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Plant Cell, Tissue and Organ Culture(PCTOC)Journal of Plant Biotechnology ISSN 0167-6857 Plant Cell Tiss Organ CultDOI 10.1007/s11240-016-0973-x

Elite hairy roots of Ocimum basilicumas a new source of rosmarinic acid andantioxidants

Shivani Srivastava, Xavier A. Conlan,Alok Adholeya & David M. Cahill

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ORIGINAL ARTICLE

Elite hairy roots of Ocimum basilicum as a new sourceof rosmarinic acid and antioxidants

Shivani Srivastava1,2 • Xavier A. Conlan2 • Alok Adholeya1 • David M. Cahill2

Received: 18 November 2015 / Accepted: 9 March 2016

� Springer Science+Business Media Dordrecht 2016

Abstract This study reports Agrobacterium rhizogenes-

mediated transformation of three cultivars of Ocimum basili-

cum for hairy root establishment, screening and selection for

the production of rosmarinic acid and antioxidants. Hairy root

development was found to be explant-specific and virulence-

dependent. Distinct inter-cultivar morphological variability

was found between the seven axenically developed hairy root

lines and morphological traits were found to be correlated with

the presence of aux2 genes, their expression and endogenous

IAA content. Further inter-cultivar variability in the content of

total phenolics, rosmarinic acid and caffeic acid was also

found. Production of rosmarinic acid was found to be age-

dependent and cultivar-specific. Chemiluminescence analysis

showed the hairy roots to be rich in antioxidants and that

rosmarinic acid was the major antioxidant molecule. The

concentration of rosmarinic acid was found to be positively

correlated with the total antioxidant potential of the hairy root

extracts. On the basis of origin, morphology and metabolite

content, three elite hairy root lines were selected that had

significantly higher rosmarinic acid production, biomass and

antioxidant potential than non-transformed roots. These new

lines are rich reserves of both antioxidants and rosmarinic acid.

Keywords Antioxidants � Chemiluminescence � Hairyroots � Ocimum basilicum � Morphotyping � Rosmarinic

acid

Abbreviations

ABTS�? 2,2-Azinobis-(3-ethylbenzothiazoline-6-

sulphonic acid) radical cation

CA Caffeic acid

dNTPs Nucleoside triphosphate

DPPH� 2,2-Diphenyl-1-picrylhydrazyl radical

GAE Gallic acid equivalents

HPLC High performance liquid chromatography

HR Hairy root

IAA Indole acetic acid

M Minimal medium

MS Murashige and Skoog media

MW Modified white’s medium

NAA Naphthalene acetic acid

NAM Naphthalene acetamide

OPA Ortho phosphoric acid

PCR Polymerase chain reaction

RA Rosmarinic acid

rpm Rotation per minute

Smr Streptomycin resistant

T-DNA Transferred DNA

TL-DNA Left subfragment of the transferred DNA

TR-DNA Right subfragment of the transferred DNA

YMA Yeast Mannitol Agar

YMB Yeast Mannitol Broth

Introduction

Hairy roots are acknowledged as an efficient and viable

resource of secondary metabolites (Georgiev et al. 2012).

Species such as Ocimum basilicum that belong to the

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11240-016-0973-x) contains supplementarymaterial, which is available to authorized users.

& David M. Cahill

david.cahill@deakin.edu.au

1 TERI–Deakin Nanobiotechnology Centre, The Energy and

Resources Institute (TERI), DS Block, India Habitat Centre,

Lodhi Road, New Delhi 110003, India

2 Deakin University, Geelong, Australia. Centre for Chemistry

and Biotechnology, School of Life and Environmental

Sciences, (Waurn Ponds Campus), 75 Pigdons Road,

Locked Bag 20000, Geelong, VIC 3220, Australia

123

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DOI 10.1007/s11240-016-0973-x

Author's personal copy

family Lamiaceae are rich reserves of polyphenolics that

have a range of biological activities (Srivastava et al.

2014). Rosmarinic acid (RA), for example, is one of the

major polyphenolics that has found application as an anti-

inflammatory, chemoprotective, neuroprotective, hypo-

glycemic and anti-proliferative agent (Khojasteh et al.

2014). Transformed (hairy) roots derived from species

belonging to the family Lamiaceae such as Salvia milti-

orrhiza (Xiao et al. 2011; Yan et al. 2006), Agastache

rugosa (Lee et al. 2008; Nourozi et al. 2014), Coleus for-

skohlii (Li et al. 2005) and Coleus blumei (Bauer et al.

2009) have been extensively studied for RA production, its

elicitation, biosynthesis and bioactivity. In contrast to the

above species hairy roots of O. basilicum (Tada et al. 1996;

Bais et al. 2002) have not been comprehensively assessed

for RA production even though this species is known as a

reliable and potentially rich source of RA, especially when

grown in vitro (Srivastava et al. 2014).

The selection of hairy roots is conventionally based on

morphological and biochemical characteristics and a distinct

inter- and intra-clonal variability has been reported in hairy

roots derived from individuals of the same species, for

example, in Panax ginseng, Catharanthus roseus, Tylophora

indica, Withania somnifera, Beta vulgaris and Draco-

cephalummoldavica (Batra et al. 2004; Chaudhuri et al. 2005;

Bandyopadhyay et al. 2007; Mallol et al. 2001; Thimmaraju

et al. 2008; Weremczuk-Je _zyna et al. 2013). The morpho-

logical diversity found among hairy roots has been correlated

with TL-(Left subfragment of the transferred DNA) and TR-

DNA (Right subfragment of the transferred DNA) insertion,

their copy number and position (Amselem and Tepfer 1992;

Bandyopadhyay et al. 2007; Chriqui et al. 1996). Further, a

distinct relationship also exists between root morphology and

metabolite content (Mallol et al. 2001). In addition to the

morphological traits, the metabolic diversity and content is

also affectedbyphysiological state, endogenous auxin content

and nutrient level (Tada et al. 1996; Bais et al. 2001; Thim-

maraju et al. 2008) and all of these traits may be used for

selection of elite hairy roots.

The presence of antioxidants and the total antioxidant

potential have been also used as selective elements for

establishment of elite hairy root lines and their quantitation

has drawn upon a number of assays including the DPPH�

(2,2-diphenyl-1-picrylhydrazyl radical), ABTS�? (2,20-azi-nobis-[3-ethylbenzothiazoline-6-sulphonic acid] radical

cation) and the phosphomolybdenum reduction assay

(Grzegorczyk et al. 2007; Nopo-Olazabal et al. 2013, 2014;

Thiruvengadam et al. 2014; Weremczuk-Je _zyna et al.

2013). While useful for the determination of total antiox-

idant activity a clear disadvantage of these assays lies in

their inability to measure the antioxidant activity of indi-

vidual compounds (Srivastava et al. 2016). In contrast,

online acidic potassium permanganate chemiluminescence-

based assays, which have not been reported for the

assessment of antioxidants in hairy roots, offer several

advantages over conventional assays including higher

sensitivity, ease in chemical preparation and their longer

shelf life, fast analysis times and the requirement of only

simple instrumentation (McDermott et al. 2011). Further,

chemiluminescence assay also shows positive correlation

with the conventional antioxidant assays (DPPH�, ABTS�?)

and bioactivity (Bellomarino et al. 2009; Conlan et al.

2010; Francis et al. 2010) showing its suitability for

application on hairy roots.

Non-transformed roots of different cultivars of O. basi-

licumwere identified as potential reserves of RA (Srivastava

et al. 2014) and transformation for hairy root production is

likely to further enhance RA yield. Here we have developed

elite transformed root lines of three different cultivars of O.

basilicum that produce high quantities of RA and antioxi-

dants in a cultivar and age-specific manner.

