1 3

Acta Physiologiae Plantarum

ISSN 0137-5881

Acta Physiol Plant
DOI 10.1007/s11738-014-1572-2
Agrobacterium-mediated genetic
transformation of Withania somnifera
using nodal explants
Rajangam Udayakumar, Sampath
Kasthurirengan, Thankaraj Salammal
Mariashibu, Jesudass Joseph Sahaya
Rayan, Andy Ganapathi, et al.
1 3
Your article is protected by copyright and
all rights are held exclusively by Franciszek
Grski Institute of Plant Physiology, Polish
Academy of Sciences, Krakw. This e-offprint
is for personal use only and shall not be self-
archived in electronic repositories. If you wish
to self-archive your article, please use the
accepted manuscript version for posting on
your own website. You may further deposit
the accepted manuscript version in any
repository, provided it is only made publicly
available 12 months after official publication
or later and provided acknowledgement is
given to the original source of publication
and a link is inserted to the published article
on Springer's website. The link must be
accompanied by the following text: "The final
publication is available at link.springer.com.
ORI GI NAL PAPER
Agrobacterium-mediated genetic transformation of Withania
somnifera using nodal explants
Rajangam Udayakumar

Sampath Kasthurirengan

Thankaraj Salammal Mariashibu

Jesudass Joseph Sahaya Rayan

Andy Ganapathi

Sei Chang Kim

Jae Jin Kim

Chang Won Choi
Received: 22 November 2013 / Revised: 8 March 2014 / Accepted: 30 April 2014
Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2014
Abstract Withania somnifera is an important medicinal
plant and used to cure many diseases. Direct regeneration
method was standardized for the nodal explants of W.
somnifera. In this method, the maximum of 42.4 2.68
shoots produced per explant was achieved at 1.5 mg l
-1
BAP with 0.3 mg l
-1
IAA in the second subculture.
Transformation was performed in the nodal explants of W.
somnifera via direct regeneration using Agrobacterium
tumefaciens strain EHA105 that harbored a binary vector
pGA492, which carrying kanamycin resistant (nptII),
phosphinothricin resistant (bar) and an intron containing b-
glucuronidase (gus-intron) genes. The sensitivity of nodal
explants to kanamycin (300 mg l
-1
) was determined for
the selection of transformed plants. Transformation was
conrmed by histochemical b-glucuronidase (GUS) assay
and amplication of the nptII gene by polymerase chain
reaction (PCR). PCR and southern blot analyses conrmed
the integration of nptII gene in the genome of W. somnifera
and the transformation frequency of 3.16 % has been
achieved. This is the rst report on the genetic
transformation of W. somnifera using nodal explants,
which may aid in the transformation of any other character
gene for improving therapeutic value.
Keywords Withania somnifera Agrobacterium
tumefaciens Genetic transformation nptII gene
Gus assay
Abbreviations
AMGT Agrobacterium-mediated genetic transformation
BAP 6-Benzyl amino purine
IAA Indole-3-acetic acid
NAA Naphthalene acetic acid
IBA Indole-3-butyric acid
GA
3
Gibberellic acid
CaMV Cauliower mosaic virus
GUS b-Glucuronidase
nptII Neomycin phosphotransferase II
PCR Polymerase chain reaction
Introduction
Withania somnifera (L.) Dunal is a perennial plant, belongs
to the natural order of Solanaceae. It is a potential
medicinal plant which has been used in the Indian tradi-
tional systems of medicine like the agent of anti-bacterial,
anti-fungal, antitumor, anti-aging, anti-inammatory, anti-
stress, anticonvulsant and tonic and central nervous system
depressant, and in the treatment of tuberculosis, arthritis,
rheumatism and cardiac diseases (Uma Devi et al. 1993;
Tripathi et al. 1996; Mishra et al. 2000). The pharmaco-
logical effect of W. somnifera may be attributed to
Communicated by J.-H. Liu.
R. Udayakumar
Department of Biochemistry, Government Arts College
(Autonomous), Kumbakonam 612 001, Tamil Nadu, India
R. Udayakumar S. Kasthurirengan T. S. Mariashibu
J. J. Sahaya Rayan A. Ganapathi (&)
Department of Biotechnology, Bharathidasan University,
Tiruchirappalli 620 024, Tamilnadu, India
e-mail: aganapathi2003@rediffmail.com
R. Udayakumar S. C. Kim J. J. Kim C. W. Choi (&)
Department of Biology and Medicinal Science,
Pai Chai University, Daejeon 302-735, Korea
e-mail: choicw@pcu.ac.kr
1 3
Acta Physiol Plant
DOI 10.1007/s11738-014-1572-2
Author's personal copy
withanolides, a group of steroidal lactones (Budhiraja and
Sudhir 1987). Recently, hypoglycemic and hypolipidemic
effects of W. somnifera root and leaf extracts have been
reported on alloxan-induced diabetic rats (Udayakumar
et al. 2009). It suggested that phenolic compounds
including avonoids in W. somnifera root and leaf extracts
(Udayakumar et al. 2010a) and their antioxidant activity
may be responsible for the reduction of blood glucose level
in alloxan-induced diabetic rats (Udayakumar et al. 2010b).
The production of W. somnifera roots through conven-
tional methods of cultivation (seed) is limited due to sev-
eral reasons such as poor yield, poor viability of seeds,
susceptibility of the seeds and seedlings to fungal infec-
tions like seedling mortality and blight, leaf blight, seed
rotting etc. Besides, over exploitation of this plant species
leads to the extinction in natural habitat. To meet this and
increasing its demand, tissue culture technology is a viable
alternative for the mass production of this medicinal plant.
Many reports are available on in vitro regeneration of W.
somnifera species using callus, leaf, stem, epicotyl and
hypocotyl explants (Baburaj and Gunasekaran 1994;
Abhyankar and Chinchanikar 1996; Kulkarni et al. 1996,
2000; Rani and Grover 1999; Pawar et al. 2001; Uda-
yakumar et al. 2013). Recently, micropropagation of W.
somnifera species using various explants was also reported
(Kanungo and Sahoo 2011; Nayak et al. 2013). But there
are only a very few reports (Sangwan et al. 2007; Kumar
et al. 2011) available on regeneration and shoot multipli-
cation using nodal explants of W. somnifera.
Agrobacterium-mediated genetic transformation would
be a powerful tool for enhancing the productivity of novel
secondary metabolites (Cucu et al. 2002). Initially, W. som-
nifera transformation has been reported through A. rhizoge-
nes, resulted in the formation of hairy roots by in vitro
culture. The transformed root cultures of W. somnifera
increased biomass production with increase in production of
secondary metabolites such as withanolide D (Ray et al.
