Scientia Horticulturae 122 (2009) 649653

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Scientia Horticulturae
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Short communication

Efcient production of transgenic plants using the bar gene for herbicide
resistance in sweetpotato
Ning Zang a, Hong Zhai a,b, Shang Gao a, Wei Chen a, Shaozhen He c, Qingchang Liu a,b,c,*
a

Key Laboratory of Crop Genomics and Genetic Improvement, Ministry of Agriculture, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing 100193, China
Beijing Key Laboratory of Crop Genetic Improvement, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing 100193, China
c
Laboratory of Crop Heterosis and Utilization, Ministry of Education, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing 100193, China
b

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 27 February 2009
Received in revised form 18 June 2009
Accepted 18 June 2009

Efcient production of transgenic sweetpotato (Ipomoea batatas (L.) Lam.) plants using the bar gene for
herbicide resistance was achieved through the use of embryogenic suspension cultures and
Agrobacterium tumefaciens-mediated transformation. Cell aggregates from embryogenic suspension
cultures of sweetpotato cv. Lizixiang were cocultivated with A. tumefaciens strain EHA 105 harboring a
binary vector pCAMBIA3300 with the bar gene and uidA gene. Selection culture was conducted using
0.5 mg/l PPT. A total of 1431 plants were produced from the inoculated 870 cell aggregates via somatic
embryogenesis. GUS assay and PCR analysis of the regenerated plants randomly sampled showed that
86.5% of the regenerated plants were transgenic plants. Stable integration of the bar gene into the
genome of transgenic plants was conrmed by Southern blot analysis and transgene expression was
demonstrated by Northern blot analysis. The copy number of integrated bar gene ranged from 1 to 3.
Transgenic plants exhibited functional expression of the bar gene by in vivo assay for herbicide
resistance. This study also provides a simple and efcient transformation system of sweetpotato based
on the use of bar gene as a selectable marker gene, which can be combined with other agronomically
important genes for the improvement of sweetpotato.
2009 Published by Elsevier B.V.

Keywords:
Agrobacterium tumefaciens
bar gene
Embryogenic suspension culture
Herbicide resistance
Ipomoea batatas (L.) Lam.
Transgenic plant

1. Introduction
Sweetpotato, Ipomoea batatas (L.) Lam., is an important food
and industrial material crop in the world. It is also an alternative
source of bio-energy as a raw material for fuel production. The
improvement of this crop by conventional hybridization is limited
because of its high male sterility, incompatibility and the
hexaploid nature (Dhir et al., 1998). Genetic engineering offers
great potential for the improvement of sweetpotato. There have
been several reports on this subject in the literature. Transgenic
plants expressing cowpea trypsin inhibitor (CpTI), snowdrop lectin,
delta-endotoxin, soybean kunitz trypsin inhibitor (SKTI-4), oryzacystatin-I (OCI), sweetpotato feathery mottle virus (SPFMV-S) coat
protein, granule-bound starch synthase I (GBSSI), tobacco microsomal v-3 fatty acid desaturase (NtFAD3) or starch branching
enzyme II (IbSBEII) gene have been produced (Newell et al., 1995;
Moran et al., 1998; Cipriani et al., 1999, 2001; Okada et al., 2001;
Kimura et al., 2001; Wakita et al., 2001; Shimada et al., 2006). But,

* Corresponding author at: Laboratory of Crop Heterosis and Utilization, Ministry

of Education, China Agricultural University, No. 2 Yuanmingyuan West Road,
Beijing 100193, China. Tel.: +86 10 62733710; fax: +86 10 62733710.
E-mail address: liuqc@cau.edu.cn (Q. Liu).
0304-4238/$ see front matter 2009 Published by Elsevier B.V.
doi:10.1016/j.scienta.2009.06.023

