International Journal of

Molecular Sciences

Article
H2O2-Based Method for Rapid Detection of
Transgene-Free Rice Plants from Segregating
CRISPR/Cas9 Genome-Edited Progenies
Tsung-Meng Wu 1,2,† , Jian-Zhi Huang 1,† , Hui-Min Oung 1,‡ , Yi-Ting Hsu 3 , Yu-Chang Tsai 4, *
and Chwan-Yang Hong 1, *
1 Department of Agricultural Chemistry, College of Bioresources and Agriculture, National Taiwan University,
Taipei 10617, Taiwan
2 Department of Aquaculture, National Pingtung University of Science and Technology,
Pingtung 91201, Taiwan
3 Department of Agronomy, College of Agriculture and Natural Resources, National Chung-Hsing University,
Taichung 10617, Taiwan
4 Department of Agronomy, College of Bioresources and Agriculture, National Taiwan University,
Taipei 10617, Taiwan
* Correspondence: yuchangt@ntu.edu.tw (Y.-C.T.); cyhong@ntu.edu.tw (C.-Y.H.)
† These authors contributed equally to this work.
‡ Current address: Institute of Biological Chemistry, Washington State University, Pullman,
WA 99164-6340, USA.




Received: 27 May 2019; Accepted: 5 August 2019; Published: 9 August 2019


Abstract: Genome-editing techniques such as CRISPR/Cas9 have been widely used in crop functional
genomics and improvement. To efficiently deliver the guide RNA and Cas9, most studies still
rely on Agrobacterium-mediated transformation, which involves a selection marker gene. However,
several limiting factors may impede the efficiency of screening transgene-free genome-edited plants,
including the time needed to produce each life cycle, the response to selection reagents, and the
labor costs of PCR-based genotyping. To overcome these disadvantages, we developed a simple and
high-throughput method based on visual detection of antibiotics-derived H2 O2 to verify transgene-free
genome-edited plants. In transgenic rice containing hygromycin phosphotransferase (HPT), H2 O2 content
did not change in the presence of hygromycin B (HyB). In contrast, in transgenic-free rice plants
with 10-h HyB treatment, levels of H2 O2 and malondialdehyde, indicators of oxidative stress, were
elevated. Detection of H2 O2 by 3,30 -diaminobenzidine (DAB) staining suggested that H2 O2 could be
a marker to efficiently distinguish transgenic and non-transgenic plants. Analysis of 24 segregating
progenies of an HPT-containing rice plant by RT-PCR and DAB staining verified that DAB staining is
a feasible method for detecting transformants and non-transformants. Transgene-free genome-edited
plants were faithfully validated by both PCR and the H2 O2 -based method. Moreover, HyB induced
overproduction of H2 O2 in leaves of Arabidopsis, maize, tobacco, and tomato, which suggests the
potential application of the DAB method for detecting transgenic events containing HPT in a wide
range of plant species. Thus, visual detection of DAB provides a simple, cheap, and reliable way to
efficiently identify transgene-free genome-edited and HPT-containing transgenic rice.

Keywords: DAB (3,30 -diaminobenzidine); H2 O2 ; hygromycin; hygromycin phosphotransferase; rice;

CRISPR; reactive oxygen species

1. Introduction
The hygromycin phosphotransferase gene (HPT) from E. coli is a positive selection marker for
plant transformation [1]. HPT confers resistance to hygromycin B (HyB) in transgenic plants by