Materials and methods

Seed surface sterilization and in vitro germination

Seeds of the three cultivars of O. basilicum (B3, SUBJA;

B12, HOLY GREEN and B13; RED RUBIN) were surface

sterilized, germinated and grown in vitro as previously

described (Srivastava et al. 2014). Young leaves, hypoco-

tyls and cotyledons from 4-week-old plants of each cultivar

were then used for Agrobacterium rhizogenes mediated

transformation.

Bacterial strains

Four strains of A. rhizogenes (A4, ARqua1-pTSC5, 8196

and 11325) were used. The glycerol stocks of all strains

were revived on Yeast Mannitol Broth (YMB) (all culture

reagents and agar from Himedia, Mumbai, India) and kept

at 28 �C in an incubator-shaker (Kuhner Shaker, Basel,

Switzerland) for 24 h. The revived cultures were then

streaked clockwise on Yeast Mannitol Agar (YMA) and

incubated for 2 days at 28 �C in an incubator (ET-650-8,

Lovibond, Dortmund, Germany). The YMA plates used for

growth of ARqua1-pTSC5 were supplemented with

100 mg/L of kanamycin and streptomycin.

A. rhizogenes mediated transformation: direct

injury method

A direct injury method (Georgiev et al. 2007) was used for

infection of all the explants (young leaf, hypocotyl and

cotyledon). Briefly, a single bacterial colony from a 2 day

old plate of A. rhizogenes was picked using a sterile needle

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(25 gauge, DispoVan, New Delhi, India) and all explants

were gently infected with the bacterial colony. To enable

the establishment of hairy roots, the A. rhizogenes infected

explants (10–15) were placed on a 85 mm diameter filter

disc (Whatman no. 1) that was positioned on the surface of

an MS media plate supplemented with 3 % (w/v) sucrose

(Srivastava et al. 2014) and 100 mM acetosyringone

(Sigma-Aldrich, St. Louis, USA). The Petri plates con-

taining explants were then incubated in the dark at

25 ± 2 �C at 60 % relative humidity. Similar steps were

repeated for all cultivars of O. basilicum with all strains of

A. rhizogenes. After 10–20 days the infected explants were

observed for hairy root induction.

Removal of redundant bacteria from the positive

explants

The positive explants showing root induction at the site of

infection were transferred to MS broth (composition sim-

ilar to MS media without 2.5 % phytagel; Srivastava et al.

2014) supplemented with 3 % (w/v) sucrose and 500 mg/L

cefotaxime (Omnatax, Abbott Healthcare Pvt. Ltd., Mum-

bai, India) for 24 h at 28 �C in an incubator-shaker. For the

next 2 days, the cefotaxime concentration was gradually

decreased to 250 and 100 mg/L and treated explants were

then transferred to MS broth and observed for any bacterial

growth. Bacteria-free positive explants were then trans-

ferred to MS media, pH 5.8 and were kept in the dark at

26 �C. After 2 weeks, the putative roots were transferred to

fresh MS medium with no growth hormones and grown

further for 4 weeks.

Stabilization of the developed roots

After 4 weeks of growth on MS medium, the putative root

tips were excised from the positive explants and transferred

to Modified White’s (MW) medium supplemented with

2 % (w/v) sucrose (Puri and Adholeya 2013). The roots

were stabilized on MW medium for 4 weeks and later

transferred to Minimal (M) medium supplemented with

1 % (w/v) sucrose (Puri and Adholeya 2013). Hairy roots

were maintained on M medium and used for further

studies.

Confirmation of putative transformed roots and test

for gene functionality

Total genomic DNA was isolated [DNeasy Plant Mini Kit

(QIAGEN, GmbH, D-40724, Hilden, Germany)] from

100 mg of freshly harvested transformed and non-trans-

formed (negative control) root samples. The genomic DNA

was quantified [NanoDrop 2000 (Thermo Scientific,

Wilmington, USA)] and for the positive control plasmid

DNA from A. rhizogenes A4 was isolated (High-Speed

Plasmid Mini Kit, Geneaid, PD 100/300, Taiwan). Poly-

merase chain reaction (PCR) was performed using rolB

(forward-50 TGA CTA TAG CAA ACC CCT CCT 30 andreverse-50 ACT TGC GAA AAT GGC GAT GA 30) andaux2 (forward-50 CGA ATC GCT CTG ACA ACC TC 30

and reverse-50 ATA GTT CCG GTA AGC CCC AC 30)primers (Xcelris Genomics, Ahmedabad, India). Absence

of the bacterium in the transformed root samples was

confirmed by the absence of the virC region using an

appropriate primer (forward-50 ATC ATT TGT AGC GAC

T 30 and reverse-50 AGC TCA AAC CTG CTT C 30). Allthe primers were designed using Primer 3 software. The

PCR mixture (20 lL) contained approximately 50 ng of

DNA as the template, 19 PCR buffer, 1 lM of each

primer, 2.5 mM of dNTPs and 1 unit of Taq DNA

polymerase (Invitrogen, Thermo Scientific, Bangalore,

India). PCR was carried out by initial denaturation at

94 �C for 3 min followed by 35 cycles of 35 s denatu-

ration at 94 �C, 30 s annealing at 60 �C and 35 s exten-

sion at 72 �C with a final extension at 72 �C for 5 min

using a thermal cycler (Applied Biosystems� Veriti�,

Life technologies, New Delhi, India). The bands were

visualized by resolving 5 lL of the PCR product on

ethidium bromide stained 1 % agarose gel (Himedia,

Mumbai, India) and documented on FluorChem E and M

Imagers (Protein simple, San Jose, USA).

To confirm the presence of active aux2 gene, the con-

firmed roots were inoculated on M medium supplemented

with 0.2 mg/L Naphthalene acetamide (NAM; Sigma,

Bangalore, India) for 15 days. The developed roots were

observed for callus formation (Amselem and Tepfer 1992).

Growth kinematics and root morphotyping study

For the selection of elite hairy roots three to four root tips

of each line was inoculated on M media plates (90 mm;

35 mL) and incubated in the dark at 26 �C for 20, 30, 40,

50 and 60 days respectively. Non-transformed plant roots

were used as the control at all ages and obtained from

plants grown on M media in an in vitro system (Srivastava

et al. 2014). At each age roots were harvested into 10 mM

sodium citrate buffer (Doner and Becard 1991) to deionise

phytagel and shaken at 25 �C for 60 min at 100 rpm.

Deionised roots were further washed in distilled water, blot

dried and were then recorded for root diameter, root length

and number of root tips using an image analysis software

WinRHIZO� (version Pro2007; Regent Instruments Inc,

Quebec, Canada) and a scanner (EPSON Perfection V 700,

Delhi, India). Roots were also recorded for hairiness,

density of hairiness and color. Harvested roots were then

lyophilized (Labconco lyophilizer, Kansas City, USA),

their dry weight recorded and used for extraction.

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Extraction for estimation of total phenolics,

polyphenolics and antioxidant content

The extraction method described by Srivastava et al. (2014)

was used in the current study. Briefly, 25 mg of the lyophi-

lized roots and 60 % (v/v) methanol (AR grade, Merck,

Mumbai, India) in water was used and extraction was per-

formed twice for fifteen min using a sonicator (B3510E-

DTH, Branson Ultrasonics, Danbury, USA) on a final vol-

ume of 25 mL. The extractswere then filtered using a syringe

filter (Millipore Millex HN, 0.45 lm; Merck, Darmstadt,

Germany) and used for quantitative assessments.

Quantification of total phenolics

A modified Folin–Ciocalteau colorimetric assay (Singleton

and Rossi 1965) was used for estimation of total phenolics.

