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Application of sonication- and vacuum infiltration-assisted Agrobacterium-mediated transformation of rice embryo (Oryza sativa L.)

doi: 10.2306/scienceasia1513-1874.2020.052
ScienceAsia 46 (2020): 412419
Application of sonication- and vacuum infiltration-
assisted Agrobacterium-mediated transformation of
rice embryo (Oryza sativa L.)
Touchkanin Jongjitvimola, Kawee Sujipulib, Sittichai Urtgama,
aBiology Program, Faculty of Science and Technology, Pibulsongkram Rajabhat University,
Phitsanulok 65000 Thailand
bDepartment of Agricultural Science, Faculty of Agriculture, Natural Resources and Environment,
Naresuan University, Phitsanulok 65000 Thailand
Corresponding author, e-mail:
Received 5 Dec 2019
Accepted 6 Jun 2020
ABSTRACT: Rice transgenics could be performed based on Agrobacterium-mediated transformation which could
improve the rice breeding programs to settle for diseases, insect pests and the climate changes. In this study, the new
strategy of combination of sonication-assisted Agrobacterium-mediated transformation (SAAT) and vacuum infiltration-
assisted Agrobacterium-mediated transformation (VIAAT) system for producing transgenic rice was developed. This
research demonstrated an Agrobacterium-mediated transformation protocol for whole rice seeds by employing both
sonication and vacuum infiltration. Two Agrobacterium tumefaciens strains AGL1 and EHA105, carrying a binary vector
pCAMBIA1301 (with a gusA-intron) or pCAMBIA1304 (with a gusA), were used to examine for embryo transformation
efficiency of the three rice grain cultivars, RD41, RD47 and PSL2. The results show that (1) SAAT for 10 min
was proved to be the best for increasing microwound regions on the embryo surface; (2) EHA105-pCAMBIA1304
gave higher transformation efficiency of dehusked seeds (87.5 ±7.5%) than AGL1-pCAMBIA1304 (37.5 ±2.5%); (3)
the combination of SAAT for 10 min followed by another 10 min of VIAAT gave the transient GUS expression in
coleoptile (18%) and primary leaf (2%) of husked seedling; and (4) the use of transformation protocol with EHA105-
pCAMBIA1301 showed that the rice cultivar PSL2 gave significantly higher transformation efficiency than RD41 and
RD47. This work thus suggests that genetic transformation in plants could be achieved by SAAT and VIAAT techniques
without tissue culture process.
KEYWORDS:Oryza sativa,Agrobacterium, sonication-assisted transformation, vacuum infiltration-assisted transforma-
tion, gusA
Rice (Oryza sativa L.) is one of the most econom-
ically important cereal crops of Thailand, but the
main reduction of rice (O. sativa L.) quality and
quantity has been greatly affected by diseases, in-
sect pests and the climate changes. Many con-
ventional breeding programs have made a signifi-
cant contribution to create and improve desirable
agronomic traits in rice; however, these require
tremendous, labor-intensive and time-consuming
process. To solve these problems, the develop-
ment of modern biotechnology techniques such as
Agrobacterium-mediated transformation technology
has made the generation of transgenic rice simpler
and more reliable [1]. This is because Agrobac-
terium-mediated transformation provides a high op-
portunity to transfer large DNA fragments, carrying
genes without any rearrangement, which is very
useful to study their cumulative and interactive
effects on polygenic traits. Moreover, it has high
inserted transgene as a single or low copy into
plant genome [2], which may give high efficiency
for gene expression in the next generation [3,
4]. Mostly, Agrobacterium-mediated transformation
protocols for plants are based on tissue culture tech-
niques, which usually have some limitation such as
long process, skilled labor and expensive laboratory
equipment [5]. In contrast, in planta transformation
such as sonication- and vacuum infiltration-assisted
Agrobacterium-mediated transformation (SAAT and
VIAAT) can directly transfer gene into a plant with
no requirement of tissue culture techniques [6]. The
SAAT and VIAAT methods have been successfully
ScienceAsia 46 (2020) 413
employed in enhancing transformation rates in ba-
nana cv. Rasthali [7],Vigna unguiculata [6],Lens
culinaris [8]and Indian soybean [9].
In this study, for the first time, we report the
development of the use of the combination of SAAT
and VIAAT systems in rice embryo, so that this proto-
col can be potentially applied in routine experiments
for further producing transgenic rice.
Rifampicin, kanamycin, 6-benzylaminopurine
(BAP), MS medium and 5-bromo-4-chloro-3-
indolyl-β-D glucuronidase (X-gluc) were purchased
from Sigma, USA. Taq DNA polymerase was from
Vivantis, Malaysia.
