ORIGINAL ARTICLE
‘AGRODATE’: a rapid Agrobacterium-mediated transient expressiontool for gene function analysis in leaf discs
Veda Krishnan1,3 • Joshna Jose1 • Monica Jolly1 • T. Vinutha1 • Raja Kumar2 • Markandan Manickavasagam3•
Shelly Praveen1 • Archana Sachdev1
Received: 11 January 2019 / Accepted: 11 September 2019� Society for Plant Biochemistry and Biotechnology 2019
AbstractTransient gene expression utilizing syringe mediated agro infiltration offers a simple yet efficient technique for various
transgenic applications. Although soybean is commonly used as a model plant for functional genomic studies, till date
there have been no reports available on a high throughput agro infiltration protocol for transient gene expression. In
the present study, we developed a simple transient expression system, named Agrobacterium-mediated disc assay for
transient expression (AGRO DATE) in mature soybean leaves involving the use of an optimized infiltration buffer
[10 mM 2-(N-morpholino) ethane sulfonic acid sodium salt, 10 mM MgCl2, 0.2 mM acetosyringone, 400 mg L-1, L-
cysteine, 0.5 mM dithiothreitol and 0.01% Tween 20] under vacuum using a needle-less syringe. This not only limits
the genetic pollution but can also be conducted without the intervention of any specialized equipments. Model protein
b-glucuronidase (GUS) was used for optimizing various parameters and the synergized composition delivered 58%
transformation efficiency within 4 days of infiltration. We demonstrated the versatile applicability of the method for
examining reporter genes (GUS and bar), over expression (GmIPK1) and antisense suppression of GmMIPS in
soybean. In addition, AGRODATE offers a viable approach for application of agro infiltration in other recalcitrant
plant species and may become a useful tool for the research community.
Electronic supplementary material The online version of thisarticle (https://doi.org/10.1007/s13562-019-00536-w) con-tains supplementary material, which is available to autho-rized users.
& Archana Sachdev
1 Division of Biochemistry, ICAR-Indian Agricultural
Research Institute (IARI), New Delhi, India
2 Department of Applied Science, Konkuk University, Seoul,
South Korea
3 Department of Biotechnology and Genetic Engineering,
Bharathidasan University, Trichy, India
123
Journal of Plant Biochemistry and Biotechnologyhttps://doi.org/10.1007/s13562-019-00536-w(0123456789().,-volV)(0123456789().,-volV)
Graphic abstract
Keywords Agrobacterium � Soybean � Transient transformation � Agro-infiltration � Leaf discs � Gene expression
Journal of Plant Biochemistry and Biotechnology
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AbbreviationsAGRO
DATE
Agrobacterium-mediated disc assay for
transient expression
DTT Dithiothreitol
GUS-b Glucuronidase
IIP Initial infiltration protocol
SAAT Sonication-assisted Agrobacterium-
mediated transformation
Introduction
Soybean (Glycine max. L) is a multifaceted high value
grain legume known for its versatility and exceptional
nutraceutical value (Krishnan et al. 2018). World soybean
meal and oil consumption have grown steadily (at 5–6%
per year on average) during the last 15 years and currently
soya meals and soybean oil account for about 65 and 25%
of global consumption of cake and oil respectively. In
addition, it is one of the important candidate crop taken
into consideration for genetic modification globally with a
repertoire of strategies and tools developed. Despite having
such enormous agro-economic relevance as well as tech-
nological advancement, this crop suffers from various
widespread diseases/abiotic stresses as well as handicapped
with certain potential antinutrients, which decrease the
crop yield and consumer preferences (Kumari et al. 2014;
Krishnan et al. 2016). The existing improvement pro-
grammes in this direction has majorly been limited due to
the notorious recalcitrance of soybean to Agrobacterium
mediated transformation and regeneration. Even though
few reports are present till date, but the unavailability of
stable mutants and lack of rapid and efficient tools for
transformation is a serious limitation which prevents this
species from being used as a ‘versatile model for gene
functional studies’ (Hada et al. 2016). The need for an
efficient transformation protocol is more imperative than
ever in this crop, as whole genomic sequence is also since
long available which widens the scope for genome editing.