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adding a phosphate to position seven of the destomic acid ring in HyB [1,2]. More importantly, HyB
does not affect regeneration and fertility of transgenic plants [3]. Therefore, HPT is broadly used
in the transformation of monocot plants, which usually show high levels of natural resistance to
another antibiotic, kanamycin [4,5]. Plants effectively selected by using HPT include rice [6], maize [1],
wheat [7,8], brachypodium [9,10], and barley [11–13].
HyB is an aminoglycoside antibiotic isolated from Streptomyces hygroscopicus [14] that has
broad-spectrum activity against both prokaryotic and eukaryotic cells by interfering with translocation
of the 70S ribosome and causing misreading of the mRNA template [15,16]. HyB significantly inhibits
plant growth and development. Different plant genotypes exhibit variable HyB sensitivity. Culture
media containing 50 mg L−1 HyB could fully inhibit the growth of rice callus [17] and cotyledon and
leaves of Arabidopsis seedlings [18]; as low as 2.5 mg L−1 HyB was able to restrict the growth of maize
cells [19].
Rice is a staple food crop feeding more than half the world’s population [20]. To accelerate
biotechnological applications, the rice transformation system was established more than two decades
ago [6,21]. During rice transformation, HPT is used for screening putative transgenic events and
screening massive transgenic progenies or homozygous transgenic lines, thereby replacing the costly
and labor-intensive PCR-based or Southern blot analysis [22–24]. For efficient HyB screening, seeds
are germinated on medium containing HyB [24,25]; however, the germination rate may be confusing
in seeds with dormant or low germination vigor, which affects the determination of homozygosity.
CRISPR/Cas technology has emerged as a powerful and promising method to precisely modify
the plant genome and efficiently generate transgene-free crops [26,27]. The introduction of a
CRISPR/Cas-mediated genome-editing cassette into the plant genome allows for integrating the
transgene into one locus and performing the editing at another locus. Therefore, traits can be segregated
by sexual reproduction, generating progenies free of the transgene [26–28]. The transgene-free crops
thus contain biallelic/homozygous mutations and are free of selection markers. A number of useful
methods have been developed for screening biallelic/homozygous mutations. However, quick screening
of marker-free transgenic plants from a genome-edited (GE) plant population remains a challenge
because PCR-based screening is labor-intensive; furthermore, plants cannot survive on selection
medium without selection markers. Thus, a reliable, inexpensive, and non-lethal selection method is
needed to efficiently distinguish GE plants with or without selection markers.
Leaf painting assay has been used to facilitate the selection of transgenic plants tolerant to
antibiotics or herbicides. In cotton, leaves of transgenic cotton treated with 750 mg/L kanamycin
exhibited chlorosis after five to seven days and then necrotic patches after 10 days [29]. Transgenic
rice [30] and maize [31] expressing the bar gene were able to tolerate the herbicide phosphinothricin.
In transgenic maize, more than 95% of transgenic events can be verified by leaf painting assay, with
results agreeing with PCR results [31]. Leaf painting assay is simple and efficient, but it takes almost
one week to observe the wilt symptoms. Therefore, developing a simple, efficient, and rapid leaf
painting assay is needed for high throughput screening of transgenic progenies.
3,3-Diaminobenzidine (DAB) staining is one of the most commonly used methods for H2 O2
detection. After being taken up by plants, DAB reacts with H2 O2 to form a dark-brown reaction product
in the presence of peroxidase [32]. Our recent research indicated that HyB significantly and rapidly
enhanced the accumulation of H2 O2 in rice leaves [33]. Taking advantage of the high production of
H2 O2 in plants induced by HyB, we aimed to develop a simple and quick, selection-independent,
H2 O2 -based assay system for identifying transgenic rice.
In the present study, transgenic and non-transgenic rice could be easily distinguished by the
H2 O2 -based assay system. The visual selection method provides a quick and reliable way for
screening transgene-free GE plants after genome editing in rice. Moreover, we found HyB-induced
overproduction of H2 O2 in a wide range of plant species, so the H2 O2 DAB method may be applicable
for efficiently distinguishing a wide range of transgenic and non-transgenic plants.
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2. Results
2. Results

2.1. Hygromycin
2.1. Hygromycin (HyB)
(HyB) Significantly
Significantly Increased
Increased the
theProduction
ProductionofofH
H22O
O22 in Leaves of Rice Seedlings
Toexamine
To examinethetheeffect
effectof
ofHyB
HyBononthe theaccumulation
accumulationofofHH22OO22,, leaf
leaf segments
segments ofof rice
rice seedlings
seedlings were
were
treated with
treated with or
orwithout
without50 50mgmgmLmL −1−1 HyB
HyB for
forvarious
varioustimes.
times. H
H22O O22 level increased
increased with
with increasing
increasing
treatmenttime.
treatment time.HH22O
O22 level slightly increased
increased after
after 6-h
6-htreatment
treatmentand andsignificantly
significantlyincreased
increasedafter
after10-h
10-
h treatment
treatment (Figure
(Figure 1).1).

Figure 1. Temporal
Figure 1. Temporal accumulation
accumulation of
of hygromycin
hygromycin BB (HyB)-induced
(HyB)-induced hydrogen
hydrogen peroxide
peroxide (H
(H22O
O22)) in
in rice
rice
leaves. Leaf segments of 2-week-old rice seedlings were incubated with or without 50 mg L −1-1HyB for
leaves. Leaf segments of 2-week-old rice seedlings were incubated with or without 50 mg L HyB
the
for indicated time.time.
the indicated DataData
are mean
are mean SE=(n4).= 4).
± SE± (n * P *<P0.05 compared
< 0.05 with
compared –HyB.
with –HyB.

2.2. Accumulation of H2 O2 in Transgenic Rice Overexpressing HPT

2.2. Accumulation of H2O2 in Transgenic Rice Overexpressing HPT
To examine levels of H2 O2 in rice containing a hygromycin-detoxifying gene, we generated
To examine levels of H2O2 in rice containing a hygromycin-detoxifying gene, we generated
transgenic rice harboring HPT. HPT was overexpressed in three transgenic lines (OE-HPT-1, OE-HPT-2,
transgenic rice harboring HPT. HPT was overexpressed in three transgenic lines (OE-HPT-1, OE-
OE-HPT-3), with no transcripts detected in non-transgenic wild-type (WT) plants (Figure 2A,B).
HPT-2, OE-HPT-3), with no transcripts detected in non-transgenic wild-type (WT) plants (Figure
Cultivation of seeds on a hydroponic solution containing 50 mg L−1 HyB showed that the three
2A,B). Cultivation of seeds on a hydroponic solution containing 50 mg L−1 HyB showed that the three
transgenic lines germinated and grew normally, but germination was inhibited in seeds from WT
transgenic lines germinated and grew normally, but germination was inhibited in seeds from WT
plants (Figure 2C). Numbers of germinated plants of WT and three transgenic lines were almost 100%
plants (Figure 2C). Numbers of germinated plants of WT and three transgenic lines were almost 100%
without HyB treatment. In contrast, the germination rate of WT and the three transgenic lines was 0%,
without HyB treatment. In contrast, the germination rate of WT and the three transgenic lines was
72%, 68%, and 76%, respectively, when seeds were germinated on a hydroponic solution containing
0%, 72%, 68%, and 76%, respectively, when seeds were germinated on a hydroponic solution
50 mg L−1 HyB (Figure 2D). Upon treatment with HyB, H2 O2 level significantly increased only in
containing 50 mg L−1 HyB (Figure 2D). Upon treatment with HyB, H2O2 level significantly increased
non-transgenic rice seedlings and not in the three transgenic lines containing HPT (Figure 3A). Then,
only in non-transgenic rice seedlings and not in the three transgenic lines containing HPT (Figure
we used DAB treatment to distinguish transgenic and non-transgenic rice. Without HyB treatment, leaf
3A). Then, we used DAB treatment to distinguish transgenic and non-transgenic rice. Without HyB
segments of both non-transgenic and transgenic rice showed very little brown color. Conversely, after
treatment, leaf segments of both non-transgenic and transgenic rice showed very little brown color.
DAB treatment, leaves of WT but not transgenic rice showed a dark brown color (Figure 3B). Moreover,
Conversely, after DAB treatment, leaves of WT but not transgenic rice showed a dark brown color
HyB treatment significantly increased the production of malondialdehyde in WT but not OE-HPT-1
(Figure 3B). Moreover, HyB treatment significantly increased the production of malondialdehyde in
plants (Figure 3C), so HyB caused severe oxidative stress, thereby enhancing lipid peroxidation.
WT but not OE-HPT-1 plants (Figure 3C), so HyB caused severe oxidative stress, thereby enhancing
lipid peroxidation.
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Figure 2. Generation of transgenic rice overexpressing hygromycin phosphotransferase. (A)

