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The Plant Journal (2007) 52, 157–166 doi: 10.1111/j.1365-313X.2007.03230.x

Molecular breeding of a novel herbicide-tolerant rice by

gene targeting
Masaki Endo1, Keishi Osakabe1, Kazuko Ono1, Hirokazu Handa2, Tsutomu Shimizu3 and Seiichi Toki1,*
1
Plant Genetic Engineering Research Unit, Division of Plant Sciences, National Institute of Agrobiological Sciences,
2-1-2, Kannondai, Tsukuba, Ibaraki 305-8602, Japan,
2
Plant Genome Research Unit, Division of Genome and Biodiversity Research, National Institute of Agrobiological Sciences,
2-1-2, Kannondai, Tsukuba, Ibaraki 305–8602, Japan, and
3
Life Science Research Institute, Kumiai Chemical Industry Co. Ltd, 3360 Kamo, Kikugawa, Shizuoka 439-0031, Japan

Received 6 June 2007; accepted 12 June 2007.

*For correspondence (fax +81 29 838 8450; e-mail stoki@affrc.go.jp).

Summary
We have previously reported the production of a rice cell line tolerant to the acetolactate synthase (ALS)-
inhibiting herbicide bispyribac (BS), and demonstrated that the BS-tolerant phenotype was due to a double
mutation in the rice ALS gene. We further indicated that while changing either of the two amino acids (W548 L
or S627I) individually resulted in a BS-tolerant phenotype, conversion of both amino acids simultaneously
conferred increased tolerance to BS. As the BS-tolerant cell line had lost the ability to regenerate during two
years of tissue culture selection, we attempted to introduce these two point mutations into the rice ALS gene
via gene targeting (GT). Using our highly efficient Agrobacterium-mediated transformation system in rice, we
were able to regenerate 66 independent GT rice plants from 1500 calli. Furthermore, two-thirds of these plants
harbored the two point mutations exclusively, without any insertion of foreign DNA such as border sequences
of T-DNA. The GT plants obtained in the present study are therefore equivalent to non-GM herbicide-tolerant
rice plants generated by conventional breeding approaches that depend on spontaneous mutations.
Surprisingly, GT rice homozygous for the modified ALS locus showed hyper-tolerance to BS when compared
to BS-tolerant plants produced by a conventional transgenic system; ALS enzymatic activity in plants
homozygous for the mutated ALS gene was inhibited only by extremely high concentrations of BS. These
results indicate that our GT method has successfully created novel herbicide-tolerant rice plants that are
superior to those produced by conventional mutation breeding protocols or transgenic technology.

Keywords: gene targeting, rice, acetolactate synthase, herbicide tolerance, homologous recombination.

Introduction
Acetolactate synthase (ALS) catalyzes the initial step com-
mon to the biosynthesis of the branched-chain amino acids
leucine, isoleucine and valine (Chipman et al., 1998). ALS is
the primary target site of action for at least four structurally
distinct classes of herbicides (sulfonylureas, imidazolinones,
triazolopyrimidine sulfonamides and pyrimidinyl carboxy
herbicides) (for review, see Corbett and Tardif, 2006). ALS-
inhibiting herbicides control a broad spectrum of grass and
broadleaf weeds in crops, including weeds that are closely
related to the crop itself and some key parasitic weeds. A
major advantage of these compounds is that they are non-
toxic to animals, highly selective, and very potent, thereby
requiring only low application rates. Thus, ALS-inhibiting
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd
herbicides are an essential part of the multi-billion dollar
weed control market.
Rotating the use of different combinations of several
herbicides and corresponding herbicide-tolerant crops in the
same field is one approach to overcoming the problem of
herbicide-tolerant weeds. Several variant ALS genes confer-
ring tolerance to ALS-inhibiting herbicides have been
discovered in various crops (maize, wheat, rice, oilseed rape
and sunflower) through conventional breeding methods
such as mutagenesis and selection. However, only a single
combination of an imidazolinone herbicide and the corre-
sponding mutant, which was developed by a conventional
breeding method, has been developed for commercializa-
157