Rice sample preparation
Three rice cultivars (standard paddy rice RD41,
RD47 and PSL2) used in this study were obtained
from Phitsanulok Rice Research Center, Thailand.
Mature rice seeds at least 2 kg were dehusked
by rice-milling machine model NW 1000 TURBO
(Natawee Technology, Thailand), and only complete
seeds (with embryo) were selected to use for trans-
formation experiments whereas husked seeds were
used as a control group.
Binary plasmid, Agrobacterium strain and
culture condition
Two strains of Agrobacterium tumefaciens, AGL1
and EHA105 harboring pCAMBIA1301 or pCAM-
BIA1304 (kindly provided by Prof. Sompong
Techato), were used for rice transformation ex-
periments. Both Agrobacteria contained rifampicin
and chloramphenicol resistant genes in bacterial
chromosome. The T-DNA region of both binary
vectors contains a marker gene (hygromycin phos-
photransferase II; hptII) and a reporter gene (beta-
glucuronidase; gusA in pCAMBIA1304 or gusA-
intron in pCAMBIA1301). All these genes were
driven by the cauliflower mosaic virus (CaMV) 35S
promoter (Fig. 1). The Agrobacterium was main-
tained on LB medium (1% (w/v) tryptone, 0.5%
(w/v) yeast extract and 1% (w/v) NaCl) supple-
mented with 40 µg/ml rifampicin and 50 µg/ml
kanamycin, and then incubated at 28 °C for 48 h.
A single bacterial colony was cultured into 25 ml
of liquid LB medium supplemented with 25 µg/ml
rifampicin and 50 µg/ml kanamycin and incubated
at 28–30 °C for 24–48 h with shaking at 200 rpm
(Shaking Incubator model SI-23MC; Bioer Technol-
ogy, China). The culture was measured at 600 nm
(OD600) until it reached 0.8. The cell pellets were
harvested by centrifugation at 3512 ×gfor 3 min at
25 °C and resuspended in a final volume of 100 ml
standard inoculation medium (SIM), modified from
Clough et al [10](½ MS medium, 5% sucrose,
44 nM 6-benzylaminopurine (BAP) and 0.075%
Tween-20, adjusted pH to 5.7).
Primer sets of gusA-intron and gusA detection
using PCR
Three primer sets used to amplify the gusA-
intron and gusA region were designed from en-
tire sequences of pCAMBIA-1301 (AF234297.1) and
pCAMBIA-1304 (AF234300.1), respectively, which
are available on NCBI website. The primer set no. 1
giving PCR product of 989 bp length in both
pCAMBIA-1301 and pCAMBIA-1304, primer set
no.2 was gusA-F2 (50-TGCGTCACAGCCAAAAGC-30)
and gusA-R2 (50-CTCGCATTACCCTTACGC-30), giv-
ing PCR product of 245 bp length only in pCAMBIA-
1304 and primer set no. 3 was gusA-F1 and gusA-
R2 giving PCR product of 597 bp length only in
PCR condition
Genomic DNA (gDNA) was extracted from a single
colony of each Agrobacterium strain, and the PCR
reaction was performed in a total volume of 20 µl
containing 100 ng gDNA, 200 µM dNTP mix, 1X
ViBuffer A, 1.5 mM MgCl2, 1 unit Taq DNA poly-
merase and 200 mM of each specific primer. The
PCR was carried out in T100™ Thermal Cycler (Bio-
Rad, USA) with optimal condition profile as pre-
denaturation at 95 °C for 3 min, amplified by 35
cycles of denaturing at 95 °C for 30 s, annealing at
55 °C for 30 s and extension at 72 °C for 60 s, and
followed by final extension at 72 °C for 5 min. The
PCR products were electrophoresed on 1% (w/v)
agarose gel and visualized by 0.001% ethidium
bromide staining.
Transformation using SAAT and VIAAT
The rice seeds were soaked in water overnight.