1996), withaferin A (Bandyopadhyay et al. 2007) and wit-
hanolide A (Murthy et al. 2008). In these cases, no report is
available on the successful regeneration of plants from the
transgenic tissue. A. tumefaciens-mediated transformation
(AMGT) of W. somnifera was initiated using wild-type
strain, resulted in only shooty teratomas and induced the
synthesis of withanolides in the transformed tissues (Ray
and Jha 1999). To date, only a few reports are available on
gene transfer studies of W. somnifera using A. tumefaciens.
Recently, an efcient AMGT protocol of W. somnifera was
developed using leaf segment, which showed that transfor-
mation efciency was 1.67 % and established transgenic
lines (Pandey et al. 2010). Successful AMGT of plant spe-
cies of interest has been mainly achieved by matching the
inoculated plant tissue to the suitable Agrobacterium strains,
and by tissue culture techniques with proper concentration
of phytohormones and transgene selection (Herrera-Estrella
et al. 2005). Therefore, the production of transformed W.
somnifera with various transgenes and different tissues is
absolutely required.
In the present study, we aimed to develop an efcient
and rapid direct regeneration protocol from nodal explants
of W. somnifera due to its genetic transformation. This
study was also aimed to establish an efcient method for
transformation using nodal explants of W. somnifera with
direct regeneration by A. tumefaciens strain EHA105
transformed with pGA492 containing neomycin phospho-
transferase (nptII) and gus (uid A) genes. Optimization of
the transgenic production of this species would be valuable
to produce stably transformed shoots and roots.
Materials and methods
Plant material
The nodal explants of 50- to 60-day-old seedlings were
used for transformation studies with direct regeneration
protocol. Seeds of W. somnifera were procured from the
Central Institute of Medicinal and Aromatic Plants (CI-
MAP), Lucknow, India. The seeds were washed with dis-
tilled water and soaked in 2.5 % (v/v) commercial bleach
Teepol (5.25 % Sodium hypochlorite; Reckitt and
Benckiser, Ltd., Kolkata, India) for 5 min. The seeds were
washed 34 times in distilled water and transferred to an
inoculation chamber and surface sterilized with 0.1 %
mercuric chloride (w/v) solution for 15 min. Then the
solution was decanted and the seeds were washed thor-
oughly in sterilized distilled water to ensure that the last
traces of mercuric chloride were removed. After steriliza-
tion, the seeds were inoculated in culture tubes
(2.5 9 15 cm, Borosil, India) containing sterile cotton
moistened with sterile water. After inoculation, the tubes
were placed in darkness for 8-10 days to render uniform
germination and then they were placed in light. The
intensity of light was 30 lmol m
-2
s
-1
, 16 h/day. The
temperature was maintained for germination at 25 2 C,
and 60 % humidity for in vitro growth and development.
After germination, the seedlings (25 to 30 day old) were
transferred into 150-ml conical ask containing half
strength MS medium and maintained for another 4 weeks.
The pH of the half strength MS medium was adjusted to 5.7
before autoclaving and then autoclaved at 1.06 kg cm
-2
and at 121 C for 20 min.
Tissue culture system-direct regeneration
Nodal explants (0.5 cm) were excised from 50- to 60-day-
old in vitro raised seedlings with sterile scalpel and used as
Acta Physiol Plant
1 3
Author's personal copy
explants. The nodal explants from in vitro seedlings were
individually inoculated in MS medium (Murashige and
Skoog 1962) supplemented with various concentrations of
cytokinin (BAP) alone and in combination with auxin
(IAA), 3 % sucrose (w/v) and 0.8 % agar (w/v) (shoot
induction medium). The effect of different concentrations
of BAP alone and in combination with IAA was studied to
nd the optimum concentration for multiple shoot induc-
tion. The nodal explants with emerging shoots were sub-
cultured in MS medium supplemented with BAP
(0.52.0 mg l
-1
) alone and BAP (1.5 mg l
-1
) in combi-
nation with IAA (0.10.5 mg l
-1
). The explants were
transferred to fresh medium with the same composition at
4-week intervals during the subculture.
Sensitivity of nodal explants to kanamycin
The sensitivity tests of election agent were carried out to nd
the inhibitory concentration which arrests nodal explant
growth. The sensitivity of nodal explants to kanamycin was
determined by culturing the explants in shoot induction
medium [MS ? BAP (1.5 mg l
-1
) ? IAA (0.3 mg l
-1
)]
along with kanamycin (Sigma, USA) at different concentra-
tions ranging from 50 mg l
-1
to 400 mg l
-1
. The minimum
inhibitory concentration (MIC) of the selection marker was
usedthroughout the selectionprocedure of transformedshoots
fromexplants. A positive control without selection agent was
also maintained.
Agrobacterium strain, plasmid vector, growth
and maintenance of microbial cultures
Transformation was performed using A. tumefaciens strain
EHA105, harboring the binary vector pGA492 (kindly
provided by Rafael Perl Treves, Bar Ilan University,
Israel). The binary vector contains nptII gene under the
contol of nos promoter and the bar as well as gus genes
under the control of CaMV 35S promoter (Fig. 1). Short-
term storage of pure bacterial culture was achieved by
maintaining it in the form of single colonies on plates of
respective solid medium at 4 C. When it needed for
experimentation, the strain was grown routinely from sin-
gle colonies on respective liquid medium in dark condition
and maintained it in an incubator shaker with 200 rpm at
28 C. The culture in the log phase of growth (usually
grown for 2448 h) was used for all the experiments
(OD = 1.0 at wavelength of 600 nm).
Agrobacterium infection and co-cultivation
A single colony of A. tumefaciens strain EHA105 suspended
in 50 ml of Luria-Bertani (LB) mediumcontaining50 mg l
-1
kanamycin (Sigma, USA) and 20 mg l
-1
tetracycline (Sigma,
USA) was incubated at 28 Con a shaker at 200 rpmfor 24 h.
The suspension of the Agrobacterium strain was diluted with
half strength MS liquid mediumto obtain 5 9 10
8
cells ml
-1
.
The nodal explants were gently pricked for about ten times to
make wounds using a sterile needle, and then immersed in the
bacterial culture for 10 min. After that the explants were
removed, blotted dry using sterile Whatman no.1 lter paper
and inoculated (one explant/culture tube) on MS medium
containing BAP (1.5 mg l
-1
) and IAA (0.3 mg l
-1
). The co-
cultivation was performed for 0, 1, 2, 3, 4 and 5 days under a
16-hphotoperiodwitha light intensityof 30lmol m
-2
s
-1
and
kept at 25 2 C. After co-cultivation, the infected explants
were washedthree times with sterile distilled water containing
cefotaxime (300 mg l
-1
) followed by hormone-free MS
liquid medium containing cefotaxime for three times with
vigorous stirring using sterile forceps and then blotted dry on
sterile lter paper. The co-cultivated explants were subjected
to selection. Then the explants were transferred to MS med-
ium containing BAP, IAA, cefotaxime and kanamycin
(300 mg l
-1
) for shoot bud induction.