As a prerequisite for functional genomics and molecular
breeding, stable genetic transformation has been achieved
by particle bombardment and Agrobacterium mediated
transformation in soybean. Although several reports have
suggested the feasibility of stable transformation using
particle bombardment method (Finner and McMullen
1991), this option demands expensive resources and
intensive work with a miniscule yield and is therefore,
potentially un-usable for small scale laboratories. As an
alternative, Agrobacterium mediated transformations using
rhizogenes as well as tumefaciens have been reported as a
more preferred technique with better transformation effi-
ciency (Thilip et al. 2015). Nevertheless, agro-mediated
transformation is time consuming, labor intensive and its
output efficiency majorly depends on the bi-directional
compatibility. More over the level of transgene expression
may vary among the events due to either the difference in
the location of transgene integration or due to some off-
target silencing phenomena, thus multiple transgenic
events are generally required for reliable analysis. Com-
pared to stable transgenic approach, transient gene
expression assays are convenient alternative in analyzing
gene functions by virtue of time required; ease of doing and
in terms of labor efficiency. Transient assays have dra-
matically fastened the pace of research by allowing mul-
tiple constructs to be assayed in parallel within a short time
frame for transgenic complementation (Bendahmane et al.
2000; Van der Hoorn et al. 2000), promoter analysis (Yang
et al. 2000) and protein production (Vaquero et al. 1999).
Generally transient assays are carried out in model plants
like Nicotiana but it is crucial to study genes with in the
same species as the native activity or subcellular distribu-
tion of the proteins may vary in a heterologous system.
Among the transient assays, needle-less syringe infil-
tration, vacuum infiltration and protoplast mediated trans-
formation are being routinely exploited. Pilot efforts in
agro-infiltration i.e. forcing the bacterial suspension
through the stomata of soybean leaf have demonstrated
low-frequency of success with great variation (King et al.
2015). Even though few studies have been conducted for
analyzing transitivity and cell to cell movement of protein,
it has been observed that the major limitations to agro
infiltration in dicots like soybean is contributed by the
morphology of leaf especially the structure of leaf epider-
mis which prevents the infiltration of bacterial suspension
in cells by simple pressure (Wroblewski et al. 2005;
Manavella and Chan 2009; Andrieu et al. 2012; Van der
Hoorn et al. 2000). In addition, infiltrating in different
leaves or sampling spots has been observed with variation
in recombinant protein accumulation due to leaf hetero-
geneity in terms of age and position. Recently, sonication-
assisted Agrobacterium-mediated transformation (SAAT)
which involves subjecting the plant tissue to a brief period
of ultrasound in the presence of bacterial suspension fol-
lowed by vacuum was validated in soybean which facili-
tated the penetration up to several layers deep and thus
reported to enable efficient delivery with better transfor-
mation efficiency (Trick and Finer 1997; Arun et al. 2015;
King et al. 2015). But it is very difficult to conduct as it
requires mass culture of bacterial suspension as well as
high cost of vacuum infiltration system. In addition, Mat-
suo et al. (2016) also reported that the whole seedlings and
plantlets transiently infiltrated for GUS expression has been
found to be genetically polluted and hence further inves-
tigation of those plants were difficult.
Journal of Plant Biochemistry and Biotechnology
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We had previously reported an improved Agrobacterium
mediated transformation in soybean using cotyledonary
node (Hada et al. 2016) as well as half seed (Hada et al.
2018) explants with improved transformation efficiency by
synergizing various parameters. In the present study, taking
a step further for quick validation, we have developed a
simple and high throughput transient gene expression
system based on soybean leaf disc vacuum infiltration.
Materials and methods
Plant materials and growth conditions
Mature soybean seeds of cultivar DS-9712 were obtained
from Division of Genetics, Indian Agricultural Research
Institute, New Delhi, India were utilized for agro infiltra-
tion studies.