After air drying for an hour, the rice sam-
ples were immersed in SIM individually contain-
ing Agrobacterium strain EHA105-pCAMBIA1301,
AGL1-pCAMBIA1301, EHA105-pCAMBIA1304 or
414 ScienceAsia 46 (2020)
Hyg-R lacZ gus first exon gusA 6xHis
LB CaMV35S-T CaMV35S-P MCS lacP CaMV35S-P cat-intron NOS-T RB
A: pCAMBIA 1301
Hyg-R lacZ mgfp5 gusA 6xHis
B: pCAMBIA 1304
Fig. 1 T-DNA mapping in (A) pCAMBIA1301 [11]and (B) pCAMBIA1304 [12]binary vectors. Arrow indicates
the direction of RNA transcription; RB =right border; LB =left border; gusA =beta-glucuronidase reporter gene;
CaMV35S-P =Cauliflower mosaic virus 35S RNA gene promoter; cat-intron =catalase intron received from castor bean
catalase gene; NOS-T =Nopaline synthase terminator (0.25 kb); Hyg-R =Hygromycin B phosphotransferase gene;
CaMV35S-T =poly A terminator (0.3 kb), mgfp5 =modified green fluorescence protein reporter gene; and MCS =the
multiple cloning sites pUC18 polylinker.
AGL1-pCAMBIA1304. They were placed in a bath-
type sonicator model WUC-D22H (Daihan Scien-
tific, Korea) and then subjected to ultrasound at
a frequency of 30 kHz for 5–10 min. After that,
the rice samples were placed in a vacuum oven
model VWR 1410 (Sheldon Manufacturing, Inc.,
USA) consisting of a vacuum pump at 27 inch of
mercury for 5–10 min. At the end of treatments,
explants were removed from the tubes, placed on
sterile filter paper surface to blot off excess bacteria
and then transferred to co-cultivation in SIM for
1 day [13].
Histochemical GUS assays
Transient gusA (or gusA-intron) expression was ex-
amined using GUS activity according to histochem-
ical assay [14]. The seed samples were immersed
in 90% acetone for 1 h and removed to incubate
at 37 °C overnight in GUS staining solution con-
taining 50 mg/ml 5-bromo-4-chloro-3-indolyl-β-D
glucuronidase (X-gluc), 10 mM EDTA (pH 7), 0.1 M
Na2PO4, 0.5 mM K4(F4(CN)6), 10% Triton X-100
and 20% methanol. After removal of GUS solution,
the stained seeds were washed once with mixed
solution of methanol:acetic acid (3:1) at room tem-
perature (25–28 °C) overnight. The samples were
transferred into 70% ethanol and stored at 4 °C at
least 2 h. The transient GUS activity of seeds was
determined as blue spots under the stereo micro-
scope model Olympus S2H10 with camera model
Olympus SZX7 (Olympus, Japan). The transforma-
tion efficiency (%) was calculated according to the
Efficiency (%) =
No. of GUS-stained seeds
Total no. of seeds ×100.
To develop the gene transformation method in
rice seed, two strains of Agrobacterium (AGL1
and EHA105) carrying individual pCAMBIA1301 or
pCAMBIA1304 were used to examine transforma-
tion efficiency in rice embryo by using SAAT fol-
lowed by VIAAT treatments.
Confirmation of gusA-intron and gusA in T-DNA
region of binary vectors
The gusA-intron in pCAMBIA1301 and gusA in
pCAMBIA1304 of Agrobacterium strains AGL1 and
EHA105 were detected by using colony PCR with
three sets of specific primers (as given above). The
results showed that primer set no. 1 gave an ex-
pected PCR-product size of approximately 989 bp
length in both binary vectors (Fig. 2; lanes 2–5). For
primer set no. 2, the amplified fragment gave an ex-
pected band at approximately 245 bp length in only
pCAMBIA-1304 but not in pCAMBIA-1301 (Fig. 2;
lanes 6–7). For primer set no. 3, the amplified
fragment gave an expected band at approximately
597 bp length in only pCAMBIA-1301 but not in
pCAMBIA-1304 (Fig. 2; lanes 8–9). These results
ScienceAsia 46 (2020) 415
Fig. 2 The confirmation of the presence of gusA-intron and gusA gene in pCAMBIA1301 and pCAMBIA1304, respectively,
using colony PCR with 3 primer sets. Lane M represents 100 bp DNA ladder. The Agrobacterium strains AGL1-
pCAMBIA1301 (lanes 2 and 8), AGL1-pCAMBIA1304 (lanes 3 and 6), EHA105-pCAMBIA1301 (lanes 4 and 9), and
EHA105-pCAMBIA1304 (lanes 5 and 7). PCR products were separated on a 1% agarose gel, and DNA bands were
visualized under UV light. The PCR fragments of approximately 989 bp (lanes 2–5), 245 bp (lanes 6–7) and 597 bp
(lanes 8–9) were observed using primer pair F1-R1, F2-R2 and F1-R2, respectively. Lane 1 represents a negative control.