Selection of transformants
After 23 weeks, the explants with emerging shoots were
transferred to fresh MS medium containing BAP, IAA,
Fig. 1 Linear map of T-DNA in plasmid vector pGA492 present
within the Agrobacterium tumefaciens strain EHA105. BR border
right. BL border left, nptII neomycin phosphotransferase II gene, 35S
cauliower mosaic virus (CaMV) 35S promoter, pAnos NOS
terminator, GUS b-glucuronidase. bar phosphinothricin acetyl trans-
ferase driven by 35S
Acta Physiol Plant
1 3
Author's personal copy
kanamycin and cefotaxime for shoot proliferation. Two
subcultures were done on the same medium at 4-week
interval. The regenerated shoots were excised from the
explants and transferred to MS medium supplemented with
GA
3
(1.0 mg l
-1
), kanamycin (300 mg l
-1
) and cefotax-
ime (300 mg l
-1
) for shoot elongation. The elongated
shoots were then transferred to MS medium fortied with
IBA (0.8 mg l
-1
), kanamycin and cefotaxime for rooting.
The rooted plants were transferred to pots containing
sterilized sand, soil and vermiculite (1:2:1, v/v/v) mixture
and were acclimatized in the green house for 30 days.
GUS assay
Transformedshoots, leaves andnodal segments were subjected
to assay for the expression of gus A int gene following the
histochemical procedure of Jefferson et al. (1987). Trans-
formed plant parts like shoot, leaf and nodal segments were
washed three times using distilled water and incubated for
10 min in phosphate buffer (0.5 mM NaH
2
PO
4
and 0.5 mM
Na
2
H PO
4
) at pH 7.0 containing 0.5 mM potassium ferri- and
ferrocyanide and 10 mMNa
2
EDTA. The buffer was removed
and fresh phosphate buffer containing 1 % (v/v) Triton X-100
was added to the plant tissues and incubated for 1 h at 37 C.
After draining the solution, fresh phosphate buffer containing
1.0 mM X-gluc (5-bromo-4-chloro-3-indolyl b-D glucuro-
nide) and 20 % of 95 % methanol was added. The reaction
mixture was placed under a mild vacuum for 5 min and incu-
bated over night at 37 C and then they were examined visu-
ally. Following the incubation the chlorophyll was removed
and xed in 95 % (v/v) ethanol: 1 % (v/v) glacial acetic acid.
Statistical analysis
Each treatment consisted of at least 10 explants and each
treatment was repeated six times. A complete randomized
design was used in all experiments and a one-way analysis
of variance (ANOVA) and comparisons between the mean
values of treatments were carried out using Duncans Mul-
tiple Range Test (DMRT) with signicance determined at
5 % level. The percentage of GUS expression was calcu-
lated by the number of explants showing GUS positive
expression divided by the number of explants subjected to
co-cultivation and multiplied by 100 (Cao et al. 1998). The
percentage of GUS expression was expressed as mean SE
of 10 experiments and each consisted of 60 explants.
Molecular conrmation of transformants
Isolation of genomic DNA
Leaf samples collected from the transformed plants were
used for the isolation of DNA (Bernatsky and Tanksley
1986). About 2 g of leaf tissue was frozen with liquid
nitrogen and ground into a ne powder and then added
10 ml of preheated (65 C) extraction buffer containing
1.5 % (w/v) hexadecyl or cetyl trimethyl ammonium bro-
mide (CTAB), 10 mM Tris HCl (pH 8.0), 1.4 M sodium
chloride, 20 mM EDTA and 0.1 % (v/v) 2-mercap-
toethanol. The mixture was incubated in a water bath for
30 min at 65 C. Equal volume of chloroform:isoamyl
alcohol mixture (24:1, v/v) was added, then gently mixed
for 15 min and centrifuged at 10,000 rpm for 20 min at
room temperature. The aqueous phase was collected and an
equal volume of ice-cold isopropanol was added and mixed
gently until the DNA was precipitated out (1020 s). The
precipitated DNA was dissolved in 200-500 ll of TE
buffer (10 mM TrispH 8.0, 1 mM EDTApH 8.0). The
contaminant RNA was eliminated from DNA by treating
with RNase to a nal concentration of 20 lg ml
-1
. To the
DNA sample, an equal volume of phenol:chloroform:iso-
amyl alcohol mixture was added and mixed well, then
centrifuged at 10,000 rpm for 10 min at room temperature.
The aqueous phase was collected and an equal volume of
chloroform was added and mixed then it was centrifuged at
10,000 rpm for 2 min. To the collected aqueous phase
1/10th volume of 3 M sodium acetate (pH 8.0) and two
volumes of absolute ethanol were added and thoroughly
mixed and then kept at -20 C for half an hour. DNA was
pelleted by centrifugation at 10,000 rpm for 5 min. The
resulting pellet was dissolved in nuclease-free water after
that the quality and quantity of the DNA were checked
using spectrophotometer and agarose gel (1.5 %) electro-
phoresis. The absorbance ratio of DNA sample between
260 and 280 nm was recorded and the quality of the
genomic DNA was conrmed. Finally, the DNA sample
was stored at 4 C for further analysis.
PCR conrmation
For PCR analysis, DNA samples from putative transfor-
mants were amplied by specic primers. The nptII gene
fragment (0.69 kb) was amplied using primers 5
0
-GCCG
CTTGGGTGGAGAGGCTAT-3
0
and 5
0
-GAGGAAGCGG
TCAGCCCATTCG-3
0
. All PCR reactions were performed
using a Peltier effect thermal cycler (MJ Research Co.,
USA). Samples containing 50 ng genomic DNA were rst
denatured at 94 C for 5 min followed by 30 cycles at
94 C for 30 s, 55 C and 72 C for 30 s followed by
7-min nal extension at 72 C. Fifty ng of plasmid DNA
was used as positive control. The PCR reactions contained
10 qM of each primer, 10 mM dNTPs mix, 15 mM MgCl
2
,
50 mM KCl, 10 mM Tris HCl (pH 9.0), 0.1 % (v/v) Triton
X-100, 2 U of Taq DNA polymerase and 50 ng of template
DNA in 2X reaction buffer. The amplied DNA was
analyzed using 1.5 % agarose gel electrophoresis at 100 v
Acta Physiol Plant
1 3
Author's personal copy
for 90 min followed by staining with sterile distilled water
containing 1 lg l
-1
ethidium bromide (Sigma, USA) for
15 min.