158 Masaki Endo et al.
tion in rice. Hence the goal of this study was to produce a
novel rice plant tolerant to the pyrimidinyl carboxy herbicide
bispyribac (BS).
In a previous study, as part of a two-year screen of
cultured rice cells, we isolated BS-tolerant rice cells that
showed hyper-tolerance to BS (Shimizu et al., 2005). BS
tolerance was found to be linked to two point mutations in
the ALS gene: a tryptophan (TGG) to leucine (TTG) change at
amino acid 548 (W548 L), and a serine (AGT) to isoleucine
(ATT) change at amino acid 627 (S627I). This double
mutation in the rice ALS gene represents a novel combina-
tion of spontaneous mutations, with the substitution at the
serine position not having been observed previously.
Recombinant ALS proteins with each mutation singly, or
the combined double mutation, were expressed in Escher-
ichia coli, and colonies were examined for their sensitivity to
BS. Although each individual amino acid change in ALS
resulted in a BS-tolerant phenotype, conversion of both
amino acids conferred increased tolerance to BS (Shimizu
et al., 2005). As no plants could be recovered from the BS-
tolerant rice cells because of prolonged tissue culture, we
decided to directly produce rice plants containing these
double mutations. Although chimeric RNA/DNA oligonucleo-
tides have been used to induce site-specific base changes in
several plant species (rice, Okuzaki and Toriyama, 2004;
tobacco, Beethman et al., 1999; maize, Zhu et al., 1999), this
technique can introduce only a single point mutation at a
time. In this study, we used a highly efficient T-DNA-
mediated gene targeting (GT) system to introduce W548 L
and S627I mutations into the ALS gene. The procedure
described here may facilitate the site-specific modification of
genes in crop plants via GT.
Results and discussion
Experimental design of gene targeting
As shown in Figure 1(a), the GT vector D5¢ ALS contains a
partial ALS coding sequence (BAC clone OSJNBa0052 M16,
137 116–139 050 bp) with two point mutations: tryptophan
(TGG) to leucine (TTG) at amino acid 548 (W548 L), and
serine (AGT) to isoleucine (ATT) at amino acid 627 (S627I).
Both mutations generate recognition sites for the restriction
enzyme MfeI (CAATTG), which allows rapid detection of GT
events. The ALS sequence on D5¢ ALS lacks the 5¢ coding
region corresponding to the first 55 amino acids of ALS,
which include the chloroplast-targeting signal (Pang et al.,
2004), but contains 6.3 kb of the region downstream region
of the ALS gene (BAC clone OSJNBa0052 M16, 139 051–
145 372 bp) to allow efficient homologous recombination
(HR). This region also contains unknown expressed genes
of 2.6 and 0.7 kb (OSJNBa0052 M16.39 and OS-
JNBa0052 M16.40). We found a silent mutation in the coding
region of the ALS gene (C fi T at +612) and another silent
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mutation in the downstream region of the ALS gene (T fi C
at +2658) in D5¢ ALS. These silent mutations were generated
by PCR amplification during the construction of D5¢ ALS. To
facilitate molecular analysis of the recombination events, we
retained these mutations. The ALS gene on D5¢ ALS is ex-
pected to be non-functional, thus recovery of BS-tolerant
plants is expected only after HR between D5¢ ALS and the
chromosomal ALS target locus.
After co-cultivation of Agrobacterium carrying D5¢ ALS
with approximately 1500 rice scutellum-derived calli
(2–5 mm in diameter), and subsequent selection of BS-
tolerant calli (Figure 1b), 72 independent T0 BS-tolerant
plants (BSR 1 to BSR 72) were selected. As a positive control
for BS selection, a second batch of rice calli was transformed
with a full-length rice ALS gene comprising the 2.3 kb ALS
gene coding region harboring the W548 L and S627I muta-
tions flanked 5’ and 3’ by 2.2 and 0.5 kb, respectively, of the
surrounding genomic sequence (Osakabe et al., 2005). In
this latter transformation, almost all transformed calli
produced considerable numbers of BS-tolerant colonies
(Figure 1c).
Molecular characterization of the ALS locus in
BS-tolerant T0 plants
A total of 72 BS-tolerant T0 plants obtained as above were
analyzed to confirm the GT event. As shown in Figure 1(a),
PCR amplification using primer F1, which anneals to the ALS
promoter region (not present on the targeting construct),
and primer R1, which anneals downstream of the ALS gene,
yields a PCR product of 2287 bp. PCR products were further
analyzed by digestion with MfeI. The presence of an uncut
DNA fragment (2287 bp) as well as the three subfragments
generated by cutting at the two restriction sites (1751, 299
and 237 bp) may be explained by HR at the target locus
creating a heterozygous condition in primary T0 transformed
plants. Figure 1(d) shows an example of this PCR–MfeI
digestion analysis. Patterns indicating HR (non-digested
2287 bp, digested 1751, 299 and 237 bp) were found in DNA
products derived from 66 individual BS-tolerant transfor-
mants. In six cases, an unexpected band pattern (non-di-
gested 2287 bp, corresponding to the wild-type ALS locus,
1751 and 536 bp; Figure 1d, BSR 70 and 72) was seen. This
result suggests that only one (W548 L) of the two mutations
was present in the endogenous ALS locus of these plants.
This result could be explained by spontaneous mutation at
W548 L. Indeed, rice plants tolerant to BS owing to a W548 L
mutation have been recovered by somaclonal variation
(Okuzaki and Toriyama, 2004). Alternatively, HR might have
occurred at a point between the W548 L and S627I
mutations.
Although BS-tolerant mutations can occur spontaneously,
the combination of the double mutation W548 L/S627I in the
rice ALS gene was discovered only after two years of
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Herbicide-tolerant rice generated by gene targeting 159

(a)

(b) (c)

(d) (e)

