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

arcs_bio@yahoo.com

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

123

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

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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

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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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