Fig. 3 Histochemical assay of GUS transient expression after gene transformation of gusA gene via SAAT, followed by
VIAAT (A) a control group of dehusked seed, (B) GUS-stained in embryo (Em), endosperm (En) and seed coat (Sc) of
dehusked seed, (C) a control group of husked seed and (D) GUS-stained trichome (TC) and pericarp (PC) of husked
indicated that pCAMBI-1301 and pCAMBIA-1304
contained gusA-intron and gusA gene, respectively.
The gusA-intron can be expressed only in eukaryotic
cells but not in prokaryotic cells including A. tume-
faciens cells. Therefore, they can be used to monitor
efficiency of gene transformation.
Effect of husked and dehusked mature seeds of
rice on gene transformation efficiency
The dehusked or husked seeds were immersed
in Agrobacterium strain AGL1-pCAMBIA1304 and
subjected to SAAT for 10 min and followed by
VIAAT for 10 min. The result showed that the
gusA gene was expressed and located in seed
coat (97 ±1%), endosperm (6 ±2%) and embryo
(4 ±2%) of dehusked seeds, but expressed only in
pericarp (100%) and trichome (100%) of husked
seeds (Fig. 3). This indicated that the pericarp
can be resistant to Agrobacterium infection into the
endosperm and embryo of husked seeds because
the pericarp has a hard structure consisting of high
cellulose, hemicellulose and lignin contents [15].
Moreover, this result may indicate that Agrobac-
terium can infect the dehusked seeds which causes
microwounds within and on the tissue [16], induc-
ing phenolic-compound synthesis [17]and vacuum
infiltration might allow better access and infection
of plant cells by Agrobacterium [8].
416 ScienceAsia 46 (2020)
Table 1 Influence of dehusked and husked seeds on
transformation efficiency using Agrobacterium-mediated
transformation via SAAT and VIAAT combination.
Agrobacterium strain Transformation efficiency (%)
Control SAAT & VIAAT
AGL1-pCAMBIA1304 17.0 ±2.0a37.5 ±2.5a
EHA105-pCAMBIA1304 35.0 ±5.0b87.5 ±7.5b
Values are mean ±SD, n=100 with triplicated exper-
iments. Letters indicate significant difference at 95%
confidence (p<0.05) via t-test within column.
Effect of Agrobacterium strains on the
transformation efficiency in dehusked seeds
Two Agrobacterium strains (AGL1 and EHA105),
carrying the binary vector pCAMBIA1304, were
used to evaluate a potential of the transfor-
mation efficiency in dehusked-rice seeds. The
histochemical assay of GUS transient expression
in rice embryo demonstrated that the treatment
of SAAT and VIAAT combination for Agrobac-
terium strains AGL1 (37.5 ±2.5) and EHA105
(87.5 ±7.5) gave significantly higher transforma-
tion efficiency than untreated AGL1 (17.0 ±2) and
EHA105 (35.0 ±5) (used as a control). Moreover,
the result revealed that the Agrobacterium strain
EHA105-pCAMBIA1304 (87.5 ±7.5) gave signifi-
cantly higher transformation efficiency than AGL1-
pCAMBIA1304 (37.5 ±2.5) at p<0.05 (Table 1).
Although both strains of Agrobacterium (EHA105
and AGL1) contained supervirulent (vir) genes from
oncogenic strain A281, which is able to transfer and
insert a T-DNA region into various plant species, this
result showed that Agrobacterium strain EHA105
was more efficient than AGL1 to transfer a T-DNA re-
gion containing gusA and nptII genes into dehusked-
rice seeds. It has also been reported that Agrobac-
terium strain EHA105 gave higher transformation
efficiency in tobacco than AGL1 [18]. Therefore,
the use of this protocol should be evaluated for
transformation efficiency in other rice cultivars.
Effect of sonication-treatment period on
microwound induction
To investigate the influence of SAAT treatment pe-
riod on microwound induction, the dehusked seeds
were immersed in SIM medium and sonicated for
different time durations of 5 and 10 min (un-
sonicated treatment was used as a control). All
seeds were dried at 45 °C for 2 days in an oven.
The microwounds on seed surfaces were evaluated
Table 2 Transformation efficiency in different organs of
germinated seeds suspended in EHA105-pCAMBIA1304
and treated with SAAT followed by VIAAT.