Figure 1. Strategy for T-DNA-mediated gene targeting of the rice acetolactate synthase (ALS) locus and analysis of homologous recombination events in T0 plants.
(a) Schematic representation of gene targeting (GT) events. The blue boxes represent the coding region of the ALS gene. The thick lines represent flanking plant
genomic DNA. A sequence encoding 55 amino acids, including the chloroplast-targeting signal, is deleted in the GT construct D5¢ ALS, rendering the gene non-
functional. The two mutations (W548 L and S627I) that confer herbicide bispyribac (BS) tolerance are marked by solid red vertical lines. Two silent mutations (C fi T
at +612, T fi C at +2658) are marked by solid or dashed red vertical lines. The W548 L and S627I mutations create novel MfeI restriction sites (CAATTG). The positions
of primers (F1 and R1) used for polymerase chain reaction (PCR), and the expected sizes of PCR-amplified fragments and their MfeI endonuclease digestion products
are shown. The position of probe A used for Southern analysis is indicated on the targeted ALS locus. LB, left border; RB, right border; S, SphI restriction site.
(b, c) Selection of BS-tolerant calli on callus induction medium containing 0.75 lM of BS after infection of rice scutellum-derived calli with Agrobacterium carrying
D5¢ ALS (b) or with Agrobacterium carrying a binary vector harboring a full-length rice ALS gene containing the W548 L and S627I mutations (c).
(d) Example of an initial DNA analysis of BS-tolerant plants by PCR–MfeI digestion analysis. PCR was performed using primers F1 and R1. A band corresponding to
536 bp indicates introduction of only the W548 L mutation but not the S627I mutation into the ALS locus. If true GT has occurred, three fragments (1751, 299 and
237 bp) appear in the T0 generation in addition to the non-digested wild-type band of 2287 bp.
(e) Example of Southern blot analysis with probe A of SphI-digested DNAs from the individual wild-type (WT) and BS-tolerant T0 plants analyzed in (d).

selection of BS-tolerant rice calli in liquid culture medium
(Shimizu et al., 2005). The possibility that these two point
mutations were acquired spontaneously during only two
weeks of cell culture before BS selection is minimal.
Furthermore, the presence of the non-selectable silent
mutations, C fi T at +612 and C fi T at +2658, in the BS-
tolerant rice plants demonstrated that the W548 L and S627I
mutations were integrated into the rice genome via GT and
not by the occurrence of spontaneous mutation (Table 2 and
Figure 3c). In the case of true GT, no modification of genomic
DNA other than point mutations should have occurred. To
verify that this was the case, we performed Southern blot
analysis on DNA from 21 randomly selected plants from the
66 individual T0 plants found to possess two MfeI sites in the
ALS locus. Southern blot analysis of SphI-digested DNA
with probe A (Figure 1e) and DraI-digested DNA with

ª 2007 The Authors

Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 52, 157–166

160 Masaki Endo et al.
(a)
(b)
probe C (Figure 2b) revealed that 14 of the 21 T0 plants had
the same band pattern as wild-type rice cv. Nipponbare,
confirming that no rearrangements had occurred other than
HR between the targeting construct (D5¢ ALS) and the
chromosomal copy of the ALS gene.
Seven of the plants analysed by Southern blotting
showed extra bands. As PCR–MfeI digestion analysis indi-
cated that GT events had occurred in these seven plants, at
least in the 5’ flanking region of D5¢ ALS, additional bands in
Southern blot analysis might result from random integration
of T-DNA. However, these additional bands could also be
explained by the occurrence of HR at one end of the T-DNA
and non-homologous end-joining at the other end, or by
ectopic GT (Endo et al., 2006a; Hanin et al., 2001; Hohn and
Puchta, 2003; Offringa et al., 1993). We eliminated these
plants from candidates of the ‘clean GT plants’.
Confirmation of GT modification at the ALS locus
in true GT candidates
Analysis of the progeny of primary BS-tolerant recombi-
nants (T1) allows distinction between true GT and ectopic GT
events, as T1 generation individuals homozygous for the
modified copy of the chromosomal ALS locus would seg-
regate only in the case of true GT. Detailed analysis of true
GT candidates was thus performed in T1 progeny. Figures 3
and 4 show examples of such analyses. We analyzed eight
randomly chosen T1 progeny plants derived from one true
GT T0 plant (BSR 9). Genotyping analysis by PCR–MfeI
digestion as described above showed segregation of the
wild-type and mutated ALS genes in the T1 generation; one
plant homozygous for the modified ALS locus (BSR 9-3), six
heterozygous plants (BSR 9-1, -2, -4, -6, -7, -8), and one plant
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Figure 2. Southern blot analysis of the ALS locus
in T0 plants.
(a) Diagram showing the T-DNA region of the
targeting vector D5¢ ALS and the targeted ALS
locus. The expected band size after Southern blot
analysis using probe C is shown. LB, left border;
RB, right border; D, DraI restriction site.
(b) Southern blot analysis with probe C of DraI-
digested individual wild-type (WT) and BS toler-
ant T0 plants. Bands other than 3.4 kb indicate
the occurrence of random integration of T-DNA,
ectopic GT, or unexpected rearrangements of the
ALS locus.
with a wild-type ALS locus (BSR 9-5) were identified
(Figure 3b). These results confirm the occurrence of precise
gene replacement at the ALS locus of BSR 9. Genomic DNAs
from the homozygote (BSR 9-3), one of the heterozygotes
(BSR 9-4), and the segregated wild-type plant (BSR 9-5) were
isolated and sequenced. Direct sequencing of PCR frag-
ments F1–R1 (see Figure 3a) revealed that the W548 L (TGG
to TTG) and S627I (AGT to ATT) mutations co-segregated in
these plants (Figure 3c), confirming the results of PCR–MfeI
digestion analysis (Figure 3b). As mentioned previously, a
silent mutation in the ALS gene (C fi T at +612) and another
silent mutation in the downstream region of the ALS gene
(T fi C at +2658) exist in D5¢ ALS. To confirm the GT event in
true GT candidates, we analyzed the existence of these
mutations in T1 progeny plants. The T fi C mutation at
+2658 also co-segregated with W548 L and S627I mutations
in BSR 9-3, -4 and -5 (Figure 3c). The C fi T mutation at +612
was not detected in these plants (Figure 3c). This means that
the C fi T mutation at +612 was not integrated into the
genome of the BSR 9 plant during GT. The C fi T mutation at
+612 is located in the vicinity of the left border of D5¢ ALS,
and thus might be excluded during the process of HR. These
results indicate that a crossover occurred between +612 and
+1643 (W548 L), and that another crossover occurred
between +2658 and +8257 in BSR 9. Neither insertion of
T-DNA border sequence nor deletion of rice genomic
sequences at the positions of the right and left borders were
detected in PCR fragments F1–R1 and F2–R2 of the three
plants analyzed (data not shown). Southern blot analysis of
MfeI-digested genomic DNA also showed segregation of the
S627I mutation (Figure 3d). Furthermore, in Southern blot
analysis using probe D, which covers D5¢ ALS, we detected
the same band pattern in the wild-type and the plant
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Herbicide-tolerant rice generated by gene targeting 161