Southern hybridization
The blotting procedures suggested by Southern (1975)
were followed. Total DNA (approximately 10 lg) from
plant samples were digested with the restriction enzyme
HindIII for 10 h, electrophoresed through a 0.8 % agarose
gel, and blotted on nylon membrane (Amersham Biosci-
ences, UK). The blot was baked at 80 C for 30 min under
vacuum. The plasmid (pGA492) used for Agrobacterium-
mediated transformation was isolated and digested with the
restriction enzyme HindIII for 4 h. The digested plasmids
were electrophoresed through agarose gel. The nptII frag-
ments (1.9 kb) were isolated and placed in the dialysis
membrane bag (Himedia Laboratories Ltd., India) and
electro-eluted. The eluted fragments were used as probes.
The probes were labeled by non-radioactive labeling kit
(ECL random labeling and detection system) (Amersham
Biosciences, UK). The blot was prehybridized at 60 C for
1 h in hybridization buffer [5X SSC, blocking agent (0.5 %
(w/v), SDS (0.1 %) and dextran sulfate (5 %)]. The dena-
tured labeled probe was added to the hybridization oven
(Amersham Biosciences, UK). Post-hybridization was
performed under high stringency conditions. The blot was
washed in an excess of 1X SSC, 0.1 % for 15 min, then in
SSC (0.5 %) and SSC (0.1 %) with SDS (0.1 %). After
hybridization, the blot was rinsed briey in antibody wash
buffer (TrisHCl 100 mM, NaCl 150 mM and pH 7.0). The
blot was blocked with blocking agent (0.5 %) (supplied by
the manufacturer of ECL kit) at room temperature for 1 h
with continuous agitation. After a brief rinse in antibody
wash buffer, the blot was incubated with antibody diluted
1,000 fold in BSA (0.5 %) (fraction V) (w/v) in antibody
wash buffer and incubated for 1 h at room temperature
with continuous agitation. The membrane was washed with
antibody wash buffer with Tween 20 (0.1 %, v/v) for
210 min followed by washing in 5 min, two times at room
temperature with continuous agitation. After removal of
excess antibody, the blot was wetted with the detection
solutions 1 and 2 (supplied by ECL manufactures). Excess
of detection solution was drained from the membrane and
the blot was covered by saran wrap. The covered blot was
immediately placed in cassettes with X-ray lm. After 1 h,
the X-ray lm was developed and the signal was detected.
Results and discussion
Successful DNA transfer through AMGT system is
dependent on the complex interaction of number of factors
like Agrobacterium strain, infection, co-cultivation
method, selection condition, type of tissue used for infec-
tion, and genotype of the plant (Hiei et al. 1997). In
addition, the age of the explant is a critical factor, which
inuences transformation efciency (De Bondt et al. 1996).
In the present study, 50- to 60-day-old in vitro seedlings
were used as the source of explants, which showed higher
GUS expression. In vitro regeneration of plants from young
seedlings has received considerable attention over the
years, because of their easy accessibility, quick responses
and high ability for shoot organogenesis (Tabei et al.
1991). Genetic transformation of different explants like
leaf, root and stem of W. somnifera (L.) Dunal using A.
tumefaciens (MTCC-431) was carried out for the analysis
of variation in the production of secondary metabolites of
the transformed plants (Ishnava et al. 2012). An efcient
and reproducible AMGT of W. coagulans was achieved
using leaf explants of in vitro multiple shoot culture
(Mishra et al. 2013).
Plant regeneration and multiple shoot formation
On MS medium supplemented with BAP (0.52.0 mg l
-1
),
multiple shoots were produced from nodal explants. BAP
increased the number of shoots formation with increase in
its concentration up to 1.5 mg l
-1
. But the number and
mean heights of shoots per explant were markedly sup-
pressed, when the concentration increased above
1.5 mg l
-1
. Maximum number of direct adventitious shoot
buds (10.4 0.77) was induced in the second subculture
from nodal explant at 1.5 mg l
-1
BAP. In earlier studies,
BAP alone improved in multiple shoot induction of similar
explants of other plants like Vigna species (Saini and Jai-
wai 2005; Chaudhury et al. 2007). In another set of
experiments, MS medium supplemented with BAP
(1.5 mg l
-1
) in combinations with different concentrations
of IAA (0.10.5 mg l
-1
) produced a higher number of
shoots than BAP alone. The maximum of 42.4 2.68
shoots per nodal explant was achieved at 1.5 mg l
-1
BAP
with 0.3 mg l
-1
IAA in the second subculture (Table 1;
Fig. 2). Thus, the presence of BAP in the medium was
essential for enhanced multiple shoot induction, and more
effect was found when it was treated with IAA.
The shoots were separated from the nodal explant and
then transferred to MS medium with different concentra-
tions of GA
3
(0.52.0 mg l
-1
) for elongation. The MS
medium containing GA
3
at 1.0 mg l
-1
showed maximum
shoot elongation response (86.7 %) with a mean shoot
length of 5.5 0.46 cm (Table 2; Fig. 2). The elongated
shoots were transferred to root induction medium con-
taining different concentrations of NAA (0.21.0 mg l
-1
),
IAA (0.21.0 mg l
-1
) and IBA (0.21.0 mg l
-1
). Addition
of IBA to rooting medium improved rooting efciency of
Acta Physiol Plant
1 3
Author's personal copy
shoots, which was superior to NAA and IAA. Under
treatment of IBA at 0.8 mg l
-1
in elongated shoots, 91.7 %
of shoots produced roots (7.2 0.72 roots/shoot) with a
mean root length of 4.6 0.36 cm (Table 3; Fig. 2). The
protocol developed in this study offers a simple and
improved in vitro method to regenerate W. somnifera.
Therefore, these conditions may be useful in the genetic
transformation of W. somnifera.
Sensitivity of nodal explants to kanamycin
The sensitivity of the nodal explants to kanamycin was rst
established before initiation of the transformation experi-
ment to determine the maximum concentration of kana-
mycin that permits the regeneration of transformed plants,
while preventing the growth of non-transformed shoots. In
control, shoots developed normally in the antibiotic-free
medium. The maximum number of shoot buds was
obtained at 50 mg l
-1
kanamycin. At 200 mg l
-1
kana-
mycin, 50 % of explants showed chlorosis. Further
increase in the level of kanamycin led to a corresponding
decrease in the shoot bud production. Kanamycin at
300 mg l
-1
caused almost total inhibition of bud induction
and regeneration from nodal explants (Table 4). Therefore,
in the present study, kanamycin at 300 mg l
-1
was used as
a selection agent for the transformation of W. somnifera.
Inoculation and co-cultivation
Agrobacterium density and co-cultivation period also
played an important role in improving the transformation
frequency. Evaluation of inoculum density revealed that
1.0 OD was optimal for co-cultivation of nodal explants
(data not shown). To determine the best duration of co-
cultivation period, the co-cultivation period of 0, 1, 2, 3, 4
and 5 days was assessed. The co-cultivation period of
2 days for nodal explants in MS medium containing BAP
(1.5 mg l
-1
) and IAA (0.3 mg l
-1
) produced higher rate of
transformants as compared to other co-cultivation experi-
ments (data not shown). Optical density greater than 1.0
OD and co-cultivation period beyond 2 days led to over
growth of bacteria, and this result is in agreement with the
observations of the earlier studies in other plant species
(Soniya and Das 2002; Vasudevan et al. 2007; Lee et al.