Table 1 Trouble shooting for rapid Agrobacterium mediated transient expression in mature soybean leaf discs
Section Problem Possible reason Solution
Agrobacterium tumefaciens growth
(2 days)
Low growth rate
of
Agrobacterium
tumefaciens
Overgrowth with
visibly clear
lumps of cells
Agrobacterium culture was old
which affects the motility and
infectivity
Possibly contamination/initial high
inoculum/extend incubation
Always use an inoculum directly from fresh
glycerol stock or streak on fresh agar plate
and use single colony for inoculation
Light affects the growth and motility, hence
keep culture in dark at optimum temperature
for growth
Don’t proceed and use a fresh inoculation from
single colony
Agrobacterium tumefaciens
maintenance by cryo-
conservation (1 day)
Low viability in
future
Not properly stored Properly mix the bacterial suspension and
glycerol to ensure a uniform solution prior
placing in liquid N2 and store immediately at
- 80 �CExplant preparation (1 day) Low infiltration
rate
Age of soybean leaves
Infiltration solution
Time lag or delay
Eight week old leaves are optimum for syringe
infiltration
Components of infiltration buffer have to be
freshly prepared. DTT, Tween 20, L-cysteine
and acetosyringone should be added just
prior to incubation
Leaf discs should be incubated in infiltration
buffer containing agro suspension without
time delay
Infiltration of Agrobacterium
tumefaciens to leaf disc using
syringe (Syringe infiltration
protocol) (5 days)
Spilling of
Agrobacterium
culture
Presence of trapped air
Improper sealing of syringe tip
Remove the air from syringe by holding the
syringe tip upward and carefully depress the
plunger
Seal the syringe tip using multiple layers of
parafilm
Verification of transient
transformation assay using GUS
assay (1 day)
Low GUS
expression
High GUS
expression
around the disc
Browning of leaf
discs
Should be careful that the leaf discs
should be immersed in infiltration
solution
GUS activity around the disc
indicates the susceptibility of
explants to Agrobacterium
mediated transformation
Vacuum promotes entry and
infection of Agrobacterium. But
excessive operation cause necrosis
Excess bacterial load mediated
hypersensitive responses
Contamination of samples
Shake well in between to immerse leaf discs
into the infiltration solution
Wounding promotes infiltration and becomes
positive transformants
Limit the vacuum application as mentioned
Wash the leaf discs properly to remove excess
bacterial load
Use sterile materials and laminar airflow
cabinet to ensure
Verification by expression analysis
using Real time PCR (2 days)
Low
transformation
efficiency
Oxidative stress
Various parameters like OD,
infiltration buffer, time of
infiltration etc
Keep explants under darkness to avoid
oxidative stress
Strictly follow the OD of Agrobacterium,
concentration of various additives and
infiltration time
Journal of Plant Biochemistry and Biotechnology
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Agrobacterium for transient gene expression
The binary vector pCAMBIA1305.1 containing the uidA
(b-glucuronidase) gene, driven by Cauliflower Mosaic
Virus35S (CaMV35S) promoter and the nopaline synthase
(Nos) terminator were used for optimization studies. To
confirm the efficacy of the protocol, the constructs—
(a) Cas9MDC123 possessing Bar gene under 2X35S pro-
moter. (b) pCAMBIA—GmIPK1 with 1032 bp IPK1 ORF
introduced in sense orientation under CaMV 35S promoter
at BglII/SpeI site (c) pBin MIPS-AS (1.5 kb MIPS1 ORF
introduced in antisense orientation under CaMV 35S pro-
moter at BamHI/XbaI site were used (Kumar et al. 2019).
Agrobacterium tumefaciens strain EHA105 was trans-
formed with different expression vectors and used for the
transient expression analysis.
Agrobacterium infiltration into soybean leaf discs
For AGRODATE assay, we followed an ‘‘initial infiltration
protocol’’ (IIP) as described subsequently. To prepare
Agrobacterium infection medium, a single colony of EHA
105 harbouring binary constructs was inoculated into
10 mL Luria–Bertani (LB) medium containing rifampicin
(30 mg L-1) and kanamycin (50 mg L-1) and incubated at
28 �C for 24 h on an orbital shaker at 200 rpm to get the
mother culture. The day before the explant inoculation,
0.2 mL of A. tumefaciens mother culture was transferred to
200 mL Erlenmeyer flask containing 50 mL LB liquid
medium supplemented with rifampicin and kanamycin,
grown at 28 �C (200 rpm) in an orbital shaker. The
Agrobacterium cells were harvested by centrifugation at
6000 rpm for 10 min at 28 �C, the harvested cells were
collected and suspended to a final OD600 of 0.6 in an
infiltration medium consisting 10 mM 2-(N-mor-
pholino)ethane sulfonic acid sodium salt, 10 mM MgCl2(pH 5.4). The Agrobacterium suspension solutions were
placed at room temperature for at least 1 h before use.