(a)

(b) (d)

(c)

Figure 3. Molecular analysis of T1 progeny of a gene targeting (GT) candidate.

(a) Diagram showing the T-DNA region of the targeting vector D5¢ ALS and the targeted ALS locus. The expected band sizes after Southern blot analysis using
probe B are shown. Pink lines indicate the polymerase chain reaction (PCR) products used for direct sequencing. Triangles indicate the primers used for
amplification of these PCR products. LB, left border; RB, right border; M, MfeI restriction site.
(b) PCR–MfeI digestion analysis of wild-type (WT) and eight randomly selected progeny of BSR 9 (BSR 9-1 to BSR 9-8). PCR was performed using primers F1 and R1.
The genotypes of each T1 progeny suggested by this experiment are: homozygous for the modified ALS locus, BSR 9-3; heterozygous for modified ALS locus, BSR
9-1, -2, -4, -6, -7 and -8; homozygous for the wild-type ALS locus, BSR 9-5.
(c) Sequencing chromatograms of the mutation sites. F1 and R1 were used as primers for PCR. Reverse primer R1 was used for direct sequencing analysis of W548 L
and S627I mutations, thus the complementary sequences of each mutation points are shown in the chromatograms. Direct sequencing indicated converted
nucleotides (TTG at amino acid 548 and ATT at amino acid 627) in BSR 9-3 (homo) or unconverted nucleotides (TGG at amino acid 548 and AGT at amino acid 627, i.e.
identical to wild-type) in BSR 9-5 (WT). Two overlapping peaks (TGG and TTG at amino acid 548, and AGT and ATT at amino acid 627) were observed in the
heterozygous plant BSR 9-4 (hetero). The reverse primer R4 was used for direct sequencing analysis of the silent mutation C fi T at +612. All three plants contained
the non-converted nucleotide C at +612. For sequencing analysis of the silent mutation T fi C at +2658, F3 and R3 were used as primers for PCR, and forward primer
F4 was used for sequencing. Direct sequencing indicated the presence of the converted nucleotide C in BSR 9-3 (homo) and the unconverted nucleotide T in BSR 9-5
(WT). Two overlapping peaks, C and T, were observed in BSR 9-4 (Hetero).
(d) Southern blot analysis of MfeI-digested wild-type (WT) and BSR 9-3, -4 and -5 using probe B.

homozygous for the modified ALS locus (Figure 4b).
The results of Southern blot analysis shown in Figure 4(c,d)
also confirmed that no modification other than the intro-
duction of the W548 L and S627I mutations had occurred in
the ALS locus of these T1 progeny. From these T1 analyses,
we concluded that true GT had indeed occurred in BSR 9. As
expected, there was no change in ALS gene transcription
levels between plants homozygous (GT homo) or heterozy-
gous (GT hetero) for the modified ALS locus and wild-type
plants (Figure 4e). For three true GT lines, T2 seeds from GT
homo/GT hetero/segregated wild-type T1 plants were ger-
minated on medium containing 1 lM BS. These plants
showed the expected segregation ratio of the BS-tolerant
trait (Table 1). We also analyzed the progeny of 12 true GT
candidates by PCR–MfeI digestion. All 12 lines showed
segregation of homozygous plants, plants heterozygous for
the modified ALS locus, and plants with the wild-type ALS
locus (Table 2). These results confirm the occurrence of
precise gene replacement at the ALS locus (true GT) in these
plants. All these true GT plants possessed the silent

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162 Masaki Endo et al.