Briefly, to 1 mL of the methanolic extract, 4 mL of distilled

water, 2.5 mL of Folin–Ciocalteau reagent (SRL, Ranbaxy,

Delhi, India), 1.25 mL of 2.1 % aqueous sodium carbonate

(Qualigens, Mumbai, India) was added and incubated in the

dark for 30 min and absorbance of the resulting mixture was

taken at 735 nm using a UV–Vis Spectrophotometer (UV–

Vis 2450, Shimadzu, Kyoto, Japan) against the samemixture

without sample. The total phenolic concentration was

reported as gallic acid equivalents (GAE mg/g DW).

High performance liquid chromatography (HPLC)

for the quantification of individual polyphenolics

Chromatographic analysis of the root extracts was carried

out using HPLC (CBM-20A, Shimadzu, Kyoto, Japan)

with a C18 Phenomenax column (Gemini-NX

250 mm 9 4.6 mm 9 5 lm particle diameter). For the

separation of individual polyphenolics, the mobile phase

used was HPLC grade water ? 0.1 % (v/v) ortho phos-

phoric acid (OPA) in water (Mobile phase A) and Metha-

nol (HPLC grade, Merck, Mumbai, India) ? 0.1 % OPA

(v/v) in methanol (Mobile phase B). A gradient program

similar to Srivastava et al. (2014, 2016) was used for the

determination of caffeic acid (CA) and RA. The flow rate

of the mobile phase was 1.0 mL/min and the wavelength

used for detection was 280 nm with an injection volume of

20 lL. Unknown samples were identified and quantified by

comparison with the retention time and standard calibration

curve of RA and CA (Sigma, Bangalore, India) over a

concentration range from 20 to 100 mg/L.

Determination of total antioxidant potential

and individual compound linked antioxidant activity

Methodology used for the estimation of total and individual

compound linked antioxidant potential using a

chemiluminescence assay was similar to that described in

Srivastava et al. (2014, 2016). Unknown samples were iden-

tified and quantified by comparison with the retention time

and standard calibration curve of commercial standards. An

Apollo TM C18 column (150 mm 9 4.60 mm 9 5 lm par-

ticle diameter) was used.

Extraction and quantification of endogenous indole

acetic acid (IAA) in hairy root extracts

To examine the correlation of root morphology with IAA

content, endogenous IAA was extracted from hairy roots

using the method of Thimmaraju et al. (2008). IAA was

quantified by HPLC (Shimadzu, Kyoto, Japan) on a C18

column (Gemini-NX 250 mm 9 4.6 mm 9 5 lm particle

diameter) using an isocratic method with Metha-

nol ? 0.1 % OPA and HPLC grade water ? 0.1 % OPA

(40:60), flow rate 1.0 mL/min and absorbance of 280 nm

(Malhotra and Srivastava 2006; Tansupo et al. 2010). IAA

concentration in the samples was determined in compar-

ison to a standard curve of a commercial standard (Hime-

dia, Mumbai, India). We used UV detection as a rapid and

relatively sensitive method for estimation of IAA in root

extracts but acknowledge that other methods could be used

to detect more precisely other forms of IAA.

Statistical analysis

All data is expressed in terms of mean ± SEM. Raw data

was analyzed using a commercial statistical package

(GraphPad Prism 6). One way analysis of variance with a

Tukey’s HSD test of significance at p B 0.05, p B 0.01

and p B 0.001 was used to determine the variation between

the hairy roots for the quantified morphological traits (root

length, diameter, number of tips), biochemical traits (total

phenolic content, CA and RA content), endogenous IAA

content and total and individual compound antioxidant

potential at specific age and among different ages. Corre-

lation between total antioxidant potential and antioxidant

potential of RA and CA, endogenous IAA content and root

length, and total phenolic and RA content was tested using

the Pearson test at a significance level of 0.0001 and 0.05.

Results

Establishment of transformed roots, transformation

efficiency and characterization of transformed

explants

Based on preliminary experiments (data not shown)

4-week-old explants and 2-day-old bacterial cultures were

used for transformation. No hairy root induction was found

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using A. rhizogenes 8196 and it was removed from further

experimentation. Out of the three remaining strains A.

rhizogenes A4 was found to induce hairy roots within

7 days in B3 and in 10 days in B12 and B13 while A.

rhizogenes ARqua1-pTSC5 and 11325 showed root

induction after 15 days in all three cultivars. Young leaf

explants were found to be more rapidly induced in com-

parison with hypocotyls and cotyledons. B3 showed the

highest transformation efficiency for each of the three A.

rhizogenes strains used (Fig. 1). In the B3 cultivar, all

strains of A. rhizogenes showed significantly similar

transformational efficiency for young leaves and cotyle-

dons and a similar observation was made for the B12

cultivar for all explants. In contrast to B3 and B12, no root

induction was found in B13 for hypocotyl and cotyledon

explants with ARqua1-pTSC5 and 11325.

Positive leaf explants showed root induction at the site

of infection (Fig. 2a). Callusing was also observed in some

of the leaf explants of all cultivars. For hypocotyl explants,

necrosis and callusing was observed in those that were

induced to form roots (Fig. 2b). On later subculture it was

observed that the roots excised from the induced hypoco-

tyls were half the diameter of that derived from other

explant types and were very slow growing.

Hairy roots obtained after infection with 11325 were

very thin and slow growing and did not survive after

antibiotic treatment and the stabilization process. Thus,

11325-derived hairy roots were not used in further exper-

iments. After antibiotic treatment and sub-culturing, in

total 11 (6 from A4 and 5 from ARqua1-pTSC5) roots were

obtained from B3, 4 (2 from A4 and 2 from ARqua1-

pTSC5) from B12 and 7 from B13 (3 from A4 and 4 from

ARqua1-pTSC5). Since ARqua1-pTSC5 is a Smr (Strep-

tomycin resistant) derivative of A4 and possesses a GUS

reporter gene (Cseke et al. 2007), the ARqua1-pTSC5

derived transformed roots were not used for characteriza-

tion studies. All putative hairy roots derived from A4

showed profuse, ageotropic and hormone-independent

types of root growth (Fig. 2c). Further, on the basis of

growth, in total three hairy roots (HR 1, 2 and 3) from B3,

one (HR 4) from B12 and three (HR 5, 6 and 7) from B13

were selected for confirmation, growth kinetics, morpho-

typing, metabolite production and chemiluminescence

based antioxidant studies.

Confirmation of putative transformants and test

of gene functionality

Seven putatively transformed roots were used for confir-

mation studies and specific amplicons for rolB (394 bp)

and aux2 (380 bp) were found in all the samples (Fig. 3) as

in bacterial (B) samples (positive control). No amplifica-

tion was observed in the non-transformed (U) root samples

(negative control). Presence of redundant bacteria was

examined using a primer specific to the virC region of A.

rhizogenes. No amplification of the virC was found in the

hairy root samples; however positive (bacteria) control

showed the amplification at the expected size of *700 bp

(data not shown).

All seven transformed roots showed callusing after

15 days on NAM supplemented medium (Online Resource

1), confirming the presence of active aux2 genes by for-

mation of naphthalene acetic acid (NAA). The highest

amount of callusing was observed in HR 4 suggesting that

the level of aux2 expression may depend on the cultivar

used for the transformation.

Root morphotyping

Root length increased gradually in all hairy roots from 20

to 60 days (Online Resource 2; a). HR 2 and 4 showed

the significantly highest root length while HR 1 and HR 5

showed the lowest root length (60 days). On the basis of

root diameter HR 2, 3 and 4 were identified as thick roots

while HR 6 and 7 were identified as thin roots (Online

Resource 2; b). At 60 days HR 4 was observed to show the

highest number of tips at all ages followed by HR 7 and 3

(Online Resource 2; c).