Plant organ Transformation efficiency (%)
Coleoptile (CT) 18
Radicle (R) 2
Coleorhiza (CR) 14
Primary leaf (PL) 2
Embryo (Em) 0
Endosperm (En) 0
CT and CR 6
CT and R and PL 4
CT and CR and PL 2
CT and R and CR and PL 0
CT and R and CR 6
CT and R 6
CT and PL 4
R and PL 2
R and PL and CR 2
R and CR 10
PL and CR 0
The number of seeds is 100 seeds with 3 biological
replicates in each treatment.
under photoelectron microscope (PEM). Among the
different time durations, the SAAT treatment for
10 min was proved to be the best for increasing
microwound regions on the embryo surface (Fig. 4).
These microwounds may facilitate Agrobacterium
to deeply penetrate into plant tissues, increasing
transformation efficiency [7,19].
Effect of different genotypes of rice on
transformation efficiency
The transformation efficiency of the dehusked seeds
of three rice cultivars (RD41, RD47 and PSL2)
was examined using Agrobacterium strain EHA105-
pCAMBIA1301 with combination of SAAT and VI-
AAT techniques. The successful transformation was
determined by GUS assay. The result showed that
the PSL2 (8.25 ±0.35%) gave significantly higher
transformation efficiency in its embryo than RD41
(1%) and RD47 (1%) at p<0.05 (Duncan’s new
multiple range test).
Influence of husked-seed germination on
transformation efficiency
To evaluate the influence of husked-seed germina-
tion on transformation efficiency, the husked seeds
of rice cultivar RD41 were immersed in water for
3 days until germinated, and then dried on air for
6 h. The germinated seeds were suspended to
ScienceAsia 46 (2020) 417
Fig. 4 Photoelectron microscope (PEM) image of microwounds on the rice-embryo surface of SAAT-treated dehusked
seed (A) no sonication, (B) SAAT-treated dehusked seed for 5 min and (C) 10 min. Bar indicates 20 µm.
Fig. 5 Influence of husked-seed germination on transformation efficiency. Germinated seeds of RD41 rice were
immersed in EHA105-pCAMBIA1304 and treated with SAAT and VIAAT followed by GUS assay in (A) control (no
gusA expression) and (B) gusA expression in radicle, coleorhiza, coleoptile and primary leaf of treated germinated
seeds. Bar represents 2 mm.
EHA105-pCAMBIA1304 and treated with SAAT for
10 min, followed by VIAAT for 10 min. The result
revealed that transient GUS expression was found
in coleoptile (CT), radicle (R), coleorhiza (CR) and
primary leaf (PL) of germinated seeds (Table 2,
Fig. 5). The expression was high in coleoptile,
coleorhiza, radicle and coleorhiza, as 18%, 14% and
10%, respectively, whereas there was no expression
of transient GUS in Em, En, CT and R and CR and PL,
and PL and CR. This result indicated that the seed
germination may induce wounds on surface of plant
tissue that could be stimulated bacterial infection.
Determination of the success of transformation
by SAAT and VIAAT combination
To determine the transient gusA expression in
seedlings of rice cultivar RD41, six hundred
dehusked seeds were immersed in EHA105-
pCAMBIA1301 and treated with SAAT for
10 min, followed by VIAAT for 10 min. Three-
hundred-dehusked seeds were determined for the
transformation efficiency by GUS assay. The GUS
stained areas were observed in embryo (Em) and
endosperm (En) with the same level of 0.67% in
one-day-old seedlings (Fig. 6A-B), and in root (Ro)
and shoot (So) with 1% and 2%, respectively, in
two-day-old seedlings (Fig. 6C-D).
These results indicated that this protocol has the
potential for the successful gene transformation by
VIAAT method, especially into embryo and shoot.
Embryo and shoot could be developed into germ line
in both male and female that can be inherited to the
next generation during the fertilization.
This work demonstrates that SAAT and VIAAT tech-
niques could be alternatively used for direct rice-
embryo transformation to avoid plant tissue culture
system. This will reduce the requirement of labor-
intensive and time-consuming process. We found
that Agrobacterium strain EHA105 gave significantly
higher transformation efficiency than the strain
418 ScienceAsia 46 (2020)
Fig. 6 Histochemical localization of gusA activity in different tissues of transformed dehusked-rice seedling at (A and
B) one-day old, and (C and D) two-day old. Dehusked seeds of RD41 rice were immersed in EHA105-pCAMBIA1301
and treated with SAAT and VIAAT, followed by GUS assay. Blue-stained seedling tissues indicate gusA expression in
transformed regions such as embryo (Em) (A), endosperm (En) (B, C and D), roots (Ro) (C and D) and shoot (So) (C).
Bar equals to 2 mm.