(a) Figure 4. Southern and Northern blot analysis of

the acetolactate synthase (ALS) locus in T1
plants.
(a) Diagram showing the T-DNA region of the
targeting vector D5¢ ALS and the targeted ALS
locus. The expected band sizes after Southern
blot analysis using probes A, C and D are shown.
LB, left border; RB, right border; D, DraI restric-
tion site; C, ClaI restriction site.
(b) Southern blot analysis of ClaI-digested wild-
type (WT) and BSR 9-3 (homozygous for the
modified ALS locus) with probe D.
(c,d) Southern blot analysis of DraI-digested
wild-type (WT) and BSR 9-1 to BSR 9-8 using
(b) (c) (d) probe A (c) or probe C (d). Several additional
bands other than 4.3 kb in (c) or 3.4 kb in (d) were
due to non-specific hybridization, as these addi-
tional bands were also seen in the WT lane.
(e) Northern blot analysis with probe C of wild-
type (WT) plants, and plants homozygous
(homo) and heterozygous (hetero) for the mod-
ified ALS locus. The upper panel shows the
position of the Oryza sativa ALS transcript
(OsALS). The lower panel shows ethidium
bromide-stained rRNA as a loading control.
(e)

mutation downstream of the ALS gene on the GT vector genome. Because 14 of 21 randomly chosen T0 plants
D5¢ ALS (T fi C at +2658), and the C fi T mutation at +612 showed the same band pattern as the wild-type in Southern
was detected in three lines (BSR 13, 26 and 54). blot analysis (Figures 1e and 2b), we estimated that true or
Summarizing the results of T0 and T1 analysis, we Table 2 Segregation of the acetolactate synthase (ALS) locus in the
obtained 72 independent BS-tolerant plants from approxi- progeny of true gene targeting candidates
mately 1500 transformed rice calli. Of these 72 BS-tolerant
Polymerase chain reaction
plants, 66 plants possessed (functional ALS with W548 L and
(PCR)–MfeI digestiona
S627I mutations), via either true or ectopic GT in the
Wild type CfiT TfiC
T0 Homo Hetero (WT) (+612)b (+2658)c
Table 1 Segregation of bispyribac (BS) sensitivity in the T2 gener-
ation of true gene targeting plants BSR 9 2 6 2 C C
BSR 13 2 8 2 T C
Number of Expected BSR 15 4 5 2 C C
T1 T2 seeds ratio BSR 16 2 4 4 C C
T0 genotypinga tested BSR BSS (BSR:BSS) BSR 17 5 3 4 C C
BSR 18 2 1 7 C C
BSR 9 Homo 26 26 0 1:0 BSR 26 1 3 1 T C
Hetero 28 21 7 3:1 BSR 27 2 7 2 C C
Wild-type (WT) 30 0 30 0:1 BSR 38 5 3 2 C C
BSR 54 Homo 24 24 0 1:0 BSR 49 1 6 4 C C
Hetero 26 20 6 3:1 BSR 54 4 3 4 T C
WT 24 0 24 0:1 BSR 59 1 7 4 C C
BSR 59 Homo 24 24 0 1:0
a
Hetero 30 23 7 3:1 Homo, homozygous for the modified ALS locus; hetero, heterozy-
WT 24 0 24 0:1 gous for the modified ALS locus; wild type, segregated-out WT ALS
locus as determined by PCR–MfeI digestion analysis.
a b
Genotypes of the T1 seeds were determined by polymerase chain Nucleotide at position +612 of the modified ALS locus in homozy-
reaction–MfeI digestion analysis. Homo, homozygous for the mod- gous plants.
c
ified acetolactate synthase (ALS) locus; hetero, heterozygous for the Nucleotide at position +2658 of the modified ALS locus in homozy-
modified ALS locus; WT, segregated-out WT ALS locus. gous plants.

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Herbicide-tolerant rice generated by gene targeting 163

ectopic GT without random integration of T-DNA occurred in (a)

two-thirds of BS-tolerant rice plants. PCR–MfeI digestion
analysis of 12 T1 progeny of true ‘clean’ GT candidates
confirmed that true GT had occurred in all 12 lines analyzed
(Figure 3b and Table 2). Thus, the estimated number of true
GT plants without any modification of the genome is two-
thirds of the 66 GT plants obtained in this experiment. It is
difficult to estimate GT efficiency precisely because a
considerable number of transformation events occurred in
each callus, judging from both the positive control experi-
ments (Figure 1c) and an Agrobacterium-mediated transfor-
mation study with a GFP reporter gene construct (Toki et al.,
2006). Based on the transformation efficiency of the GT
vector (D5¢ ALS) and that of positive control experiments,
the estimated GT efficiency in our study was roughly 1 event (b)
in 2000–3000 potential transformants.