Bacterial concentrations were determined by measuring
OD at 600 nm. In a typical experiment, 10 mL of the
bacterial suspension and a 20 mL plastic syringe (no nee-
dle) were used for each individual operation. Leaf discs
were cut from Glycine max leaves approximately
7–8 weeks after germination using a cork borer
(& 8.5 mm). The explants were immersed in the infiltra-
tion agro suspension for 2 h immediately after cutting.
After incubation, plunger was removed from the 20-mL
plastic syringe and leaf discs (15–20 discs per syringe)
were placed into the body of the syringe followed by
insertion of the plunger back. Agrobacterium suspension
solution was poured into a petri dish, and the tip of the
syringe was inserted into the Agrobacterium suspension
solution to draw all solution (10 mL) into the syringe. Air
was removed from the syringe by holding the syringe tip
upward and carefully depressing the plunger. The tip of the
syringe was then sealed using parafilm tightly in multiple
layers, and the syringe was shaken vigorously to remove
any discs from the wall of the syringe. Next, the plunger
was pulled to create a small vacuum in the syringe (in a
typical experiment, the plunger was pulled 1 mL). After
vigorous shaking for 2 min, the plunger was rapidly
released. These infiltration steps were able to be repeated
up to five times, but excessive operation was noted to cause
necrosis. After infiltration, the parafilm was removed after
pulling the plunger a little to prevent scattering of bacterial
suspension. The bacterial suspension was then discarded,
and the infiltrated leaf discs removed from the syringe,
subsequently washed to remove excess bacterial suspen-
sion and incubated in petri dishes with MS medium con-
taining 3% sucrose, sodium azide (10 mg L-1), and 0.8%
agar under lighting programs (16/8 h; light/dark) at
26–28 �C, 80� humidity. Trouble shooting for rapid
Agrobacterium mediated transient expression in mature
soybean leaf discs is in Table 1.
Experimental design
IIP was used further for optimizing five key parameters like
Agrobacterium concentration, infiltration time, pH, and
concentration of media supplements like acetosyringone
and L-cysteine. The varied OD of Agrobacterium culture
from 0.4–1.5 at A600 was studied. For further accelerating
the process of infection, the leaf discs were infiltrated using
buffer at varied pH (5.0–6.2) and subjected to infiltration
for varied time durations (1–8 days). Optimum concen-
trations of infiltration media like acetosyringone
(0–0.5 mM) and L-cysteine (100–1000 mg L-1) were also
optimized using IIP. Further holding the above variables
constant, the role of surfactant Tween 20 (0.01%) and
reducing agent DTT (0.5 mM) was tested on transient
transformation efficiency alone and in combination using
GUS histochemical assay and reported as percentage of the
explants transformed. Each experiment was replicated
thrice with 10 explants per treatment.
GUS histochemical assay
Histochemical staining for GUS was performed according
to Jefferson et al. 1987. The Agrobacterium infiltrated
soybean leaf discs were washed twice with 50 mM Tris–
HCl buffer (pH 7.5) containing carbenicillin
(500 mg mL-1) and twice with distilled water. Subse-
quently the leaf discs were prefixed with chilled 90% (v/v)
aqueous acetone and then washed with chilled water. They
were then immersed in GUS staining solution consisting of
Journal of Plant Biochemistry and Biotechnology
123
50 mM phosphate buffer (pH 7.2), 5 mM K3Fe(CN)6,
5 mM K4Fe(CN)6, 0.2% (v/v) Triton X-100, and 2 mM
X-Gluc (5-bromo-4-chloro-3-indoxyl-beta-D-glucuronide
cyclohexylammonium salt) and following an overnight
incubation at 37 �C. The stained leaf discs were succes-
sively submerged in 25%, 50%, 70%, and 95% (v/v)
ethanol to remove chlorophyll completely. GUS expression
level in the infiltrated leaf discs were analyzed using
stereomicroscope and the image analysis was performed
using Image J (National Institutes of Health, Bethesda,
MD, USA). The tissues of positive transformants show
blue coloration. Percentage was calculated based on the
equation = (number of leaf discs with blue spots or col-
oration/number of leaf discs infiltrated) 9 100.