2006). Thus, 1.0 OD culture and 2 days of co-cultivation
period for W. somnifera were selected and found optimal
for the effective infection and regeneration of transgenic
shoots. In case of W. coagulans, the optimal conditions for
the AMGT of leaf explants were found as the agrobacterial
inoculum at 0.4 OD culture and 3 days of co-cultivation on
medium supplemented with BAP and NAA (Mishra et al.
2013).
Selection of transformants
In the present study, the vector used for transformation
contains two selection markers (nptII and bar) and a GUS
reporter gene system. Similarly, the Agrobacterium strain
LBA4404 harboring binary vector pIG121Hm containing
GUS reporter gene (gusA) under the control of CaMV35S
promoter was used in the development of transformation
protocol (Mishra et al. 2013). Inclusion of selection agent
Table 1 Effect of different concentrations of BAP alone and in combination with IAA on multiple shoot formation from 50- to 60-day-old nodal
explants of W.somnifera on MS medium
Plant
growth
regulators
(mg/l)
Number of
cultures
developing
shoots*
Explants
developing
shoots (%)
Initial culture First subculture Second subculture
Number of
shoots/
explant
Height of
shoots (cm)
Number of
shoots/
explant
Height of
shoots (cm)
Number of
shoots/
explant
Height of
shoots (cm)
BAP IAA
0 8 13.3 1.0 0.00 a 1.0 0.09 a 1.2 0.23 a 1.2 0.09 a 1.2 0.23 a 1.4 0.09 a
0.5 13 21.7 2.1 0.12 b 1.2 0.14 b 3.8 0.27 c 1.3 0.14 ab 5.2 0.40 b 1.5 0.14 a
1.0 17 28.3 3.0 0.27 c 1.3 0.18 bc 4.6 0.35 d 1.5 0.14 bc 7.6 0.73 c 1.8 0.14 b
1.5 25 41.7 4.5 0.55 d 1.6 0.14 e 6.8 0.40 e 1.8 0.18 e 10.4 0.77 d 2.2 0.18 e
2.0 14 23.3 2.3 0.23 b 1.4 0.14 cd 2.9 0.36 b 1.6 0.14 de 4.1 0.53 b 2.0 0.18 cd
1.5 0.1 33 55.0 5.6 0.54 e 1.5 0.14 de 9.5 0.77 f 1.6 0.14 cd 18.4 1.35 f 1.8 0.09 bc
0.2 46 76.7 8.3 0.60 f 1.6 0.09 e 14.6 1.00 g 1.8 0.09 e 26.2 1.80 g 1.9 0.14 b
0.3 54 90.0 13.0 0.89 g 2.0 0.15 g 20.8 1.43 h 2.1 0.18 f 42.4 2.68 h 2.4 0.23 f
0.4 26 43.3 4.4 0.36 d 1.8 0.09 f 7.4 0.46 e 1.8 0.14 e 12.7 0.89 e 2.1 0.18 de
0.5 21 35.0 3.0 0.25 c 1.7 0.14 ef 5.4 0.42 d 1.8 0.09 e 10.5 0.90 d 2.0 0.18 cd
Data shown are mean SD of six experiments each experiment consisted of 10 replicates
Values with the same letter within columns are not signicantly different using Duncans multiple range test (DMRT) at 5 % level (p B 0.05)
* Out of 60 explants inoculated
Acta Physiol Plant
1 3
Author's personal copy
in the regeneration and rooting media selectively permits
the development of putative transformed shoots, which
stably expressed transgenes. Shoot bud induction as well as
differentiation occurred on MS medium supplemented with
BAP, IAA, and kanamycin after selection (Mishra et al.
2013). The transformation frequency was evaluated by
histochemical GUS activity (Fig. 3) and PCR analysis.
Sixty explants were infected with Agrobacterium for each
experiment, and a total of 600 explants from 10 separate
experiments were co-cultivated for 2 days and
Fig. 2 Direct regeneration of
shoots from 50- to 60-day-old
nodal explants of Withania
somnifera on MS medium.
a Shoot bud initiation BAP
(1.5 mg l
-1
) ? IAA
(0.3 mg l
-1
). bd Multiple
shoots production BAP
(1.5 mg l
-1
) ? IAA
(0.3 mg l
-1
) in 4, 8 and
12 weeks of culture. e Shoot
elongation GA
3
(1.0 mg l
-1
).
f Rooting of shoot IBA
(0.8 mg l
-1
). g Acclimatized
plant
Acta Physiol Plant
1 3
Author's personal copy
3.16 0.384 % of regenerated shoots exhibited GUS
positive and kanamycin resistance. In the present study, the
co-cultivation period of 2 days with A. tumefaciens strain
EHA105 resulted in increased transient GUS expression on
MS medium fortied with kanamycin (300 mg l
-1
), BAP
(1.5 mg l
-1
), IAA (0.3 mg l
-1
) and cefotaxime
(300 mg l
-1
) as a selection medium (Fig. 3). Similar
results have been reported in many plants with different
selection media (Kondo et al. 2000; Tsukazaki et al. 2002;
Lee et al. 2006), while signicant differences in the
efciency of GUS expression were reported in other plants
(De Bondt et al. 1996; Cao et al. 1998). Histochemical
GUS expression was also reported in the putative trans-
genic W. coagulans (Mishra et al. 2013).