Schematic representation of the pPZP/HPH binary vector used for rice transformation. 35S,
Figure 2. Generation
Cauliflower of transgenic
mosaic virus rice overexpressing
35S promoter; HPT, hygromycin hygromycin phosphotransferase.
phosphotransferase (HPT); 3’Nos, (A)3’UTR
Schematic
of
Figure 2. Generation of transgenic rice overexpressing hygromycin phosphotransferase. (A)
representation
Nopaline of the
synthase. (B) pPZP/HPH
RT-PCR binaryof
analysis vector
mRNA used for rice transformation.
expression of HPT in 35S, Cauliflower
wild-type (WT) and mosaic
HPT-
Schematic representation of the pPZP/HPH binary vector used for rice transformation. 35S,
virus 35S promoter;
overexpressing HPT, hygromycin
rice plants. OsActin1, phosphotransferase
rice actin1hygromycin (HPT); 3’Nos,
gene. (C) Phenotypes 3’UTR
of rice of Nopaline
seedlings grown synthase.
on of
Cauliflower mosaic virus 35S promoter; HPT, phosphotransferase (HPT); 3’Nos, 3’UTR
(B) RT-PCR
hydroponic analysis
solution of mRNA expression of HPT in wild-type (WT) and HPT-overexpressing rice
(B)with (+HyB) or without (−HyB) 50 mg LofHyB HPTfor 7 days. (bar = 1 cm). (D) The
-1
Nopaline synthase. RT-PCR analysis of mRNA expression in wild-type (WT) and HPT-
plants.
number OsActin1, rice
of germinated actin1 gene. (C)
and OsActin1, Phenotypes
non-germinated plantsof rice
in WT seedlings grown on hydroponic
and HPT-overexpressing solution
ricegrown
plants on with
overexpressing rice plants. rice actin1 gene. (C) Phenotypes of rice seedlings
(+HyB)
grown or without
on hydroponic(−HyB)
solution L−1 (+HyB)
50 mgwith HyB foror7 days.
without (bar = 1 cm).
(−HyB) 50 (D)
mg The
L number
-1 HyB for 7of germinated
days (n =(D) and
50).The
hydroponic solution with (+HyB) or without (−HyB) 50 mg L-1 HyB for 7 days. (bar = 1 cm).
non-germinated
Absolute percentplants in WT and
germination is HPT-overexpressing
reported as the average rice
ofplants
the grown
mean of on hydroponic
three independent solution with
number of germinated and non-germinated plants in WT and HPT-overexpressing rice plants
(+HyB) or without
experiments with (−HyB)
the error50bars L−1 HyB for 7SD.
mg representing days (n = 50). Absolute percent germination is reported
grown on hydroponic solution with (+HyB) or without (−HyB) 50 mg L-1 HyB for 7 days (n = 50).
as the average of the mean of three independent experiments with the error bars representing SD.
Absolute percent germination is reported as the average of the mean of three independent
experiments with the error bars representing SD.