Qualitatively thick and hairy (sparse), thick and hairy

(dense), thin and callusing and thin, callusing and hairy

(dense) root types were obtained (Online Resource 3). HR

2, 3 and 4 derived from B3 and B12 cultivars of O. basi-

licum were observed to be thick in morphology while HR 1

from B3 and HR 5, 6 and 7 from B13 cultivar were thin in

Fig. 1 Transformation efficiency. The percentage of hairy root

induction in three different explants of three cultivars (B3, B12 and

B13) of O. basilicum using three different strains of A. rhizogenes.

Data is represented as the mean of the positive explants

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diameter. Callus formation was distinctly observed in all

roots derived from B13 which can be correlated with high

aux2 activity. All roots were white in color while callus in

HR 5, 6 and 7 was pink.

Root biomass

Root dry weight increased from 20 to 60 days in all samples

and the highest biomass was found in samples collected at

60 days. At 60 days, HR 2 (114.33 ± 2.11 mg) and HR 4

(116.60 ± 3.44 mg) were found to show highest biomass

(Table 1) in comparison to the other five and the lowest was

recorded for HR 6 (59.47 ± 3.89 mg). B13 derived HR 5, 6

and 7 showed the lowest biomass at all ages. HR 2 and HR 4

were identified as high biomass producers.

Endogenous IAA content and its correlation

with root morphology

IAA was detected in all hairy root lines (Fig. 4a). Variation

in the content of IAA was found among the hairy roots

derived from the same cultivar (Fig. 4b). HR1 (0.011 lg/mg

Fig. 2 Representative

figures showing positive leaf

and hypocotyl explants after A.

rhizogenes A4 mediated

transformation of B3 cultivar.

a Positive leaf (L) explant

showing hairy root induction at

the site of infection, arrows

show dense hairs on root

surface, b positive hypocotyl

(H) explants showing callusing

(C) and necrosis (N) and root

emergence (very hairy and very

thin) at the infected site,

c positive leaf (L) explant

showing hairy ageotropic and

profuse type of root growth (as

indicated by arrows). Scale for

a = 0.2 cm, b, c = 0.5 cm

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FW) showed a significantly lower amount of IAA in com-

parison to HR 2 and HR 3 (0.039; 0.034 lg/mg FW re-

spectively) derived from the same cultivar (B3). Similarly in

B13 derived hairy roots significantly lower amount of IAA

was found inHR 5 (0.008 lg/mg FW) in comparison toHR 6

and 7 (0.031; 0.034 lg/mg FW respectively). Significantly

higher levels of IAA found in HR 2, 3 and 4 in comparison to

HR 1 and 5 were found to be correlated with their thicker

morphology and greater root length.

Total phenolics

In general, the total phenolics content increased in all hairy

roots with age (Table 2). HR 4 showed higher levels of

total phenolics at all ages. The level of total phenolic

content in B13 derived roots (HR 5, 6 and 7) increased

significantly after 40 days of subculture. The total pheno-

lics content ranged from 247.33 ± 35.73 GAE (mg/g DW;

HR 2) to 378.80 ± 26.74 GAE (mg/g DW; HR 4) in

60 days old samples.

Individual polyphenolic content: rosmarinic acid

and caffeic acid

RA was observed as the major polyphenolic in all the

samples at 15.28 min. Variation in the levels of RA content

was found in extracts of all hairy roots. RA content was

found to increase till 40 days in HR 1, 2 and 3 (Table 3a).

Fig. 3 Confirmation of the transgenic nature of the developed hairy

roots using rolB (a) and aux2 (b) specific primers. ‘‘M’’ = DNA

marker (100 bp ladder), ‘‘B’’ = positive control (A. rhizogenes A4),

U = Negative control (non-transformed root), 1–7 = HR 1–7 (test

samples). Sizes of bands for rolB and aux2 was 394 and 380 bp in

positive (bacterial) and test (HR) samples

Table 1 Dry weight (mg) of the seven transformed hairy roots after plating onto minimal (M) medium for five different time periods

Days (d) HR 1 HR 2 HR 3 HR 4 HR 5 HR 6 HR 7

Dry weight (mg)

20 27.03 ± 1.46d 34.37 ± 1.67d 24.97 ± 2.11c 24.03 ± 1.07d 4.50 ± 0.15e 3.70 ± 0.45c 4.17 ± 0.26d

30 41.33 ± 3.28 cd 57.03 ± 0.59c 27.90 ± 1.85c 54.67 ± 3.46c 20.13 ± 0.91d 14.33 ± 1.43c 20.00 ± 0.91c

40 48.97 ± 2.51c 66.90 ± 2.10b 51.10 ± 3.27b 72.80 ± 6.01b 44.30 ± 1.96c 26.13 ± 1.19b 34.13 ± 2.70b

50 80.21 ± 8.72b 74.23 ± 3.02b 64.60 ± 4.45b 103.87 ± 2.47a 52.03 ± 1.82b 53.40 ± 3.21a 43.07 ± 2.42b

60 106.87 ± 2.45a 114.33 ± 2.11a 95.00 ± 3.08a 116.60 ± 3.44a 60.53 ± 1.11a 59.47 ± 3.89a 65.80 ± 2.16a

Data represented as mean ± SEM of each of three replicates (n = 3) in mg. Different letters indicate significant differences (p B 0.05, p B 0.01)

between ages for specific hairy roots according to Tukey’s HSD

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HR 5, 6 and 7 showed the highest RA content at 60 days.

The highest RA content were found in HR 1 (40 days;

71.03 ± 12.67 mg/g DW), HR 4 (50 days; 69.49 ±

1.79 mg/g DW) and HR 5 (60 days; 76.41 ± 3.71 mg/g

DW) at different ages and this clearly indicated that RA

synthesis is age-dependent in hairy roots derived from the

three different cultivars of O. basilicum. On comparison

between hairy roots derived from the same cultivar, no

significant difference was found between HR 1, 2 and 3 at

40 and 60 days and similar observations were made for HR

5, 6 and 7 at 40, 50 and 60 days. Forty days can be con-

sidered to be the optimum age for RA production in HR 1,

2, 3 and 4.

Other than RA, caffeic acid (CA) was also detected in

all samples at 12.08 min. The presence of CA in hairy roots

of O. basilicum is reported for the first time. The trend of

CA content with age differed between hairy root lines. An

increase in the level of CA (Table 3b) was observed in HR

2, 5, 6 and 7 while a decrease in CA content after 40 days

in HR 1 and 3 and after 50 days in HR 4 was found. HR 4

was observed to have significantly higher concentrations of

CA at all ages. CA was identified as a minor metabolite in

our study.