Real-time reverse transcriptase-polymerase chainreaction
Total RNA was extracted from the leaf discs (5–6 discs)
after four days of infiltration with RNeasy plant mini kit
(Qiagen, Hilden, Germany) according to manufacturer’s
instruction. Contaminated genomic DNA in the total RNA
was degraded with DNase (Sigma Aldrich, USA). One
microgram of total RNA was used to generate first-strand
cDNA using a Verso First-strand cDNA synthesis kit
(Thermoscientfic, USA) with oligo dT primers. Real time
expression analyses of the target and reference gene were
carried out in PikoReal 96 Real Time PCR System
(Thermo Scientific, USA). The experimentation was
Fig. 1 Leaf disc infiltration method. a Cut leaf discs (u 8.5 mm,
10–50 discs) from 8 week old Glycine max (DS9712 cv.). b Binary
vector [pCambia1305 (Genbank accession no. AF354045; 11.8 Kb; in
this E. coli GusA gene has been replaced by GUSPlus). The GUSPlus
gene contains an intron from the castor bean catalase gene to prevent
expression by bacteria and ensure detection of plant-expressed
glucuronidase activity]. c Incubation of leaf discs in infiltration
buffer only (control) and in Agro-suspension harbouring binary vector
(test). d Place the leaf discs into the body of the 20-mL plastic
syringe, and then insert the plunger. e Draw up 10 mL of Agrobac-
terium solution suspended with 10 mM MES-KOH buffer. f Remove
air from the syringe, and then seal the tip of the syringe using stacked
parafilm or a plastic cap. Shake the syringe vigorously to release the
discs from the inside wall of the syringe. g Pull the plunger
approximately 1 mL to create a small vacuum in the syringe, shake
vigorously then release the plunger rapidly. h Incubate leaf discs on
MS agar plate (26–28 �C, 16 h light/8 h dark)
Journal of Plant Biochemistry and Biotechnology
123
performed according to the standard protocol using
DyNAmo Flash SYBR Green qPCR Kit (Thermo Scien-
tific, USA). The three biological samples were run in
triplicates under the following conditions: 1 cycle of 3 min
at 94 �C followed by 35 cycles of 95 �C for 30 s and 30 s
at 60 �C with a final extension of 30 s at 72 �C. The primer
pair Bar (Fp 50-TGCCTCCAGGGAATTACAAG-30; Rp-50-GTATAAAAGCCCGTCGGTGTC-30); IPK1 (Fp 50-ACGCGTCGACTTTTGATCTTGTTCCTGTG-30; Rp 50-CCATCGATGGTAAAAGAA GGTGAGGATCCAGC-30)and MIPS (Fp-50-CGGGATCCCGACCACCGAATCTTGTTCAC-30; Rp-50TCCCCGCGGGGAAAATCTCAGCCTCATTTC-30). The cDNA was normalized by using the
soybean housekeeping gene PEP carboxylase (Fp 50-CATGCACCAAAGGGTGTTTT-30 and Rp 50-TTTTGCGGCAGC TAT CTC TC-30). Amplified products were subjected
to melt-curve analysis and the specificity of the
amplification was assessed by dissociation curve analysis.
A unique peak on the dissociation curve was confirmed for
the gene (Fig. S1). The thermal profile for melt curve
determination began with an incubation of 1 min at 60 �C
Fig. 2 Optimization of various
parameters on transient
transformation efficiency.