Figure 3. H2 O2 content in WT and HPT-overexpressing seedlings under HyB treatment. (A) changes
Figure
in H2 O23.content
H2O2 content
in leaf in WT andof
segments HPT-overexpressing seedlings under
WT and HPT-overexpressing HyB treatment.
2-week-old seedlings (A) changes
treated with
in H 2O2 −1
content in leaf segments of WT and HPT-overexpressing 2-week-old seedlings
50 mg L HyB for 12 h. Data are mean ± SE (n = 4). * P < 0.05 compared with WT. (B) Histochemical treated with
50 mg L3.−1 H
Figure
detection HyB
of H22for
2O O2 12
content h.in
with Data
WTare
DAB andmean ± SE (nsegments
= 4). * P <seedlings
HPT-overexpressing
staining in leaf 0.05
of WTcompared
under
and with
HyBWT. (B) Histochemical
HPT-Overexpressing
treatment. (A) changes
rice plants
detection
(OE-HPT-1) of
in H2O2 content H 2O2 with DAB staining in leaf segments of WT and HPT-Overexpressing rice plants
treated with
in leaf or without
segments HyB.
of WT and(C)HPT-overexpressing
Quantification of DAB staining.seedlings
2-week-old The histogram
treatedtool
within
(OE-HPT-1)
ImageJ was treated
used to with
record or without
the HyB.
grayscale (C)
values Quantification
of all pixels of DAB
within the staining.
brown The
areas
50 mg L HyB for 12 h. Data are mean ± SE (n = 4). * P < 0.05 compared with WT. (B) Histochemical
−1 histogram
of eight-bit tool in
images.
ImageJ
(D) was used
Malondialdehyde
detection to record
of H2O2 with(MDA) the grayscale
content in
DAB staining values
in leaf of all pixels within
and HPT-overexpressing
WT segments the brown areas of eight-bit
rice plants (OE-HPT-1)
of WT and HPT-Overexpressing treated
rice plants
images.
with
(OE-HPT-1)(D) Malondialdehyde
or without 50 mg
treated withL−1orHyB (MDA)
for 12
without content
h. Data
HyB. in
areWT
meanand±HPT-overexpressing
(C) Quantification SEof = 4). staining.
(nDAB rice
Bars withThe plants
a same (OE-HPT-
letter
histogram are
toolnot
in
1) treated
significantly
ImageJ waswith or to
without
different
used at P <50
record mg
0.05.
the L−1 HyBvalues
grayscale for 12 h.
of Data are mean
all pixels within± SE
the(n = 4). Bars
brown areaswith a same letter
of eight-bit
are not significantly
images. different at(MDA)
(D) Malondialdehyde P < 0.05.
content in WT and HPT-overexpressing rice plants (OE-HPT-
1) treated with or without 50 mg L−1 HyB for 12 h. Data are mean ± SE (n = 4). Bars with a same letter
are not significantly different at P < 0.05.
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2.3. DAB–H2O2 Method is Efficient for Detecting HPT-Containing Transgenic Rice

2.3. DAB–H2 O2 Method is Efficient for Detecting HPT-Containing Transgenic Rice
To test the accuracy of the DAB–H2O2 method for distinguishing transgenic plants, we used RT-
To test the accuracy of the DAB–H O method for distinguishing transgenic plants, we used
PCR and the DAB method to examine 242 T12 segregants from one transgenic plant carrying the HPT
RT-PCR and the DAB method to examine 24 T1 segregants from one transgenic plant carrying the HPT
transgene (OE-HPT-1). On RT-PCR, HPT was detected in transgenic progenies but not null
transgene (OE-HPT-1). On RT-PCR, HPT was detected in transgenic progenies but not null segregants
segregants (Figure 4A). T1 segregants without HPT transcripts showed significant DAB–H2O2
(Figure 4A). T1 segregants without HPT transcripts showed significant DAB–H2 O2 accumulation.
accumulation. Conversely, plants expressing HPT showed no significant DAB–H2O2 accumulation
Conversely, plants expressing HPT showed no significant DAB–H2 O2 accumulation (Figure 4B). Thus,
(Figure 4B). Thus, the DAB–H2O2 method was as reliable as RT-PCR for detecting transgenic plants
the DAB–H2 O2 method was as reliable as RT-PCR for detecting transgenic plants containing an
containing an HyB-resistant gene.
HyB-resistant gene.