in most cases only a low transformation efciency was obtained,

which limits the successful application of genetic engineering in
sweetpotato improvement.
The bar gene is widely used for producing herbicide-resistant
plants in many crop species. The enzyme phosphinothricin (PPT)
acetyltransferase encoded by the bar gene inactivates PPT, the
active ingredient of herbicides such as Basta and Buster, by
acetylating its free ammonium group, thereby rending it non-toxic
(De Block et al., 1987; Strauch et al., 1988). Otani et al. (2003), Yi
et al. (2007) and Choi et al. (2007) obtained only a few herbicideresistant plants expressing the bar gene from sweetpotato
embryogenic calluses using Agrobacterium tumefaciens-mediated
transformation or particle bombardment. Moreover, embryogenic
calluses are not readily available target tissues for most of
sweetpotato cultivars due to low frequencies of embryogenic
callus formation in apical meristem cultures (Al-Mazrooei et al.,
1997; Liu et al., 1997; Wang et al., 1998).
The availability of protocol to achieve high-frequency plant
regeneration from cultured cells or tissues is a prerequisite for the
application of genetic engineering. We succeeded in developing an
efcient system of embryogenic suspension cultures for a wide
range of sweetpotato genotypes especially for commercial
cultivars (Liu et al., 2001). Using embryogenic suspension cultures
of sweetpotato and hptII/hygromycin selection system, we have

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N. Zang et al. / Scientia Horticulturae 122 (2009) 649653

also established an efcient A. tumefaciens-mediated transformation system (Yu et al., 2007). This paper describes efcient
production of herbicide-resistant sweetpotato plants with the bar
gene through the use of embryogenic suspension cultures and
direct bar/PPT selection.

uorescent light at 54 mmol/(m2 s). The regenerated plantlets were

further transferred to the basal medium and developed into whole
plants at 27  1 8C under 13 h of cool-white uorescent light at
54 mmol/(m2 s).
2.5. GUS assay

2. Materials and methods

2.1. Plant material
Sweetpotato cv. Lizixiang used in this study is one of
commercial cultivars planted in China. Embryogenic suspension
cultures of Lizixiang were prepared according to the method of Liu
et al. (2001). Sixteen weeks after initiation, cell aggregates 0.7
1.3 mm in size from embryogenic suspension cultures of 3 days
after subculture were used for the transformation.
2.2. Bacterial strain and plasmid
The A. tumefaciens strain EHA 105 harboring a binary vector,
plasmid pCAMBIA3300/uidA was used in the present study. This
vector contains the bar gene driven by a CaMV 35S promoter and
the fragment of uidA gene with the CaMV 35S promoter excited
from pBI121 in the following order: 35S-bar-35S-uidA.
2.3. Sensitivity of cell aggregates to PPT
The sensitivity of the uninoculated cell aggregates to PPT was
tested in order to determine the optimal concentration of PPT in
the selective medium. The uninoculated cell aggregates were
cultured on Murashige and Skoog (1962) (MS) medium supplemented with 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D),
100 mg/l carbencillin (Carb) and different concentrations of PPT (0,
0.1, 0.3, 0.5, 0.8, 1.0, 2.0, 5.0, 10.0, 15.0, 20 mg/l) in the dark at
27  1 8C for 6 weeks. The growth of cell aggregates was observed.
The experiments were repeated three times with 50 cell aggregates
per treatment.
2.4. Transformation, selection and plant regeneration
The Agrobaterium single colony was cultured in 25 ml Luria
Bertani (LB) liquid medium containing 50 mg/l kanamycin and
50 mg/l rifampycin on a reciprocal shaker (200 rpm) at 28 8C for
1618 h until OD600 nm = 0.5 was reached. The bacteria were
collected by centrifugation at 5000 rpm for 5 min, washed with LB
liquid medium and further with MS liquid medium containing
2.0 mg/l 2,4-D, and then were resuspended in 25 ml MS medium
containing 2.0 mg/l 2,4-D for the inoculation. Cell aggregates were
infected for 5 min in the bacteria suspensions at room temperature. Following inoculation, the cell aggregates were blotted on
sterile lter paper and placed on lter paper in a Petri dish
containing 25 ml solid MS medium with 2.0 mg/l 2,4-D and 30 mg/
l acetosyringone (AS) and cocultivated for 3 days in the dark at
27  1 8C. After cocultivation, the cell aggregates were washed twice
with liquid MS medium containing 2.0 mg/l 2,4-D and 500 mg/l Carb
and maintained in liquid MS medium with 2.0 mg/l 2,4-D and
100 mg/l Carb on a reciprocal shaker (100 rpm) at 27  1 8C under
13 h of cool-white uorescent light at 10 mmol/(m2 s) for 1 week, and
then were cultured at 2-week interval on MS solid medium
supplemented with 2.0 mg/l 2,4-D, 100 mg/l Carb and 0.5 mg/l PPT
for the selection culture in the dark at 27  1 8C.
Six to eight weeks after selection, the obtained PPT-resistant
embryogenic calluses were transferred to MS medium supplemented with 1.0 mg/l abscisic acid (ABA), 100 mg/l Carb and
0.5 mg/l PPT to induce the formation of somatic embryos and
regeneration of plantlets at 27  1 8C under 13 h of cool-white

