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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,
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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.
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.
652
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).
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.
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.
653
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