Total and individual compound linked antioxidant

potential analysis

HR 2, HR 4 and HR 5 were selected for chemilumines-

cence-based total antioxidant and individual compound

antioxidant potential studies as they represented variability

in terms of origin (derived from three different cultivars),

morphology and had high RA content. Chemiluminescence

based analysis was performed using an HPLC system

equipped with or without a column (to model a flow

Fig. 4 Quantification of endogenous IAA content in the developed

hairy roots (60 days old). a HPLC chromatogram showing IAA peak

in standard (dotted line) and HR4 (black), b IAA levels (lg/mg FW)

detected in 60 days old HR (HR 1–7) samples. Data is represented as

mean ± SEM (n = 3). Different letters indicate significant differ-

ences (p B 0.05, p B 0.01) according to Tukey’s HSD between

different hairy roots

Table 2 Levels of total phenolics (expressed as GAE (mg/g DW) detected in hairy roots after five different ages of growth on M medium

Hairy roots (HR) Days after subculturing (d)

20 30 40 50 60

Total phenolic content; GAE (mg/g DW)

HR 1 127.70 ± 16.64ab 246.30 ± 37.56ab 254.77 ± 11.39a 285.90 ± 6.40a 331.60 – 13.23ab

HR 2 161.47 ± 17.07a 204.27 ± 15.54ab 243.60 ± 0.46a 252.93 – 11.24a 247.33 ± 35.73b

HR 3 156.33 ± 12.67a 161.87 ± 14.31bc 233.73 ± 17.47a 312.40 ± 19.42a 354.97 – 15.83ab

HR 4 139.10 ± 24.76a 282.00 ± 12.76a 315.33 ± 6.76a 377.47 ± 2.82a 378.80 – 26.74a

HR 5 62.93 ± 6.35bc 107.93 ± 14.13bc 239.73 ± 14.21a 297.00 ± 5.15a 374.97 – 31.65a

HR 6 62.38 ± 5.99bc 96.07 ± 3.52 cd 183.37 ± 12.71a 284.60 ± 36.34a 339.17 – 9.12ab

HR 7 55.75 ± 3.68c 89.95 ± 6.66 cd 161.60 ± 15.62a 255.47 ± 18.11a 319.77 – 8.44ab

Data represented as mean ± SEM of each of three replicates (n = 3). Different letters indicate significant differences (p B 0.05) according to

Tukey’s HSD between different hairy roots at specific age. Bold emphasis shows highest value for each root

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injection system) for total and individual compound

antioxidant studies. A representative chromatogram of UV

and chemiluminescence signal for HR 2 (Online Resource

4) shows RA as the major peak in the extract of HR 2.

The HPLC signal of HR 2 showed retention times of

14.5 min for RA and 10.6 min for CA. The slight differ-

ence in retention time of the same sample to that reported

in the quantification of individual polyphenolic section

(RA, 15.28 min and CA, 12.08 min) can be attributed to

the use of different columns and HPLC systems. In total 16

peaks were observed in the chemiluminescence signal of

HR 2 (Online Resource 4) and following comparison with

standards, peaks 9 and 13 were identified as CA and RA

respectively.

No significant difference was found in total antioxidant

potential among all three hairy roots at the three different

ages (Fig. 5a). An increase in total antioxidant potential

was found from 20 to 60 days in extracts of all roots. There

was a 14-fold increase in total antioxidant potential in

extracts of HR 2 and HR 4 from 20 to 60 days while HR 5

showed a 45-fold increase over the same time period.

The antioxidant potential of RA and CA was found to

increase from 20 to 60 days in all hairy roots. No significant

differencewas found in antioxidant potential ofRAat 20 days

(2.95–5.37 mM/100 g DW) and 40 days (11.47–14.19 mM/

100 g DW) for all roots (Fig. 5b). A significantly higher

antioxidant potential was detected for RA in HR 2

(49.16 mM/100 g DW) in comparison to HR 4 and 5

(27.18–28.79 mM/100 g DW) after 60 days of growth. RA

contributed to 60, 40 and 53 % (HR 2), 8.5, 27 and 39 % (HR

4), 12, 50 and 51 % (HR 5) to total antioxidant potential after

20, 40, 60 days respectively confirming it as the major

antioxidant active molecule in the hairy root extracts.

For CA, no significant difference was found among all

the samples at 20 and 60 days (Fig. 5c). At 40 days HR 2

and HR 4 showed a significantly higher antioxidant

potential of CA in comparison to HR 5. CA contributed

2.6, 5.6, 1.7 % (HR 2), 4.5, 4.2, 2 % (HR 4) and 3.9, 2.7,

2.8 % (HR 5) to total antioxidant potential after 20, 40 and

60 days respectively. Correlation analysis showed that RA

and CA content were positively correlated (p B 0.0001) to

total antioxidant potential of all three hairy roots at all ages

(Fig. 5d). The highest correlation was found between RA

and total antioxidant potential (R2 = 0.8798) in the root

extracts at all ages. Similar to RA, positive correlation

(R2 = 0.6114) was obtained for CA also.

Comparison between non-transformed

and transformed roots: RA, biomass

and antioxidant potential

The RA content found in the hairy roots was compared

with that found in non-transformed roots and the age

selected for comparative studies was based on that at which

Table 3 Amount of rosmarinic acid and caffeic acid detected in hairy roots after five different ages of growth on M medium

Hairy

roots

(HR)

Days after subculturing (d)

20 30 40 50 60

(a) Rosmarinic acid (mg/g DW)

HR 1 10.53 ± 1.76b 22.62 ± 2.39b 71.03 – 12.67a 47.06 ± 3.63b 38.22 ± 4.62 cd

HR 2 28.83 ± 1.65a 42.31 ± 2.22a 69.02 – 2.78a 67.21 ± 5.95a 41.44 ± 5.82bcd

HR 3 4.63 ± 0.65c 23.76 ± 2.20b 66.24 – 5.48ab 42.92 ± 1.27b 24.23 ± 1.32c

HR 4 11.10 ± 1.75b 23.31 ± 1.89b 68.93 ± 3.97a 69.49 – 1.79a 57.98 ± 4.24abc

HR 5 11.34 ± 1.03b 15.54 ± 2.09bc 45.03 ± 7.32abc 55.15 ± 1.67ab 76.41 – 3.71a

HR 6 4.11 ± 0.14c 5.48 ± 0.21d 29.76 ± 4.61c 48.82 ± 1.24b 67.17 – 9.38ab

HR 7 3.13 ± 0.21c 9.70 ± 2.24 cd 36.75 ± 3.07bc 49.59 ± 2.44b 68.47 – 7.21ab

(b) Caffeic acid (mg/g DW)

HR 1 0.15 ± 0.02b 1.05 – 0.05b 1.02 ± 0.01b 0.72 ± 0.04b 0.15 ± 0.01d

HR 2 0.27 ± 0.03b 0.62 ± 0.04c 0.67 ± 0.01 cd 0.73 ± 0.04b 0.74 – 0.04c

HR 3 0.12 ± 0.02b 0.44 ± 0.02 cd 0.93 – 0.07bc 0.15 ± 0.00c 0.11 ± 0.01d

HR 4 0.93 ± 0.16a,#,## 1.30 ± 0.07a,#,## 1.61 ± 0.14a,#,## 1.74 – 0.17a,#,## 1.53 ± 0.01a,#,##

HR 5 0.14 ± 0.02b 0.41 ± 0.01d 0.50 ± 0.01d 0.70 ± 0.02b 0.78 – 0.01c

HR 6 0.12 ± 0.01b 0.26 ± 0.04de 0.78 ± 0.08bcd 0.89 ± 0.04b 1.03 – 0.03b

HR 7 0.11 ± 0.00b 0.18 ± 0.00e 0.51 ± 0.05d 0.78 ± 0.01b 0.98 – 0.01b

Data represented as mean ± SEM of three replicates (n = 3). Different letters indicate significant differences (p B 0.05, p B 0.01) according to

Tukey’s HSD between different hairy roots at specific age for RA and CA content. HR 4 showed significantly higher CA content in comparison

to all other hairy roots at all ages (p B 0.01 and p B 0.001) and is represented as ‘‘#’’ and ‘‘##’’ in the section b of the table. Bold emphasis shows

highest value for each root

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the highest amount of RA was obtained. HR 1, 2 and 3

showed fivefold higher levels of RA compared with non-

transformed roots while in B12 and B13 derived hairy

roots, the production rate was double that of non-trans-

formed roots (Fig. 6a). HR 1, 2 and 3 showed twice the

biomass of non-transformed roots while 1.5-fold higher

biomass compared with non-transformed roots was found

in HR 4, 5, 6 and 7 (Fig. 6b). At 60 days the total

antioxidant potential of selected HR 2, 4 and 5 was twice

the level found in non-transformed roots (Fig. 6c) showing

the distinct difference between transformed versus non-

transformed roots.