The PPT-resistant calluses, and leaves, stems and roots of

transgenic plants were tested for GUS expression using histochemical GUS assay as described by Jefferson et al. (1987). The
explants were incubated in GUS assay buffer at 37 8C for 12 h. Blue
staining of the cells or tissues denoted positive reaction.
2.6. PCR and Southern blot analyses
Genomic DNA was extracted from fresh leaf tissues of in vitrogrown GUS-positive/-negative plants and untransformed control
plants by the cetyltrimethylammonium bromide (CTAB) method
(Saghai-Maroof et al., 1984). Specic primers for the bar gene: ATG
AGC CCA GAA CGA CGC and TCT CAA ATC TCG GTG ACG were used.
These primers were expected to give products of 550 bp. PCR
analysis was done according to the method of Yu et al. (2007).
For Southern blot analysis, 25 mg of transgenic plants and
control plants DNA was digested with EcoRI. The restriction
fragments were size-fractionated by 0.8% (w/v) agarose gel
electrophoresis and transferred to a Hybond-N+ nylon membrane
(Amersham Pharmacia Biotech, UK). Coding sequence of the bar
gene was used as probe. The labeling of probe, prehybridization,
hybridization and detection were performed by the protocol of DIG
High Prime DNA Labeling and Detection Starter Kit II (Roche
Diagnostics GmbH, Germany).
2.7. Northern blot analysis
Total leaf RNA was extracted from in vitro-grown plants of the
transgenic plants and the untransformed control plants using the
plant RNAtrip kit (Applygen Technologies Inc, China). Equal
amounts of total RNA (20 mg) were separated by a denaturing
1.2% (w/v) formaldehydeagarose gel (Sambrook et al., 1989). The
RNA was blotted to a Hybond-N+ nylon membrane. The labeling of
probe, prehybridization, hybridization and detection were performed by the protocol of DIG High Prime DNA Labeling and
Detection Starter KitII.
2.8. In vivo assay for herbicide resistance
The transgenic plants and the untransformed control plants
were transplanted to pots with a mixture of soil and vermiculite
(1:1) in a greenhouse for the domestication. They were propagated
by cutting, and individuals from 3 cuttings per plant were
evaluated for in vivo herbicide resistance at each Basta concentration under greenhouse conditions. The plants were sprayed
directly to leaves with 1000 mg/l (normal eld dosage) and
2000 mg/l PPT aqueous solution of commercial product Basta
(Bayer Crop Science, Germany). Symptoms of the plants were
continuously observed for 4 weeks.
3. Results and discussion
3.1. Sensitivity of cell aggregates to PPT
The sensitivity of cell aggregates from embryogenic suspension
cultures of sweetpotato cv. Lizixiang to PPT was tested in order to
establish an efcient selection system. The PPT concentrations
tested ranged from 0.1 mg/l to 20 mg/l. The results showed that
PPT concentrations signicantly inuenced the growth and

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651

Fig. 1. Production of transgenic plants from sweetpotato cv. Lizixiang cell aggregates and evaluation of herbicide resistance. (A) Embryogenic suspension cultures. (B) PPTresistant calluses formed on MS medium with 2.0 mg/l 2,4-D, 100 mg/l Carb and 0.5 mg/l PPT after 8 weeks of selection (bar = 10 mm). (C) GUS expression in a piece of PPTresistant calluses and no GUS expression in untransformed control callus (CK) (bar = 1 mm). (D and E) Formation and germination of somatic embryos from PPT-resistant
calluses on MS medium with 1.0 mg/l ABA, 100 mg/l Carb and 0.5 mg/l PPT. (F) Whole regenerated plants obtained on the basal medium. (G, H and I) GUS expression in leaf,
stem and root of a transgenic plant and no GUS expression in the control (CK). (J) Transgenic plants grown in a greenhouse. (K) Transgenic plants displaying complete Basta
resistance and the untransformed control plants with necrotic leaves (CK) 10 days after spraying with 1000 mg/l PPT of Basta. (L) Transgenic plants with the high Basta
resistance and the died untransformed control plants (CK) 10 days after spraying with 2000 mg/l PPT of Basta.