Plants homozygous for the modified ALS gene

exhibit hyper-tolerance to BS

We confirmed the acquisition of BS tolerance in GT rice

plants by spraying segregated T1 plants with BS (1 kg active
ingredient (a.i.) ha)1) (Figure 5a). Rice plants homozygous
and heterozygous for the modified ALS locus exhibited tol-
erance to BS and grew normally.
Next, we analyzed the enzymatic activity of ALS. Protein
extracts from wild-type plants, plants homozygous or het-
erozygous for the modified ALS locus, and plants over-
expressing the ALS gene containing the same two mutations
(W548 L and S627I), were examined for their sensitivities to
BS. In plants over-expressing the ALS gene containing the
two point mutations, the mutated ALS gene was driven by a
strong 35S promoter, thus the transcriptional level of this
mutated ALS gene was much higher than that in wild-type or
GT plants (approximately 20-fold higher than wild-type, data
not shown). In this experiment, extracts of plants homozy-
gous for the modified ALS locus were extremely tolerant to
BS; BS had almost no effect on the enzyme even at a
concentration of 100 lM (Figure 5b). Interestingly, the BS
tolerance level of GT homozygous plants exceeded that of
plants over-expressing the mutated ALS gene. The BS
tolerance levels of plants heterozygous for the modified
ALS locus and that of plants over-expressing the ALS gene
containing the same two mutations were almost the same.
The crystal structure of the catalytic subunit of Arabidop-
sis ALS in complex with ALS-inhibiting herbicides was
recently published (McCourt et al., 2006). The Arabidopsis
ALS tetramer consists of four identical subunits with an
overall folding similar to that of ALS from Saccharomyces
cerevisiae. In plants heterozygous for the modified ALS
gene, or in plants over-expressing the mutated ALS gene,
the putative ALS complexes would be composed of
a mixture of BS-tolerant and BS-sensitive subunits.
Presumably chimeric ALS complexes containing both types
Figure 5. BS sensitivity of segregated T1 plants.
(a) Plants at the 5–6-leaf stage were sprayed with bispyribac (BS) solutions
(1 kg a.i. ha)1). T1 plants with a modified ALS locus (either homozygous or
heterozygous) showed BS tolerance. T1 plants carrying the wild-type ALS
gene showed BS sensitivity, i.e. withered leaves.
(b) Sensitivity of ALS enzyme to BS. The inhibition rates of ALS enzymatic
activities at various BS concentrations are shown. ‘Over-expression’ refers to
plants over-expressing the mutated ALS gene containing the same two
mutations as GT plants.
of subunit would be inhibited by BS. Thus, exclusion of the
wild-type, BS-sensitive, ALS allele is important to produce
novel crops, which are hypertolerant to BS. In this regard,
GT can directly replace a wild-type gene with its mutated
counterpart, making it a powerful tool for this kind of gene
modification.
Concluding remarks
We were able to obtain clean GT plants with high efficiency
(approximately two-thirds of 66 GT plants from 1500 trans-
formed rice calli weighing approximately 30 g). This success
can be ascribed to several factors. Firstly, the efficient GT
system presented here is highly dependent on our efficient
transformation system in rice (Toki et al., 2006). In
our transformation system, several to hundreds of inde-
pendent transformation events occur in each small piece
of callus (Toki et al., 2006). Secondly, in contrast to a
negative–positive selection GT system (Terada et al., 2002),