Figure 4. Detection of non-transformants and HPT-overexpressing transgenic rice by RT-PCR and DAB
Figure 4. Detection
staining. of non-transformants
(A) Total RNA was isolated fromand HPT-overexpressing
leaves of 24 progenies oftransgenic rice by RT-PCR
the T1 segregation and of
population
DAB staining. (A) Total RNA was isolated from leaves of 24 progenies of the T1 segregation
OE-HPT-1. Rice Actin1 was an internal control. (B) H2 O2 levels were detected by DAB staining in leaf
population
segments ofof24 OE-HPT-1.
T1 progeny Rice
of Actin1 was treated
OE-HPT-1 an internal
withcontrol.
or without 2O2 levels were detected by DAB
(B) HHyB for 12 h. (C) quantification of
staining
DAB staining results. The histogram tool in ImageJ was used to record thewithout
in leaf segments of 24 T1 progeny of OE-HPT-1 treated with or HyB
grayscale for 12
values of h.
all(C)
pixels
quantification of DAB staining results. The histogram tool in ImageJ was used to record the
within the brown areas of eight-bit images. Each experiment was carried out in 4 technical replicates
grayscale values replicates.
and 3 biological of all pixelsOne
within the brown experiment
representative areas of eight-bit images.
is shown. DataEach experiment
are mean was with a
± SE. Bars
carried out in 4 technical replicates and 3 biological
same letter are not significantly different at P < 0.05. replicates. One representative experiment is
shown. Data are mean ± SE. Bars with a same letter are not significantly different at P < 0.05.
2.4. Detection of Transgene-Free Genome-Edited Plants by DAB Method
2.4. Detection of Transgene-Free Genome-Edited Plants by DAB Method
To screen transgene-free genome-edited rice, the T1 population of Agrobacterium-mediated
CRISPR/Cas9
To screenrice was evaluated
transgene-free with the DABrice,
genome-edited method.
the T1The CRISPR/Cas9
population binary vector encoded
of Agrobacterium-mediated
an HPT gene for
CRISPR/Cas9 ricetransformation
was evaluated selection
with the DABin themethod.
T0 generation [34,35]. After
The CRISPR/Cas9 the CRISPR/Cas9
binary vector
vector encoded an
HPT gene
is used forforgene
transformation
editing at the selection
targetinregions,
the T0 generation
the vector [34,35].
can be After the CRISPR/Cas9
removed vector is
in the T1 segregation
used for geneFour
population. editing at the target regions,
T1 genome-edited mutantsthe (osrr6/osrr11#2-3-1,
vector can be removed osrr6/osrr11#5-1-11, osrr9/osrr10#9-2-4,
in the T1 segregation population.
and osrr9/osrr10#10-3-6)
Four T1 genome-editedfrom mutants (osrr6/osrr11#2-3-1,
two transgenic events wereosrr6/osrr11#5-1-11,
used to examineosrr9/osrr10#9-2-4,
the elimination ofand the
osrr9/osrr10#10-3-6)
transgene vector. The fromtwotwo transgenicevents
transgenic eventstargeted
were used to examine
multiple the elimination
cytokinin of the transgene
two-component signaling
vector. The two regulators,
type-A response transgenic OsRR6,
events OsRR11,
targeted OsRR9,
multipleandcytokinin
OsRR10.two-component
The mutations atsignaling
the targettype-A
region
response regulators,
were verified by SangerOsRR6, OsRR11, OsRR9,
sequencing and OsRR10.
(Supplementary Table The
S1mutations at the
[34,36]). The target region
presence of the were
HPT
verified
selectablebymarker
Sangerwas sequencing
visually (Supplementary
selected by the DAB Table S1 [34,36]).
method The presence of the HPT selectable
and RT-PCR.
marker was visuallygenome-edited
In osrr6/osrr11 selected by thelines,
DAB single
method and RNAs
guide RT-PCR. (sgRNAs) specific to editing conserved
In osrr6/osrr11
sequences of OsRR6genome-edited
and OsRR11 were lines, single guide
designed. RNAs (sgRNAs)
In osrr9/osrr10 specificlines,
genome-edited to editing
sgRNAs conserved
specific
sequences of OsRR6 sequences
to editing conserved and OsRR11 were designed.
of OsRR9 and OsRR10 In osrr9/osrr10
were designed. genome-edited
No significantlines, sgRNAs
DAB–H 2 O2
specific to editing
accumulation wasconserved
detected sequences of OsRR9HyB
in leaves without and treatment
OsRR10 were designed.
(Figure No significant
5A). After DAB–
HyB treatment,
H2O2 accumulation was detected in leaves without HyB treatment (Figure 5A). After HyB treatment,
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a number of segregants of osrr6/osrr11#5-1-1, osrr9/osrr10#9-2-4, and osrr9/osrr10#10-3-6 showed slight

a number of segregants of osrr6/osrr11#5-1-1, osrr9/osrr10#9-2-4, and osrr9/osrr10#10-3-6 showed slight
DAB–H2O2 accumulation, which indicated the presence of HPT. In contrast, significant DAB–H2O2
DAB–H2 O2 accumulation, which indicated the presence of HPT. In contrast, significant DAB–H2 O2
accumulation could be detected in all tested segregants of osrr6/11#2-3-1, which strongly suggests
accumulation could be detected in all tested segregants of osrr6/11#2-3-1, which strongly suggests that
that osrr6/osrr11#2-3-1 is a transgene-free homozygous null mutant (Figure 5A).
osrr6/osrr11#2-3-1 is a transgene-free homozygous null mutant (Figure 5A).