survival of Lizixiang cell aggregates and a 0.5 mg/l concentration of

PPT was enough to inhibit the growth of cell aggregates. This
concentration of PPT was lower than that reported by Otani et al.
(2003), Yi et al. (2007) and Choi et al. (2007) who suggested that
5 mg/l PPT completely inhibited the growth of sweetpotato
embryogenic calluses. Thus, sweetpotato cell aggregates are more
sensitive to PPT than the embryogenic calluses. This allowed us to
conrm the selective medium used in the present study.
3.2. Transformation, selection and plant regeneration
Cell aggregates of Lizixiang (Fig. 1A) cocultivated with the A.
tumefaciens were cultured on the selective medium with 0.5 mg/l
PPT. Six to eight weeks after selection, a total of 165 PPT-resistant
embryogenic calluses were formed from the inoculated 870 cell

aggregates (Fig. 1B). Pieces of 20 randomly sampled PPT-resistant

calluses were detected for GUS expression. It was found that all of
the tested pieces of calluses expressed the uidA gene (Fig. 1C).
The PPT-resistant calluses were transferred to MS medium
supplemented with 1.0 mg/l ABA, 100 mg/l Carb and 0.5 mg/l PPT,
after 5 weeks 139 of them produced somatic embryos which
further germinated into plantlets on the same medium (Fig. 1D and
E). The regenerated plantlets developed into whole plants on the
basal medium (Fig. 1F). A total of 1431 putatively transgenic plants
were obtained.
3.3. GUS assay of transgenic plants
Of the 1431 regenerated plants, 200 were randomly sampled for
detecting GUS expression in leaf, stem and root tissues; 173

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N. Zang et al. / Scientia Horticulturae 122 (2009) 649653

Fig. 2. PCR analysis of transgenic plants. Lane M: DNA marker; Lane P: plasmid pCAMBIA3300 as positive control; Lane C: the untransformed control plant as negative control;
Lanes 1, 2, 47, 9, 1122, 2431, 3337: transgenic plants (GUS-positive plants); Lanes 3, 8, 10, 23, 32: non-transgenic plants (GUS-negative plants).

(86.5%) had visible GUS activity in these tissues (Fig. 1 GI),

indicating stable uidA gene integration into the genome of the
plants. The remaining 27 plants and untransformed control plants
showed no GUS expression (Fig. 1 GI). These 27 plants showing no
GUS expression probably came from non-transgenic cells of the
transgenic calluses which possibly contained a few non-transgenic
cells as reported by Yu et al. (2007). To inhibit the growth of nontransgenic cells and reduce the escape rate of non-transformed
plants effectively, it is suggested that a little higher concentration
of PPT might be used in future transformation experiments of
sweetpotato.

Fig. 4. (A) Northern blot analysis of transgenic plants. The 550 bp bar fragment was
used as a probe. (B) Ethidium bromide-stained total RNA as a loading control. Lane
C: the untransformed control plant; Lanes 16: transgenic plants produced from six
independent PPT-resistant calluses.

3.4. Molecular analysis of transgenic plants

To verify presence of the bar gene, the 200 GUS-positive/negative plants and the untransformed control plants were
analyzed by PCR amplication. A specic 550 bp band of the bar
gene was observed in all the 173 GUS-positive plants, while no
amplication occurred with the DNA from the 27 GUS-negative
plants and untransformed control plants (Fig. 2). These results
demonstrated that the 173 plants were transgenic.
The DNA of six transgenic plants regenerated from independent
PPT-resistant calluses and the untransformed control plant was
digested with EcoRI, which has a unique cleavage site in the T-DNA
region in the vector, and hybridized with a bar gene probe. Each
hybridizing band can therefore be interpreted as a separate
integration event. These six transgenic plants displayed different
patterns and the copy number of integrated bar gene varied from 1
to 3 (Fig. 3). No hybridizing band was observed in the control plant
as expected (Fig. 3).