ª 2007 The Authors

Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 52, 157–166

164 Masaki Endo et al.
our GT vector did not contain a negative selection marker, so
random integration and/or transient expression of the GT
vector was not thought to inhibit growth of potential tar-
geted transformants. Thirdly, we speculate that the condi-
tion of the chromatin structure of rice callus cells might
enhance HR. Recent reports indicate that an open chromatin
structure stimulates HR in higher plants (Endo et al., 2006b;
Shaked et al., 2005), and patterns of DNA methylation and
histone H3, H4 acetylation have been shown to vary during
growth of asynchronous potato cell suspensions (Law and
Suttle, 2005).
As the first report of the targeted reconstruction of a
transgene in the tobacco genome (Paszkowski et al., 1988),
GT events have been described at several loci in dicotyle-
donous plants (Endo et al., 2006a; Hanin et al., 2001; Kempin
et al., 1997; Lee et al., 1990; Miao and Lam, 1995; Shaked
et al., 2005). However, in monocots, which represent many
important crop plants, only one successful GT event has
been reported – a targeted disruption in rice of the Waxy
gene, a key enzyme in amylose synthesis, by an exogenous
selection marker (hygromycin phosphotransferase) using a
negative–positive selection GT system (Terada et al., 2002).
In this study, we succeeded in introducing several point
mutations (W548 L and S627I as well as silent mutations) by
a T-DNA-mediated GT system. GT plants obtained in this
study are equivalent to non-GM herbicide-tolerant rice
plants generated by conventional breeding approaches that
depend on spontaneous mutations. Furthermore, BS toler-
ance of GT plants homozygous for the mutated ALS gene
exceeded that of conventional transgenic plants over-
expressing an ALS gene containing W548 L and S627I
mutations, indicating that the GT approach might be supe-
rior to the conventional transgenic system in this respect.
Many agronomically valuable phenotypes and natural
variations are caused by single, or a small number of, point
mutations. In addition to herbicide tolerance, salt tolerance
and pigmentation are also potential selectable phenotypes.
Beneficial mutations are often discovered under long peri-
ods of cell culture, but few plants can be recovered from
these cells because of the prolonged cell culture. From this
point of view, GT is a powerful tool for producing plants
possessing these mutations in situ. Our GT strategy could be
applied directly to the improvement of these traits. On the
other hand, other agronomically valuable phenotypes
caused by single, or a small number of, point mutations
are non-selectable at the stage of transformation using
current methods (photoperiod sensitivity, Takahashi et al.,
2001; spotted leaf phenotype, Yamanouchi et al., 2002;
short-day promotion of flowering, Doi et al., 2004; seed
shattering, Konishi et al., 2006). If the GT frequency can be
improved substantially, co-transformation of a selectable
marker gene and a non-selectable GT construct and
subsequent identification of desirable targeted events using
suitable techniques such as a TILLING (Till et al., 2003) will
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cope with this problem. In fact, we observed that one-third of
GT plants possessed randomly integrated T-DNA (Fig-
ures 1e and 2b). Previous attempts to improve GT or HR
efficiency have included the induction of double-strand
breaks at the target site (Lloyd et al., 2005; Puchta et al.,
1996; Urnov et al., 2005), modification of proteins involved
in HR (e.g. Rad54, Shaked et al., 2005), and remodeling of
chromatin structure (INO80, Fritsch et al., 2004; CAF-1, Endo
et al., 2006b). Establishment of a GT system generally
applicable to any gene represents the next step in the
practical molecular breeding of crops.
Experimental procedures
Vector construction
The residues to be mutated were selected based on analysis of a
BS-tolerant rice callus (Oryza sativa L. cv. Kinmaze) produced by
somaclonal mutation during tissue culture (Shimizu et al., 2005). In
the ALS gene of this BS-tolerant callus, tryptophan 548 was mutated
to leucine (W548 L) and serine 627 was mutated to isoleucine
(S627I). Both mutations produce novel MfeI sites (Figure 1a). To
construct the targeting vector D5¢ ALS harboring the 5¢ truncated
ALS gene carrying these two point mutations, genomic DNA from
BS-tolerant rice callus was used as a template for PCR with primers
Asc-ALS (5¢-GGCGCGCCGCCGGCCACGCCGCTCCGGCCGT-3¢) and
Pac-ALS (5¢-TTAATTAAAAACTTGATATTATTCACACAGTGCCCCA-
3¢). An 8 kb fragment of the PCR product containing the W548 L and
S627I mutations was cloned into the AscI/Pac I sites of the binary
vector pPZP2028 (M. Kuroda, National Agricultural Research Center,
Hokuriku Research Center, Niigata, Japan, and S. Toki, unpublished
data), which is a derivative of pPZP202 (Hajdukiewicz et al., 1994),
with the addition of rare restriction (AscI/PacI) sites to both ends of
the multi-cloning site of pPZP202.
Rice transformation and BS selection
Agrobacterium-mediated transformation of rice (O. sativa L. cv.
Nipponbare) was performed as described previously (Toki, 1997;
Toki et al., 2006). After co-cultivation of Agrobacterium carrying the
binary vector D5¢ ALS with approximately 1500 rice scutellum-
derived calli (approximately 30 g; pre-cultured for 25 days, 2–5 mm
in diameter) for 3 days, infected calli were transferred to fresh callus
induction medium (Toki, 1997) containing 0.75 lM BS (Kumiai
Chemical Industry Co.; http://www.kumiai-chem.co.jp/english/
index.html), and BS-tolerant cells were selected over 25 days. Calli
that grew vigorously on the selection medium were transferred to
regeneration medium containing the same concentration of BS. The
regenerated plants were further grown on hormone-free medium to
facilitate root growth, and then planted in soil and grown to matu-
rity. These transgenic plants (T0 generation) were self-fertilized, and
T1 seeds were collected for detailed analysis.
PCR–MfeI digestion analysis
To confirm the occurrence of GT in BS-tolerant rice plants, we
performed PCR analysis coupled with MfeI digestion. We amplified
a 2287 bp PCR fragment corresponding to the N-terminal region of
ALS using primers F1 (5¢- CGTCACCGCGCGCGGACAAAACACC-
CAC-3¢), which anneals to the promoter region of the ALS
ª 2007 The Authors