Figure 5. Detection of transgene-free CRISPR/Cas9 genome-edited plants by DAB staining in rice. Leaf
painting
Figure 5.assay and quantification
Detection of transgene-freeof DAB staining results
CRISPR/Cas9 were carried
genome-edited out by
plants in leaf
DABsegments
staining treated
in rice.
without (A,B) or
Leaf painting withand
assay (C,D) 50 mg L−1 HyB,
quantification of DABrespectively. (E) Total
staining results wereRNA was out
carried isolated from
in leaf leaves
segments
of CRISPR mutant lines (osrr6/11#2-3-1, osrr6/11#5-1-1, osrr9/10#9-2-4, and osrr9/10#10-3-6).
treated without (A,B) or with (C,D) 50 mg L HyB, respectively. (E) Total RNA was isolated from
-1 HPT was
detected by RT-PCR. Rice Actin 1 was an internal control. Leaf segments of CRISPR mutant
leaves of CRISPR mutant lines (osrr6/11#2-3-1, osrr6/11#5-1-1, osrr9/10#9-2-4, and osrr9/10#10-3-6). HPT lines treated
with or without
was detected HyB for 12
by RT-PCR. Riceh Actin
were 1used
was for DAB staining.
an internal control. Each experiment
Leaf segments was carried
of CRISPR out lines
mutant in 4
technical replicates
treated with and 3HyB
or without biological
for 12replicates.
h were usedOneforrepresentative
DAB staining.experiment is shown.was
Each experiment Data are mean
carried out
± SE. Bars with a same letter within each line are not significantly different at P < 0.05.
in 4 technical replicates and 3 biological replicates. One representative experiment is shown. Data are
mean ± SE. Bars with a same letter within each line are not significantly different at P < 0.05.
Further RT-PCR analysis was conducted to validate the presence of the HPT gene in genome-edited
HPT could
lines.Further be detected
RT-PCR analysisinwas
several segregants
conducted of osrr6/11#5-1-1,
to validate osrr9/10#9-2-4,
the presence of the HPT and osrr9/10#10-3-6
gene in genome-
(No. 1–5). In contrast, no HPT product could be detected in five segregants of osrr6/11#2-3-1,
edited lines. HPT could be detected in several segregants of osrr6/11#5-1-1, osrr9/10#9-2-4, which
and
indicates lack of(No.
osrr9/10#10-3-6 the transgene in osrr6/11#2-3-1
1 – 5). In contrast, no HPT(Figure
product5C).could
However, segregants
be detected of genome-edited
in five segregants of
plants containing the HPT gene in osrr6/11#5-1-1 (No. 1 and 5), osrr9/10#9-2-4 (No. 3–5), and
osrr6/11#2-3-1, which indicates lack of the transgene in osrr6/11#2-3-1 (Figure 5C). However,
osrr9/10#10-3-6 (No. 1 and 2) all showed slight DAB–H2 O2 staining (Figure 5B). In contrast, strong
segregants of genome-edited plants containing the HPT gene in osrr6/11#5-1-1 (No. 1 and 5),
DAB–H2 O2 accumulation was detected in segregants of genome-edited plants without the HPT gene:
osrr9/10#9-2-4 (No. 3–5), and osrr9/10#10-3-6 (No. 1 and 2) all showed slight DAB–H2O2 staining
Osrr6/11#2-3-1 (No. 1–5), osrr6/11#5-1-1 (No. 2–4), osrr9/10#9-2-4 (No. 1 and 2), and osrr9/10#10-3-6
(Figure 5B). In contrast, strong DAB–H2O2 accumulation was detected in segregants of genome-
(No. 3–5). Hence, DAB–H2 O2 staining is a reliable method for detecting transgenic plants free of
edited plants without the HPT gene: Osrr6/11#2-3-1 (No. 1–5), osrr6/11#5-1-1 (No. 2–4), osrr9/10#9-2-4
a transgene (Figure 5B,C). No HPT product could be detected in WT plants. Transgenic OE-HPT-1
(No. 1 and 2), and osrr9/10#10-3-6 (No. 3–5). Hence, DAB–H2O2 staining is a reliable method for
containing an HPT gene were a positive control.
detecting transgenic plants free of a transgene (Figure 5B,C). No HPT product could be detected in
WT HyB-Induced
2.5. plants. Transgenic OE-HPT-1
Overproduction of containing an HPT
H2 O2 Observed gene were
in Monocot and aDicot
positive control.
Plants
To examine Overproduction
2.5. HyB-Induced whether H2 O2 ofcan
H2be
O2 significantly inducedand
Observed in Monocot byDicot
HyB Plants
in other plants, we examined
plants such as Arabidopsis, tobacco, tomato, and maize. DAB–H2 O2 staining showed that H2 O2 was
Toinduced
highly examineinwhether H2plants
all tested O2 can after
be significantly induced
HyB treatment by HyB
(Figure in othershowed
6). Tobacco plants, we
the examined
strongest
plants such as Arabidopsis, tobacco, tomato, and maize. DAB–H2O2 staining showed that H2O2 was
highly induced in all tested plants after HyB treatment (Figure 6). Tobacco showed the strongest
Int. J. Mol. Sci. 2019, 20, 3885 7 of 11
Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 7 of 11

accumulation of
of H
H2OO2, followed by Arabidopsis and tomato. The monocot plant maize showed a slight
accumulation 2 2 , followed by Arabidopsis and tomato. The monocot plant maize showed a slight
enhancement of
enhancement of H
H2OO2 after
after HyB
HyB treatment.
treatment.
2 2

Figure 6. Detection of HyB-induced H2 O2 accumulation by DAB staining in different plant species.

Figure 6. Detection of HyB-induced H2O2 accumulation by DAB staining in different plant species.
(A) Arabidopsis leaves or leaf discs of (B) tomato, (C) tobacco, and (D) maize were treated with or
(A) Arabidopsis leaves or leaf discs of (B) tomato, (C) tobacco, and (D) maize were treated with or
without 50 mg L−1 HyB for different times as indicated. Ten leaf or leaf discs were used for DAB
without 50 mg L HyB for different times as indicated. Ten leaf or leaf discs were used for DAB
−1
staining per sample, with 4 biological replicates for each treatment. Representative results are shown.
staining per sample, with 4 biological replicates for each treatment. Representative results are shown.
Scale bars: 0.5 cm.
Scale bars: 0.5 cm.
3. Discussion
3. Discussion
Genome-editing is a powerful tool for precision breeding in crops. Several countries will not
Genome-editing
regulate is a powerful tool
the use of genome-editing for precision
techniques breeding
in plants, whichinindicates
crops. Several countries
that there will be will not
more
regulate
GE cropsthe in use
the of genome-editing
future. Therefore, techniques
developinginaplants, which indicates
high-throughput that there
screening will be
platform formore GE
rapidly
crops in the future. Therefore, developing a high-throughput screening
distinguishing transgenic or non-transgenic plants is required to efficiently generate transgene-free platform for rapidly
distinguishing transgenic
genome-edited plants. Our or previous
non-transgenic
study plants
showed is required to efficiently
that rice seedlings generate
treated withtransgene-free
HyB for 12 h
genome-edited
markedly plants.
increased H2 O Our
2
previous
level in study showed
non-transformants that
but rice
not inseedlings
transgenic treated
rice with HyB
containing for HPT
the 12 h
markedly increased H O level in non-transformants but not in transgenic
gene [33]. In this study, we further verified that genome-edited rice progenies containing HPT can
2 2 rice containing the HPT
genebe
also [33]. In this study,
faithfully reflectedweby further
visualverified
detection thatofgenome-edited
the DAB–H2 O2rice progenies
complex containing
in leaves HPT can
and validated
also be faithfully reflected by visual detection of the DAB–H O complex
by RT-PCR, which indicates that the H2 O2 -based leaf painting assay is a rapid and reliable method.
2 2 in leaves and validated by
RT-PCR, which indicates that the H O -based leaf painting assay is a rapid
As compared with previous studies of cotton [29], rice [30], and maize [31], our H2 O2 -based method in
2 2 and reliable method. As
compared
rice largely with previous
decreased studies
the time fromoffive
cotton [29], days
to seven rice [30],
to 12 and
h, somaize
the H2[31], our Hleaf
O2 -based 2O2painting
-based method
assay isin
a
rice largely decreased the time from five to seven days to 12 h, so the H O
high-throughput system for screening transgene-free genome-edited crops from segregating progenies.
2 2 -based leaf painting assay
is a high-throughput system for screening transgene-free genome-edited crops from segregating
4. Materials and Methods
progenies.

