5-7 0.30 5 0 0 0 5
T2-38-5-11 0.31 7 0 0 0 6
T2-38-5-19 0.30 6 0 0 0 6
T2-64-4-8 0.31 7 0 0 0 6
T2-64-4-9 0.31 6 0 0 0 6
T2-64-4-17 0.30 8 0 0 0 7
UTC 0.00 7 4 1 5 2
The whole-plant bioassay was initiated at the mid-tillering stage, using neonate YSB larvae (25 larvae/plant). The number of
dead hearts was recorded 25 days after infestation, and white heads were recorded at the post-panicle emergence stage. Num-
bers of tillers and panicles were recorded during final observations.

support (Senior Research Fellowship) to S Kumar
from the University Grant Commission, New Delhi,
is gratefully acknowledged.
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