Herbicide-tolerant rice generated by gene targeting 165
gene (absent in D5¢ ALS), and R1 (5¢-ACATGATATCTTGTGA-
TGCATATGCCTAC-3¢), present in D5¢ ALS (Figure 1a). The PCR
products were digested with MfeI.
Southern blot analysis
Genomic DNA was extracted from leaves of seedlings using the
Nucleon Phytopure extraction kit (Amersham Pharmacia Biotech;
http://www5.amershambiosciences.com/) according to the manu-
facturer’s instructions. After SphI, MfeI, DraI or ClaI endonuclease
digestion and electrophoresis on a 1% agarose gel, DNA fragments
were transferred onto a positively charged nylon membrane
(Roche; http://www.roche.com). Probes were prepared using a PCR
DIG probe synthesis kit (Roche) and hybridization was performed
according to the DIG Application Manual (Roche). Hybridization was
performed at 42C, and washing was performed under high-strin-
gency conditions at 68C.
Sequence analysis
Amplification using primers F1 and R1 yielded a PCR product of
2287 bp. Primer F1 was used to check the integration of left border
sequence or the deletion of any rice genomic sequence, and primer
R1 was used to check the presence of the W548 L and S627I muta-
tions. Primer R4 (5¢-AGGCGTGCGATGTACCCTGGTAGATT-3¢) was
used to check the presence of the C fi T mutation at +612. Amplifi-
cation using primers F2 (5¢-AGCCTGTCTGCTGGGAGACCACT-3¢)
(present in D5¢ ALS) and R2 (5¢-AGGATGCTTCTCTC-
TTCCACCGATCCA -3¢) (absent in D5¢ ALS) yielded a PCR product of
887 bp. Primer F2 was used to check the integration of right border
sequence or the deletion of rice genomic sequence. Amplification
using primers F3 (5¢-TGTCTTCGGCTGGTCTGGGCGCA-3¢) and R3
(5¢-TCCATTGGCCTAGGTAGTACACACTTACATCA-3¢) yielded a PCR
product of 1859 bp. Primer F4 (5¢-TGGGTACTACTATAGAGA-
GAGGCTGCATGAAGT-3¢) was used to check the presence of the
T fi C mutation at +2658. In all sequencing analyses, PCR products
were used directly as templates for sequencing.
Northern blot analysis
Total RNA was prepared from 6-week-old seedlings using an
RNeasy Plant Mini Kit (Qiagen; http://www.qiagen.com/). Total RNA
(10 lg) was loaded and separated on a 1% agarose gel, and trans-
ferred onto a positively charged nylon membrane (Roche). Probe C
was prepared using a PCR DIG probe synthesis kit (Roche), and
hybridization was performed according to the DIG Application
Manual (Roche). Hybridization was performed at 50C, and washing
was performed under high-stringency conditions at 50C.
BS sensitivity test of T1 progeny
Plants at the 5–6-leaf stage were sprayed with BS solutions (1 kg
a.i. ha)1) containing 5000-fold diluted Kumiten (Kumiai Chemical
Industry Co.) as an adhesive. Photographs were taken 9 days after
BS treatment.
Enzymatic assay of ALS
Proteins including ALS were extracted from rice shoots. The shoots
were homogenized in five volumes of 0.1 M potassium phosphate
buffer (pH 7.5) containing 0.5 mM MgCl2, 10% v/v glycerol. The
ª 2007 The Authors
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1365313x, 2007, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1365-313X.2007.03230.x by Kong Ju National University, Wiley Online Library on [11/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
homogenates were filtered through one layer of nylon gauze, and
centrifuged at 15 000 g for 20 min. ALS was precipitated from the
supernatant with ammonium sulfate at 50% saturation. After cen-
trifugation, at 15 000 g for 20 min, the pellets were dissolved in 0.1 M
potassium phosphate buffer (pH 7.5) containing 10% glycerol and
0.5 mM MgCl2. The solution was centrifuged at 15 000 g for 20 min
to remove insoluble substances. The supernatant fluids were then
desalted on a Sephadex G-25 column (GE Healthcare Bio-Sciences;
http://www.gehealthcare.com) equilibrated with the same buffer
and stored at )80C. All operations were carried out at 0–4C.
Enzyme activity of ALS was assayed at 37C in 0.5 ml of assay
mixture containing 20 mM potassium phosphate buffer (pH 7.5),
20 mM sodium pyruvate, 0.5 mM thiamine pyrophosphate,
0.5 mM MgCl2, 10 lM FAD, and various concentrations of BS
(10)9 to 10)4 M). Assays were initiated by adding 100 ll of crude
enzyme solution, and terminated after 30 min by the addition of
50 ll of 6 N H2SO4. The amount of acetolactate produced by the
enzyme reaction was determined as described previously (Ray,
1984) with the following modifications. The acidified reaction
mixtures were heated for 10 min at 60C, after which 0.5 ml of
0.5% w/v creatine and 0.5 ml of 2-naphtol solution (5% in 2.5 N
NaOH), both freshly prepared, were added to the solution, which
was then heated for 10 min at 60C. The absorbance at 525 nm
was then determined using a double beam spectrophotometer.
Acknowledgements
We thank R. Aoto, E. Ozawa, A. Nagashii, Y. Nomura and F. Suzuki
for their technical help. We also thank P. Maliga for providing
pPZP202, H. Rothnie for editing, and B. Hohn for critical reading of
the manuscript. This work was supported by the Ministry of
Agriculture, Forestry and Fisheries of Japan. This work was also
supported by a Program for Promotion of Basic Research Activi-
ties for Innovative Biosciences (PROBRAIN) grant to S.T. A part of
this study was also financially supported by the Budget for
Nuclear Research of the Ministry of Education, Culture, Sports,
Science and Technology, based on screening and counseling
by the Atomic Energy Commission. M.E. was supported by a
Japan Society for the Promotion of Science (JSPS) grant-in-aid
fellowship.
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