Among the different concentrations of kanamycin tes-
ted, 300 mg l
-1
was found effective in selection of trans-
formants for W. somnifera. The maximum number of
transgenic shoot development was achieved in the medium
containing kanamycin at 300 mg l
-1
with a transformation
frequency of 3.16 0.384 %. This frequency is much
higher than that of leaf explants from in vitro-grown shoots
for transformation (Pandey et al. 2010). The inoculum
density at 1.0 OD showed decrease in regeneration fre-
quency, but increase in the number of GUS positive shoot
Table 2 Effect of GA
3
on shoot elongation from regenerated shoots
of W. somnifera
Plant
growth
regulator
(mg/l)
Number of
cultures
responding
elongation*
Shoot
elongation
response
(%)
Mean shoot
length (cm)
Mean
number of
nodes
GA
3
0 13 21.7 1.6 0.09 a 2.4 0.18 a
0.5 35 58.3 3.6 0.27 c 5.8 0.54 c
1.0 52 86.7 5.5 0.46 e 7.6 0.72 d
1.5 43 71.7 4.6 0.36 d 7.1 0.63 d
2.0 27 45.0 2.9 0.27 b 4.4 0.45 b
Data shown are mean SD of six experiments each experiment
consisted of 10 replicates
Values with the same letter within columns are not signicantly
different using Duncans multiple range test (DMRT) at 5 % level
(p B 0.05)
* Out of 60 explants inoculated
Table 3 Effect of NAA, IAA
and IBA on root induction from
regenerated shoots of
W.somnifera
Data shown are mean SD of
six experiments each
experiment consisted of 10
replicates
Values with the same letter
within columns are not
signicantly different using
Duncans multiple range test
(DMRT) at 5 % level
(p B 0.05)
* Out of 60 explants inoculated
Plant growth
regulators (mg/l)
Number of cultures
producing roots*
Cultures
producing roots
(%)
Mean number of
roots/shoot
Mean root
length (cm)
NAA
0.2 14 23.3 2.3 0.23 a 1.0 0.10 a
0.4 21 35.0 2.8 0.27 b 1.4 0.14 b
0.6 28 46.7 3.4 0.36 cd 2.0 0.18 cd
0.8 36 60.0 4.8 0.45 f 2.6 0.27 f
1.0 24 40.0 3.2 0.27 bc 1.9 0.45 cd
IAA
0.2 26 43.3 2.8 0.23 b 1.2 0.14 ab
0.4 34 56.7 3.4 0.31 cd 1.8 0.18 c
0.6 39 65.0 4.6 0.36 ef 2.5 0.23 ef
0.8 47 78.3 5.8 0.54 g 3.6 0.36 h
1.0 36 60.0 3.8 0.18 d 2.2 0.18 de
IBA
0.2 19 31.7 3.6 0.31 cd 2.0 0.18 cd
0.4 25 41.7 4.3 0.40 e 3.2 0.27 g
0.6 47 78.3 5.8 0.54 g 4.0 0.45 i
0.8 55 91.7 7.2 0.72 h 4.6 0.36 j
1.0 29 48.3 4.6 0.45 ef 3.3 0.27 gh
Table 4 Kanamycin sensitivity of nodal explants of W. somnifera on
MS medium containing BAP (1.5 mg/l) and IAA (0.3 mg/l)
Antibiotic
(kanamycin)
concentration
(mg/l)
Total
number of
explants
cultured
Number of
explants
producing
shoots
Explants
developing
shoots (%)
Total
number of
shoots
produced
0 60 54 90 705
50 60 42 70 466
100 60 30 50 342
150 60 25 41.7 275
200 60 14 23.3 132
250 60 5 8.3 25
300 60 0 0 0
Acta Physiol Plant
1 3
Author's personal copy
Fig. 3 Transformation of
Withania somnifera nodal
explants by Agrobacterium
tumefaciens strain EHA 105.
a Shoot bud initiation from
nodal explants [MS ? BAP
(1.5 mg l
-1
) ? IAA
(0.3 mg l
-1
) ? kanamycin
(300 mg l
-1
) ? cefotaxime
(300 mg l
-1
)]. b Non-
transformed plant (control).
c, d Proliferation of shoots
[MS ? BAP
(1.5 mg l
-1
) ? IAA
(0.3 mg l
-1
) ? kanamycin
(300 mg l
-1
) ? cefotaxime
(300 mg l
-1
)]. e Elongation of
shoots [MS ? GA
3
(1.0 mg l
-1
) ? kanamycin
(300 mg l
-1
) ? cefotaxime
(300 mg l
-1
)]. f Rooting of
elongated shoots [MS ? IBA
(0.8 mg l
-1
) ? kanamycin
(300 mg l
-1
) ? Cefotaxime
(300 mg l
-1
)]. g Hardening of
transformed plant
(sand:soil:vermiculite 1:1:1
v/v/v). h, i Transformed shoots
showing Gus expression after
4 weeks of culture on MS
medium containing BAP
(1.5 mg l
-1
) ? IAA
(0.3 mg l
-1
) ? kanamycin
(300 mg l
-1
) ? cefotaxime
(300 mg l
-1
)
Acta Physiol Plant
1 3
Author's personal copy
development (data not shown). The reduction of regener-
ation frequency caused by inoculation with bacterium may
be related to hypersensitive response of explants to bacte-
rial infection (Selvaraj 2002). Therefore, the transforma-
tion efciency is depending strongly on explants sources
and genotypes of the plant.
Molecular conrmation of transformants
To prove the integration of the foreign DNA into W.
somnifera genome, PCR amplication of the nptII gene
was performed. The size of the amplied band (0.69 kb)
indicates the integration of the nptII fragment. Molecular
analysis through PCR amplication conrmed the presence
of nptII gene in the putative transformants developed by
co-cultivation experiments. The DNA isolated from puta-
tive transformed shoots, control plants and plasmid
pGA492 was used as template DNA. The presence of
amplied bands at 0.69 kb in transformed shoots conrmed
the presence of the nptII gene (Fig. 4, lanes 39). The
amplication of the same band was also observed from the
lanes loaded with plasmid DNA pGA492 (Fig. 4, lane 1).
However, the amplication was not seen in non-trans-
formed control shoot (Fig. 4, lane 2).
Southern hybridization was performed with genomic
DNA isolated from putative transformants which was
digested with HindIII. The nptII DNA was detected as a
HindIII fragment of expected size (1.9 kb) in the shoots
transformed with EHA105. The nptII DNA fragment
probes hybridized with digested DNA from transgenic
shoots (Fig. 5, lanes 310), but they did not digest DNA
from non-transformed control leaves and no hybridization
signals were detected (Fig. 5, lane 2). In Fig. 5, lane 1
indicated plasmid DNA suggesting that the vector DNA
carried nptII fragments which cleaved under digestion with
HindIII. The size of the bands from HindIII digested plant
DNA identied with the nptII probes conrmed the inte-
gration of the nptII gene into the plant nuclear genome. The
Fig. 4 PCR conrmations of
transformed shoots. Arrow
indicates the amplication of
nptII gene (0.69 kb). M Marker
(100 bp ladder). Lane 1 Plasmid
DNA of vector pGA492
(positive control). Lane 2 DNA
sample from non-transformed
control shoot. Lanes 39 DNA
samples from transformed
shoots
Fig. 5 Southern hybridization. Arrow indicates the presence of npt II
gene (1.9 kb). Lane 1 Plasmid DNA (pGA492) undigested. Lane 2
DNA sample from non-transformed control shoot. Lanes 310 DNA
samples from transformed shoots
Acta Physiol Plant
1 3
Author's personal copy
number and intensity of the bands of lanes 4, 8, 9 and 10 in
Fig. 5 indicated that the transgenic shoot carries integrated
nptII gene at one site, but the lanes 3, 5, 6 and 7 showed the
multiple insertion of nptII gene in W. somnifera genome.