4.1. Plant Materials

4. Materials and Methods
Rice (Oryza sativa, L. cv. Tainung 67) seedlings were grown hydroponically in half-strength Kimura
B solutionMaterials
4.1. Plant in a phytotron (Agricultural Experimental Station, National Taiwan University, Taipei,
Taiwan) ◦ C day/25 ◦ C night and 90% relative humidity. Arabidopsis thaliana
Ricewith natural
(Oryza light
sativa, L. at
cv.30Tainung 67) seedlings were grown hydroponically in half-strength
ecotype Columbia (Col-0), maize (Zea
Kimura B solution in a phytotron (Agricultural mays L. cv.Experimental
Tainan-White), tobacco
Station, (Nicotiana
National benthamiana
Taiwan L.),
University,
and tomato
Taipei, Taiwan) withlycopersicum
(Solanum natural lightL.atcv.30Moneymaker)
°C day/25 °Cwere
nightgrown in a relative
and 90% mixturehumidity.
of perlite:vermiculite
Arabidopsis
(1:1) in a growth chamber at 25 ◦ C and light intensity 400 µmol m−2 s−1 with a 16-h light/8-h dark cycle
thaliana ecotype Columbia (Col-0), maize (Zea mays L. cv. Tainan-White), tobacco (Nicotiana
and relative humidity
benthamiana 80%. Two-week-old
L.), and tomato seedlingsL.were
(Solanum lycopersicum used for HyB treatment.
cv. Moneymaker) were grown in a mixture of
perlite:vermiculite (1:1) in a growth chamber at 25 °C and light intensity 400 μmol m−2s−1 with a 16-h
4.2. Generation of Hygromycinb (HyB)-Resistant Transgenic Rice
light/8-h dark cycle and relative humidity 80%. Two-week-old seedlings were used for HyB treatment.
The pPZP/HPH binary vector [21] was used for transformation of HyB-resistant rice plants.
4.2. plasmid
The Generation of Hygromycinb
pPZP/HPH (HyB)-Resistant
was introduced Transgenic Rice
into Agrobacterium tumefaciens strain EHA105, and embryogenic
calli derived from immature
The pPZP/HPH binaryseeds of[21]
vector Tainung 67 were
was used fortransfected as described
transformation [21]. Putative
of HyB-resistant ricetransformed
plants. The
plasmid pPZP/HPH was introduced into Agrobacterium tumefaciens strain EHA105, and embryogenic
Int. J. Mol. Sci. 2019, 20, 3885 8 of 11

calli were selected on HyB (Invitrogen, Carlsbad, CA, USA). Regenerated transgenic plants were
grown and self-pollinated for two generations. For generating CRISPR/Cas9-mediated mutants,
20-bp sgRNAs complementary to rice cytokinin signaling type-A response regulators, osrr6, osrr9,
osrr10, and osrr11, were designed and cloned into a pAS3 binary vector harboring an sgRNA
cassette, a maize UBQ10-drived Cas9, and a hygromycin selection marker for Agrobacterium-mediated
transformation [34–36]. The transformed calli were first selected on HyB media and the surviving
calli were regenerated to T0 plantlets. At the T1 generation, the GE target regions may segregate with
CRISRP/Cas9 vectors. To identify the mutants at the T1 generation, the sgRNA target sequences were
amplified by PCR and subjected to one-step PAGE [37], high-resolution melt analysis (HRM) [38], or
Sanger sequencing [39].

4.3. HyB Treatment

Leaf segments of 1 cm from 2-week-old rice seedlings were cut and cultured in sterile distilled
water containing HyB. Leaf segments without any treatment were controls. For Arabidopsis, leaves of
2-week-old seedlings were treated with or without HyB. For tomato, tobacco, and maize, leaf discs
were used for HyB treatment. All leaf segments/discs were treated with 50 mg L−1 HyB, and plants
were incubated in a growth chamber at 27 ◦ C under light for 12 h. We used 10 leaf segments or leaf
discs per sample, with 4 biological replicates for each treatment. Representative results are shown.