Fig. 3. Southern blot analysis of transgenic plants to detect the copy number of bar
gene. DNA was digested with EcoRI and hybridized with the DIG-labeled bar gene
probe. Lane C: the untransformed control plant; Lanes 16: transgenic plants
produced from 6 independent PPT-resistant calluses. Arrows indicate light bands.

Northern blot analysis demonstrated that all these six

transgenic plants gave the expression of the bar gene, while the
transgene expression was not observed in the untransformed
control plant (Fig. 4). The transgene expression varied among the
transgenic plants according to different band intensity, and Lane 4
and Lane 6 plants showed a low level of the transgene expression
(Fig. 4). Transgenic plants with multiple copies of inserted genes
displayed a low transgene expression level in our experiment
(Figs. 3 and 4).
3.5. In vivo assay for herbicide resistance
All the 173 PCR-positive transgenic plants were transferred to
the soil and showed 100% survival (Fig. 1J). No morphological
variations were observed. The response of the transgenic plants to
herbicide (Basta) applied directly to leaves provides convincing
evidence for functional activity of the transgene. After spraying
with 1000 mg/l PPT of Basta (normal eld dosage) directly to the
leaves, the leaves of control plants turned black or brown within a
few days and eventually the whole plant died (Fig. 1K). In contrast,
the leaves of 162 of the 173 transgenic plants remained green and
healthy and the plants ourished (Fig. 1K) even after 4 weeks
except for 11 transgenic plants (including two plants showing a
low level of the transgene expression by Northern blot analysis)
which showed necrotic lesions on leaves and/or wrinkled leaves,
but eventually recovered. The spraying of 2000 mg/l PPT of Basta
directly to the leaves of control plants resulted in the death of
whole plant within a few days (Fig. 1L). Whereas at this dosage of
Basta all the 173 transgenic plants could survive (Fig. 1L), and 114
of them displayed necrotic lesions on leaves and/or wrinkled
leaves, but necrosis did not spread throughout the leaves and in
most cases the plants eventually recovered fully.
In the present study, a large number of transgenic plants
expressing the bar gene were obtained using embryogenic
suspension cultures of sweetpotato cv. Lizixiang and A.
tumefaciens-mediated transformation. Most of the obtained
transgenic plants showed complete herbicide resistance at a
normal eld dosage of Basta. Thus, introduction of the bar gene
into sweetpotato cultivars can allow efcient production of
herbicide-resistant transgenic sweetpotatoes with commercial
potential.

N. Zang et al. / Scientia Horticulturae 122 (2009) 649653

The present results exceed all transformation experiments

reported so far in the literature in quantity of independent events
per transformation experiment in sweetpotato. Otani et al. (2003)
and Yi et al. (2007) obtained only a few herbicide-resistant plants
expressing the bar gene from embryogenic calluses of sweetpotato.
Using direct bar/PPT selection system, Choi et al. (2007) obtained
18 transgenic plants from the infected 644 embryogenic calluses of
sweetpotato cv. Yulmi. The present transformation frequency was
also signicantly higher than that reported by Yu et al. (2007) who
used embryogenic suspension cultures of sweetpotato cv. Lizixiang
and hptII/hygromycin selection system. To explain the large
increase in transformed plants per experiment using the new
protocol reported here, we postulate that the combination of
embryogenic suspension cultures (not embryogenic callus) and bar
(not hptII) gene as selectable marker gene greatly inuences the
transformation efciency of sweetpotato. In addition, a possibility
exists that some transgenic plants were from the division of the
same transformed cells at the early stage of the selection culture,
which contributed to the increased transformation efciency in the
present study. This problem will be discussed in further study. In
general, the present study provides a simple and efcient
transformation system of sweetpotato based on the use of bar
gene as a selectable marker gene, which can be combined with
other agronomically important genes for the improvement of
sweetpotato.
Acknowledgements
This work was supported by the National Science and
Technology Project of China (no. 2006BAD01A06), the National
Natural Science Foundation of China (no. 30671321), the National
Project for Public Industry of China (no. nyhyzx07-012) and 948
Project for China Agriculture (no. 2006G21). We thank Prof. Wang
T., State Key Laboratory of Agrobiotechnology, Beijing, China, and
Prof. Zhu Z., Institute of Genetics and Developmental Biology,
Chinese Academy of Sciences, Beijing, China, for providing Strain
EHA 105 and bar gene, respectively.
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