The kanamycin selection with the PCR and southern blot
results provides strong evidence for the transgenic status of
the W. somnifera. All plants that exhibited GUS positive
based on histochemical staining revealed the expected nptII
gene fragment in southern blot analysis and it conrmed
that the nptII was stably integrated with plant genome. The
result indicates that nodal explant of W. somnifera is an
excellent source of AMGT.
AMGT protocol assists the genetic improvement of W.
somnifera to create new variety with desirable properties.
The present study has established that the optimization of
culture parameters may enhance AMGT to produce valu-
able compounds in W. somnifera. Up to now, this may be
the rst report on transformation studies of W. somnifera
using nodal explants by Agrobacterium strain EHA105
with kanamycin (nptII gene) as a selectable marker in
combination with other marker bar gene and GUS reporter
gene. W. somnifera and Solanum surattense, the two
medicinally important members of family Solanaceae,
were reported for induction of hairy roots using A. rhiz-
ogenes and the transformed roots were used as a source of
secondary metabolites (Pawar and Maheshwari 2004).
Transgenic hairy roots were induced from W. somnifera by
infecting leaf explants with A. rhizogenes and reported the
presence of antioxidant principles in genetically modied
roots (Kumar et al. 2005). So, the AMGT is the most
commonly used method for plant genetic transformation
because of relatively high efciency. The advantages of
using AMGT over other transformation methods are the
reduction in transgene copy number and intact stable
integration of the transgene into the plant genome (Jones
et al. 2005). The development of transformation procedures
based on A. tumefaciens-mediated gene transfer for
medicinally important species like W. somnifera is advis-
able to produce transgenic plants with more production of
therapeutically important phytocompounds. In the present
study, an efcient, rapid and simple AMGT protocol was
developed for W. somnifera using in vitro-derived nodal
explants. In summary, an efcient system of genetic
transformation and plant regeneration in W. somnifera was
established.
Conclusion
In conclusion, the present protocol produced more number
of transgenic shoots directly from the nodal explants of W.
somnifera within a short period. The transformation pro-
tocol developed in this study is a simple, reliable and an
efcient system of AMGT and plant regeneration using
nodal explants of W. somnifera with nptII gene as a
selectable marker. So, the transformation protocol of this
study will be helpful to produce transgenic plants of W.
somnifera with a desirable character gene in a short period.
Author contribution This work was performed through
international collaboration between seven authors. R. Uda-
yakumar performed experiments, statistical analysis and
wrote the initial draft of the manuscript. S. Kasthurirengan,
T.S. Mariashibu, J.J. Sahaya Rayan provided all the tech-
nical support and assistance. A. Ganapathi as a co-corre-
sponding author designed the study and leads this project.
J.J. Kim and S.C. Kim guided the rst authors experiment
and analyzed the data. C.W. Choi as a co-corresponding
author funded a grant and made the nal draft of the man-
uscript. All authors read and approved the nal manuscript.
Acknowledgments This work was supported by the facility from
Bharathidasan University, (CSIR-Govt. of India) India and Pai Chai
University, Korea and by the grant (111074-3) from Bio-industry
Technology Development Program of Ministry for Food, Agriculture,
Forestry and Fisheries, Republic of Korea through the IPET.
Conict of interest Authors have declared that no competing
interests exist.
References
Abhyankar GA, Chinchanikar GS (1996) Response of Withania
somnifera (L.) Dunal leaf explants in vitro. Phytomorphol
46:249252
Baburaj S, Gunasekaran K (1994) Regeneration of plants from leaf
callus cultures of Solanum pseudocapsicum L. Ind J Expl Biol
32:141143
Bandyopadhyay M, Jha S, David T (2007) Changes in morphological
phenotypes and withanolide composition of Ri-transformed roots
of Withania somnifera. Plant Cell Rep 26:599609
Bernatsky R, Tanksley SD (1986) Toward a saturated linkage map in
tomato based on isozymes and random cDNA sequences.
Genetics 112:887898
Budhiraja RD, Sudhir S (1987) Review of biological activity of
withanolides. J Sci Ind Res 42:488491
Cao X, Liu Q, Rowland LJ, Hammerschlag FA (1998) GUS
expression in blueberry (Vaccinium spp.): factors inuencing
Agrobacterium mediated gene transfer efciency. Plant Cell Rep
18:266270
Chaudhury D, Madanpotra S, Jaiwal R, Saini R, Ananda Kumar P,
Jaiwal PK (2007) Agrobacterium tumefaciens-mediated high
frequency genetic transformation of an Indian cowpea (Vigna
unguiculata L. Walp.) cultivar and transmission of transgenes
into progeny. Plant Sci 172:692700
Cucu N, Gabriela P, Gavrila L (2002) Genetically modied medicinal
plants. II. Transfer and expression of a marker kanamycin
resistance gene in Atropa belladonna plants. Rom Biotechnol
Lett 7:869874
De Bondt A, Eggermont K, Penninckx I, Goderis I, Broekaut WF
(1996) Agrobacterium mediated transformation of apple (Malus
domestica Borkh): an assessment of factors affecting regenera-
tion of transgenic plants. Plant Cell Rep 15:549554
Acta Physiol Plant
1 3
Author's personal copy
Herrera-Estrella L, Simpson J, Martinez-Trujillo M (2005) Trans-
genic plants: an historical perspective. Methods Mol Biol
286:332
Hiei Y, Komari T, Kubo T (1997) Transformation of rice mediated by
Agrobacterium tumefaciens. Plant Mol Biol 35:205218
Ishnava K, Patel T, Chauhan JB (2012) Study of genetic transforma-
tion of medicinal plants, Withania somnifera (L.) Dunal by
Agrobacterium tumefaciens (MTCC-431). Asian J Exp Biol Sci
3(3):536542
Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusion: b-
glucuronidase as a sensitive and versatile gene fusion marker in
higher plants. EMBO J 6:39013907
Jones DH, Doherty A, Wu H (2005) Review of methodologies and a
protocol for the Agrobacterium-mediated transformation of
wheat. Plant Methods 1:5
Kanungo S, Sahoo SL (2011) Direct organogenesis of Withania
somnifera L. from apical bud. Int Res J Biotechnol 2:5861
Kondo T, Hasegawa H, Suzuki M (2000) Transformation and
regeneration in garlic (Allium sativum L.) by Agrobacterium
mediated gene transfer. Plant Cell Rep 19:989993
Kulkarni AA, Thengane SR, Krishnamurthy KV (1996) Direct
in vitro regeneration of leaf explants of Withania somnifera
(L.). Plant Sci (Limerick) 119:12
Kulkarni AA, Thengane SR, Krishnamurthy KV (2000) Direct shoot
regeneration from node, internode, hypocotyls and embryo
explants of Withania somnifera. Plant Cell Tiss Org Cult
62:203209
Kumar V, Chidambara Murthy KN, Bhamid S, Sudha CG, Ravi-
shankar GA (2005) Genetically modied hairy roots of Withania
somnifera Dunal: a potent source of rejuvenating principles.