AGL1. Using SAAT for 10 min was proved to be
the best for increasing microwound regions on the
embryo surface. SAAT in combination with VIAAT
led to a transfer and insert of a T-DNA region into
microwound of dehusked-rice embryo induced by
milling machine or sonication and into microwound
of husked-rice embryo induced by seed germination.
In addition, the rice cultivar PSL2 was the best for
transformation efficiency when compared to RD41
and RD47.
Acknowledgements:The authors would like to thank
Faculty of Agriculture, Natural Resources and Environ-
ment, Naresuan University, Faculty of Science and Tech-
nology, Pibulsongkram Rajabhat University and The Sci-
ence Center, Faculty of Science and Technology, Pibul-
songkram Rajabhat University. We would like to give
our sincere appreciation to Prof. Dr. Sompong Te-chato
from Prince of Songkla University for kindly providing
A. tumefaciens strains AGL1. Thanks also go to Dr. Orawan
Chatchawankanphanich from the National Center for Ge-
netic Engineering and Biotechnology, Kasetsart University
for proving A. tumefaciens strains EHA105. This research
work was financially supported by a grant from National
Research Council of Thailand (2558A14202028).
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... Agrobacterium-mediated transformation method has been widely used in gramineous crops, such as rice (O. sativa) (Touchkanin et al. 2020), maize (Zea mays) (Soyza et al. 2017), wheat (Triticum aestivum) , barley (H. vulgare) (Nataliya et al. 2013), and sorghum (Sorghum bicolor) (Wu et al. 2014). ...
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Alkaligrass (Puccinellia tenuiflora) is a monocotyledonous halophyte pasture, which has strong tolerance to saline-alkali, drought, and chilling stresses. We have reported a high-quality chromosome-level genome and stress-responsive proteomic results in P. tenuiflora. However, the gene/protein function investigations are still lacking, due to the absent of genetic transformation system in P. tenuiflora. In this study, we established a higher efficient Agrobacterium-mediated transformation for P. tenuiflora using calluses induced from seeds. Agrobacterium strain EHA105 harbors pANIC 6B vectors that contain GUS reporter gene and Hyg gene for screening. Ten mg·L−1 hygromycin was used for selecting transgenic calluses. The optimized condition of vacuum for 10 min, ultrasonication for 10 min, and then vacuum for 10 min was used for improvement of conversion efficiency. Besides, 300 mg·L−1 timentin was the optimum antibiotics in transformation. PCR amplification exhibited that GUS gene has been successfully integrated into the chromosome of P. tenuiflora. Histochemical GUS staining and qRT-PCR analysis indicated that GUS gene has stably expressed with ß-glucuronidase activity in transgene seedlings. All these demonstrated that we have successfully established an Agrobacterium-mediated transformation system of P. tenuiflora, which provides a good platform for further gene function analysis and lays a solid foundation for molecular breeding.
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Soybean is a recalcitrant crop to Agrobacterium-mediated genetic transformation. Development of highly efficient, reproducible, and genotype-independent transformation protocol is highly desirable for soybean genetic improvement. Hence, an improved Agrobacterium-mediated genetic transformation protocol has been developed for cultivar PK 416 by evaluating various parameters including Agrobacterium tumefaciens strains (LBA4404, EHA101, and EHA105 harboring pCAMBIA1304 plasmid), sonication duration, vacuum infiltration pressure, and vacuum duration using cotyledonary node explants of soybean prepared from 7-day-old seedlings. The transformed plants were successfully developed through direct organogenesis system. Transgene expression was assessed by GUS histochemical and gfp visual assays, and integration was analyzed by PCR and Southern blot hybridization. Among the different combinations and durations evaluated, a maximum transformation efficiency of 18.6 % was achieved when the cotyledonary node explants of cv. PK 416 were sonicated for 20 s and vacuum infiltered for 2 min at 250 mmHg in A. tumefaciens EHA105 suspension. The amenability of the standardized protocol was tested on four more soybean cultivars JS 90-41, Hara Soy, Co 1, and Co 2 in which all the cultivars responded favorably with transformation efficiency ranging from 13.3 to 16.6 %. The transformation protocol developed in the present study would be useful to transform diverse soybean cultivars with desirable traits.