Discussion

We have successfully demonstrated through our current

study that A. rhizogenes mediated transformation of three

cultivars of O. basilicum led to the development of seven

axenic hairy root lines which (1) show cultivar specific

morphological variability and polyphenolic (RA and CA)

and antioxidant content, (2) produce RA (the major

polyphenolic) in an age-dependent manner and (3) are

richer reserves of RA and antioxidants than non-trans-

formed roots. Hairy root establishment studies showed that

hairy root induction in three different cultivars of O.

basilicum was explant- and bacterial strain-dependent

(Grzegorczyk et al. 2006; Thiruvengadam et al. 2014). We

found, as have others, that following infection with A.

rhizogenes the greatest (50–70 % approximately) root

induction response was by the young leaf which has been

attributed to its active physiological state, high regenera-

tion capacity and large wounding surface area availability

(Bansal et al. 2014; Chaudhuri et al. 2005; Nourozi et al.

2014). Differences obtained in transformation efficiency

between the three different strains of A. rhizogenes may be

attributed to differences in their virulence level as sug-

gested by Bansal et al. (2014) and Nourozi et al. (2014).

Similar to the reports of Batra et al. (2004) and Chaudhuri

et al. (2005), the agropine producing strain A4 was iden-

tified as the most potent bacterial strain and its wild origin

may account for such an observation. The callus formation

near the induction site in the leaf and hypocotyl explants of

B12 and B13 was likely the result of wound induced

phytohormone production that stimulated cell proliferation

(Triplett et al. 2008). The lowest infection potential of

11325 observed in our study may be related to the differ-

ence in chromosomal virulence genes (Tiwari et al. 2008).

The developed hairy roots showed highly branched and

plagiotropic type growth showing similarity to typical

hairy root morphology (Bansal et al. 2014). Intra-cultivar

morphological variability obtained in B3 and B13 derived

Fig. 5 Acidic potassium permanganate based chemiluminescence

detection of total and individual compound linked antioxidant

potential in the three selected hairy roots. a Total antioxidant

potential, b, c antioxidant potential of RA and CA, d correlation

between total antioxidant potential and antioxidant potential of RA

and CA in hairy root extracts at three different ages. The Pearson

correlation coefficient (r) for RA and CA with total antioxidant

potential content response was 0.938 and 0.781 respectively.

Antioxidant potential detected by chemiluminescence assay is

expressed as mM/100 g DW. Data is represented as mean ± SEM

(n = 3) for the hairy root extracts. Similar letters show no significant

difference between the three hairy roots at different ages (p B 0.05)

by Tukey’s HSD

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hairy roots suggest that differential expression of

T-DNA (Transferred DNA) genes and positional integra-

tion of T-DNA occurred in their host genome (Thimmaraju

et al. 2008; Bansal et al. 2014). The presence of callus in

the B13 derived hairy roots can be attributed to the addi-

tional expression of aux genes present on TR-DNA (Batra

et al. 2004; Bandyopadhyay et al. 2007). Callus formation

by all hairy roots on NAM-supplemented medium con-

firmed the formation of NAA that inhibits root growth

(Amselem and Tepfer 1992; Chriqui et al. 1996). The

highest response in callus formation that was obtained for

HR 4 may be correlated with its thick morphology. Auxin

levels affect root length, branching pattern and number of

laterals in hairy roots (Thimmaraju et al. 2008) and a

positive correlation (R2 = 0.7271) found between

endogenous IAA content and root length for 60 day old

hairy roots of O. basilicum in our study also confirmed that

auxin levels impact root morphology. The thin root mor-

phology of HR 1 and 5 may be correlated with their low

endogenous IAA concentration.

RA production was found to be cultivar specific and

growth-related as the highly prolific, fast growing lines

(HR 1, 2 and 3) derived from B3 produced higher RA

levels at a younger age (40 days) than B13 derived hairy

root lines (60 days; HR 5, 6 and 7) that were slower

growing. The decrease in RA levels of HR 1, 2 and 3 after

40 days was likely due to nutrient limitation in the growth

medium (a minimal ‘M’ medium was used) which indi-

cated that RA production was nutrient-dependent. We have

established that A. rhizogenes mediated transformation is

an efficient method to improve RA production in low RA

producing (B3; Srivastava et al. 2014) cultivars of O.

basilicum. The higher RA levels in B3 derived roots rather

than in B12 and B13 roots can be also correlated with its

earlier transformation (7 days) and higher transformation

efficiency (70 %) in comparison to B12 (59.9 %) and B13

(55 %). The low content of CA showed that it is a minor

polyphenolic in hairy roots as in non-transformed roots

(Srivastava et al. 2014). To our knowledge there are only

two reports on RA production in hairy roots of

O. basilicum (Tada et al. 1996; Bais et al. 2002) on MS,

WP and B5 medium while there are no reports on pro-

duction on the M medium used in the current study. We

used M medium in the current study to develop hairy roots

for mycorrhization studies. Tada et al. (1996) reported

development of five hairy root lines for RA production and

the highest (14.1 % DW) was found in J1 clone after

8 weeks of growth on MS broth, twice the highest amount

reported in our study. Use of a different bacterial strain for

hairy root induction, cultivar of O. basilicum, broth (MS)

and shaking condition may account for such differences.

Further, on comparison with hairy roots of other species

of the family Lamiaceae, RA levels found in our study

were similar to the amount reported in transformed roots of

Salvia officinalis and D. moldavica L. (Grzegorczyk et al.

2006; Weremczuk-Je _zyna et al. 2013) and more than fifty

times greater (1500 lg/g DW) than that claimed by Fattahi

et al. (2013) in Dracocephalum kotschyi Boiss.

Antioxidants of plant origin have played a significant

role in nutritional and pharmaceutical industries as an

excellent alternative to chemically-derived antioxidants

(Srivastava et al. 2016). For the standardized, environment-

independent and improved production of antioxidants,

hairy roots have been identified as an excellent alternative

to in planta, callus and suspension culture-like techniques

(Grzegorczyk et al. 2007; Weremczuk-Je _zyna et al. 2013;

Thiruvengadam et al. 2014). An acidic potassium per-

manganate-based chemiluminescence assay was chosen in

the current study to determine antioxidant potential of hairy

root extracts because it readily reacts with polyphenolics

found in O. basilicum, shows good agreement with con-

ventionally used assays and provides information on indi-

vidual compound-linked antioxidant activity (Bellomarino

et al. 2009; Conlan et al. 2010; Francis et al. 2010;

Fig. 6 Comparison between non-transformed and hairy roots of three

cultivars of O. basilicum. a RA, b dry weight and c antioxidant

potential after 60 days. Solid black line show boundary between three

different cultivars in a, b. Data represented as mean ± SEM (n = 3).

Different letters indicate significant differences (p B 0.05) between

non-transformed and hairy roots according to Tukey’s HSD test

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Srivastava et al. 2014, 2016). Similar levels of total

antioxidant potential found in elite hairy roots indicated

that all hairy roots were equally good reserves of antioxi-

dants irrespective of their origin. In line with our earlier

reports (Srivastava et al. 2014, 2016), RA was found to be

the major contributor to total antioxidant potential in hairy

root extracts likely due to its chemical structure that

includes the presence of two catechol rings conjugated with

a carboxylic acid. High correlation between RA and total

antioxidant potential showed that the antioxidant properties

of hairy root extracts was tightly RA-linked. In contrast to

RA, CA is a minor antioxidant with low antioxidant

potential in hairy root extracts and was found at higher

levels to that in non-transformed roots (Srivastava et al.