4.4. Visual Detection of H2 O2

H2 O2 was visually detected in leaves by using DAB as a substrate [40]. Leaf segments or leaf
discs were treated with or without 50 mg L−1 HyB. After 12 h, leaves were first rinsed with distilled
water and then supplied with DAB solution (1 mg mL−1 ) through the cut ends for 12 h under light at
27 ◦ C. Leaves were decolorized in boiling ethanol (95%) for 0.5 h. This treatment decolorized the leaves
except for the brown polymerization. The H2 O2 staining was repeated 4 times with similar results.

4.5. Quantification of H2 O2 , DAB–H2 O2 , Malondialdehyde, and Protein Content

To measure H2 O2 content, the reaction mixture consisted of 2 mL of 50 mM phosphate-buffered
(pH 6.8) leaf extract supernatant and 1 mL reagent [0.1% (v/v) TiCl4 in 20% (v/v) H2 SO4 ]. The blank
reaction consisted of 50 mM phosphate buffer without leaf extract. H2 O2 content was measured
spectrophotometrically after a reaction with TiCl4 [41] with absorbance measured at 410 nm. The amount
of H2 O2 was calculated by using a standard curve prepared with known concentrations of H2 O2 in
some experiments, H2 O2 content was measured spectrophotometrically after a reaction with DAB
solution [42]. The reaction mixture consisted of 2 mL of 50 mM phosphate buffer (pH 6.8) leaf extract
supernatant and 1 mg mL−1 DAB solution. The blank reaction consisted of 50 mM phosphate buffer
without leaf extract. The absorbance was measured at 465 nm. To quantify DAB staining results, brown
areas of the DAB–H2 O2 reaction were scanned and measured with ImageJ (https://imagej.nih.gov/ij/).
The ImageJ was used to record the grayscale values of all pixels within the brown areas of eight-bit
images. An identical noise threshold was used for all analyses and was visually inspected for accurate
representation of the particles.
Malondialdehyde, routinely used as an indicator of lipid peroxidation, was extracted with 5% (w/v)
trichloroacetic acid, and the content was determined by the thiobarbituric acid reaction as described [43]
and expressed on the basis of fresh weight. For protein determination, leaves were homogenized in
50 mM sodium phosphate buffer (pH 6.8). Extracts were centrifuged at 17,600× g for 20 min, and the
supernatant was used for determining protein level by the Bradford method [44].

4.6. RT-PCR Analysis

For molecular analysis of HPT-overexpressing transgenic rice and CRISPR mutant lines
(osrr6/osrr11#2-3-1, osrr6/orss11#5-1-1, osrr9/osrr10#9-2-4, and osrr9/osrr10#10-3-6), total RNA was
isolated by using TRIzol solution (Invitrogen, Carlsbad, CA, USA) from leaves of wild-type and
Int. J. Mol. Sci. 2019, 20, 3885 9 of 11

putative transformants. For RT-PCR analysis, HPT was amplified with the primer sequences
HPT-F, 50 -GTGCTTGACATTGGGGAGTT-30 , and HPT-R, 50 -ACATTGTTGGAGCCGAAATC-30 . PCR
conditions were 94 ◦ C for 5 min, then 32 cycles at 94 ◦ C for 30 s, 50 ◦ C for 30 s, and 72 ◦ C for 1 min.
Rice Actin1 gene, amplified with the primer sequences Actin1-F, 50 -ATGCTCTCCCCCATGCTATC,
and Actin1-R, 50 -TCTTCCTTGCTCATCCTGTC-30 , was an internal control. RT-PCR was performed in
triplicate for each individual line; results from one repeat are shown in the figure.

4.7. Statistical Analysis

Data are expressed as mean ± SE. Each experiment was carried out in 4 technical replicates and 3
biological replicates. One representative experiment is shown. Differences between measurements
were analyzed by Student t test or Duncan’s multiple range test. p < 0.05 was considered
statistically significant.

5. Conclusions
We show that the notable difference in H2 O2 levels induced by HyB between non-transformant
and transgenic rice harboring HPT can be a convenient system for distinguishing transgenic plants.
Extraction of DNA or RNA is not necessary in this protocol. Instead, we used a simple DAB histochemical
staining method, which was convenient, cheap, quick, and reliable for analysis of transgene-free
genome-edited plants. The procedure was feasible for determining transgenic rice containing HPT.
More importantly, HyB-induced overproduction of H2 O2 in other plants demonstrated its broad
application for a wide range of plant species.

Supplementary Materials: The following are available online at http://www.mdpi.com/1422-0067/20/16/3885/s1.

Author Contributions: T.-M.W., J.-Z.H., Y.-T.H., Y.-C.T., and C.-Y.H. conceived and designed the experiments.
T.-M.W., J.-Z.H., Y.-T.H., and H.-M.O. performed the experiments. J.-Z.H., Y.-C.T., and C.-Y.H. wrote the paper.
Acknowledgments: We thank Laura Smales for English editing. This research was supported in part by the
Ministry of Science and Technology (MOST), Taiwan (MOST 105-2628-B-002-036-MY3) to C.Y. Hong and Ministry
of Science and Technology (MOST), Taiwan (MOST 107-2313-B-002-023-) to Y.-C.T.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations
CRISPR Clustered Regularly Interspaced Palindromic Repeat
DAB 3,30 -diaminobenzidine
HPT Hygromycin phosphotransferase
HyB Hygromycin B
WT Wild-type

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