8 week old soybean leaf discs
were infiltrated using
Agrobacterium EHA105
harbouring binary vector
pCAMBIA1305.1 containing
the uidA (b-glucuronidase)gene, driven by Cauliflower
Mosaic Virus35S (CaMV35S)
promoter and the nopaline
synthase (Nos) terminator in
MES infiltration buffer [10 mM
2-(N-morpholino] ethane
sulfonic acid sodium salt,
10 mM MgCl2) for
optimization. Various
parameters affecting transient
transformation like a optical
density of Agrobacterium
culture (OD 0.4–1.5), b pH of
infiltration medium (5–6.2),
c infiltration time (1–8 days),
d concentration of
acetosyringone (0.1–0.5 lM),
e concentration of L-cysteine
(100–1000 mg L-1) were
optimized. Frequency of GUS
expression levels in the
infiltrated leaf discs were
analyzed using
stereomicroscope followed by
image analysis
Table 2 Summary of infiltration conditions used for optimization
Infiltration conditions IF1 IF2 IF3 IF4
Bacterial solution (OD600 = 0.6) - ? ? ?
pH-5.5 ? ? ? ?
Infiltration time (4 days) ? ? ? ?
Acetosyringone (0.1–0.5 mM) ? ? ? ?
L-cysteine
(100–800 mg L-1)
? ? ? ?
DTT (0.5 mM) - ? - ?
Tween 20 (0.01%) - - ? ?
Journal of Plant Biochemistry and Biotechnology
123
with a gradual increase in temperature (1 �C/15 s) to
95 �C, during which time changes in fluorescence were
monitored. Normalized expression of target genes was
calculated using the absolute values normalized to
endogenous reference gene. The specificity of the PCR
amplification reaction was analyzed on 1.2% agarose gel.
Calculations and data analysis
Experiments were performed with at least six discs with in
each treatment. The entire experiment was repeated thrice
and the data are represented as means ± standard error.
Mean histochemical GUS expression was calculated per
leaf disc and the frequency of GUS expression is expressed
as percentage.
Results and discussion
Optimization of AGRODATE for transientexpression in soybean
Syringe mediated agro-infiltration has largely been unsuc-
cessful in dicots like soybean due to its peculiar leaf
architecture in terms of high density of palisade and spongy
mesophyll cells as well as low density and/or small
Fig.3 GUS based transient expression in leaf discs under different
conditions. a Leaf discs infiltrated with four different kinds of buffer
compositions (IF1-MES buffer alone without Agrobacterium suspen-
sion; IF2-4 MES buffer with Agrobacterium solution for GUS
expression (OD600 = 0.6) under respective conditions shown in
Table 2, b GUS expression level in leaf discs were analyzed using
stereomicroscope and the image analysis was performed using Image
J (National Institutes of Health, Bethesda, MD, USA). GUS
expression frequency in %, c T-DNA regions of plant expression
vectors used for transient expression. To validate AGRODATE,
expression vectors harboring transgenes were studied
(i) Cas9MDC123 (Addgene plasmid ID 59184; 10.2 K) having Bar
gene, (ii) pCAMBIA-GmIPK1 (modified pCAMBIA 1302, Genbank
accession no: AF234298.1 where 1032 bp GmIPK1 ORF introduced
at BglII/ SpeI site), (iii) pBIN-MIPS-AS (1.5 kb MIPS1 ORF
introduced in antisense orientation at BamHI/XbaI site). 35S, CaMV
35S promoter; poly A, CaMV poly A; nptII, neomycin phospho-
transferase II gene; MCS, multiple cloning site; Noster, Nos
terminator; Bar, phosphinothricin acetyl transferase gene; LB, left
border; RB, right border, d relative quantification of transgene
expression in soybean leaf discs infiltrated with Agrobacterium
harbouring different binary constructs (i) control—only infiltration
buffer; Bar-harbouring Cas9MDC123, (ii) control—pCAMBIA1302
mock vector infiltrated sample; IPK1-pCAMBIA-GmIPK1 mock
vector infiltrated sample, (iii) control—pBIN mock vector infiltrated
sample; pBIN MIPS-AS construct infiltrated sample. Normalization
factors were calculated as the geometric mean of the expression levels
of PEP carboxylase (PEPCo) gene. Error bars represent mean
standard error calculated from three biological replicates with
*P\ 0.05 significance
Journal of Plant Biochemistry and Biotechnology
123
aperture of stomatal pores (Van der Hoorn et al. 2000; King
et al. 2015). Vacuum-aided agro infiltration which over-
come this anatomical barrier reported by Tague and Mantis
(2006) and King et al. (2015) required about 400–500 mL
of bacterial suspension to soak the entire plant, custom-
made leaf disc holder and infiltration tanks, that made the
application of the method difficult and cumbersome. Here,
we report a new approach where 15–20 leaf discs sub-
merged in 10 ml of agrobacterial suspension in syringe and
the plunger was used to create sufficient vacuum. The
pressure rapidly increases when the vacuum gets conked
out which allows the Agrobacterium cells to get driven into
the explant enabling transient expression of transgenes
(Fig. 1).