2014) in our current study. Other than RA and CA many

other peaks offering antioxidant potential were also

observed in the hairy root extracts and future mass spectral

analysis of the these extracts may fruitfully identify several

of the unknowns. The application of the acidic potassium

permanganate based chemiluminescence assay for the

rapid assessment of antioxidants in in vitro raised callus

and hairy root cultures is thus proposed through our study.

In the present study we found that, compared with non-

transformed roots, elite hairy root lines produced signifi-

cantly higher levels of biomass, rosmarinic acid and

antioxidants. For rosmarinic acid, other reports have indi-

cated two to three-fold higher levels in transformed rather

than in non-transformed roots of O. basilicum (Bais et al.

2002) and S. officinalis (Grzegorczyk et al. 2006). Inter-

estingly, our chemiluminescence based assay also showed

elite hairy root lines to be richer reserves of antioxidants

than non-transformed roots as was also shown by Grze-

gorczyk et al. (2007) and Thiruvengadam et al. (2014)

using DPPH� and phosphomolybdenum assays. Taken

together these results suggest that transformation positively

impacts on the biosynthesis of antioxidant molecules in

root cells. Hairy roots result from the integration of the Ri

plasmid of A. rhizogenes into the plant genome and then

expression of aux (TR-DNA) and rol (TL-DNA) genes

(Amselem and Tepfer 1992). Integration of T-DNA indu-

ces endogenous auxin and secondary metabolite biosyn-

thesis that enables hairy roots to show hormone

independent excessive growth and increased secondary

metabolite production (Sharma et al. 2013). The rol genes

(rolA, rolB, and rolC) have been identified as potential

activators of secondary metabolite pathways with rolB

being the most powerful inducer of secondary metabolism,

followed by rolC (Bulgakov 2008). To date the impact of

the expression of individual rol genes on RA production by

hairy roots of O. basilicum has not been examined but

would be useful for selecting hairy roots for RA

production.

Conclusion

Hairy roots derived from three different cultivars of O.

basilicum were identified as new, potential and advanta-

geous reserves of RA and antioxidants. On the basis of

origin (cultivar), morphological parameters and high RA

content HR 2, HR 4 and HR 5 were selected as elite roots

for co-culture development, elicitation and scale up studies.

The strategic approach adapted in our study showed the

importance of morphological characterization, phyto-

chemical screening and antioxidant studies for the selection

of elite hairy roots of species that produce compounds of

medicinal and commercial importance.

Acknowledgments We acknowledge Dr. Pushplata Singh for

assistance with primer design and Ms. Deep Rajni for HPLC analysis.

Infrastructure support provided by TERI, India and Deakin Univer-

sity, Australia is also duly acknowledged. Deakin University provided

a postgraduate scholarship to SS.

Funding This study was funded by Deakin University, Australia.

Author’s contribution DC and AA conceived the work and pro-

vided comments on all drafts of the manuscript. XC provided tech-

nical expertise on total antioxidant and individual antioxidant

chemiluminescence analysis. SS designed and carried out all the

experiments, analyzed the results, prepared all the figures and

tables and drafted the manuscript.

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflicts

of interest.

Ethical approval This article does not contain any studies with

human or animal subjects.

References

Amselem J, Tepfer M (1992) Molecular basis for novel root

phenotypes induced by Agrobacterium rhizogenes A4 on

cucumber. Plant Mol Biol 19(3):421–432. doi:10.1007/

BF00023390

Bais HP, Sudha G, George J, Ravishankar GA (2001) Influence of

exogenous hormones on growth and secondary metabolite

production in hairy root cultures of Cichorium intybus L. cv.

Lucknow local. In Vitro Cell Dev Plant 37(2):293–299. doi:10.

1007/s11627-001-0052-8

Bais HP, Walker TS, Schweizer HP, Vivanco JM (2002) Root specific

elicitation and antimicrobial activity of rosmarinic acid in hairy

root cultures of Ocimum basilicum. Plant Physiol Biochem

40(11):983–995. doi:10.1016/S0981-9428(02)01460-2

Bandyopadhyay M, Jha S, Tepfer D (2007) Changes in morphological

phenotypes and withanolide composition of Ri-transformed roots

of Withania somnifera. Plant Cell Rep 26(5):599–609. doi:10.

1007/s00299-006-0260-0

Bansal M, Kumar A, Sudhakara Reddy M (2014) Influence of

Agrobacterium rhizogenes strains on hairy root induction and

‘bacoside A’ production from Bacopa monnieri (L.) Wettst.

Plant Cell Tiss Organ Cult

123

Author's personal copy

Acta Physiol Plant 36(10):2793–2801. doi:10.1007/s11738-014-

1650-5

Batra J, Dutta A, Singh D, Kumar S, Sen J (2004) Growth and

terpenoid indole alkaloid production in Catharanthus roseus

hairy root clones in relation to left- and right-termini-linked Ri

T-DNA gene integration. Plant Cell Rep 23(3):148–154. doi:10.

1007/s00299-004-0815-x

Bauer N, Kiseljak D, Jelaska S (2009) The effect of yeast extract and

methyl jasmonate on rosmarinic acid accumulation in Coleus

blumei hairy roots. Biol Plant 53(4):650–656. doi:10.1007/

s10535-009-0118-8

Bellomarino SA, Conlan XA, Parker RM, Barnett NW, Adams MJ

(2009) Geographical classification of some Australian wines by

discriminant analysis using HPLC with UV and chemilumines-

cence detection. Talanta 80(2):833–838. doi:10.1016/j.talanta.

2009.08.001

Bulgakov VP (2008) Functions of rol genes in plant secondary

metabolism. Biotechnol Adv 26(4):318–324. doi:10.1016/j.

biotechadv.2008.03.001

Chaudhuri KN, Ghosh B, Tepfer D, Jha S (2005) Genetic transfor-

mation of Tylophora indica with Agrobacterium rhizogenes A4:

growth and tylophorine productivity in different transformed

root clones. Plant Cell Rep 24(1):25–35. doi:10.1007/s00299-

004-0904-x

Chriqui D, Guivarc’h A, Dewitte W, Prinsen E, van Onkelen H (1996)

Rol genes and root initiation and development. Plant Soil

187(1):47–55. doi:10.1007/BF00011656

Conlan XA, Stupka N, McDermott GP, Barnett NW, Francis PS

(2010) Correlation between acidic potassium permanganate

chemiluminescence and in vitro cell culture assay: physiologi-

cally meaningful antioxidant activity. Anal Methods 2(2):171–

173. doi:10.1039/B9AY00242A

Cseke LJ, Cseke SB, Podila GK (2007) High efficiency poplar

transformation. Plant Cell Rep 26(9):1529–1538. doi:10.1007/

s00299-007-0365-0

Doner L, Becard G (1991) Solubilization of gellan gels by chelation

of cations. Biotechnol Tech 5(1):25–28. doi:10.1007/BF001

52749

Fattahi M, Nazeri V, Torras-Claveria L, Sefidkon F, Cusido RM,

Zamani Z, Palazon J (2013) A new biotechnological source of

rosmarinic acid and surface flavonoids: Hairy root cultures of

Dracocephalum kotschyi Boiss. Ind Crop Prod 50:256–263.

doi:10.1016/j.indcrop.2013.07.029

Francis PS, Costin JW, Conlan XA, Bellomarino SA, Barnett JA,

Barnett NW (2010) A rapid antioxidant assay based on acidic

potassium permanganate chemiluminescence. Food Chem

122(3):926–929. doi:10.1016/j.foodchem.2010.02.050

Georgiev MI, Pavlov AI, Bley T (2007) Hairy root type plant in vitro

systems as sources of bioactive substances. Appl Microbiol

Biotechnol 74(6):1175–1185. doi:10.1007/s00253-007-0856-5

Georgiev MI, Agostini E, Ludwig-Muller J, Xu J (2012) Genetically

transformed roots: from plant disease to biotechnological

resource. Trends Biotechnol 30(10):528–537. doi:10.1016/j.

tibtech.2012.07.001

Grzegorczyk I, Krolicka A, Wysokinska H (2006) Establishment of

Salvia officinalis L. hairy root cultures for the production of

rosmarinic acid. Z Naturforsch C 61(5–6):351–356

Grzegorczyk I, Matkowski A, Wysokinska H (2007) Antioxidant

activity of extracts from in vitro cultures of Salvia officinalis L.