Rejuvenation Res 8(1):3745
Kumar OA, Jyothirmayee G, Tata SS (2011) Multiple shoot
regeneration from nodal explants of Ashwagandha (L.) Dunal
Withania somnifera. Asian J Exp Biol Sci 2:636640
Lee S-H, Lee D-G, Woo H-S, Lee K-W, Kim D-H, Kwak S-S, Kim
J-S, Kim H, Ahsan N, Choi MS, Yang J-K, Lee B-H (2006)
Production of transgenic orchardgrass via Agrobacterium-med-
iated transformation of seed-derived callus tissues. Plant Sci
171:408414
Mishra LC, Singh BB, Dagenais S (2000) Scientic basis for the
therapeutic use of Withania somnifera (Ashwagandha): a review.
Altern Med Rev 5:334346
Mishra S, Sangwan RS, Bansal S, Sangwan NS (2013) Efcient
genetic transformation of Withania coagulans (Stocks)
Dunal mediated by Agrobacterium tumefaciens from leaf
explants of in vitro multiple shoot culture. Protoplasma
250(2):451458
Murashige T, Skoog F (1962) A revised medium for rapid growth and
bioassays with tobacco tissue culture. Physiol Plant 15:73497
Murthy HN, Dijkstra C, Anthony P, White DA, Davey MR, Power
JB, Hahn EJ, Paek KY (2008) Establishment of Withania
somnifera hairy root cultures for the production of Withanolide
A. J Integr Plant Biol 50:975981
Nayak SA, Kumar S, Satapathy K, Moharana A, Behera B, Barik DP,
Naik SK (2013) In vitro plant regeneration from cotyledonary
nodes of Withania somnifera (L.) Dunal and assessment of
clonal delity using RAPD and ISSR markers. Acta Physiol
Plant 35:195203
Pandey V, Misra P, Chaturvedi P, Mishra MK, Trivedi PK, Tuli R
(2010) Agrobacterium tumefaciens-mediated transformation of
Withania somnifera (L.) Dunal: an important medicinal plant.
Plant Cell Rep 29:133141
Pawar PK, Maheshwari VL (2004) Agrobacterium rhizogenes med-
iated hairy root induction in two medicinally important members
of family Solanaceae. Indian J Biotechnol 3(3):414417
Pawar PK, Teli NP, Bhalsing SR, Maheswari VL (2001) Microprop-
agation and organogenic studies in Withania somnifera (L.)
Dunal. J Plant Biol 28:217221
Rani G, Grover IS (1999) In vitro callus induction and regeneration
studies in Withania somnifera. Plant Cell Tiss Org Cult 57:2327
Ray S, Jha S (1999) Withanolide synthesis in cultures of Withania
somnifera transformed with Agrobacterium tumefaciens. Plant
Sci 146:17
Ray S, Ghosh B, Sen S, Jha S (1996) Withanolide production by root
cultures of Withania somnifera transformed with Agrobacterium
rhizogenes. Planta Med 62:571573
Saini R, Jaiwai PK (2005) Efcient transformation of a recalcitrant
grain legume Vigna mungo L. Hepper via Agrobacterium-
mediated gene transfer into shoot apical meristem cultures. Plant
Cell Rep 24:164171
Sangwan RS, Chaurasiya ND, Lal P, Misra L, Uniyal GC, Tuli R,
Sangwan NS (2007) Withanolide A biogeneration in in vitro
shoot cultures of ashwagandha (Withania somnifera DUNAL), a
main medicinal plant in Ayurveda. Chem Pharm Bull (Tokyo)
55:13711375
Selvaraj N (2002) In vitro culture and Agrobacterium-mediated
genetic transformation in cucumber (Cucumis sativus L.). Ph.D
Thesis, Bharathidasan University, Tiruchirappalli, Tamilnadu,
India
Soniya EV, Das MR (2002) In vitro organogenesis and genetic
transformation in popular Cucumis sativus L. through Agrobac-
terium tumefaciens. Ind J Expl Biol 40:329333
Southern EM (1975) Detection of specic sequences among DNA
fragments separated by gel electrophoresis. J Mol Biol
98:503517
Tabei Y, Kanno T, Nishio T (1991) Regulation of organogenesis and
somatic embryogenesis by auxin in melon Cucumis melo (L.).
Plant Cell Rep 10:225229
Tripathi AK, Shukla YN, Sushilkumar T (1996) Ashwagandha
[Withania somnifera (L.) Dunal (Solanaceae)]: a status report.
J Med Arom Plant Sci 18:4662
Tsukazaki H, Kuginuki Y, Aida R, Suzuki T (2002) Agrobacterium-
mediated transformation of a double haploid line of cabbage.
Plant Cell Rep 21:257262
Udayakumar R, Kasthurirengan S, Mariashibu TS, Rajesh M, Ramesh
Anbazhagan V, Kim SC, Ganapathi A, Choi CW (2009)
Hypoglycaemic and hypolipidaemic effects of Withania somnif-
era root and leaf extracts on alloxan-induced diabetic rats. Int J
Mol Sci 10:23672382
Udayakumar R, Kasthurirengan S, Mariashibu TS, Sahaya Rayan JJ,
Kim SC, Choi CW, Ganapathi A (2010a) Antioxidant activity of
phenolic compounds extracted from the roots and leaves of
Withania somnifera (L.) from different geographical locations in
India. Func Plant Sci Biotech 4:2833
Udayakumar R, Kasthurirengan S, Vasudevan A, Mariashibu TS,
Sahaya Rayan JJ, Choi CW, Ganapathi A, Kim SC (2010b)
Antioxidant effect of dietary supplement Withania somnifera L.
reduce blood glucose levels in alloxan-induced diabetic rats.
Plant Foods Hum Nutr 65:9198
Udayakumar R, Choi CW, Kim KT, Kim SC, Kasthurirengan S,
Mariashibu TS, Sahaya Rayan JJ, Ganapathi A (2013) In vitro
plant regeneration from epicotyl explant of Withania somnifera
(L.) Dunal. J Med Plants Res 7:4352
Uma Devi P, Sharada AC, Solomon FE (1993) Anti-tumor and
radiosensitizing effects of Withania somnifera (Ashwagandha)
on a transplantable mouse tumor sarcoma 180. Ind J Exp Biol
31:607611
Vasudevan A, Selvaraj N, Ganapathi A, Choi CW (2007) Agrobac-
terium-mediated Genetic Transformation in Cucumber (Cucumis
sativus L.). Am J Biochem Biotechnol 3(1):2432
Acta Physiol Plant
1 3
Author's personal copy