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Agrobacterium mediated transformation has been widely used for research in plant molecular biology and for genetic improvement of crops exploiting its tremendous ability to transfer foreign DNA to plants. In this study, the transformation efficiency of five Agrobacterium tumefaciens strains GV2260, LBA4404, AGL1, EHA105, and C58C1 was evaluated in Nicotiana tabacum L. cultivar Samsun. The Agrobacterium strains contained the recombinant binary vector pBin19 harboring beta-glucuronidase uidA gene under 35S promoter. Neomycin phosphotransferase (nptII) gene was used as a selectable marker at a concentration of 100 mg L-1 kanamycin. The expression of uidA gene in regenerated TO plants was firstly analyzed by GUS histochemical analyses and later on confirmation of presence of the nptII and uidA genes in regenerated plants was determined by PCR. The highest transformation rate (20%) was obtained with the Agrobacterium strain LBA4404, followed by EHA105, GV2260, C58C1 and AGL1. The higher transformation efficiency achieved in our studies make LBA4404 Agrobacterium strain optimal for functional genomics and biotechnological applications in tobacco plants.
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For the first time we have developed a reliable and efficient vacuum infiltration-assisted Agrobacterium-mediated genetic transformation (VIAAT) protocol for Indian soybean cultivars and recovered fertile transgenic soybean plants through somatic embryogenesis. Immature cotyledons were used as an explant and three Agrobacterium tumefaciens strains (EHA 101, EHA 105, and KYRT 1) harbouring the binary vector pCAMBIA1301 were experimented in the co-cultivation. The immature cotyledons were pre-cultured in liquid somatic embryo induction medium prior to vacuum infiltration with the Agrobacterium suspension and co-cultivated for 3 days on co-cultivation medium containing 50 mg l−1 citric acid, 100 µM acetosyringone, and 100 mg l−1l-cysteine. The transformed somatic embryos were selected in liquid somatic embryo induction medium containing 10 mg l−1 hygromycin and the embryos were germinated in basal medium containing 20 mg l−1 hygromycin. The presence and integration of the hpt II and gus genes into the soybean genome were confirmed by GUS histochemical assay, polymerase chain reaction, and Southern hybridization. Among the different combinations tested, high transformation efficiency (9.45 %) was achieved when immature cotyledons of cv. Pusa 16 were pre-cultured for 18 h and vacuum infiltrated with Agrobacterium tumefaciens KYRT 1 for 2 min at 750 mm of Hg. Among six Indian soybean cultivars tested, Pusa 16 showed highest transformation efficiency of 9.45 %. The transformation efficiency of this method (VIAAT) was higher than previously reported sonication-assisted Agrobacterium-mediated transformation. These results suggest that an efficient Agrobacterium-mediated transformation protocol for stable integration of foreign genes into soybean has been developed.
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OsWRKY76 encodes a group IIa WRKY transcription factor of rice. The expression of OsWRKY76 was induced within 48h after inoculation with rice blast fungus (Magnaporthe oryzae), and by wounding, low temperature, benzothiadiazole, and abscisic acid. Green fluorescent protein-fused OsWRKY76 localized to the nuclei in rice epidermal cells. OsWRKY76 showed sequence-specific DNA binding to the W-box element in vitro and exhibited W-box-mediated transcriptional repressor activity in cultured rice cells. Overexpression of OsWRKY76 in rice plants resulted in drastically increased susceptibility to M. oryzae, but improved tolerance to cold stress. Microarray analysis revealed that overexpression of OsWRKY76 suppresses the induction of a specific set of PR genes and of genes involved in phytoalexin synthesis after inoculation with blast fungus, consistent with the observation that the levels of phytoalexins in the transgenic rice plants remained significantly lower than those in non-transformed control plants. Furthermore, overexpression of OsWRKY76 led to the increased expression of abiotic stress-associated genes such as peroxidase and lipid metabolism genes. These results strongly suggest that OsWRKY76 plays dual and opposing roles in blast disease resistance and cold tolerance.
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A sonication-assisted, Agrobacterium-mediated, co-cultivation technique was used in an attempt to increase the transformation efficiency of flax. Hypocotyls and cotyledons excised from about 10-day-old flax seedlings grown invitro were placed into a 10mM MgSO4 solution, and inoculated with an A. tumefaciens vector bearing the mgfp5-ER gene driven by the CaMV 35S promoter. The explants were subjected to pulses of ultrasound delivered by a sonicator apparatus (35kHz) for 0–150s and co-cultivated for 2h at 27°C. The dried hypocotyls and cotyledons were grown on a selective MS medium to promote shoot regeneration. An electron microscopic study showed that the sonication treatment resulted in thousands of microwounds on and below the surface of the explants. A stereo microscope Leica MZ 12 equipped with a GFP adaptor was used to assess the infection and transformation of plant tissues in real time. After only 48h and for at least 30days after bacteria elimination, signs of transgene expression could be seen as a bright fluorescence. Our results show that treatment with ultrasound facilitates an enhanced uptake of plasmid DNA into the cells of flax hypocotyls and cotyledons and that its efficiency depends on the duration of the treatment and the frequency used. SAAT could be a promising tool for enhancing transformation efficiency in flax.