Food Chem 104(2):536–541. doi:10.1016/j.foodchem.2006.12.

003

Khojasteh A, Mirjalili MH, Hidalgo D, Corchete P, Palazon J (2014)

New trends in biotechnological production of rosmarinic acid.

Biotechnol Lett 36(12):2393–2406. doi:10.1007/s10529-014-

1640-0

Lee S, Xu H, Kim Y, Park S (2008) Rosmarinic acid production in

hairy root cultures of Agastache rugosa Kuntze. World J

Microbiol Biotechnol 24(7):969–972. doi:10.1007/s11274-007-

9560-y

Li W, Koike K, Asada Y, Yoshikawa T, Nikaido T (2005) Rosmarinic

acid production by Coleus forskohlii hairy root cultures. Plant

Cell Tiss Organ Cult 80(2):151–155. doi:10.1007/s11240-004-

9541-x

Malhotra M, Srivastava S (2006) Targeted engineering of Azospir-

illum brasilense SM with indole acetamide pathway for

indoleacetic acid over-expression. Can J Microbiol 52(11):

1078–1084. doi:10.1139/w06-071

Mallol A, Cusido RM, Palazon J, Bonfill M, Morales C, Pinol MT

(2001) Ginsenoside production in different phenotypes of Panax

ginseng transformed roots. Phytochemistry 57(3):365–371

McDermott GP, Conlan XA, Noonan LK, Costin JW, Mnatsakanyan

M, Shalliker RA, Barnett NW, Francis PS (2011) Screening for

antioxidants in complex matrices using high performance liquid

chromatography with acidic potassium permanganate chemilu-

minescence detection. Anal Chim Acta 684(1–2):134–141.

doi:10.1016/j.aca.2010.10.046

Nopo-Olazabal C, Hubstenberger J, Nopo-Olazabal L, Medina-

Bolivar F (2013) Antioxidant activity of selected stilbenoids

and their bioproduction in hairy root cultures of muscadine grape

(Vitis rotundifolia Michx.). J Agric Food Chem 61(48):11744–

11758. doi:10.1021/jf400760k

Nopo-Olazabal C, Condori J, Nopo-Olazabal L, Medina-Bolivar F

(2014) Differential induction of antioxidant stilbenoids in hairy

roots of Vitis rotundifolia treated with methyl jasmonate and

hydrogen peroxide. Plant Physiol Biochem 74:50–69. doi:10.

1016/j.plaphy.2013.10.035

Nourozi E, Hosseini B, Hassani A (2014) A reliable and efficient

protocol for induction of hairy roots in Agastache foeniculum.

Biologia 69(7):870–879. doi:10.2478/s11756-014-0382-8

Puri A, Adholeya A (2013) A new system using Solanum tuberosum

for the co-cultivation of Glomus intraradices and its potential for

mass producing spores of arbuscular mycorrhizal fungi. Sym-

biosis 59(2):87–97. doi:10.1007/s13199-012-0213-z

Sharma P, Padh H, Shrivastava N (2013) Hairy root cultures: a

suitable biological system for studying secondary metabolic

pathways in plants. Eng Life Sci 13(1):62–75. doi:10.1002/elsc.

201200030

Singleton VL, Rossi JA (1965) Colorimetry of total phenolics with

phosphomolybdic–phosphotungstic acid reagents. Am J Enol

Vitic 16(3):144–158

Srivastava S, Cahill DM, Conlan XA, Adholeya A (2014) A novel

in vitro whole plant system for analysis of polyphenolics and their

antioxidant potential in cultivars of Ocimum basilicum. J Agric

Food Chem 62(41):10064–10075. doi:10.1021/jf502709e

Srivastava S, Adholeya A, Conlan XA, Cahill DM (2016) Acidic

potassium permanganate chemiluminescence for the determina-

tion of antioxidant potential in three cultivars of Ocimum

basilicum. Plant Food Hum Nutr. doi:10.1007/s11130-016-

0527-8

Tada H, Murakami Y, Omoto T, Shimomura K, Ishimaru K (1996)

Rosmarinic acid and related phenolics in hairy root cultures of

Ocimum basilicum. Phytochemistry 42(2):431–434. doi:10.1016/

0031-9422(96)00005-2

Tansupo P, Suwannasom P, Luthria DL, Chanthai S, Ruangviriyachai

C (2010) Optimised separation procedures for the simultaneous

assay of three plant hormones in liquid biofertilisers. Phytochem

Anal 21(2):157–162. doi:10.1002/pca.1172

Thimmaraju R, Venkatachalam L, Bhagyalakshmi N (2008) Mor-

phometric and biochemical characterization of red beet (Beta

vulgaris L.) hairy roots obtained after single and double

Plant Cell Tiss Organ Cult

123

Author's personal copy

transformations. Plant Cell Rep 27(6):1039–1052. doi:10.1007/

s00299-008-0527-8

Thiruvengadam M, Praveen N, Maria John KM, Yang Y-S, Kim S-H,

Chung I-M (2014) Establishment of Momordica charantia hairy

root cultures for the production of phenolic compounds and

determination of their biological activities. Plant Cell Tissue Org

118(3):545–557. doi:10.1007/s11240-014-0506-4

Tiwari RK, Trivedi M, Guang ZC, Guo GQ, Zheng GC (2008)

Agrobacterium rhizogenes mediated transformation of Scutel-

laria baicalensis and production of flavonoids in hairy roots.

Biol Plant 52(1):26–35. doi:10.1007/s10535-008-0004-9

Triplett B, Moss S, Bland J, Dowd M (2008) Induction of hairy root

cultures from Gossypium hirsutum and Gossypium barbadense to

produce gossypol and related compounds. In Vitro Cell Dev

Plant 44(6):508–517. doi:10.1007/s11627-008-9141-2

Weremczuk-Je _zyna I, Grzegorczyk-Karolak I, Frydrych B, Krolicka

A, Wysokinska H (2013) Hairy roots of Dracocephalum

moldavica: rosmarinic acid content and antioxidant potential.

Acta Physiol Plant 35(7):2095–2103. doi:10.1007/s11738-013-

1244-7

Xiao Y, Zhang L, Gao S, Saechao S, Di P, Chen J, Chen W (2011)

The c4h, tat, hppr and hppd genes prompted engineering of

rosmarinic acid biosynthetic pathway in Salvia miltiorrhiza hairy

root cultures. PLoS One 6(12):e29713. doi:10.1371/journal.

pone.0029713

Yan Q, Shi M, Ng J, Wu JY (2006) Elicitor-induced rosmarinic acid

accumulation and secondary metabolism enzyme activities in

Salvia miltiorrhiza hairy roots. Plant Sci 170(4):853–858. doi:10.

1016/j.plantsci.2005.12.004

Plant Cell Tiss Organ Cult

123

Author's personal copy