This IIP was used to validate the transient transforma-
tion efficiency of Agrobacterium harbouring the binary
construct pCAMBIA 1305.1 containing the GUS gene,
driven by CaMV35S into soybean leaf discs. GUS?ve
indicated positive transformants, while no colour change
was observed in the control un-infected leaf discs. Tran-
sient transformation efficiency was found to depend on
various parameters viz Agrobacterium concentration, pH,
infiltration time, acetosyringone concentration and L-cys-
teine which was optimized prior (Fig. 2). In this study, we
systematically investigated the above mentioned parame-
ters to define a combination that might contribute to the
unprecedentedly high transient transformation. The optimal
OD600 of Agrobacterium for agro infiltration, among those
tested, was found to be 0.6. Lower concentrations resulted
in lower infection efficiency, while higher concentrations
impaired plant survival (Fig. 2a). Leaf-discs of grape
showed high tissue necrosis when co-cultivated with a
higher density of A. tumefaciens and longer infection
duration (Das et al. 2002). Increased OD resulted with
higher secretion of antimicrobial substances which poten-
tially might reduce A. tumefaciens colonization and
T-DNA transfer (Goodman and Novacky 1994).
pH of the infiltration medium being another critical
bottle neck in transformation, the effect of different pH
(5.0–6.2) of infiltration buffer on transient transformation
was investigated. It was interesting to observe that opti-
mum pH directly affected the transformation efficiency and
the highest transformation efficiency was observed at pH
5.4, at which 43% leaf discs showed GUS activity
(Fig. 2b). At higher pH, low intensity of GUS expression
was observed. To determine the optimum infiltration time
duration, infected leaf discs were subjected to different
time periods ranging from 1–8 days and the transient
transformation efficiency was determined based on GUS
assay. GUS expression observed positively correlated with
the increased time duration till 4 days. Maximum per-
centage of GUS positive leaf disc explants (46%) was
observed at 4 days after syringe infiltration (Fig. 2c).
Further decrease in GUS positives to 21% and 17%
respectively was observed after 6 and 8 days of agro
infiltration, while 28% was observed at 2 days after infil-
tration. Therefore, we concluded that the optimal maximal
infiltration period as 4 days.
Further to improve the infection efficiency of infiltration
medium, varied concentrations of thiol compounds like ace-
tosyringone (0–0.5 mM) and L-cysteine (100–1000 mg L-1)
were incorporated into the infiltration medium. At a con-
centration of 0.3 mM acetosyringone, the maximum GUS
expression of 46% was observed (Fig. 2d, e) while
400 mg L-1 of L-cysteine was found optimum with maxi-
mum GUS expression. Lower concentrations were less
effective and even higher concentrations revealed a decrease
in expression. Acetosyringone, known to induce the virulence
genes of Agrobacterium, is necessary for effective transfer
and incorporation of T-DNA into the host plant, significantly
improved the transient transformation efficiency up to
0.2 mM, indicating its critical role in soybean transformation.
Higher concentration was observed with decrease in trans-
formation efficiency, this could be due to the fact that higher
concentrations contain a higher amount of alcohol; the sol-
vent used for its preparation and hence might be toxic to the
explants (Sreeramanan et al. 2005). Previous research have
shown that thiols like L-cysteine have role in scavenging
reactive oxygen species (ROS) by inhibiting copper and iron
containing enzymes active in plant defense mechanism such
as polyphenol oxidases (PPOs) and have been used success-
fully to increase recovery of transgenic events in soybean
using cotyledonary nodes (Olhoft et al. 2001), half seeds
(Hada et al. 2016) and proliferative embryogenic tissues
(Finer and Larkin 2008).