The discovery that the bacterial phytopathogen Agrobacterium is a natural expert at interkingdom gene transfer has proven to be rewarding in areas far afield from plant pathology. It has provided researchers with a powerful means of dissecting and eventually modifying the genomes of many kinds of plants. It has led to landmark discoveries about the molecular basis of plant-pathogen interactions. It has furthered our understanding of plant gene function and regulation involved in the physiological and developmental processes in plants. Agrobacterium has played a key role in many of the recent advances in plant science, and future exploitation of this microbe is limited only by the imagination of its scientific practitioners. Access to a wide array of genes with good potential for crop improvement is now intensifying interest in development of a plant transformation system with consistent, optimal and cost-effective performance. Once again, Agrohacterium seems destined for a central role.
In this study, a protocol was developed to obtain stable lines of the Spring Dendrobium cultivar 'Sanya' via sonication assisted Agrobacterium-mediated transformation (SAAT) of protocorm-like bodies (PLBs). Agrobacterium tumefaciens strain LBA4404 was used with the binary vector AG205 containing the chalcone synthase (CHS) gene for flower color change and the neomycin phosphotransferase II gene (NPTIIgene) as a selectable marker for kanamycin resistance. PLBs were treated for 10 min using ultrasound (40 KHz) and subsequently immersed in the Agrobacterium suspension for 60 min. The addition of 100 μM acetosyringone (AS) during the pre-culture and co-culture achieved the highest efficiency of gene transformation (0.5%). Following co-cultivation, PLBs were cultured on selective medium containing 200 mgL -1 kanamycin and 250 mgL -1 cefotaxime. Proliferating PLBs were repeatedly selected for kanamycin resistance and stable transformed lines were generated. Incorporation and expression of transgenes were confirmed by PCR analysis, southern blot analysis and reverse transcription-quantitative real-time PCR (RT-PCR). A total of five southern blot positive lines were obtained from co-cultivated PLBs.
The regeneration and transformation protocol for lentil employing both sonication and vacuum infiltration of whole seed explants is described. This protocol overcomes the non-competence of whole seed explants to Agrobacterium mediated transformation though the regeneration potential of this explant is high as compare to other agro-competent explants. The decoated seeds of four genotypes were cultured on regeneration media that consisted of Murashige and Skoog (MS) salts and Gamborg (B5) vitamins (MSB5V) supplemented with different concentrations of 6-benzylaminopurine (2.5–20 μM) and thidiazuron (0.1–10 μM) separately. Best regeneration response with an average of 9 shoots per explant was observed in 95% of the cultures on MSB5V medium fortified with 5.0 μM thidiazuron. The protocol was found independent to the effect of genotype. Among the effects of physical treatments, it was found that sonication at 20 KHz for 60 s followed by vacuum infiltration at −660 mm of Hg for 5 min resulted in highest transient GUS expression (68%) with no significant effect on regeneration. Rooting was observed in 45% of shoots on rooting medium containing half strength MS salts and full strength MS vitamins supplemented with 2.5 μM IBA. Regenerated plantlets were established in soil with 50% survival rate and grown to maturity. GUS activity in leaves and roots of primary transformants recovered on kanamycin containing selection media confirmed the expression of transgene. These plants were tested and found positive for nptII gene by PCR and Southern analysis with an overall transformation frequency of 0.66%. PCR analysis of T0 progeny revealed the inheritance of transgene.
TheAgrobacteriumvacuum infiltration method has made it possible to transformArabidopsis thalianawithout plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified transformation method was developed. The labor-intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containingAgrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77. Sucrose and surfactant were critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate. Plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, andAgrobacteriumcould be applied to plants at a range of cell densities. Repeated application ofAgrobacteriumimproved transformation rates and overall yield of transformants approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold. Multiple ecotypes were transformable by this method. The modified method should facilitate high-throughput transformation ofArabidopsisfor efforts such as T-DNA gene tagging, positional cloning, or attempts at targeted gene replacement.
Plant transformation technology has become a versatile platform for cultivar improvement as well as for studying gene function in plants. This success represents the culmination of many years of effort in tissue culture improvement, in transformation techniques and in genetic engineering. The next challenge is to develop technology that minimizes or eliminates the tissue culture steps, and provides predictable transgene expression.