Holding these variables constant, further the effect of
DTT (0.5 mM) and Tween 20 (0.01%) in improving the
transient transformation efficiency was investigated indi-
vidually and in combination (Table 2). Maximum GUS
expression of 58% was observed in IF4 (Fig. 3a) which
underlines the role of DTT in suppressing the hypersensi-
tive responses and nonionic surfactants like Tween 20 in
assisting bacterial infiltration (Shamloul et al. 2014).
Above 0.01% of Tween 20, was observed with deleterious
effects on transient transformation by Matsuo et al. (2016)
and 0.5 mM DTT was optimized prior under lab conditions
to understand the synergistic role of various parameters in
transient expression in soybean (Fig. 3a, b). In our study,
the transient transformation efficiency improved with
infiltration time up to 4 days, after which there was a
drastic decrease in transient transformation efficiency.
Moreover, extending the duration of infiltration beyond a
threshold time (4 days) reduced the transformation effi-
ciency, possibly because excessive infection by Agrobac-
terium causes cell death. This report suggests that pH,
Journal of Plant Biochemistry and Biotechnology
123
media composition and infiltration time have synergistic
effects in agro infiltration.
Validation of AGRODATE for transient expression
We developed a highly efficient and robust Agrobacterium-
mediated transient expression system, named AGRODATE
(Agrobacterium-mediated disc assay for transient expres-
sion), which can achieve versatile analysis of diverse gene
functions in intact soybean leaf discs with in limited time
frame. The high throughput applicability of the developed
protocol was further validated using Agrobacterium har-
boring different binary constructs—Cas9MDC123,
pCAMBIA-GmIPK1 and pBINMIPS-AS (Fig. 3c). Rela-
tive quantification analysis revealed a significant up-regu-
lation (3.4 fold) of bar gene expression in Agrobacterium
(Cas9MDC123) infected soybean leaf discs compared to
control (Fig. 3d). Similarly a 5.2 fold up-regulation of the
IPK1 gene expression was observed in pCAMBIA-
GmIPK1 infiltrated leaf discs samples (Fig. 3d). Using
pBIN-MIPS antisense construct, we validated the down
regulation of MIPS gene using the present method and
observed a 2.1-fold decrease in MIPS expression compared
to pBIN only control (Fig. 3d). The results validated the
versatility and utility of the present methodology—
AGRODATE, for infiltrating various binary constructs
with reasonable efficiency. The variation in the expression
stability of the transgenes observed might be possibly due
to size difference of binary vectors used which varied the
chance of integration or expression. Thus this present
protocol allows a high throughput evaluation of multiple
expression vectors without consuming large number of
plants as well as without mass culturing of bacterial sus-
pensions. The combined use of vacuum, detergent, thiols
and reducing agent allowed better delivery, penetration and
infection of Agrobacterium into interior leaf tissues with-
out much mechanical injury compared to sonication
strategies. In addition, the discs were extracted from single
leaf for technical replicates and in a way were able to tease
apart the variations in transient agro infiltration experi-
ments mainly associated with leaf heterogeneity in terms of
age and position. This not only limits the genetic pollution
i.e. unwanted diffusion of genetically modified bacterium
into the environment, but can also be conducted without
the intervention of any specialized equipments. As this leaf
disc based agro-infiltration approach meets the needs for
rapidity, convenience and efficiency for high throughput
screening, it can also be attempted for other possible
downstream applications through transient expression
analysis in other recalcitrant species too.
Acknowledgements Authors are grateful to Indian Council of Agri-
cultural Research-National Agricultural Science Fund (Grant-RNAi-
2011) for supporting with financial assistance. We are thankful to
Division of Genetics, Indian Agricultural Research Institute, New
Delhi, India for soybean samples (DS-9712 cv) which were utilized
for the agro infiltration studies.
Author contributions KV conceived the experiments. KV, JM and
JJ performed the experiments and analyzed data together with SA and
MM. KV wrote the paper and editing was carried out by SA, RK, VT
and SP. All authors discussed the results and commented on the
manuscript.
Compliance with ethical standards
Conflict of interest The authors declare that they do not have any
conflict of interest.
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